diff --git "a/deduped/dedup_0273.jsonl" "b/deduped/dedup_0273.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0273.jsonl" @@ -0,0 +1,45 @@ +{"text": "Anopheles arabiensis for selected houses in the Lower Moshi irrigation scheme.Malaria control measures were initiated from in October 2005 to August 2006 in the Lower Moshi irrigation schemes, Tanzania. This manuscript reports on the entomological evaluation of the impact of pyrethroid-treated cattle in reducing the population of the Cattle were sprayed with the pyrethroid (deltamethrin) acaricide. Grazing and non-grazing cattles were compared and assessed for difference in knockdown resistance (kdr) time using cone or contact bioassay and residual effect . In experimental huts, mortality was compared between the huts with treated and untreated cattle.An. arabiensis, which are conducive for zooprophylaxis.Results from contact bioassays of cattle treated with deltamethrin showed a knockdown effect of 50% within 21 days for grazing cattle and 29 days for non-grazing cattle. Residual effect at 50% was achieved within 17 days for grazing cattle compared to 24 days for inshed cattle. In discussing the results, reference has been made to the exophilic and zoophilic tendencies of An. arabiensis and Culex spp collected from huts with different baits, i e. human, untreated cow and treated cow. Results indicate higher mortality rates in mosquitoes collected from the hut containing the treated cow (mean = 2) compared to huts with untreated cow (mean = 0.3) and human (mean = 0.8). A significantly higher number of Culex spp. was recorded in huts with treated cows compared to the rest.Experimental studies in Verandah huts at Mabogini compared An. arabiensis population, cattle should be placed close to dwelling houses in order to maximize the effects of zooprophylaxis. Protective effects of cattle can further be enhanced by regular treatment with pyrethroids at least every three weeks. This paper demonstrates that cattle can be considered as Insecticide-Treated Material (ITM) as long as acaricide treatment is conducted regularly.This study has demonstrated the role of cattle treated with pyrethroid in the control of malaria and reduction of vector density. It showed that, in areas with a predominant Plasmodium falciparum is predominant, accounting for more than 25% of childhood mortality outside the neonatal period[The greatest impact of malaria in terms of morbidity and mortality is in sub-Saharan African, where al period. Recent al period. In manyal period. It is eCurrent mosquito control methods rely heavily on the use of insecticides through larviciding, residual house spraying, insecticide-treated nets and other personal protection methods ,7. This Anopheles arabiensis [There is, therefore, an urgent need to look at novel techniques, which will complement the existing strategies; one of such method is the use of zooprophylaxis in areas where large numbers of livestock exist and where is the predominant vector of abiensis An. arabiensis on treated cow by using contact or cone bioassay, (ii) The behavioural aspect of mosquitoes on treated cows as compared to mosquitoes on humans and untreated cows.In this study the effect of deltamethrine applied on cattle, on the target mosquito species and on natural mosquito species were thoroughly assessed. The acaricide used was a deltamethrin formulation, commonly used to control tsetse and ticks in the study area. This study focused on two aspects in particular: (i) the knockdown and residual effect of An. arabiensis, by means of killing rather than simply diverting host-seeking mosquitoes in areas with a high population of An. arabiensis.The study observed that in-shade treated animals have higher protection than treated grazing animals. This study assessed the impact of insecticide-treated cattle with pyrethroids in reducing the population of the This study was carried out in Lower Moshi area, Kilimanjaro region, Northern Tanzania. The average population per village was 2,842,12. LoweA total of eight cows were selected, half of them were left untreated (control) and the rest were treated with deltamethrin, the normal practice of cattle protection against ticks and tsetse flies. Of the four cattle in each group, two were kept under shelter at all times (in-shed), while the other two were grazed outdoors during the day as per recommendation of Hewitt and Rowland .To avoid possible insecticide contamination, the experimental sites for untreated and treated cattle were approximately one km from each other and during the day time they were grazed separately. Residual insecticide on the animals was carried out by contact bioassay method as per WHO guidelines .An. arabiensis mosquitoes were exposed on the treated and untreated animals (control) for three minutes and then transferred into a clean paper cup containing a piece of cotton soaked in 10% sugar solution as food source. Immediate (KDR) and delayed mortality rates were recorded after one hour and 24 hours respectively.Five unfed, 2\u20135 days old An. arabiensis was also assessed in the experimental huts as follows: the treated cow, human and untreated cow were rotated according to a latin square design in three experimental huts in order to minimize the influencing factors such as variation in wind direction, collector ability and mosquitoes relative abundance. One cow was treated with deltamethrin according to the community normal practice and as per recommendations of the manufacturer . Before sunset, the test calves (one year old) were tethered, one in each hut and volunteer sleeper in another hut. All verandahs were left open in the evening at 6.00 pm to allow mosquitoes to enter into the hut. The verandahs were closed in the morning at 5.00 a.m in order to prevent mosquitoes from escaping. Mosquitoes were collected from the verandah and inside the hut using an aspirator. Collected mosquitoes were sorted and recorded according to their species [The effect of cattle treated with pyrethroids on species and abdoThe percentage of blood-fed, repellence and mortality were calculated according to the following formulae:-2 and P value were calculated.The data entry was done in Microsoft Excel (2000) and analysis was carried out using statistical package for social science (SPSS) version 10 program. The significance test was estimated assuming an \u03b1 (two sided) = 0.05). Other data were analysed by using EpiInfo\u2122 Version 3.2.2 programme where \u03c7Before conducting this study, ethical clearance was sought from the Kilimanjaro Christian Medical College Research Ethics Committee. Permission from the district and respective village authorities was obtained also. Both verbal and written informed consent was obtained from the head of the household of the respective households that were selected for the study. Antimalaria drugs were kept at the field station for emergency purposes, although luckily during the course of the experiment none of the sleepers contacted malaria. Among households where field experiments were conducted, the mosquito density was reduced by the pyrethrum-spraying catches. Customs and norms of the respective community at the study area were maintained and respected.An. arabiensis mosquitoes were tested on treated cows. The insecticide was found to be effective for almost a month giving more than 50% knockdown at three weeks post-treatment for grazing animals, and four weeks for in-shed animals and mortality for in-shed and grazing cows were recorded and summarized as shown in Figures s Figure . Also ths Figure 2 = 8.9, P = 0.003), and , respectively. Similarly, the chi-square in 24 hrs mortality rate for in-shed cows was , while that of grazing animal it was .The chi-square and P value for KDR and mortality rate of mosquitoes on the treated cows were calculated Table . Both KDCulex spp. mortalities were recorded in the experimental huts with treated cows compared with the rest.Higher mortality rates were observed in mosquitoes collected from the experimental hut containing treated cows (mean = 2) compared to untreated cows (mean = 0.3) and humans (mean = 0.8). However, no statistical significant difference was found in mortality in the hut with treated cows and that with untreated cows (P > 0.05). Significantly higher An. arabiensis, both through repellency and mortality. The same observation had previously been made by others [An. arabiensis re-treatment must be done after 21 days for grazing cattle and 29 days for in-shed cattle. In order to achieve 50% mortality , retreatment must be done before 17 days for grazing cattle and 25 days for in-shed cattle [An. arabiensis population because this insect has an overlapping distribution [The results from field and experimental huts have demonstrated that cattle treated with the acaricide deltamethrin can offer protection of humans against y others -19. The d cattle . The effd cattle ,16,21-23ribution ,24-27 heAn. arabiensis and Culex spp, as had been described elsewhere [Result from the experimental huts where untreated cows, acaricide-treated cows with and humans were rotated also indicates a slightly higher performance of the treated cows particularly in terms of mortality rates on lsewhere ,21,22,28An. arabiensis mosquito population can be reduced by zooprophylaxis. Cattle treated with pyrethroid acaricides will not simply divert host-seeking mosquitoes from man, but will also cause mortality of more than half of them. In this way, the negative impact of zooprophylaxis, whereby mosquito densities may increase due the presence of a readily available blood meal source, can be minimized. Cattle also serve as dead-end hosts since malaria parasites cannot develop in their red blood cells.The present studies have shed some more light on the positive impact of pyrethroid-treated cattle in reducing man-vector contact. Therefore, malaria transmission in areas with a predominant In pastoral communities, where dwelling areas are shared with cattle, routine application of acaricides on the animals for tick and tsetse control may also serve as an alternative method to Insecticide-Treated Materials (ITMs).The author(s) declare that they have no competing interests.AMM and FWM conceived and designed the study, participated in analysis and interpretation of data and contributed to the drafting of the manuscript. JMM carried out the analyses of mosquitoes assisted with data analysis and interpretation and were involved in the drafting of the manuscript. EJK designed and coordinated the study, participated in the analysis and interpretation of results, and was involved it the drafting of the manuscript and critical evaluation thereof. All authors read and approved the manuscript."} +{"text": "Knowledge of how mosquitoes respond to insecticides is of paramount importance in understanding how an insecticide functions to prevent disease transmission. A suite of laboratory assays was used to quantitatively characterize mosquito responses to toxic, contact irritant, and non-contact spatial repellent actions of standard insecticides. Highly replicated tests of these compounds over a range of concentrations proved that all were toxic, some were contact irritants, and even fewer were non-contact repellents. Of many chemicals tested, three were selected for testing in experimental huts to confirm that chemical actions documented in laboratory tests are also expressed in the field. The laboratory tests showed the primary action of DDT is repellent, alphacypermethrin is irritant, and dieldrin is only toxic. These tests were followed with hut studies in Thailand against marked-released populations. DDT exhibited a highly protective level of repellency that kept mosquitoes outside of huts. Alphacypermethrin did not keep mosquitoes out, but its strong irritant action caused them to prematurely exit the treated house. Dieldrin was highly toxic but showed no irritant or repellent action. Based on the combination of laboratory and confirmatory field data, we propose a new paradigm for classifying chemicals used for vector control according to how the chemicals actually function to prevent disease transmission inside houses. The new classification scheme will characterize chemicals on the basis of spatial repellent, contact irritant and toxic actions. Science and society label almost any chemical used against insects as an \u201cinsecticide.\u201d By definition, an insecticide is a chemical that kills insects. This single term is not adequate for meaningful discourse about chemicals, chemical actions, insect responses to chemicals, and the different ways in which chemicals are used. However, this single response is the foundation for the old paradigm that classifies chemicals sprayed on house walls for malaria control based solely on their killing action. A new paradigm is needed to take into account the behavioral actions of these chemicals in disrupting man-vector contact and thereby breaking disease transmission. The fact that repellent and irritant actions were first documented more than 60 years ago Over 45 years ago Dethier Aedes aegypti mosquitoes as a model system. Although Ae. aegypti does not transmit malaria, it is responsible for transmitting dengue and yellow fever viruses in urban environments. This species was selected as our model system because of its medical importance and because eggs can be stored dry and used when needed for producing test populations. Additionally, new colonies are easily established by bringing wild caught material from the field.The overall aim of this research was to quantify and accurately describe chemical actions and mosquito responses to those actions using We used a suite of assays to quantitatively characterize mosquito responses to toxic, contact irritant, and non-contact spatial repellent actions of insecticides Thresholds exist for when and how insects respond to these chemical actions. These thresholds are governed by intrinsic and extrinsic factors such as inherent strength of a chemical action, chemical volatility, environmental temperature, humidity, proximity and length of exposure, and a mosquito's sensitivity to a compound, to name just a few factors. The dose dependent order in which thresholds are exceeded determines whether the primary mode of chemical action is repellent, irritant or toxicant. Research described here will show that house wall residues of three important and commonly used insecticides elicit varying combinations of behavioral and toxic actions. Based on these results, we are proposing criteria for revising classifications of chemicals that are presently recommended for use in malaria control programs. This revised classification scheme is a new paradigm for disease control that emphasizes a single or the combination of multiple chemical actions to control disease transmission by breaking man-vector contact. This new paradigm will permit chemists, toxicologists, and public health scientists to discuss and characterize chemicals according to their true modes of action.1 to F3). A colony of Aedes aegypti was maintained and renewed every 6 months with field populations from Thailand. The field populations were collected as larvae from Pateuy Village, Saiyok District, Kanchanaburi Province, western Thailand . Baseline toxicity data revealed this population to be highly resistant to DDT, tolerant to alphacypermethrin and susceptible to dieldrin.Past research suggests that behavioral responses of extensively colonized mosquitoes to chemicals are diminished or non-existent. Therefore, we conducted all laboratory tests with female mosquitoes only recently brought from the field was toxic but had no repellent or irritant actions. Another had irritant and toxic actions; but had no repellent action. The third chemical (DDT) exhibited all three actions; repellency, irritancy, and toxicity.2) using a micropipette, resulting in treatment concentrations of 0.25, 2.5, 25, and 250 nmol/cm2. All netting, both control and treatment, were allowed to sit for at least 30 minutes to ensure that the acetone completely evaporated from the netting leaving only the chemical of interested (treatment) or the clean netting (control).The test compounds were DDT -2,2,2-trichloroethane, 98%)(Sigma-Aldrich), alphacypermethrin (a racemate comprising (S) alpha-cyano-3-phenoxybenzyl -3- 2,2-dimethyl cyclopropane carboxylate and (R) alpha-cyano-3-phenoxybenzyl -3- 2,2-dimethyl cyclopropane carboxylate)(BASF) and dieldrin (Sigma-Aldrich). Chemical solutions using an acetone solvent (1.5 ml) were applied evenly to nylon organdy netting strips (330 cm2 (0.03 g/m2 recommended use equates to 7.2 nmoles/cm2), DDT at 500 nmoles/cm2 (2.0 g/m2 recommended use equates to 564.2 nmoles/cm2) and dieldrin at 25 nmoles/cm2 (0.1 g/m2). Sheets of polyester netting (1 m\u00d73 m) with mesh size of 24\u00d720/inch were treated. Individual sheets of netting were saturated with treatment solution and excess solvent was allowed to evaporate.We applied each compound in hut treatments at a dose that closely approximated the WHO recommended field application rate. In this case we applied alphacypermethrin at 2.5 nmoles/cmA suite of assays was deveAssay is composed of a metal chamber that houses the netting which is connected to a clear receiving chamber. The two ends of the assay are separated by a beveled divider containing a butterfly valued gate. Ten mosquitoes were transferred into the treatment end of the assembly and, after 30 sec, the butterfly valve was opened. After 10 min, the valve was again closed, and counts were immediately made of the number of mosquitoes in the clear end (number escaping) and those remaining in the treatment end. Those knocked down were also recorded. For every two trials a control assay was run, in which the acetone-treated net was used in place of the insecticide treated one. Six replicates were performed at each treatment concentration.This assay consists of three chambers connected in unison. At one end is a treatment chamber and at the other end is a control chamber. Treatment and control chambers are connected to each other by a clear cylinder to form the complete spatial repellency assay assembly. Twenty mosquitoes were transferred into the clear chamber, and the assay covered. After a 30 sec resting period, the butterfly valves were opened. After 10 min, the valves were closed and the number of mosquitoes in each chamber was counted as well as the numbers knocked down. Between replicates, the assay is disassembled to allow volatilized chemical to be ventilated from the chamber. Nine replicates were performed for each treatment concentration.A single chamber is used as an exposure chamber to evaluate a chemical's toxicity much in the same way as the bottle assay 1 populations of Ae. aegypti in Thailand. These mosquitoes were 5\u20137 day old, mated females that were only provided a sugar meal prior to use in the field studies. These conditions were identical to those mosquitoes used in the laboratory assay. The goal was to confirm that the orderly sequence of chemical actions identified in laboratory tests would actually occur with natural mosquito population under field conditions. Two portable huts were constructed for evaluating entering and exiting behavior of Ae. aegypti. The huts were constructed in the fashion of indigenous Thai homes with wood walls and corrugated tin roof and were positioned 100 m from each other. The dimensions of the huts were 4 m wide\u00d75 m long\u00d73.5 m high with three windows and one door onto which could be affixed entrance and exit traps. Floors were adjusted and aligned with cement blocks and covered with a white sheet for detecting mosquitoes on the floor that had been knocked down. A series of aluminum panels were developed for holding treated netting which could be positioned around the interior surface of the hut. Each panel has a backing of aluminum wire mesh that prevents the netting from making contact with the hut wall.The field studies with experimental huts were conducted against FHuts were fitted with window and door traps that were positioned inside to capture entering mosquitoes. Two pools of 100 mosquitoes were placed into two separate 1-gallon cardboard containers topped with mesh netting. One container was used for the treatment population and the other contained a control population. Populations were marked with luminous marking powder using a \u00bc in. paintbrush. The paintbrush was loaded with powder then quickly brushed against the mesh netting of the container lid in a circular motion from the outside circumference to the inside center of the container. Marked mosquitoes (100 per hut) were released 10 meters outside of each hut at 0540 hr just prior to sunrise and collections were made from the traps at 20 min intervals, from 0600\u20131800 h. Two collectors were positioned in each hut immediately after mosquitoes were released. All collected mosquitoes were placed in plastic cups and were labeled with time and location of each trap.Huts were armed with window and door traps placed outside to capture exiting mosquitoes. Marked mosquitoes (100 per hut) were released inside at 0540 hr. A human host entered an untreated bed net in each hut immediately after marked females were released indoors. All mosquitoes collected from the traps were placed in holding cups labeled by time and location. Removal of mosquitoes from the traps was made by collectors located outside the huts. At the top of each hour, the collectors located on the inside of the hut exited the bed net and searched the floor for knocked down mosquitoes. All cups were provided a 10% sugar soaked cotton pad and were checked after 24 hr to record mortality. Additional cups of 25 marked mosquitoes with moist sugar pads were placed in both huts as controls.P\u200a=\u200a0.05 Contact irritancy assay data were analyzed using the Wilcoxon two-sample test cN\u2212tN)/(cN+ tN), in which cN was the number of females in the control chamber of the spatial repellency assay assembly and tN was the number of females in the treated chamber. The SAI was a measure of the proportion of females in the control chamber over the treated chamber after correcting for the proportion of females in the control chamber. The SAI varies from \u22121 to 1, with 0 indicating no response. An SAI value of \u22121 indicated that a greater proportion of mosquitoes moved into the treatment chamber than the control chamber thus indicating an attractant response. An SAI value of 1 indicated a greater proportion of mosquitoes moved into the control chamber (away from the treatment end of the assay device) indicating a repellent action.A spatial activity index (SAI), based upon the oviposition activity index of Kramer and Mulla Data from the field studies revealed that when releasing a fixed population, there are diminishing returns on the probability of recapture as higher numbers of marked mosquitoes are removed from the pool of potential responders. Therefore appropriate analysis of time-trend data of exiting mosquitoes from houses require adjustments for the numbers of mosquitoes capable of responding at a given time x. That is to say, the first half of the day is the richest period for showing the influence of chemical actions. The strength of the data declines as mosquitoes are removed from the population through recapture. Furthermore, in evaluating a contact irritant response the time when escape occurs is as important as the numbers that escape. The faster a mosquito escapes, the less is its chance of making lethal contact with a treated surface. The numbers that are knocked down in the hut must also be removed from total numbers in huts. Therefore, we focus our analysis on the first 7 hours and remove the numbers that are knocked down before they can escape.Ae. aegypti females escaping from treatment chambers was proportional to the dose of insecticide used. In general, mean number and corrected percent escaping from treated chambers increased with increasing concentrations of the chemical treatment (P<0.05) contact irritancy response to alphacypermethrin was observed at treatment concentrations of 0.25 nmoles/cm2 and higher. In other words, alphacypermethrin functioned as a contact irritant at all test concentrations. DDT produced significant contact irritancy responses at concentrations of 2.5 nmoles/cm2 and higher (Our findings from laboratory tests for contact irritancy showed that the percent of reatment . A signid higher . Dieldri2 (P\u200a=\u200a0.0010), 25 nmoles/cm2 (P\u200a=\u200a0.0005) and 250 nmoles/cm2 (P\u200a=\u200a0.0039). To more accurately express the intensity of the directional movement, a weighted spatial activity index was calculated by factoring in the percent responding. These weighted values showed an increased response to increasing doses of DDT as well as directional movement away from the treated cylinder. With alphacypermethrin, there was no statistical significance in directional movement or increase in response with increasing concentrations . On the other hand, DDT showed very little if any knockdown (1\u20132%) at all test concentrations, and the knockdown that did occur was due to handling as exhibited by mortality in control populations. Low mortality was recorded for DDT at the highest concentration of 250 nmoles/cm2 of knockdown at all treatment concentrations after a one hour exposure . The two 24 hrs) .Baseline studies conducted prior to the addition of chemical to the interior of the huts demonstrated high between day variance and low same day variance . High between day variance is attributed to differences in meteorological conditions that occurred from one day to the next. For this reason the treatment huts were evaluated day by day against their matched control. These critical findings emphasize the need for conducting extensive baseline studies through a range of environmental variables to establish movement patterns of the natural populations so that changes in these patterns can be taken into consideration upon introduction of chemical. These results also focused our attention on the need for studies that always include a control (untreated) hut paired with a treatment hut both temporally and spatially.P\u200a=\u200a0.05). Overall, of the 400 marked Ae. aegypti released at the DDT treated hut, 107 (27%) were recaptured entering the hut. In comparison, 259 (65%) of the 400 marked mosquitoes released at the control hut entered the hut. This equates to a 59% reduction in numbers entering the DDT treated hut compared to the control hut. In contrast, there were no significant differences in numbers entering the alphacypermethrin treated hut compared to the paired control hut (P\u200a=\u200a0.24). Actual values were 198 (50%) recaptured entering the alphacypermethrin treated hut compared to 153 (39%) entering the control hut. Dieldrin showed similar results in that there was no significant difference between the numbers of marked mosquitoes that were capture entering the treated hut as compared to the control hut. A total of 89 marked mosquitoes were collected entering the control hut as compared to 80 mosquitoes entering the treated hut. The slight reduction that was documented did not equate to a statistically significant difference and could be explained as normal background noise. The peak time of entering populations in both the control and treated huts occurred between 0800 and 0900 hrs for both the treated and the control huts and control huts .The time trend for the exiting populations from the DDT, alphacypermethrin and dieldrin huts with their matched controls can be seen in A total of 294 mosquitoes exited the DDT treated hut by 1200 hr with 12 knocked down inside the hut. During the first 7 hours, 207 exited the control hut, with 3 knocked down. This was an increase of 31% exiting over that observed in the control hut during the first half of the day. The results for alphacypermethrin were more dramatic. A total of 281 mosquitoes exited the alphacypermethrin hut by 1200 hr with an additional 51 mosquitoes collected in the hut as knockdown. A total of 146 marked mosquitoes were collected from the exit traps in the control huts in the first half of the collection with only 2 specimens on the floor (knocked down). This equated to a 55% increase in exiting mosquitoes by midday from the alphacypermethrin treated hut compared to the matched control hut. This same treatment of the data was not performed for dieldrin due to its extreme toxicity and lack of a behavior modifying action.After holding mosquitoes collected from the DDT hut exit traps for 24 hrs, a total of 251 remained alive compared to 278 remaining alive for the control. Of those that were on the floor (knocked down) inside the hut, only 1 (6%) remained alive. In the alphacypermethrin hut, 205 mosquitoes removed from the traps remained alive, compared to 212 that remained alive from the control traps. All the mosquitoes that were collected in the hut that were knocked down did not recover after 24 hrs .This study demonstrated that the impact of insecticides on vector populations is much more complex than just toxicity. In fact, our studies showed that, while the toxic effect of a chemical like dieldrin can have a dramatic effect on the immediate population density, it carries with it the chance for rapid build up of resistance. For this reason we feel that while toxicity has an immediate impact, the long term implications make it the least important of the chemical actions. This study clearly showed that the primary indicators of chemical actions in huts were proportions repelled , proportions stimulated to prematurely exit (contact irritancy), and proportions that died (toxicity). Estimates of proportions repelled were taken from hut entry data. For DDT, alphacypermethrin and dieldrin the proportions repelled were 59%, 0% and 0% respectively. Contact irritant actions were measured by numbers escaping into exit traps during the first 7 hours of observations. The proportions exiting were 31% (DDT), 55% and 0% (dieldrin). Toxicity was estimated from two parameters; number dead on the floor and number in exit traps that died after 24 hours. The two parameters for DDT were 5% dead on the floor, and 11% of those that escaped subsequently died. The same values for alphacypermethrin were 15% and 19%, respectively. These two parameters for dieldrin were much more dramatic at 75% dead on the floor, and 69% of those that escaped subsequently died. Numbers in exit traps that subsequently die must be separated from numbers that were irritated and exited prematurely. With no correction the mosquitoes in exit traps that died would be included in two different parameters. We address this by adding the number in exit traps that subsequently died from intoxication to the number dying from toxic actions inside the hut. Thus, toxicity included numbers that died after escaping the hut and numbers dead on the floor. To avoid inflating estimates by including mosquitoes in more than one measure, our estimate for contact irritant action included the number caught in exit traps minus the number that subsequently died.We defined composite impact of the two chemicals by assuming that a hundred mosquitoes would enter a house, bite while indoors, and escape and survive if the house were not sprayed. We can use our proportions, described above, to evaluate how spraying with one or the other chemical will impact the 100 mosquitoes.In huts sprayed with DDT, 59 of the 100 mosquitoes would not enter. Of the 41 that enter, 2 would die and fall to the floor. Of the 39 survivors, 12 would exit prematurely. One of the 12 mosquitoes that escaped would die within the next 24 hours. This leaves 27 mosquitoes that theoretically could bite and survive. However, it is important to understand that chemical is present in houses 24 hours each day, these statistics cover only 7 hours, not 24. These statistics suggest that DDT reduced risk from 100 mosquitoes by 73% within the first 7 hours.In huts sprayed with alphacypermethrin, all 100 mosquitoes would enter the house. Of the 100 that entered, 15 would die. Of the remaining 85, 46 would exit prematurely and 9 of those would die. This leaves 39 mosquitoes that theoretically could bite and survive. The spatial repellent, contact irritant, and toxic actions of alphacypermethrin sum to 61% protection.In huts sprayed with dieldrin, all 100 mosquitoes would enter the house. Of the 100 that enter, 75 would die before exiting. Of the 25 that exit, 17 would escape and subsequently die. This would leave 8 mosquitoes that could take a blood meal and survive for a summed 92% protection. While this may have an immediate impact on the population densities, it carries with it the potential for a quick build up of insecticide resistance thus rendering the chemical ineffective over time. While the failure to feed that occurs through repellency may also provide enough selection pressure to engender resistance, this phenomenon has never been documented and must be examined further.Ae. aegypti. However, our earlier research showed similar but even stronger chemical actions on malaria vector mosquitoes The data presented here are based on laboratory and field tests with populations of There has been no failure in understanding that DDT is by far the most cost-effective chemical yet discovered for sustained use in malaria control programs. However, there has been an enormous failure to accurately account for how DDT actually functions in control of malaria. As stated before, this failure has impeded the search for DDT alternatives. In a more general way, this encompasses both a failure to properly characterize and quantify the separate actions of chemicals and a failure to characterize and quantify the separate responses of vector mosquitoes to those chemical actions.The new classification scheme that we are proposing will characterize chemicals on the basis of spatial repellent, contact irritant and toxic actions. The first criterion for evaluating a chemical is the concentration at which the chemical exceeds a threshold for vector response. If mosquitoes are intoxicated at concentrations lower than that required for a behavioral response then toxicity supersedes other actions since the insect might be overcome before being stimulated through mechanisms of contact irritancy or spatial repellency. Likewise, if an irritant response occurs at a lower concentration of chemical than required for toxicity, then the irritant response precludes toxicity since the insect or some proportion of insects may move away from the chemical before acquiring a lethal dose. These relationships are even more pronounced for a spatial repellent action. If a spatial repellent response is stimulated by a lower or equal concentration of chemical than required for either contact irritancy or toxicity, then the insect or some proportion of insects will be repelled without making contact with the chemical. Thus the three chemical actions can be quantified according to proportional dose-response relationships and the relative rank order of actions can be defined.As described, the first criterion for rank ordering of chemical actions is the relative concentration of chemical required for a given response. If a significant level of toxicity, as found with dieldrin, is produced at lower concentrations than required for contact irritant or spatial repellent actions, then toxicity is the first order action. According to this definition, a first order action occurs at lowest concentration, second order action occurs at second lowest concentration, and third order occurs at third lowest concentration. That is to say, concentration for 1st order is 0.9), especially in the urban areas of Cotonou [et al in an experimental hut study showed a reduced efficacy of lambdacyhalothrin-treated nets against An. gambiae in Ladji, an outskirt area of Cotonou [In Benin, malaria is one of the most frequently recorded diseases in health centres. The incidence for both uncomplicated and complicated cases in 2006 was 139 per 1,000 inhabitants [d'Ivoire , Burkinad'Ivoire , South Ad'Ivoire and Camed'Ivoire . In Beni Cotonou . A recen Cotonou .Despite these reports on pyrethroid resistance in B\u00e9nin, the National Malaria Control Programme decided to undertake, in 2008, a distribution of LLINs and to implement IRS in the department of Ou\u00e9m\u00e9 particularly in the districts of S\u00e8m\u00e8-Kpodji, Dangbo, Miss\u00e9r\u00e9t\u00e9 and Adjohoun. The increasing emergence of resistance leads to an urgent need to investigate alternatives to pyrethroid insecticides and a coAn. gambiae and other culicinae breeding sites around the vegetable plots. Porto Novo is situated at 6. 33 N, 2.37 E in the southern part of Benin, about 30 kilometres from the Atlantic Ocean. The composition of An. gambiae s.l. is 100% An. gambiae s.s. M form the main malaria vector in this area. This vector is present all year-round and has developed a strong resistance to pyrethroid insecticides with a high frequency of kdr gene at 86.7% [The study was carried out in experimental huts located in Akron at the outskirt of Porto Novo, , a swampat 86.7% .The study was conducted in experimental huts Figure , which a2 and that of the ceiling (polyethylene), the entry slits, and the door was in total 53.13 ml/m2. The area of the walls to treat was 15 m2 and that of the roof, the doors, and the entry slits was 5.1 m2. To treat the walls of the huts, 1.7 L of water was used. For the rest of the huts, 270.8 ml of water was used. Using these measures, the five huts were treated according to WHO recommendations [A 10 cm wide moat filled with water surrounded each hut to prevent the entry of scavengers such as ants and spiders. Six identical huts were built at the station. Five huts were treated with insecticides using a backpack sprayer and the sixth was left untreated as a control. The absorption of the walls was 112 ml of insecticide per mndations ;2;Hut 1: Bendiocarb (800 g/kg) at 200 mg/m2;Hut 2: Deltamethrin (250 g/kg) at 25 mg/m2Hut 3: Alphacypermethrin (50 g/kg at 30 mg/m2Hut 4: Fenitrothion (400 g/kg) at 2 g/m2 and 25 mg/m2, respectivelyHut 5: Mixture of chlorpyriphos 250 g/L + deltamethrin 12 g/L at 560 mg/mThroughout the study, sleepers slept under untreated bed nets.An. gambiae: The wild populations of Akron area attracted to sleepers inside experimental huts and wild An. gambiae emerged from field collected larvae which were released into the experimental huts. This last sample of mosquitoes was released during the period where there was insignificant density of mosquitoes entering the huts. The released An. gambiae were collected as larvae from the study site, and reared at the same place, so that the tested mosquitoes are not different from the wild population entering the huts. On average 20-27 not-blood-fed females of An. gambiae were released three times a month at 20:00 hours for a total number of 60-80 An. gambiae per hut.The behaviour of mosquitoes in the presence of insecticidal treatments was analysed on two samples of Before the beginning of the evaluation, a blank collection of mosquitoes was carried out during two weeks in the experimental huts to compare the natural attractiveness between huts. Sleepers spent the same number of nights in each hut. The collections were done by six volunteer adult men, recruited by the CREC from the study area. The collectors were rotated between the huts, sleeping under a mosquito net from 21:00 hours to 06:00 hours. At 06:00 hours, mosquitoes were collected in the hut, using a mouth aspirator in the veranda and the hut room. By 08:00 in the morning, collection in huts was completed. All mosquitoes were put in netted plastic cups and transferred to the laboratory for identification. Mosquitoes were identified into species using Coluzzi key and reco- Deterrence rate: percentage of reduction in the number of mosquitoes caught in treated hut relative to the number caught in the control hut;- Exophily rate: percentage of mosquitoes that have escaped the hut and have taken refuge in the veranda trap divided by the total number of mosquitoes collected in the hut;- Blood-feeding rate: percentage of blood fed mosquitoes collected divided by the total of mosquitoes collected in verandah and hut;- Immediate mortality: percentage of dead mosquitoes collected in the morning compared to total mosquitoes collected in the hut;- Overall mortality: general mortality: immediate mortality + delayed mortality recorded after 24 h.Over the course of the study, interviews were conducted to identify any side effects of insecticide treatments on the collectors. These interviews were conducted at the end of the first and fourth month of collection.The statistical analysis was conducted using SPSS (Version 16.0). The effect of treatments was evaluated using analysis of variance (ANOVA) to compare treatments to the control.The present study received a formal approval from the Ministry of Health of Benin and the Entomological Research Centre of Cotonou (CREC). The consent of all volunteers was required before their participation in the study. Malaria prevention and curative treatments were provided to all sleepers in the huts who showed symptoms of the disease.An. gambiae and p = 0.257 for culicine mosquitoes).The homogeneity of attractiveness of the experimental huts was verified using ANOVA on the numbers of mosquitoes caught in each hut prior to treatment. This analysis showed Figure that theAn. gambiae of 31.3% and 23.8%, respectively compared to the control hut on wild ae Table , as wellae Table . This waAn. gambiae, the blood feeding rates in all five insecticide treatments were significantly less than that of the control (p < 0.05). In the second month, the blood feeding rates of mosquitoes in the Bendiocarb, fenitrothion and chlorpyriphos-deltamethrin mixture huts were significantly less than the control hut (p < 0.05). Over the third and fourth months, blood feeding in the chlorpyriphos-deltamethrin mixture and fenitrothion huts remained lower than that of the control (p < 0.05).The blood feeding rates of wild Anopheles entering the huts treated with deltamethrin, alphacypermethrin, and bendiocarb were not significantly different from the control hut during the first month (p > 0.05) Table . ContrarAn. gambiae in hut treated with alphacypermethrin (39.0%) was significantly higher than that treated with deltamethrin (p < 0.05). For bendiocarb, the immediate mortality during the first month was clearly higher than that of deltamethrin and alphacypermethrin, but less than that of those of fenitrothion and the mixture of chlorpyriphos and deltamethrin. During the second month, the immediate mortality in the chlropyriphos-deltamethrin hut and alphacypermethrin (3.1%). However, the insecticidal effects of fenitrothion 68.6% and 71.1%, chlorpyriphos-deltamethrin 61.2% and 28.6%, alphacypermethrin 3.4% and 29.4%, and bendiocarb 69.5% and 10.9% remain continually high . During the second month Table , the morrmethrin 9.0% was An. gambiae [An. gambiae varied throughout the study. The lowest rate (6.8%) was observed in 3rd month after treatment and the highest rate the following month (42.8%). It is very difficult to explain this variation in behaviour as both rates take place during the same period. These months correspond to the beginning of the dry season and do not seem to result from changes of behaviour in humans or mosquitoes.The deterrency or reduction of entry rates for both methrin) . HoweverAn. gambiae was the highest in huts where walls were treated with alphacypermethrin and deltamethrin as a result of the repellent effect pyrethroids, which was not observed with carbamates and organophosphates. In contrast, the induced exophily on released An. gambiae in huts treated with fenitrothion and the chlorpyriphos-deltamethrin mixture was relatively high. The strong exophily due to the carbamates and organophosphates on these specimens might be explained by the fact that the An. gambiae released in the huts had lost their vigour after being reared in the insectary. The effect of the chlorpyriphos-deltamethrin mixture could be explained by a synergy between the two products. Similar results were found when testing combinations of non-pyrethroid insecticides and a repellent (DEET), an organophosphate (chlorpyriphos methyl) and an oxadiazine (Indoxacarb) alone or in combination with pyrethroids on resistant mosquitoes [The induced exophiliy (in the treated hut) of wild squitoes -25.The treatment of the huts with insecticide did not prevent a proportion of mosquitoes from taking a blood meal. This blood meal was facilitated by the fact that the mosquito nets used to protect sleepers were not treated. The fact that deltamethrin, alphacypermethrin, and bendiocarb did not significantly reduce blood feeding compared to the control might be explained by the significant immediate mortality in the other two insecticides during the first month, fenitrothion (91.7%) and the chlorpyriphos-deltamethrin mixture (95.3%) (p < 0.05). In these cases, mosquitoes entering the huts could also have been killed before being able to blood feed.Culex and Mansonia spp. Resistance to organophosphates, carbamates, and pyrethroids has been reported in Culex quinquefasciatus in West Africa [Cx. quinquefasciatus [An. gambiae were strongly affected by fenitrothion, the mixture of chlorpyriphos-deltamethrin, and bendiocarb. The difference between carbamates, organophosphates and pyrethroid insecticides can be explained by the current emergence and widespread resistance of An. gambiae s.l. to pyrethroids that we mentioned earlier [The mortality of culicinae was only 53.4% with fenitrothion in the first month; this can be explained by the resistance of culicinae, largely t Africa . Moreoveasciatus . However earlier . Other t earlier .After four months experiment of indoor residual spraying treatments in experimental huts, fenitrothion, chlorpyriphos-deltamethrin mixture, and bendiocarb were shown to be effective insecticides for controlling pyrethroid-resistant Anopheles. They showed to be effective alternatives to pyrethroids for indoor residual spraying. Bendiocarb decayed in less than four months, showing a short-life on cement walls, but still seems a promising insecticide to control resistant vectors. A micro-encapsulation formulation of bendiocarb will make it last longer in treated supports. Reports from Equatorial Guinea, Namibia, Mozambique, Mexico, and India showed good performance of bendiocarb as an indoor residual spraying treatment against mosquito vectors. The mixture of chlorpyriphos and deltamethrin is not yet registered with the WHO and cannot be imported for public health purposes. Fenitrothion was an effective product, but the side effects were not appreciated by the sleepers, however reports on fenitrothion indicated its better personal protection effects than Bendiocarb.The authors declare that they have no competing interests.MCA conceived and designed the study, supervised fields and laboratory procedures, data analysis and interpretation, revised the manuscript and gave final approval for the version to be published. GGP carried out field experiments, collected, analysed, interpreted data and wrote the first draft of the manuscript. SI helped with translation of the manuscript and contributed to the design of the study. DG and AY contributed to the design of the study and substantially helped in drafting the manuscript. All authors read and approved the final manuscript."} +{"text": "The aim of this study was to evaluate the performances of a new mosaic long-lasting insecticidal net (LLIN), i.e. PermaNet\u00ae 3.0 (i.e. a mosaic net containing piperonyl butoxide and deltamethrin on the roof) comparatively to the WHO recommended PermaNet\u00ae 2.0 (unwashed and washed 20-times) and a conventionally deltamethrin-treated net (CTN).A multi centre experimental hut trial was conducted in Malanville (Benin), Vall\u00e9e du Kou (Burkina Faso) and Pitoa (Cameroon) to investigate the exophily, blood feeding inhibition and mortality induced by PermaNet\u00ae 2.0 were excellent (>80%) in the \"pyrethroid-tolerant\" area of Malanville. In the pyrethroid-resistance areas of Pitoa (metabolic resistance) and Vall\u00e9e du Kou (presence of the L1014F kdr mutation), PermaNet\u00ae 3.0 showed equal or better performances than PermaNet\u00ae 2.0. It should be noted however that the deltamethrin content on PermaNet\u00ae 3.0 was up to twice higher than that of PermaNet\u00ae 2.0. Significant reduction of efficacy of both LLIN was noted after 20 washes although PermaNet\u00ae 3.0 still fulfilled the WHO requirement for LLIN.The personal protection and insecticidal activity of PermaNet 3.0 and PermaNet\u00ae 3.0 for the control of pyrethroid resistant mosquito populations in Africa.The use of combination nets for malaria control offers promising prospects. However, further investigations are needed to demonstrate the benefits of using PermaNet Malaria remains a major public health problem. Last global estimates of the malaria disease burden in 2006 indicate that at least 250 million clinical cases occurred each year, with around 1 million deaths of which 90% occurred in sub-Saharan Africa -5. Eightkdr), cause resistance to DDT and/or pyrethroid insecticides [Unfortunately, pyrethroid resistance is now widespread in malaria vectors including Western , Centralcticides ,15. Overcticides . Among tcticides , Cameroocticides , Ghana [cticides and Soutcticides .Anopheles gambiae [kdr resistance [An. gambiae strain sharing oxidase-based pyrethroid tolerance was also reported in Cameroon [kdr mutation and increased levels of P450s within the same Anopheline populations [Culex quinquefasciatus, multiplicative interaction (epistasis) between these two types of resistance can lead to extremely high level of resistance to pyrethroids [There are more and more evidences in the recent literature to support that pyrethroid resistance may seriously impact on malaria vector control . An expe gambiae . A recensistance . ReducedCameroon and KenyCameroon ,26. Moreulations ,19. As dethroids ,28. Thus\u00ae 3.0, has been designed to improving efficacy against pyrethroid-resistant mosquito populations [\u00ae 3.0 is a mosaic net combining deltamethrin-coated-polyester side panels and a deltamethrin plus piperonyl butoxide (PBO) incorporated-polyethylene roof. PBO has been incorporated to the net as it showed to enhance the effects of deltamethrin against insects by inhibiting metabolic defence systems, mainly P450s [Among the new tools available in public health, PermaNetulations . PermaNely P450s .\u00ae 3.0) in comparison with the classical PermaNet\u00ae 2.0 recommended by WHO. Experimental hut trials were conducted in Malanville (Benin), Pitoa (Cameroon) and Vall\u00e9e du Kou (Burkina Faso), where An. gambiae populations showed different levels and types of pyrethroid resistance (i.e. metabolic versus target site modification). Standard WHO procedures in phase II were followed to investigate the efficacy of unwashed and 20 times washed LLINs in terms of induced exophily, blood-feeding inhibition and mortality.In this paper, a multi centre study was carried out in western and central Africa to evaluate the performances of this new LLIN technology network Figure . Two staAnopheles gambiae s.s. M cytotype is the main malaria vector in this area and presents very low levels of pyrethroid resistance [Malanville is in northern Benin, 800 km from Cotonou, in an irrigated rice-growing valley. The climate is tropical soudanian, characterized by a dry season from December to June and a rainy season from July to November. sistance .Anopheles gambiae s.l. and Anopheles funestus s.l. are the main malaria vectors in this area. Anopheles arabiensis is predominant and showed moderate level of resistance to permethrin, deltamethrin and DDT [Pitoa is a small village, with around 5,000 inhabitants, located at 15 km from Garoua, in an area of extensive cotton cultivation in Northern Cameroon (around 35 000 ha cultivated). and DDT due to i and DDT ,33.An. gambiae co-exist in sympatry but the M form is mostly present during the dry season [Anopheles gambiae showed high level of resistance to pyrethroids due to the presence of the Kdr mutation occurring at high allelic frequency among the molecular M and S forms [Vall\u00e9e du Kou is a large rice-growing area , located at 30 km Northern Bobo-Dioulasso and comprised seven villages, surrounded by wooded savannah. VK7 is a village located on the outskirts of rice fields. Both M & S molecular forms of y season . Anophel S forms .An. gambiae s.l. to pyrethroids was checked using WHO cylinder test [et al [kdr mutation.In each site and just prior to the trials, resistance of der test . Four bader test ,38. The t [et al was usedExperimental huts are specially designed to test vector control products against freely entering mosquitoes under natural but controlled conditions . The 3.5In each country, the treatments were randomly allocated to six experimental huts, which did not differ between them for their mosquito attractiveness in absence of treatment. Adult volunteers have been recruited among the inhabitants of the villages where experimental huts were implemented. They have been informed on the objective of this study and signed (or through a literate witness if illiterate) an informed consent. They entered the hut at dusk (7:00pm) and remained inside until dawn (5:30 am) of the next morning. Early in the morning, dead mosquitoes were collected from the floor of the hut as well as from the exit traps and inside the nets; resting mosquitoes were collected using aspirators from inside the net and from the walls and roof of the hut and exit traps. Mosquitoes were scored by location as dead or alive and as fed or unfed. Live mosquitoes were placed in small cups and provided with access to sugar solution for 24 hours to assess the delayed mortality. To minimize bias related to mosquito attractiveness of each volunteer and spatial variation in mosquito densities, the volunteers and bed nets were rotated between huts each day according to a Latin square design .\u00ae 3.0 comparatively to the WHO recommended PermaNet\u00ae 2.0 and a conventional deltamethrin-treated net washed to just before exhaustion . Their In each country, six treated arms were randomly allocated to huts:1. Untreated net (same fabric - polyester on the side with a strengthened 70 cm lower border/polyethylene on top)\u00ae 3.0 unwashed2. PermaNet\u00ae 2.0. unwashed3. PermaNet\u00ae 3.0 washed 20 times4. PermaNet\u00ae 2.0 washed 20 times5. PermaNet2 and washed to just before exhaustion i.e. 95% Knock down after 1 h of contact and/or 80% mortality after 24 h [6. Polyester net conventionally treated with deltamethrin at 25 mg a.i./mter 24 h .\u00ae 2.0 and PermaNet\u00ae 3.0) were provided by Vestergaard Frandsen SA, (Switzerland). PermaNet\u00ae 2.0 is a deltamethrin-coated LN, made of knitted multi-filament polyester fibres and is treated with deltamethrin at a target concentration of 55 mg/m2 . PermaNet\u00ae 2.0 received WHOPES full recommendation for LLIN in 2009. PermaNet\u00ae 3.0 product is a combination of different long-lasting technologies. The roof of PermaNet\u00ae 3.0 utilizes deltamethrin and PBO incorporated into monofilament polyethylene yarn of 100 denier at the target dosage of 4.0 g AI/kg and 25 g AI/kg of netting material, respectively. The side panels of PermaNet\u00ae 3.0 are made of multi-filament polyester fibres, treated with deltamethrin in a resin coating . The side netting has two parts: a strengthened lower part, so-called border (70 cm) by using 75 denier yarn (weight 40 \u00b1 10% g/m2) and a side panel made of 75 denier (weight of 30 \u00b1 10% g/m2). The target dose of deltamethrin in the side panels is 2.8 g AI/kg of netting material, i.e. 115 mg AI/m2 of the border and 85 mg AI/m2 of the remaining of the side panels.The LLINs . This treatment was used as a positive control. Each net was deliberately holed with six holes (4 cm \u00d7 4 cm) to simulate a torn net [The polyester net was conventionally treated with deltamethrin at 25 mg AI/mtorn net .An. gambiae Kisumu strain in WHO cone bioassays [The bio-efficacy of each treatment was determined before washing and after field testing by exposing 2 to 5 days old unfed females of the susceptible ioassays . This teioassays .\u00ae 3.0. The chromatographic determination of deltamethrin, deltamethrin R-isomer and piperonyl butoxide was performed by gas chromatography with flame ionization detection (GC-FID) after extraction by refluxing with xylene. Before the analysis of samples, the analytical method was successfully validated for its specificity, linearity of detector response, accuracy, repeatability and reproducibility.Determination of deltamethrin and PBO content on nets, before washing and after the field testing was investigated using a new method developed by the WHO Collaborating Centre for the Quality Control of Pesticides . In eachin situ bioassays were compared between each net using a Chi square test at 95% confidence interval, using the Minitab software version 12.2. In each study site, the number of mosquitoes of each species entering the huts was compared by species and analysed using the non parametric Kruskal-Wallis test. The proportion of mosquitoes that exited early (induced exophily), the proportion that was killed within the hut and the proportion that successfully blood fed (blood feeding rate) were compared and analysed using the logistic regression . The percentage personal protection (PP) was calculated as (BFC-BFT)/(BFC) * 100, where BFC is the total number of blood fed females in the control hut and BFT the total number of blood-fed female mosquitoes in the treated hut. The overall killing effect (KE) of a treatment was calculated as (DT-DC)/(TC) * 100, where DT is the total number of dead mosquitoes in the treated hut, DC the total number of dead mosquitoes in the control hut and TC is the total number of mosquitoes collected in the control hut [Data from trol hut .An. gambiae s.l. collected in the three experimental hut stations. Anopheles gambiae s.s. was predominant in Malanville (95%) and Vall\u00e9e du Kou (100%), whereas An. arabiensis was predominant (95%) in Pitoa. The composition of An. gambiae s.s. was 100% M form for the Malanville sample and 80%/20% S/M molecular forms for the Kou Valley sample. Different levels of deltamethrin resistance were reported in the three study sites; the most \"susceptible\" population was found in malanville , the most resistant in Vall\u00e9e du Kou and the population of Pitoa being intermediate . The kdr mutation was present at very high frequency (>80%) in both molecular M & S forms in Vall\u00e9e du Kou whereas it was only 16% in the M form in Malanville. In Pitoa, the kdr mutation was almost absent (<5%) and deltamethrin resistance in An. arabiensis was associated with elevated esterase and oxidase activities as described previously [Table eviously ,33.\u00ae 2.0 and PermaNet\u00ae 3.0 as measured by WHO cone bioassay tests showed no significant decrease in efficacy after washing and/or field testing , KD and mortality decreased below the WHO threshold (95% and 80% respectively) after four washes (respectively 73% and 71%). Hence, three washes were considered as the number of washes required before exhaustion. Residual activity of PermaNetAn. gambiae s.l. mosquitoes were collected in the control (untreated) huts among which 908 , 401 (eq. 5.8 bites per man per nigh) and 285 (eq.1.5 bites per man per man) were found in Vall\u00e9e du Kou, Pitoa and Malanville, respectively.The experimental hut trials were conducted from September till November 2007 in the Vall\u00e9e du Kou and from July till September 2008 in Malanville and Pitoa. Thirty-six night collections (one Latin square) were required in Vall\u00e9e du Kou and Pitoa to collect sufficient number of Anopheline mosquitoes for statistical analysis, whereas 72 nights collection (two Latin squares) were required in Malanville to obtain a correct density. In overall, 1,594 \u00ae 2.0 and 3.0 in Vall\u00e9e du Kou and Pitoa compared to the untreated (control) arm whereas no significant reduction was noted in Malanville regardless the treatments was noted with the unwashed PermaNet\u00ae 2.0 and 3.0 (washed or unwashed) was greater in Vall\u00e9e du Kou (from 67 to 80%) than in Pitoa and Malanville (from 51 to 67%). Both LLINs (washed or unwashed) induced significantly more exophily than the untreated nets, regardless of the ecological settings. PermaNet\u00ae 3.0 (washed or unwashed) did not induce significantly higher exophily than PermaNet\u00ae 2.0 (washed or unwashed), except in Vall\u00e9e du Kou where the proportion of mosquitoes found in the veranda trap was higher with PermaNet\u00ae 3.0 washed 20 times (75%) than PermaNet\u00ae 2.0 washed 20 times (67%) (P < 0.05).In the control huts, the exophily in Pitoa did not differ significantly from the two others study sites, but the exophily rate in Malanville was significantly higher than in Vall\u00e9e du Kou p = 0.018) , to Pitoa and Vall\u00e9e du Kou was significantly lower than for Permanet\u00ae 3.0 unwashed (98%) and PermaNet\u00ae 2.0 unwashed or washed 20 times (respectively 90% and 84%). In Pitoa, PermaNet\u00ae 2.0 induced a higher BFI, although this was only significant for unwashed nets. In contrast, BFI was higher with PermaNet\u00ae 3.0 over PermaNet\u00ae 2.0 in Vall\u00e9e du Kou for both unwashed and washed bed nets (P < 0.05).The blood feeding inhibition (BFI) rates induced by each treated hut are illustrated in the Figure \u00ae 2.0 and 3.0 was good when the LLIN was unwashed but much lower when the nets were washed 20 times, especially in the pyrethroid resistance area of Vall\u00e9e du Kou (44% and 62% for PermaNet\u00ae 2.0 and PermaNet\u00ae 3.0 respectively). In this resistance area of Vall\u00e9e du Kou, PermaNet\u00ae 3.0 conferred a significantly better protection than the PermaNet\u00ae 2.0 .The personal protection of PermaNetAn. gambiae s.s. was predominant. In contrast, higher mortality (13%) was recorded in Pitoa where An. arabiensis was found in higher proportion.Mortality of mosquitoes in the control huts was low (around 5%) in Malanville and Vall\u00e9e du Kou where An. gambiae killed by the treated nets greatly differed according to the entomological setting. The corrected mortality was high in Malanville (from 61% for CTN to 96% for PermaNet\u00ae 3.0) and, in a lesser extent, Pitoa (from 41% for CTN to 93% for PermaNet\u00ae 3.0). In contrast, the mortality in Vall\u00e9e du Kou ranged from 28% for CTN to 78% for PermaNet\u00ae 3.0. Regarding the LLINs only, mortality (~69%) was similar between PermaNet\u00ae 3.0 and PermaNet\u00ae 2.0 washed 20 times in Malanville whereas in Pitoa and Vall\u00e9e du Kou PermaNet\u00ae 3.0 (washed or unwashed) induced significantly more mortality than PermaNet\u00ae 2.0 (p < 0.05). In all settings, washing the nets 20 times significantly reduced the number of mosquitoes being killed by the LLINs (p < 0.05).The mortality induced by each treated arm is illustrated in Figure \u00ae 2.0 and PermaNet\u00ae 3.0 were high in Malanville and Pitoa (from 80 to 96%) comparatively to Vall\u00e9e du Kou (from 41 to 77%). This trend was stronger with washed LLINs especially in Vall\u00e9e du Kou where the insecticidal activity of PermaNet\u00ae 3.0 and PermaNet\u00ae 2.0 decreased to 46% and 26%, respectively and PermaNet\u00ae 2.0 (deltamethrin) complied with the target doses (\u00b1 25%), except for one PermaNet\u00ae 2.0 (Pitoa) for which the average deltamethrin content on the side panels (2.53 g AI/kg) was just above the upper limit (2.25 g AI/kg) of the target dose. No major loss of deltamethrin and/or PBO content (from nil to 17%) was reported for unwashed PermaNet\u00ae 2.0 (deltamethrin) and PermaNet\u00ae 3.0 (deltamethrin and PBO) after the field testing. However, the loss of deltamethrin after 20 washes was relatively important for both PermaNet\u00ae 2.0 (from 59 to 85%) and PermaNet\u00ae 3.0 in the side panels (from 71 to 87%). However, the deltamethrin and PBO content in the roof panel remained high (>60% retention) in PermaNet\u00ae 3.0 after 20 washes. The deltamethrin content for unwashed CTN (0.8 g AI/kg) complied with the initial target doses (\u00b1 25%). After 3 washes the loss of active ingredient ranged from 85 to 91%.In the three study sites, neither deltamethrin nor PBO was detected (limit of quantification = 0.01 g/kg for deltamethrin and 0.1 g/kg for PBO) in the untreated nets, hence confirming that no contamination occurred during the rotations of the nets Table . The act\u00ae 3.0 against wild pyrethroid-tolerant An. gambiae s.s. , kdr-resistant An. gambiae s.s. and pyrethroid-resistant An. arabiensis s.l. showing metabolic resistance (Pitoa). The ABC network offers ideal conditions to address this objective based on the existence of eight experimental hut stations in different ecological and entomological settings and the two others An. gambiae populations from Malanville and Vall\u00e9e du Kou (<5%). Unfortunately, this study did not allow to decipher on the causes of this difference of mortality but other authors have already reported similar mortality rates of An. arabiensis (10%) in experimental huts [An. gambiae s.s. and An. arabiensis.The results from the control huts showed that the behavioural preferences of Anopheline populations (in terms of endophily/exphily) significantly differ between the three sites as expected from literature on trophic behaviour of malaria vectors -42. It ctal huts . Neverth\u00ae 3.0 and PermaNet\u00ae 2.0 was good, a rather high loss of insecticide was noted in the side panels washed until just before exhaustion. This confirms that the PermaNet\u00ae 3.0 fulfils the WHOPES efficacy criteria of Phase II studies for LLIN.This study first demonstrated a better or equal impact of PermaNet\u00ae 3.0 also induced higher insecticidal effect than PermaNet\u00ae 2.0 in the pyrethroid resistance areas of Pitoa and Vall\u00e9e du Kou, but performed equally in the area of Malanville.Regarding the two LLINs, unwashed Permanet 3.0 induced significantly higher BFI and mortality than Permanet 2.0 in Vall\u00e9e du Kou and Malanville. In the locality of Pitoa, the BFI was however higher with Permanet 2.0 than Permanet 3.0 but the mortality was still higher with Permanet 3.0. After 20 washes, the PermaNet\u00ae 3.0 relative to 75% survival after exposure to PermaNet\u00ae 2.0. It remains to be seen if the gain of efficacy of PermaNet\u00ae 3.0 over PermaNet\u00ae 2.0 is enough to control highly pyrethroid-resistant malaria vector populations. Here, it is difficult to conclude on the benefit of using PBO on the roof because the deltamethrin content on PermaNet\u00ae 3.0 was approximately twice higher than that of PermaNet\u00ae 2.0. So the better efficacy on resistant mosquitoes could be impeded either to the higher deltamethrin concentration or to the PBO itself or both. Other field studies did not show an increase of efficacy on resistant Culex and pyrethroid susceptible An. gambiae s.s. [Anopheles epiroticus [One should note that in areas with high resistance levels 50% of resistant mosquitoes survived after exposure to PermaNetiae s.s. as well iroticus .kdr mutation frequency was high . The same trend was observed with PermaNet\u00ae 2.0, confirming that the Kdr mutation is an important predictor of pyrethroid resistance phenotype in malaria vectors as previously described [\u00ae net or PermaNet\u00ae [An. gambiae populations are sharing very high frequency of Kdr mutation [An. arabiensis show higher metabolism through elevated oxidase and esterase activity [This multi-centre study provided also more evidence that pyrethroid resistance can seriously reduce the efficacy of pyrethroid -treated materials in malaria vectors ,22. Resuescribed ,46. LoweermaNet\u00ae and alsoermaNet\u00ae . Unfortumutation -51 alonemutation ,18. In Pactivity , CTN effactivity .\u00ae 3.0 caused better efficacy against both Kdr and metabolic resistant malaria vectors than PermaNet\u00ae 2.0. Nevertheless in areas of strong resistance like the Vall\u00e9e du Kou, a large number of exposed mosquitoes survived after exposure to both LLINs. Then as a short term prospect, it seems essential to evaluate this tool in others areas of strong resistance like southern Benin, southern Nigeria and C\u00f4te d'Ivoire. It is also crucial to strengthen the collaboration between companies and Research Institutions to find alternative tools for malaria vector control , becausThe authors received financial support from Vestergard Frandsen Company to carry out the experimental huts trials in Burkina Faso and Cameroon. However, the authors have strictly followed the WHOPES procedures for testing and evaluation of the efficacy of Permanet 3.0 against malaria vectors. The Research teams involve in this study have no competing and commercial interests with the manufacturer.VC designs the study and drafted the manuscript. JC, RDD, JE, PN carried out bioassays and conducted the experimental hut trials. OP conducted the chemical analysis on nets. MA and JMH helped design the study and critically revised the manuscript. All authors read and approved the final manuscript.Anopheles gambiae in experimental huts of all countriesComparison of exophily obtained for free flying wild . Raw data from the experimental hut trials.Click here for fileAnopheles gambiae in experimental huts of all countriesComparison of blood feeding rates obtained for free flying wild . Raw data from the experimental hut trials.Click here for fileAnopheles gambiae in experimental huts of all countriesComparison of mortality rates obtained for free flying wild . Raw data from the experimental hut trials.Click here for file"} +{"text": "Combination mosquito nets incorporating two unrelated insecticides or insecticide plus synergist are designed to control insecticide resistant mosquitoes. PermaNet 3.0 is a long-lasting combination net incorporating deltamethrin on the side panels and a mixture of deltamethrin and synergist piperonyl butoxide (PBO) on the top panel. PBO is an inhibitor of mixed function oxidases implicated in pyrethroid resistance.An experimental hut trial comparing PermaNet 3.0, PermaNet 2.0 and a conventional deltamethrin-treated net was conducted in NE Tanzania using standard WHOPES procedures. The PermaNet arms included unwashed nets and nets washed 20 times. PermaNet 2.0 is a long-lasting insecticidal net incorporating deltamethrin as a single active.Against pyrethroid susceptible Anopheles gambiae the unwashed PermaNet 3.0 showed no difference to unwashed PermaNet 2.0 in terms of mortality (95% killed), but showed differences in blood-feeding rate (3% blood-fed with PermaNet 3.0 versus 10% with PermaNet 2.0). After 20 washes the two products showed no difference in feeding rate (10% with 3.0 and 9% with 2.0) but showed small differences in mortality (95% with 3.0 and 87% with 2.0). Against pyrethroid resistant Culex quinquefasciatus, mediated by elevated oxidase and kdr mechanisms, the unwashed PermaNet 3.0 killed 48% and PermaNet 2.0 killed 32% but after 20 washes there was no significant difference in mortality between the two products (32% killed by 3.0 and 30% by 2.0). For protecting against Culex PermaNet 3.0 showed no difference to PermaNet 2.0 when either unwashed or after 20 washes; both products were highly protective against biting. Laboratory tunnel bioassays confirmed the loss of biological activity of the PBO/deltamethrin-treated panel after washing.Both PermaNet products were highly effective against susceptible Anopheles gambiae. As a long-lasting net to control or protect against pyrethroid resistant mosquitoes PermaNet 3.0 showed limited improvement over PermaNet 2.0 against Culex quinquefasciatus. The development of insecticide resistance is probably the biggest threat to capacity to control malaria vectors or sustain any drive towards malaria elimination. The chemical agents that make malaria vector control feasible are the pyrethroids. The best tools for delivering pyrethroids are long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS) . Recent Resistance management has a theoretical foundation in population genetics that goes back three decades -21. SimuThe same principle has been adopted in the strategy to preserve anti-malarial drug efficacy known as combination therapy ,26. AdopAn. gambiae complex do in fact contact the top [Alternative insecticides to pyrethroids have been tested on nets for effect against wild, pyrethroid resistant mosquito populations but only under limited experimental conditions in the field -31. Most the top possiblykdr and a metabolic mechanism caused by mixed function oxidases (MFOs). MFOs are responsible for the pyrethroid resistance that evolved in Anopheles funestus and which led to the failure of IRS campaigns in South Africa [kdr to create a pyrethroid resistance, that is causing control failure of Anopheles gambiae M form in parts of West Africa [kdr together are responsible for pyrethroid resistance in Culex quinquefasciatus [Rather than use a non-pyrethroid insecticide to overcome resistance, an equally valid approach is to deploy a chemical synergist on the fibres. Synergists overcome resistance by inhibiting the enzymes responsible for certain types of resistance. Resistance to pyrethroids in Anopheline mosquitoes appears to be caused by two primary mechanisms: a target site insensitivity mechanism known as h Africa ,33. It at Africa ,14,34. Basciatus ,36. One asciatus .An. gambiae and other vector species. PermaNet 3.0 is a long-lasting insecticidal net developed by Vestergaard Frandsen in which the PBO together with the pyrethroid deltamethin are incorporated into the polyethylene fibres on the roof panel of the net. The sides of PermaNet 3.0 are made of polyester and coated with a long-lasting formulation of deltamethrin similar to the pyrethroid-based LLIN, PermaNet 2.0 but with a strengthened lower part. By restricting PBO to the roof of the net the concept of PermaNet 3.0 is to have the insect make contact with the synergist on the roof, mediated by the odour plume, before making further contact with pyrethroid on the sides during exploration.PBO has potential to combat the growing problem of pyrethroid resistance in An. gambiae and Cx quinquefasciatus in Muheza, Tanzania, together with supporting laboratory data and chemical analysis.PermaNet 3.0 was submitted by Vestergaard Frandsen to the WHO Pesticide Evaluation Scheme (WHOPES) for formal evaluation. The current paper reports upon a WHOPES-sponsored experimental hut trial conducted against wild, free flying 2) and piperonyl butoxide at 25 g/kg (approx. 1.1 g/m2), plus side panels made of multifilament polyester (75 denier) fabric with a strengthened border treated with deltamethrin at 2.8 g/kg (approx. 118 mg/m2).PermaNet 3.0 LN is a LLIN consisting of a top panel made of monofilament polyethylene (100 denier) fabric incorporating deltamethrin at 4 g/kg (approx. 180 mg/m2).PermaNet 2.0 LN is a LLIN made of multifilament polyester (75-100 denier) fabric, factory treated with a wash-resistant formulation of deltamethrin at 1.8 g/kg (for 75 denier) is a multifilament polyester (100 denier) fabric treated with deltamethrin at 25 mg/mThe size of the PermaNet 2.0 and standard nets was 130 cm wide, 190 cm long, 150 cm high. PermaNet 3.0 nets measured 120 cm wide, 190 cm long, 150 cm high. Hence the top panel of PermaNet 3.0 containing the PBO plus deltamethrin constituted 19.7% of the overall surface area whereas the remaining 80.3% on the sides contained only deltamethrin as an active ingredient.Anopheles gambiae sensu stricto Kisumu, a laboratory insecticide susceptible strain, originally from Kenya.Culex quinquefasciatus TPRI, a laboratory insecticide susceptible strain, maintained by the Tropical Pesticide Research Institute, Tanzania.Culex quinquefasciatus Masimbani, a multiple resistant strain from northeast Tanzania, containing elevated oxidase and kdr pyrethroid resistance mechanisms. In WHO resistance tests the strain showed survival after one hour exposure to test papers of permethrin deltamethrin (48%), DDT (58%), malathion (27%) and propoxur (46%).An. gambiae Kisumu (pyrethroid susceptible) on PermaNet 3.0, PermaNet 2.0 and the CTN after 0, 10 and 20 washes. Similarly Cx. quinquefasciatus Masimbani (pyrethroid resistant) was exposed in 3 min bioassay tests to netting taken from the roof and sides of the PermaNet 3.0 after 0, 10 and 20 wash intervals. Washing was carried out on entire nets using the WHO Phase II washing protocol [Three min exposure bioassay tests were carried out using protocol . Bioassaprotocol ,38. AfteAn. gambiae Kisumu (insecticide susceptible), Cx. quinquefasciatus TPRI (insecticide susceptible) and Cx. quinquefasciatus Masimbani (pyrethroid resistant). Tunnel tests were replicated three times.The tunnel tests were carried out on samples of PermaNet 3.0 netting cut from the roof and lower sides of the net after 0, 10 and 20 washes using the Phase II washing procedure . The tesThe tunnel test apparatus is comprised of a glass cuboid tube, 25 cm high, 21 cm wide, 60 cm long, divided into two chambers by a transverse netting insert fitted onto a frame which slots across the tunnel. Nine 1 cm diameter holes were cut into the netting to allow passage of mosquitoes. In the bait chamber, a guinea pig was housed unconstrained in a wire meshed cage and in the other chamber 100 unfed female mosquitoes 3-5 days old were released at dusk and left overnight in the dark as per WHO guidelines ,38. The 2 was washed until just before 'insecticide exhaustion' as defined by WHO [An. gambiae Kisumu in 10 replicates of 5 mosquitoes per replicate at each wash interval on the five panels of each net. Exposure was for 3 min and mortality was scored 24 h later.A polyester net conventionally treated with deltamethrin at dosage 25 mg/md by WHO . The convice versa, to compensate for possible selective exiting in one compass direction.The six veranda trap huts were situated at Zeneti village, Muheza district, NE Tanzania (5\u00b013'S and 38\u00b039'E). They were constructed according to a design first described by Smith [Anopheles gambiae s.s., An. funestus and Cx. quinquefasciatus are the predominant mosquito species in the area. The An. gambiae and An. funestus are susceptible to pyrethroids; Cx. quinquefasciatus is resistant to pyrethroids, mediated by enhanced oxidase and site insensitivity mechanisms [[An. gambiae and Cx. quinquefasciatus were abundant. The wild adult mosquitoes were characterized for resistance by testing with deltamethin 0.05% papers in WHO test kits.hanisms [, Malima The following six treatment arms were compared:1. Unwashed PermaNet 3.02. PermaNet 3.0 washed 20 times3. Unwashed PermaNet 2.04. PermaNet 2.0 washed 20 times2, washed until just before exhaustion5. Polyester net, conventionally treated with deltamethrin at 25 mg/m6. Untreated polyester netEach net was deliberately holed with six 4 cm \u00d7 4 cm holes to simulate a worn net. The trial took place between 7 July and 4 October 2008. The treatment arms were rotated 3 times through the huts according to a Latin Square design. A treatment was assigned at random to a particular hut for 3 nights' observation before being rotated to the next hut. Male volunteers slept on beds under the net which were tucked under the mattress. The six sleepers were rotated through the six huts on consecutive nights. Data were collected for 54 nights. Three nets were available per treatment arm and each net was tested on consecutive nights during the three-night rotation. At the end of each rotation the huts were cleaned and aired for one day and the treatments moved to the next hut.White sheets were laid over the veranda and room floors to ease the collection of knocked-down mosquitoes. Each morning after dawn, mosquitoes were collected using aspirators from the floor, walls, exit traps and inside the nets, scored as dead or alive and as fed or unfed and identified to species using a binocular microscope. Live mosquitoes were held for 24 h with sugar solution in paper cups to determine delayed mortality.The primary outcomes were:\u25aa deterrence - reduction in hut entry relative to the control huts fitted with untreated nets\u25aa treatment induced exiting - the proportion of mosquitoes found in exit traps relative to control huts\u25aa blood-feeding inhibition - the proportional reduction in blood feeding relative to untreated nets\u25aa mortality - the proportion of mosquitoes killedThe first and third of these outcomes are indicators of personal protection which can be estimated by the equation:u - Bt)/Bu% personal protection = 100/TuInsecticidal effect (%) = 100 using the internal standard calibration.The analysis of experimental hut data were carried out using logistic regression for proportional data and negative binomial regression for numeric data after adjusting for the effects of individual huts and sleepers. Data was analysed using Stata 9 software .Proportional data from laboratory bioassay tests (cone tests and tunnel tests) were normalised using arcsine square root transformation and the replicate test data analysed using analysis of variance .Approval was obtained from the ethics review committees of the London School of Hygiene and Tropical Medicine, the Tanzanian National Institute of Medical Research (Ref: NIMR/HQ/R.8a/Vol. X/86) and the World Health Organization. Each trial participant gave written informed and was offered chemoprophylaxis during and for one month after the experimental hut trial.The procedure for use of guinea pigs in tunnel tests conformed with criteria established in EC Directive 86/609/ECC regarding protection of animals used for experimental purposes. The procedure accorded with published guidelines of the World Health Organization and was approved by the Tanzanian National Institute of Medical Research Project Review Committee.An. gambiae Kisumu showed over 90% penetration through the untreated netting. All insecticide treatments inhibited passage of mosquitoes through the netting but less so for the pyrethroid resistant Cx. quinquefasciatus Masimbani (P = 0.014) through the untreated netting. The blood-feeding trends for each of the treatments Figure mirroredAn. gambiae Kisumu and Cx. quinquefasciatus TPRI susceptible strains showed low rates of feeding through the unwashed deltamethrin-treated side netting but higher rates of feeding through side netting washed 20 times (P = 0.07). The feeding rate associated with the PBO-deltamethrin impregnated netting was highly inhibited both in unwashed and in 20 washed samples.The Cx. quinquefasciatus Masimbani strain showed higher feeding rates than the susceptible Cx. quinquefasciatus TPRI strain (or An. gambiae) through treated netting at each wash interval (P = 0.01). The feeding rate of the resistant Cx. quinquefasciatus Masimbani was notably higher through the deltamethrin-treated side netting after 20 washes. The feeding rate observed with PBO/deltamethrin-impregnated top netting did not change significantly with washing (P = 0.34) and after 20 washes was only half that observed with the deltamethrin side netting.The resistant Cx. quinquefasciatus Masimbani strain showed little or no difference in the proportion feeding between the deltamethin-treated and PBO/deltamethrin-treated netting.The trend in feeding rate among mosquitoes penetrating the holed netting showed a gradual increase over the course of 20 washes regardless of whether the netting was treated with deltamethrin or PBO-deltamethrin Figure . For theAn. gambiae Kisumu than against Cx. quinquefasciatus susceptible and resistant strains (P = 0.001) . Washing the two types of netting up to 20 times did not have a significant effect on mortality of the highly susceptible An. gambiae Kisumu (P = 0.21).The untreated nets recorded zero mortality against all three strains. Both types of treated netting induced greater mortality against ) Figure . The morCx. quinquefasciatus TPRI strain recorded greater mortality than susceptible An. gambiae Kisumu on each type of netting at the corresponding wash interval (P = 0.01). The pyrethroid resistant Cx. quinquefasciatus Masimbani strain recorded greater mortality (20%) than the susceptible TPRI strain (62%) on unwashed deltamethrin-treated side netting (P = 0.047). Mortality rates in both strains decreased after washing, indicating removal of deltamethrin by the washing process. The difference in mortality between resistant and susceptible strains became progressively smaller with washing (17% difference at 10 washes) and was barely evident at 20 washes (2% difference). Hence the contact time of Cx. quinquefasciatus with the deltamethrin-treated net at 20 washes was insufficient to induce mortality in either strain.The pyrethroid susceptible Cx. quinquefasciatus, indicating that PBO was synergizing the pyrethroid resistance in Cx. quinquefasciatus Masimbani. Over the course of 20 washes of the PBO-deltamethrin netting there was a partial loss of activity against the susceptible strain and a near complete loss of activity against the resistant strain. After 20 washes there was no significant difference in mortality induced against Cx. quinquefasciatus Masimbani by the top or side nettings (P = 0.68). This indicates that the surface concentration of PBO was largely removed by washing so no further synergy was evident against the resistant Masimbani strain and any PBO replenishment from the core of the fibres after washing was insufficient to regain toxic activity.The unwashed PBO-deltamethrin top netting induced an almost identical level of mortality in susceptible (64.2%) and resistant (67.7%) An. gambiae Kisumu decreases to less than 80%. Mortality decreased below 80% after four washes, and hence CTNs washed three times were used as reference nets in the hut trials.The point of exhaustion is the number of washes at which cone bioassay mortality of An. gambiae was susceptible and Cx. quinquefasciatus was resistant .WHO resistance tests with deltamethrin 0.05% test papers on adults collected from huts at the start of the trial indicated that An. gambiae Kisumu were recorded as 100% for each treatment arm. After 20 washes, PermaNet 3.0 still scored 100% mortality whereas PermaNet 2.0 scored 96% mortality. At the end of the 36 day trial, mortality in PermaNet 3.0 and 2.0 arms stood at 96% or more and in the CTN washed to just before exhaustion mortality stood at 90%.Before washing of the trial nets, the percentage knockdown and mortality of Anopheles gambiae sensu strictu was the only member of the An. gambiae complex present at Zenet village. An. gambiae were more abundant than Cx. quinquefasciatus during the trial .l Tables and 2. TAn. gambiae (85.5%) than for Cx. quinquefasciatus (56.8%). Insecticide induced exiting from huts with treated nets relative to the huts with untreated nets was evident for Cx. quinquefasciatus but not for An. gambiae, because most of the latter exited naturally each night from the huts with untreated nets (Tables The proportion collected each morning from the veranda and window traps of huts with untreated nets was higher for s Tables and 2.Cx. quinquefasciatus than among An. gambiae (Tables Cx. quinquefasciatus and An. gambiae in huts with treated nets (P = 0.0001).In huts with untreated nets, there was a higher rate of blood-feeding among e Tables and 2. TAnopheles gambiae showed the lowest blood-feeding rate in huts with the unwashed PermaNet 3.0. However, there was no significant difference in the An. gambiae feeding rates between PermaNet 3.0 washed 20 times, PermaNet 2.0 washed 20 times, PermaNet 2.0 unwashed or CTN washed to just before exhaustion.Cx. quinquefasciatus did not differ in huts with unwashed PermaNet 3.0 or unwashed PermaNet 2.0 (P = 0.73). Feeding rates after 20 washes were 0% for both PermaNet 3.0 and PermaNet 2.0. Huts with CTN washed three times recorded a feeding rate of 15%, a significantly higher rate than that recorded for PermaNet 3.0 washed 20 times or PermaNet 2.0 washed 20 times.The feeding rates of An. gambiae . The personal protection recorded against Cx. quinquefasciatus with the PermaNet 2.0, PermaNet 3.0 and CTN were not significantly different from one another.The PermaNet 3.0 and PermaNet 2.0 washed 20 times, and the CTN washed three times, scored similar levels of personal protection against An. gambiae and Cx. quinquefasciatus than huts with untreated nets.The huts with LLINs and CTNs recorded much greater levels of mortality of An. gambiae there was no difference between the proportions killed by the unwashed PermaNet 3.0 or unwashed PermaNet 2.0 (P = 0.79); both induced greater than 95% mortality. Against Cx. quinquefasciatus, the proportion killed by the unwashed PermaNet 3.0 (51%) was greater than the proportion killed by the unwashed PermaNet 2.0 (37%) (P = 0.05). The mortality associated with PermaNet 3.0 fell to 37% after 20 washes. The mortality associated with PermaNet 2.0 remained a consistent 36% and 35% at 0 and 20 washes respectively; both these values were virtually identical to the mortality induced by PermaNet 3.0 after 20 washes (37%) (P = 0.77). This means that any synergized toxicity by PBO in PermaNet 3.0 against Cx. quinquefasciatus was no longer evident after 20 washes. The mortality associated with PermaNet 3.0 and PermaNet 2.0 after 20 washes against Cx. quinquefasciatus was similar to that of the positive control (CTN washed three times) (P = 0.85) . The difference was small (8%) but may indicate that the PBO was not completely depleted but could still exert a limited effect against highly susceptible mosquitoes. Both PermaNet 3.0 and PermaNet 2.0 washed 20 times induced significantly greater mortality against An. gambiae than the conventionally treated net washed three times (P = 0.001).Against An. gambiae (the proportion of mosquitoes killed by the treated nets relative to the number entering untreated huts) was similar in PermaNet 3.0 (59%) and PermaNet 2.0 (56%) when washed 20 times (P = 0.87). These overall killing effects were not significantly greater than that for the CTN washed three times (42%) (P = 0.89).The overall killing effect against 2 in one sample and 119 mg/m2 in a second sample. After 20 washes the quantity on the sides had decreased to 53 mg/m2. After the trial was completed the quantity had decreased still further to 28 mg/m2. The loading dosage of deltamethrin on the polyethylene top panel of PermaNet 3.0 was 136 mg/m2. After 20 washes it was recorded as 132 mg/m2 and after the trial was completed it was recorded as 135 mg/m2. The lack of any evident decrease in content was presumably due to the deltamethrin on the top panel being incorporated or locked into the polyethylene fibre as opposed to being coated on the surface of the fibre on the polyester sides during manufacture.The chemical analysis is presented in Figure 2 in one sample and 77 mg/m2 in another. After 20 washes the dose had decreased to 25 mg/m2; no further loss was recorded at the completion of the trial.The loading dose of deltamethrin in PermaNet 2.0 was 61 mg/m2 before washing, 684 mg/m2 after 20 washes, and 1013 mg/m2 at the end of the trial. The variation partly reflects the limited number of samples taken for analysis . There was no evidence of any real change in PBO content due to washing.The PBO on the top panel of PermaNet 3.0 was 1142 mg/mAn. gambiae and both susceptible and resistant Cx. quinquefasciatus relative to netting from side panels treated with deltamethrin alone. This synergy was manifested in higher mortality, reduced passage through the holes and reduced feeding rates with netting treated with PBO-deltamethrin. The synergy in tunnels against pyrethroid resistant Cx. quinquefasciatus was progressively lost over 10 washes and fully lost after 20 washes. Cone bioassays on resistant Cx. quinquefasciatus confirmed the loss of synergy over 20 washes.Laboratory tunnel tests with PermaNet 3.0 produced results consistent with experimental hut trials and help to explain the trends in blood feeding and mortality associated with PermaNet 3.0 and PermaNet 2.0 in the field. The tunnel tests demonstrated a synergistic interaction of PBO and deltamethrin on roof netting against susceptible An. gambiae at 0 and 20 washes and both rates exceeded that of the CTN washed to just before cut off point. On the basis of this result PermaNet 3.0, like PermaNet 2.0 before it, warrants interim approval by WHO as a LLIN [An. gambiae blood feeding rate associated with the zero washed PermaNet 3.0 was lower than with the zero washed PermaNet 2.0. After 20 washes, however, the feeding rates between PermaNet 3.0 and PermaNet 2.0 no longer differed, indicating a loss of activity under field conditions.In the experimental hut trial both PermaNet 2.0 and PermaNet 3.0 induced high rates of mortality against pyrethroid susceptible s a LLIN . It was Cx. quinquefasciatus in huts with zero washed PermaNet 3.0 was higher than that with zero washed PermaNet 2.0. This indicated the PBO in PermaNet 3.0 was exerting a partial synergism against Cx. quinquefasciatus. As per tunnel test results the synergism in the huts was fully lost after 20 washes. Blood feeding rates in Cx. quinquefasciatus in the huts did not differ between PermaNet 2.0 and 3.0 either in unwashed or 20 washed nets. According to Khayrandish & Wood [Cx. quinquefasciatus from this region, enhanced oxidases and a nerve insensitivity mechanism, probably kdr, are responsible for pyrethroid resistance.It was initially encouraging that the mortality rate of pyrethroid resistant h & Wood , WHO useCx. quinquefasciatus (S): deltamethrin must still be present on the surface of both PermaNet 2.0 and 3.0 and causing some mortality of susceptible Cx. quinquefasciatus but there seems little or no PBO left on the surface of PermaNet 3.0 netting to allow synergy in resistant Cx. quinquefasciatus.The roof netting showed little change in chemical content (either in deltamethrin or in PBO) after twenty washes. Certainly any small change observed was not sufficient to explain the large difference in efficacy between unwashed and 20 times washed PermaNet 3.0 in cones, tunnel tests or experimental huts. This indicates that deltamethrin, the PBO or both compounds are depleted from the surface of the fibre after 20 washes and fail to migrate sufficiently from the core to the surface to allow full regeneration. It would seem that PBO rather than deltamethrin is the compound that remains locked in the fibre. The evidence for this stems from the zero difference in mortality in the tunnel tests between the 20 times washed PermaNet 3.0 and the 20 times washed PermaNet 2.0 against resistant Cx. quinquefasciatus taken together with the higher mortality with 20 times washed PermaNet 3.0 against susceptible Cx. quinquefasciatus mortality between PermaNet 3.0 washed 20 times and PermaNet 2.0 washed 20 times suggests a failure to regenerate.It is possible insufficient time was given between washing and tunnel testing for regeneration of PBO to occur. However, the evidence from the hut trial indicates this is not the reason since over the six weeks in the huts the PermaNet 3.0 washed 20 times showed no difference in performance to the PermaNet 2.0 washed 20 times, but during this six week interval there was plenty of time for the PBO to migrate to the surface of fibres. The no difference in It is possible that the higher mortality initially seen with PermaNet 3.0 relative to PermaNet 2.0 is due more to the higher loading dose of deltamethrin than to any contribution of PBO. An appropriate control to test this hypothesis - a 'PermaNet 3.0' loaded with the same dosage of deltamethrin but containing no PBO - was not available for testing. Such a control should always be considered in future testing of combination nets.Culex in huts with unwashed PermaNet 3.0 versus unwashed PermaNet 2.0 does, however, provide some support to the concept.At present there is limited evidence that mosquitoes contact the roof of the net while seeking access to the host. This may not hold for all species, and more corroborative observations are required. Unless the majority of mosquitoes respond to host odour or convection plumes in this way, the 2-in-1 concept as a tactic for managing resistance management tactic is flawed. The higher mortality of It is important to note that the laboratory tests and Phase II trials reported here refer to efficacy before and after standardized washing rather than to performance under long term household use. There is limited temporal dimension to this work because the interval between the start of washing and the completion of the trials was only three months. Because pyrethroids used on nets have low vapour pressure a pyrethroid LLIN that showed high efficacy after 20 Phase II washes might, quite reasonably, be expected to remain efficacious for at least three years of household use, as reported recently for PermaNet 2.0 and Olyset LLINs ,42. We hAn. gambiae in huts either before or after washing would seem unlikely to provide additional control of An. gambiae populations; besides, mortality with PermaNet 2.0 was already very high. With pyrethroid resistant Cx. quinquefasciatus almost half survived exposure to PermaNet 3.0 in the huts and this proportion increased to 64% after washing. As a combination net designed to control pyrethroid resistance mediated by mixed function oxidase mechanisms the capacity of PermaNet 3.0 to control pyrethroid multiple resistant mosquitoes or prevent selection of resistance appears limited.The negligible difference in mortality between PermaNet 3.0 and 2.0 against The authors declare that they have no competing interests.PT carried out the hut trials, supervised the laboratory tests, analysed the data and drafted the report. SM supervised the field and laboratory trials and revised the report and manuscript. CM supervised the field trials. DM carried out the field trials. WS and JM carried out the laboratory tests. OP carried out the chemical analysis. MR analysed and interpreted the data and wrote the paper. All authors read and approved the final manuscript.http://www.pamverc.org.Information on the Pan-African Malaria Vector Research Consortium can be found at the following website:"} +{"text": "Cardiovascular disease (CVD) is the leading cause of death in the United States. Diets high in fat, especially saturated fat, are often linked to obesity, hypertension and hypercholesterolemia, all risk factors for CVD. The purpose of this study was to determine the association between diet and CVD risk factors in members of a university marching band, dance team and cheer squad.In 2004, 232 marching band, dance team and cheer squad members completed a self-administered survey evaluating dietary intake. Body mass index (BMI), waist-to-hip ratio (WHR), blood pressure, fasting serum glucose and cholesterol were measured. Unpaired t-test and Pearson's chi square test were used to determine baseline differences by gender. Multiple linear regression analysis was used to determine the cross-sectional association between dietary intake of various food groups such as grains, meats, fruits & vegetables, dairy, water, alcohol and risk factors for CVD namely BMI, WHR, blood glucose, total cholesterol, and blood pressure (BP).45% of the participants were overweight; 30% of females and 4.3% of males had WHR \u2265 0.80 and 0.95 respectively. Almost 8% were hyperglycemic, 10% hypercholesterolemic, 15% had high systolic and 9% had high diastolic BP. Less than 50% consumed the recommended servings of grains, fruits and vegetables, dairy and water and 58% consumed alcohol. Higher grains intake was positively associated with higher BMI and; higher alcohol intake was also positively associated with higher BMI .These results warrant the evaluation of existing college-based health programs and development of new interventions to improve dietary habits and promote a healthy lifestyle in these athletes. Dietary habits are typically developing in childhood and established by young adulthood . DevelopData suggest that caloric intake is increasing among all ethnicities, age and socioeconomic groups, and diets contain more energy-dense, nutrient-poor foods . EvidencUniversity marching band, dance team and cheer squad members perform physical activity for prolonged periods of time. It has been estimated that these athletes exercise at an intensity of 4.5\u20136 metabolic equivalents (METs) . This METhe primary purpose of this study was to assess prevalence of CVD risk factors, measure dietary intake, readiness to change dietary intake and finally, examine the relationship between dietary intake and risk factors for CVD among members of an urban university marching band, dance team and cheer squad. We hypothesized that dietary factors such as low intake of grains, FV, dairy and high intake of alcohol are significantly associated with the CVD risk factors such as body mass index (BMI), waist to hip ratio (WHR), blood pressure, blood glucose and blood cholesterol.This study examined baseline pre-season data collected in fall of 2004. This baseline analysis was part of the main study, the primary aim of which was to promote healthy eating habits and physical fitness for the prevention of obesity-related conditions and CVD among marching band, dance team and cheer squad members in an urban university .Members of a mid-sized university marching band located in the southwest part of the United States (N = 275) participated in the study. The study was approved for the use of human subjects by the university Committee for the Protection of Human Subjects. The study investigators attended practice sessions during pre-season to recruit volunteers for the study. They informed the band members of the purpose of the study and the study procedures. During this time the investigators asked all interested band members to provide their contact information and review the informed consent document. Incentives were offered to the participants in the form of free fitness testing and nutrition counseling as a part of the study. The response rate was 93%. Informed consent was obtained from all participants prior to start of the study. Band members were also informed that they could refuse to partake in any portion of the testing procedures without negative consequences. During the pre-season, which is a 2-week band camp, participants attended a testing session in the investigator's laboratory. Participants refrained from any regular exercise programs two days prior to the testing by not participating in band activities during that period. They completed a self-administered demographic, nutrition and medical history questionnaire. Since 232 participants completed the nutrition survey, the final sample for the present study utilized for the analysis was N = 232. Please note that detailed description of the study protocol is presented elsewhere . All datBody height (cm) to the nearest 0.5 cm was obtained using a standard physician's scale height stadiometer . Body weight (kg) was measured to the nearest 0.1 kg using a digital scale . Body height and weight were used to calculate BMI. BMI was classified using the CDC classification as normal (18.5\u201324.9), overweight (25.0\u201329.9), and obese (\u2265 30.0) . Waist aAfter resting for 10 minutes quietly, systolic and diastolic blood pressure was obtained using a digital automatic blood pressure monitor . Fasting blood glucose (mg/dL) and total cholesterol (mg/dL) were obtained from each participant through a fingerstick puncture. The whole blood was analyzed for total blood cholesterol and blood glucose. Blood glucose values are presented as normal (\u2264 110 mg/dL) or high (>110 mg/dL) . Blood tParticipants N = 232) completed a self-administered cross-sectional survey evaluating the number of servings of various foods eaten per day based on the recommendations of the 2000 USDA Food Guide Pyramid as well 2 completThe survey instrument also contained one question regarding their alcohol consumption: Do you drink alcohol? Subjects who answered yes on alcohol intake were asked about the number of servings of alcohol consumed per week.Pilot testing and focus groups affirmed understanding of question terminology in the survey and did not report any problems with the determination of what their perception of a serving was for the various food groups. Data from surveys were double entered and cross checked with error rates of <1%.Unpaired t-test and Pearson's chi square test were used to determine baseline differences in participant characteristics by gender and ethnicity. Data were also analyzed to determine if the participants who did not complete the nutrition survey were different in their CVD risk factor characteristics than those who did. Multiple linear regression analysis was used to determine the association between dietary intake of various food groups such as grains, meats, fruits & vegetables, dairy, water and alcohol (independent variables) and risk factors for CVD namely BMI, blood glucose, blood total cholesterol, and blood pressure (dependent variables). All CVD risk factors were used as continuous variables in the analysis. Bivariate and multivariate analysis was performed with each CVD risk factor as the outcome variable and each food group as the predictor variable. Covariates adjusted for in the multivariate analysis included age, gender, BMI and ethnicity. Results presented are those from the regression models that were statistically significant.Staging for grains, FV, meats, dairy and water was determined using a staging algorithm. While the nutrition survey provided information on all five Dietary Stages of Change categories, due to small cell sizes it was necessary to collapse the stages of change from five stages to two stages. These were named the 'cognitive stage' since these participants have not made any overt behavior changes and; the 'behavior stage' (action and maintenance) since these participants have either made overt behavior changes for less than or more than 6 months and the behavior change is observable. Significance level for all analysis was set at p < 0.05.Table Table Table not in an action or maintenance stage for fruits, vegetables, dairy and water consumption.Table Multiple linear regression analysis was performed to determine if the dietary intake of the various food groups such as grains, FV, meats, dairy as well as alcohol and water intake were significant predictors of the CVD risk factors namely BMI, systolic blood pressure, diastolic blood pressure, blood cholesterol and blood glucose. Multivariate analysis after adjusting for confounders such as age, gender and ethnicity showed that higher grains intake was positively associated with higher BMI and higher alcohol intake was also positively associated with higher BMI . Other associations of interest between the food groups and CVD risk factors were not statistically significant.Overall a high percentage of participants reported eating fewer grains, FV and drinking fewer glasses of water than recommended. Additionally, a majority of the participants were not ready to produce a change in their eating behaviors. Our results also identified a positive association between grain consumption, alcohol intake and BMI.Forty-five percent of the participants were overweight or obese. The high prevalence of overweight/obesity that was observed in our study concurs with other studies on college-age students which report that 20% to 40% of college students are overweight or obese ,25. The When compared to the 2000 Dietary Guidelines for Americans, our data indicate that less than 20% of participants in this urban marching band university were consuming 5 or more servings of FV per day. Similar results were observed in the 2006 NCHA survey, which reported that only 6.2% of the students were usually consuming 5 or more servings of FV a day ,30. AlsoThe results from the Dietary Stages of Change assessment suggest that the majority of participants are in the cognitive stages for the dietary intake of grains and FV indicating that they are not making any overt changes towards changing their eating behavior for grains and FV. These data imply that health promotion programs aimed at moving these athletes from the cognitive stages to the behavior stages by increasing knowledge and providing necessary skills to improve dietary habits are critical in this population. These data provide critical information regarding the study population's readiness to change their dietary habits which clearly indicates that, at this time, it would be counterproductive to provide action-oriented material about healthy dietary change behaviors. In fact, the focus needs to be more on raising awareness of poor dietary habits, pros of eating more whole grains, FV to move the participants towards action. It is also important to note that the Stages of Change model has potential for public health interventions as well as individual nutrition interventions by health care practitioners towards improving the dietary habits of this population. Additionally, environmental factors, such as availability of FV and whole grain foods on-campus, need to be evaluated. Psychosocial factors such as knowledge, attitudes, beliefs, self-efficacy, outcome expectations and social support influencing diet and weight status in these athletes also need to be assessed.Greater than fifty-seven percent of the participants indicated consuming 1 or more servings of alcohol per week with more than 10% of them drinking 5 or more servings of alcohol per week. These results concur with the NCHA statistics which reported that 70% of college students used alcohol in the last 30 days . While mPerhaps the most alarming finding of our study was the high percentage of participants with high WHR, elevated blood glucose, cholesterol and systolic and diastolic blood pressure. The major risk factors for CVD include levels of cholesterol , BMI, blood pressure, cigarette smoking and diabetes ,38. StudThese results could indicate the presence of components of metabolic syndrome among participants which is considered to be a risk factor for CVD . Huang eThis study is among the first to evaluate the dietary habits and examine their relationship to CVD risk factors in marching band, dance team and cheer squad members, dance team and cheer squad. The large sample size lends sufficient power to the study. Currently there is little data available on the dietary habits as well as CVD risk factors in this population. While not confirmatory, these results are an appropriate starting point for further in-depth exploration into these relationships between diet and CVD risk factors in student athletes and also pave the way for developing future research and health promotion/health education programs targeting this population.These strengths notwithstanding, our study has its limitations. We had a high response rate since the study was conducted on campus and the participants had incentives in the form of free nutrition and fitness counseling sessions conducted by exercise physiologists and licensed dietitians which can be very expensive if the participants were to obtain these on their own. This high response rate could have biased our results away from the null. Secondly, only about 84% (N = 232) of the total respondents completed the nutrition survey. Also, it appears that the prevalence of the CVD risk factors was higher in the larger sample (N = 275) as compared to those who did not complete the survey which could have biased our results towards the null. However, all attempts were made by study investigators to contact the participants who did not complete the nutrition survey.Another limitation is that this study relied on self-reported measures of dietary intake which could bias the validity . HoweverIn sum, our results suggest a trend toward significant health risks in this elite members of a marching band unless an intervention of diet and exercise are performed. A high percentage of members of this urban marching band have a poor dietary intake and demonstrate a relatively high prevalence of overweight, hyperglycemia, hypercholesterolemia, and hypertension that increase their risk for CVD as well as other lifestyle-related illnesses. These results are especially important since these athletes participate in rigorous activities often under extreme weather conditions throughout the season and need to be in sound health for optimal performance. The unhealthy eating behaviors and a relatively high prevalence of alcohol consumption identified in the current study imply the need to evaluate existing health promotion programs aimed at improving the lifestyle of college students on campus and for evidence-based programs to be implemented. Finally, ACHA tools such as Standards of Practice for Health Promotion in Higher Education and HealThe authors declare that they have no competing interests.SVS conceptualized the study question and design, conducted statistical analysis, interpretation of results, drafting of manuscript and reviewing of the manuscript. JAB is the Principal investigator of the study, conducted data collection, drafting and critically evaluating the manuscript. AJL did data collection, data entry, drafting of the manuscript and reviewing the manuscript. MK did data collection and critically reviewed the manuscript. DA did data collection and critically reviewed the manuscript. GB did data collection and critically reviewed the manuscript. DB participated in study design and data collection.Informed consent was obtained from all participants prior to the study and the study protocol was approved by the University of Houston, Committee for Protection of Human Subjects."} +{"text": "Neisseria gonorrhoeae-mediated enhancement of HIV infectivity in vitro. In this study, we dissect the molecular mechanism of the HIV enhancing effect of defensins.Concurrent sexually transmitted infections (STIs) increase the likelihood of HIV transmission. The levels of defensins are frequently elevated in genital fluids from individuals with STIs. We have previously shown that human defensins 5 and 6 (HD5 and HD6) promote HIV entry and contribute to HD5 and HD6 primarily acted on the virion to promote HIV infection. Both HD5 and HD6 antagonized the anti-HIV activities of inhibitors of HIV entry (TAK 779) and fusion (T-20) when the inhibitors were present only during viral attachment; however, when these inhibitors were added back during viral infection they overrode the HIV enhancing effect of defensins. HD5 and HD6 enhanced HIV infectivity by promoting HIV attachment to target cells. Studies using fluorescent HIV containing Vpr-GFP indicated that these defensins enhanced HIV attachment by concentrating virus particles on the target cells. HD5 and HD6 blocked anti-HIV activities of soluble glycosaminoglycans including heparin, chondroitin sulfate, and dextran sulfate. However, heparin, at a high concentration, diminished the HIV enhancing effect of HD5, but not HD6. Additionally, the degree of the HIV enhancing effect of HD5, but not HD6, was increased in heparinase-treated cells. These results suggest that HD5 and haparin/heparan sulfate compete for binding to HIV.HD5 and HD6 increased HIV infectivity by concentrating virus on the target cells. These defensins may have a negative effect on the efficacy of microbicides, especially in the setting of STIs. There were an estimated 33 million people living with HIV in 2007, and there were 2.7 million new HIV infections, with the predominant mode of infection being sexual transmission (UNAIDS 2008). Currently, there is no effective vaccine or microbicide available to prevent HIV spread. According to CDC data in 2008, approximately 56,000 people become newly infected with HIV every year in the U.S. It was estimated that more than 21% of the 1.1 million infected individuals in the U.S. are unaware of their infection. While the spread of HIV is inefficient, sexually transmitted infections (STIs) are known to increase the likelihood of HIV transmission HD5 and [Abu]HD6, in which the six cysteine residues were replaced by isosteric \u03b1-aminobutyric acid (Abu), were chemically synthesized and folded as described previously . The mol+ T cells were isolated from PBMCs by negative selection using a CD4+ T cell isolation kit from Miltenyi Biotech . The purity of cells was 98% based on flow cytometric analysis. CD4+ T cells were stimulated with phytohemagglutinin (PHA) at 5 \u03bcg/ml and maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 25 units/ml IL-2 for 3 days at 37\u00b0C prior to viral infection. HeLa-CD4-CCR5 cells were provided by David Kabat and maintained in Dulbecco's minimal essential medium (DMEM) containing 10% FBS.Peripheral blood mononuclear cells (PBMC) from normal healthy blood donors were isolated by Ficoll-Hypaque gradient centrifugation. CD4JR-FL for a single-cycle infection assay, were produced as described previously [nef and a pSV plasmid expressing the JR-FL glycoprotein. The supernatant was collected 48 hours after transfection, and filtered. Virus stocks were analyzed for HIV-1 p24 antigen by ELISA . To produce HIV-1JR-FL pseudotyped viruses in the absence of serum, transfection was performed as described above. Transfected cells were incubated for 24 h, washed with PBS, and cultured in medium without serum for an additional 24 h prior to collecting viruses.Replication-defective HIV-1 luciferase-expressing reporter viruses, pseudotyped with HIV-1eviously ,51. BrieJR-FL luciferase reporter viruses were incubated with defensins at 20 \u03bcg/ml at 37\u00b0C for 1 hour. FBS at a final concentration of 10% (v/v) was added the defensin-virus mixture before addition to HeLa-CD4-CCR5 cells, seeded at 5 \u00d7 104 in a 48-well plate and grown for overnight. After 2 h incubation, cells were washed extensively and cultured for 48 hours before measuring of luciferase activity using Luciferase Substrate Buffer (Promega Inc). Luciferase activity reflecting viral infection was measured on an EG & G (Berthold) MiniLumat LB9506 luminometer.To assess whether defensins enhanced HIV infection by acting on the virions, serum-free pseudotyped HIV-16) or HeLa-CD4-CCR5 cells (5 \u00d7 104) were treated with defensins in the presence of FBS for 1 hour at 37\u00b0C, washed, exposed to pseudotyped HIV-1JR-FL luciferase reporter viruses for 2 hours, washed, and cultured for additional 48 hours.To determine the effect of defensins on the target cell, PHA-activated primary CD4+ T cells (1 \u00d7 10JR-FL virus (~10 ng p24 per sample) was incubated with HD5 or HD6 at 20 \u03bcg/ml at 37\u00b0C for 1 hour. The virus mixture was then added to cells in the presence or absence of inhibitors for 2 hours. After washing off unbound virus, infected cells were cultured in the absence (wash off) or presence (add back) of the inhibitors for 48 hours before measurement of luciferase activity.To determine the effect of defensins on anti-HIV activity of HIV inhibitors, HeLa-CD4-CCR5 cells were pre-treated with or without TAK-779 (2 \u03bcM) or T-20 (200 nM) for 1 hour. Cells without HIV inhibitor treatment were included as a control. Serum-free pseudotyped HIV-1JR-FL pseudotyped luciferase reporter virus was incubated with or without HD5 or HD6 in the presence of soluble GAGs at varying concentrations at 37\u00b0C for 1 hour followed by HIV infection. The removal of cell-associated GAGs was performed by incubating with heparinase I (20 U/ml) for 2 hours at 37\u00b0C. Cells were washed with PBS three times before HIV infection.To determine the effect of defensins on HIV infection in the presence or absence of soluble GAGs, serum-free HIV-14 per well in 48-well plates and cultured overnight. PHA-activated primary CD4+ T cells (5 \u00d7 105 per sample) were prepared as described above. Serum-free pseudotyped HIV-1JR-FL was pre-incubated in the absence or presence of defensins for 1 h at 37\u00b0C. FBS was added the virus mixture to a final concentration to 10% (v/v) before addition to cells. Cells were then incubated with virus for 2 hours at 4\u00b0C or 37\u00b0C. Cells were washed four times and lysed with 1% Triton X-100. Cell-associated HIV p24 antigen was measured by p24 ELISA .HeLa-CD4-CCR5 cells were seeded at 5 \u00d7 10JR-FL virus containing Vpr-GFP (25 ng p24) was incubated with or without defensins for 1 hour before exposure to EDTA-suspended HeLa-CD4-CCR5 cells (5 \u00d7 105 cell per sample) at 4\u00b0C for 2 hours. After washing off unbound virus, cells were fixed with 2% paraformaldehyde and analyzed on a FACScan . Results were analyzed with FlowJo Software . To analyze the effect of defensins using microscopy, HeLa-CD4-CCR5 cells at 2.5 \u00d7 105 cells per well were seeded into a 4-well chamber slide and cultured overnight. The defensin-GFP virus mixture was added to the cells and incubated at 4\u00b0C for 2 hours. After washing off unbound virus, cells were fixed and mounted with VECTASHIELD HardSet mounting media with DAPI and visualized using Axioplan 2 . The images were analyzed using Volocity 5.2.1 .To access the effect of defensins on HIV attachment by FACS analysis, pseudotyped HIV-1The authors declare that they have no competing interests.AR performed the experiments on HIV infection and HIV attachment by HIV p24 ELISA. JD performed the experiments on HIV infection and HIV attachment by FACS and microscopy, and prepared the manuscript; BB assisted in the preparation of recombinant viruses and HIV infection; YL performed statistical analysis; MN analyzed the surface charges of dimerized defensins and prepared the manuscript; WL prepared peptides, discussed the results, and was involved in manuscript preparation; TLC oversaw the entire project, designed experiments and prepared the manuscript. All authors read and approved the final manuscript."} +{"text": "Gynura procumbens is a traditional herb used for the treatment of inflammation, rheumatism and viral infections, although the antitumor effect and its potential mechanisms of action remain unclear. In the present study, the antitumor effect of Gynura procumbens ethanolic extract (GPE) on the osteosarcoma (OS) cell line, U2-OS, was investigated in vitro. Cell proliferation and apoptosis were measured by 3--2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Transwell invasion and wound healing assays were performed to investigate the invasion and migration of the U2-OS cells. The results showed that GPE was able to inhibit U2-OS cell proliferation and metastasis and induce cell apoptosis. Furthermore, the expression of the NF-\u03baBp65 protein was detected by western blotting to evaluate the effects of GPE on the nuclear transfer of NF-\u03baB. It was demonstrated that the expression of the NF-\u03baBp65 protein was significantly decreased by GPE. This indicated that GPE was able to inhibit the nuclear transfer of NF-\u03baB. The study shows that GPE is able to induce apoptosis and suppress proliferation and metastasis in U2-OS cells via the inhibition of the nuclear translocation of NF-\u03baB. Osteosarcoma (OS) is the most common primary mesenchymal malignant tumor of the bone tissue in humans, particularly children and adolescents . It was Gynura procumbens is a decumbent perennial herb belonging to the Asteraceae family and is widely distributed in Southeast Asian countries, including China, Indonesia, Thailand and Malaysia. The stem and leaves of Gynura procumbens have been used as a food and traditional medicine, particularly in treating cancer, inflammation, rheumatism and viral infections -1 and MMP-9 expression induced by UV-B irradiation via the inhibition of pro-inflammatory cytokine mediator release and reactive oxygen species (ROS) production. It has been demonstrated that MMPs are essential in the degradation of the basement membrane and epimatrix, among which MMP-2 and MMP-9 are the most markedly correlated with tumor invasion and metastasis on cell proliferation, apoptosis and metastais was analyzed in the OS cell line, U2-OS. Subsequently, the effect of GPE on the inhibition of the nuclear transfer of NF-\u03baB in U2-OS cells was investigated.In the present study, the effect of Gynura procumbens (Lour.) Merr. plants, excluding the roots, were collected from the Yifeng county of Jiangxi, China and authenticated at the Jiangxi University of Traditional Chinese medicine, China, by Professor Luo Guangming.Whole in vacuo, with a yield of 1.4%. The extract was then reconstituted in 95% ethanol and vacuum filtered. The resulting filtrate was subjected to evaporation in vacuo, which removed the ethanol and left an aqueous solution containing an ethanol-soluble precipitate. The GPE was dissolved in dimethyl sulfoxide and the final concentration of DMSO in the culture medium was controlled at 0.1% (v/v).The leaves and stems from the fresh plant were cleaned and dried in an oven at 40\u00b0C, then ground into powdered form at 100 mesh size. A crude ethanolic extract was created by macerating the powder with 95% ethanol at 85\u00b0C for 12 h. The extract was concentrated until dry 2.The human OS cell line, U2-OS, was obtained from the American Type Culture Collection and routinely cultured in Dulbecco\u2019s modified Eagle\u2019s medium supplemented with 10% fetal bovine serum in a humidified 37\u00b0C incubator containing 5% CO\u03bcg/ml) of GPE. Subsequent to treatment for 24 h, 3--2,5-diphenyltetrazolium bromide (MTT) assays were performed in triplicate at a wave-length of 490 nm.The U2-OS cell line was cultured in 96-well tissue culture plates at a cell density of 5,000 cells per well, in DMEM containing 10% FBS and 2 mM L-glutamine. Following adherence overnight, the medium was replaced and the cells were incubated with increasing concentrations and fixed overnight in ice-cold 70% ethanol. Subsequent to fixation, the cells were washed twice with 1% bovine serum albumin (BSA) in PBS, then resuspended in 1 ml DNA-binding propidium iodide (PI) solution , incubated at room temperature in the dark for 15 min and analyzed with an EPICS XL flow cytometer . The number of apoptotic cells were measured using the control software of the flow cytometer.Human OS U2-OS cells were seeded at 5\u00d710\u03bcg/ml) for 24 h. The cells were then washed with cold PBS. Total protein from the cells was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer containing 60 \u03bcg/ml phenylmethanesulfonylfluoride (PMSF) and the protein concentration was determined using a Bradford assay. Equal amounts of protein were electrophoresed by 10% SDS-PAGE and transferred onto a pure nitrocellulose blotting membrane (0.22-\u03bcm pores). The membranes were blocked with 5% Difco skimmed milk for 1 h at room temperature (RT), then blocked with primary antibody overnight at 4\u00b0C. The membranes were then washed prior to incubation with the appropriate peroxidase-conjugated secondary antibodies . The immune complexes were detected with a Pro-light HRP kit . All experiments were repeated six times.U2-OS cells in the exponential growth phase were treated with various concentrations of GPE and added to the upper chambers simultaneously (2\u00d7104 cells/ml in 0.1 ml). The cells that passed through the Matrigel-coated membrane were stained with Diff-Quik and images were captured. Cell migration was quantified by direct microscopic visualization and counting. The values for invasion were obtained by counting three fields per membrane and the results are presented as the average of six independent experiments performed over multiple days.The invasiveness of the U2-OS cells was measured using BD BioCoat\u2122 BD Matrigel TM Invasion Chambers according to the manufacturer\u2019s instructions. The medium in the lower chamber contained 5% fetal calf serum as a source of chemoattractant. The cells were suspended in serum-free medium containing various concentrations of GPE for 24 h, the cells were denuded by dragging a rubber policeman through the center of the plate. The cultures were rinsed with PBS and fresh quiescent medium alone or with 10% FBS was added, after which the cells were incubated at 37\u00b0C for 24 h. The cells were photographed at 0 and 24 h and the migrated distance was measured. The cell migration rate was obtained by counting three fields per area and the results presented as the average of six independent experiments performed over multiple days.Cell migration was assessed by determining the ability of the cells to move into a cellular space in a two-dimensional Data are expressed as the mean \u00b1 SD. The differences in invasion and migration between cells treated with GPE and the control group were evaluated using independent-sample t-tests. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS Version 13.0 .in vitro.The effect of GPE on the growth of the U2-OS cell line was investigated using MTT assays. The growth curves indicated that the U2-OS cells were sensitive to GPE and that growth inhibition occurred in a dose- and time-dependent manner , indicatin vitro. GPE at various concentrations was added to the U2-OS cell cultures in the exponential growth phase. Subsequent to treatment for 24 h, treated and untreated cell samples were obtained and fixed for FCM analysis. The FCM analysis demonstrated that the percentages of apoptotic cells were 0, 5.5, 7.6, 24.7 and 37.94% at 0, 10, 20, 40 and 80 \u03bcg/ml GPE. This indicates that apoptosis occurred in a dose-dependent manner in cells treated with GPE Merr., which is also known as \u2018Sambung nyawa\u2019, is widely used in Southeast Asian countries as a herbal medicine in the traditional treatment of numerous ailments, including eruptive fevers, rashes, kidney disease, migraines, constipation, hypertension and diabetes mellitus. The benefits of the traditional use of Gynura procumbens have also been supported by the isolation and identification of several possible active chemical components from this plant, including flavonoids, saponins, tannins and terpenoids components, such as collagen and elastin. The degradation of ECM components has a significant role in tumor cell metastasis (Gynura procumbens extract may inhibit the nuclear translocation of NF-\u03baB.The potential molecular mechanism of the inhibition of U2-OS cell invasion and migration by GPE remains unclear. Kim et al reportedtastasis . Among atastasis \u201319. The tastasis . It has tastasis , while ttastasis . In the in vitro and that the inhibition of the nuclear translocation of NF-\u03baB appeared to be the potential molecular mechanism. However, previous studies have shown that the tumor micro-enviroment may affect tumor cell proliferation, invasion and migration. Consequently, further in vivo experiments are necessary to confirm the anticancer activity of Gynura procumbens extract. Based on the results observed in the present study, it appears that further advances in the identification of the active principles of GPE are likely to provide more solid evidence of Gynura procumbens as an antitumor agent.In conclusion, the present study showed that GPE was able to significantly inhibit U2-OS cell proliferation and metastasis"} +{"text": "Neospora caninum infection. In order to explore this in the absence of acquired host immunity to the parasite, we have tested infection in locusts (Schistocerca gregaria). We show for the first time that locusts are permissive to intra-hemocoel infection with N. caninum tachyzoites. This was characterized by alteration in body weight, fecal output, hemoparasitemia, and sickness-related behavior. Infected locusts exhibited progressive signs of sickness leading to mortality. Also, N. caninum showed neuropathogenic affinity, induced histological changes in the brain and was able to replicate in the brain of infected locusts. Fatty acid (FA) profiling analysis of the brains by gas chromatography and multi-variate prediction models discriminated with high accuracy (98%) between the FA profiles of the infected and control locusts. DNA microarray gene expression profiling distinguished infected from control S. gregaria brain tissues on the basis of distinct differentially-expressed genes. These data indicate that locusts are permissible to infection with N. caninum and that the parasite retains its tropism for neural tissues in the invertebrate host. Locusts may facilitate preclinical testing of interventional strategies to inhibit the growth of N. caninum tachyzoites. Further studies on how N. caninum brings about changes in locust brain tissue are now warranted.Neuropathogenesis is a feature of Neospora caninum is an important cause of infertility and abortion in cattle and neuromuscular disorders in dogs School of Veterinary Medicine and Science (SVMS) Ethical Review Committee. The Committee reviews all research studies involving School personnel and is chaired by Professor David Haig. The committee passed this study as good to proceed, not requiring any further ethical review as it involved invertebrates. FELASA guidelines as outlined in \u2018principles and practice in ethical review of animal experiments across Europe (2005)\u2019 and UK guidelines on the use of invertebrates in research were followed.Neospora caninum (Liverpool strain) was cultured in human brain microvascular endothelial cells (HBMECs) originally obtained from ScienCell Research Laboratories , and counted using a haemocytometer and diluted to the desired concentration, 103, 104, 105 and 106 tachyzoites suspended in 20 \u00b5l culture medium, for inoculation . Locusts were kept in a large glass cage for not less than one week after arrival to acclimatize and reduce any stress associated with transportation. Adult locusts were used throughout the study to ensure a fully developed innate immune system. Each group of 10 locusts were housed in separate plastic \u201ccritter cages\u201d with ventilation slats in the lid and solid plastic bottoms to enable feces collection. They were fed daily with fresh grass and wheat seedlings supplemented with bran. Water was provided ad libitum in petri dishes to increase hemolymph volume. The \u201ccritter cages\u201d were kept on the bench in a controlled environment at 25 \u00b0C and 65% humidity with a 12-h light cycle. During the parasite exposure period, housing and manipulation of locusts was performed within a biological safety cabinet (class II) inside an ABSL2 + containment area.Desert locusts, N. caninum tachyzoites, 103, 104, 105, or 106 tachyzoites were inoculated in a total volume of 20 \u00b5l culture medium into the hemocoel of locusts in groups G1 to G4, respectively. Locusts in G5 were mock-inoculated with medium only and used as controls. An additional control group was included . Injection of the test materials was achieved using a 26g Terumo\u00ae Neolus hypodermic needle fitted into plastic pipette tips. Inocula were injected into the hemocoel by inserting the needle horizontally into the inter-segmental membrane at the last two abdominal segments using a pipetter, followed by gentle stretching of the locust\u2019s abdomen to ensure an even distribution of parasites within the hemocoel (Locusts were randomized into five groups (G1 to G5), with 10 locusts per group and were housed in separate \u201ccritter cages\u201d to prevent cross-cage contamination after parasite challenge. To establish a suitable dose for hemocoel . IndividPost mortem analysis was performed within the safety cabinet. Control locusts were sacrificed by making an incision behind the head at the end of the experiment. Survival data was analyzed using the Gehan-Breslow-Wilcoxon test, with death as the primary variable using GraphPad Prism version 5.00 for Windows . Significance between survival curves was assessed using the Log-rank (Mantel-Cox) test.Locusts were assessed for mortality twice daily and any dead locusts were removed to prevent cannibalism. N. caninum infection on the behavior of the locusts, a range of parameters were evaluated. These include the following: (i) The number of times the insect was inactive. (2) The number of walking bouts . (3) The number of times leg movements occurred. (4) The number of times antennal movements occurred. (5) The number of times grooming occurred. (6) The number of times wing-fanning occurred. (7) The number of times jumping occurred. This experiment was divided into two 15 min periods. During the first 15 min period, individual behavioral variables, chosen after preliminary observations, were registered every 10 s for locusts in each cage.To assess the effect of N. caninum tachyzoites under X40 magnification.To determine whether locusts developed parasitemia, hemolymph was collected from one locust per group 3 h PI and daily thereafter for three consecutive days. Hemolymph was collected from locusts by inserting a 1\u201310 \u00b5l pipette tip fitted into a pipette-aid into the arthrodial membrane at the base of the legs . At leasN. caninum infection on food consumption locusts were weighed prior to infection and daily for the duration of the experiment. The body weight changes were calculated as a percentage of initial body weight. Each locust was transferred from the cage within a closed 50-ml falcon tube, weighed on a weighing balance and returned back to the cage within the same falcon tube. Results are presented as means \u00b1 SEM from three independent experiments.To assess the effect of N. caninum on food consumption where the output of fecal pellets was determined on a daily basis. Fecal pellets produced per cage over a 24 h period were separated from non-fecal materials and weighed. This figure was divided by the number of surviving locusts to give an average of fecal output per locust.Another indirect measure was used to assess the effect of exposure to 4 tachyzoites and another group was sham inoculated with medium only. One locust from each of the infected and control groups was sacrificed daily and samples from the brains of each locust were collected and preserved in 5% paraformaldehyde for histology or 70% ethanol for genetic characterization. For histological analysis, samples were fixed for at least 24 hr before they were sectioned and stained with hematoxylin and eosin. Also, PCR-DNA sequencing was used to document the presence of growing parasites in the brain. Genomic DNA was extracted from the locust\u2019s brain using a DNeasy Blood and Tissue Kit (Qiagen Inc.) following the Animal Tissues Spin Protocol supplied by the manufacturer. The DNA was eluted in 50 \u00b5l of kit elution buffer and stored at \u221220 \u00b0C. Concentration and purity of DNA in sample extracts were checked by using a Thermo Scientific NanoDrop\u2122 1,000 Spectrophotometer, prior to PCR amplification. The two most commonly species-specific primers used to diagnose N. caninum, primers Np6 plus (5\u2032CTCGCCAGTCAACCTACGTCTTCT3\u2032) and Np21 plus (5\u2032CCCAGTGCGTCCAATCCTGTAAC3\u2032), were used to amplify approximately 337-350-bp fragment of the Nc5 gene, which is commonly used to identify N. caninum infection , 17 \u00b5l of nuclease-free water (Fisher Scientific), and 0.5 \u00b5l (\u223c10 pmol) of each forward and reverse oligonucleotide primer (Eurofins). BioMix\u2122 is a complete, ready-to-use, 2 \u00d7 reaction mix containing Taq DNA polymerase. It is used to perform PCR assays of numerous genomic and cDNA templates by only adding water, template and primers. All amplifications were carried out in triplicates using the Bioer Xp Cycler. The PCR cycling conditions consisted of an initial denaturation for 5 min at 94 \u00b0C and then 40 cycles of 94 \u00b0C for 1 min, 63 \u00b0C for 1 min, and extension at 74 \u00b0C for 3.5 min, followed by a final extension at 72 \u00b0C for 10 min. A negative control (nuclease-free water instead of DNA) and a positive control (DNA from cultured-derived N. caninum) were included in each run. The quality and specificity of all the amplification products were assessed by 2% agarose gel electrophoresis followed by staining with ethidium bromide. Individual PCR product bands were visualized under ultraviolet light. Amplicons were purified using a QIAquick PCR Purification Kit (Qiagne) according to the manufacturer\u2019s instructions. Cleaned-up products were sequenced bidirectionally using the same PCR oligonucleotide primers used in initial amplification using a commercial service . To gain insight into the anatomic sites of N. caninum distribution and replication, we detected N. caninum DNA in several different body tissues of locusts using the same PCR assay described above.In a second experiment one group of five locusts were inoculated with 10nfection . Nc5 hase region . Hence, 3, 104, 105, or 106 tachyzoites (three locusts per group). At 5, 10 or 17 day PI brain was obtained from one locust from each group , and were dissected under aseptic conditions for parasite isolation. Brains were washed twice in RPMI medium, divided into small pieces by scissors and homogenized by pipetting up and down gently 10 times through a 1-ml pipette tip, followed by passing the homogenate through an 18-gauge needle and syringe. The resulting homogenate from four brains at each of the time points mentioned above were pooled and resuspended in 6 ml of RPMI medium. The 6 ml homogenate from the pooled four brains (1ml/well) was overlaid on healthy HBMECs seeded in a 6-well culture plate and topped up by a second ml of fresh RPMI medium, and plate was incubated at 37 \u00b0C. After 24 h the culture medium was decanted, and 2 ml of fresh RPMI medium was added to each well. One 6-well culture plate was seeded at 5, 10 or 17 day PI. All plates were incubated in a humidified atmosphere at 37 \u00b0C, and were examined daily for signs of parasite growth within cultured cells by inverted light microscopy.For this experiment 12 freshly dead locusts were randomly selected from groups which have been infected with 10N. caninum in a non-natural host has favoured parasite phenotypic or genotypic changes. The species identity of the locust-derived N. caninum isolate was confirmed and characterised in comparison to the original isolate. Tachyzoites from each isolate were subjected to a range of phenotypic and genetic characterisation methods. Firstly, immunofluorescence staining of tissue culture infected with each isolate was performed as described previously to identify chemical differences. The measurements were performed using 785 nm laser, and at 3 s integration time for every spectrum. Fifthly, approximately 105 tachyzoites derived from one locust-derived isolate were used for subsequent inoculation into five new locusts to assess the effect of passage into a non-natural host on the parasite\u2019s ability to retain its neurotropism. Locusts were monitored daily for 3 weeks for mortality and sickness behavior. All experiments were performed three independent times.We tested if the passage of eviously . Secondl4N. caninum tachyzoites; this dose was chosen because it produces clinical illness in the infected locusts. Negative-control locusts were injected with 20 \u00b5l of medium. One day after infection and daily thereafter, five locusts from each group (infected or control) were sacrificed by cervical dislocation and their brains were dissected within the safety cabinet, providing five replicates per group. Brain samples were immediately frozen in liquid nitrogen and homogenised using a gentleMACSTM closed homogeniser . Homogenised tissues were subjected to lipid extraction as described previously using a general linear model. The significance of each fatty acid was assessed using p-values corrected for multiple comparisons using the Bonferroni method. All fatty acids that obtained a p-value <0.05 were kept for further analysis.Data obtained from the brain phospholipid composition was filtered of poorly measured fatty acids (defined as fatty acids with percentiles below 0.01%). Afterwards, the data were evaluated using SPSS 16.0 mortar and ground with a pestle to a very fine powder with liquid nitrogen. The powdered samples were transferred to 2 ml Eppendorf microtubes and used for extraction of total RNA. Total RNA was extracted from 50 mg sample powder using the QIAGEN RNeasy Mini Kit . To verify the quality of the RNA, the yield and purity were determined spectrophotometrically and with an Agilent 2100 Bioanalyzer using RNA 6000 Nano kits . A whole transcript drosophila array was assessed for the study of gene expression in locust. The hybridization of heterologous (non-specific) nucleic acids onto arrays designed for model-organisms have been shown to be a viable genomic resource for estimating gene expression in many non-model organisms and used as described previously to conveN. caninum tachyzoites showed less survival time, where all locusts in G1, G2, G3 and G4 died by day 25, 23, 21 and 20 PI, respectively , , , or ]. The mortality rate didn\u2019t follow a dose-dependent manner, despite the increase in inoculation dose. Two locusts from the negative (uninfected) control group and one locust from the environmental control group had died during the course of the experiment, but their death was not due to infection with N. caninum based on a negative PCR result for brains of those three locusts.Locusts died gradually as the infection progressed over the course of the experiment which lasted up to 27 days PI. Locusts infected with larger number of ectively . CompareAssessment of various behavioral parameters revealed significant inter-locust variability that did not correlate with differences in the dose of infection. For example, variability in behavioral parameters in individual locusts reared in the same cage varied by as much as 5-fold, which precluded any statistical analysis to be performed. The source of this variability among locusts is not understood despite the standardization of age, food, handling, and rearing conditions. Despite this variability we observed abnormal behavior in locusts two days after infection that increased over time. Most of the locusts exhibited a sluggish mobility one day before death and some showed dark blue discoloration on the day of death.N. caninum were observed in the hemolymph collected from all infected locusts up to 48 h PI. Subsequently, no evidence of the parasites was obtained in hemolymph from any locust. Collection of 20 \u00b5l of hemolymph from each locust did not seem to have any effect on the activity or weight of the infected or control locusts.Tachyzoites of N. caninum did not cause a remarkable reduction in the body weight (3 tachyzoites), the locusts did not gain or lose weight, and there was no significant difference between locusts from the sham-infected or environmental groups. However, there was a statistically significant difference in weight change between control locusts and N. caninum-infected locusts in the other groups . The calculation of the average body weight of each group could not be performed beyond day 15 PI because only a few locusts were left in infected groups, which precluded direct quantitative comparison between different groups.Infection with y weight . HoweverA slight increase in fecal output was detected in infected locusts 1 day PI, followed by progressive decrease in the subsequent days especially in locusts given the higher doses. But, a dose\u2013response reduction in the fecal output was not detected . No diffN. caninum-infected locusts demonstrated substantial changes in the brain of infected locusts. Even though the parasite was not observed in histological section there was accumulation of many inflammatory cells, located primarily in the white matter tracts of the brain of infected locusts, whereas no changes or immune response were detected in control locusts .Using a cell culture bioassay, viable N. caninum isolate. DNA sequences from N. caninum tachyzoites of each isolate was identical, and a consensus sequence obtained from both isolates showed 100% sequence homology to N. caninum sequence accession number AY497045 in GenBank database.Immunofluorescent and TEM N. caninum isolates. Locust-brain-derived N. caninum isolate was used to infect locusts to test their neuropathogenic capacity. In vivo passage in the locust\u2019s brain did not result in alteration in neuropathogenicity. One of the locust-adapted isolates induced brain infection in 100% of five newly infected locusts, which had detectable parasite burdens in the brain at different time points PI as evidenced by PCR. But no further attempt was made to re-isolate the parasites from these locusts and use for infection again.It was important to find out if the passage of the parasite through the locust had induced any alteration in the phenotype of the parasite. Chemical profiling using Confocal Raman Spectroscopy technique and prinindividual fatty acid is strongly associated to any of the two groups. Nonetheless, our analysis using the BioHEL machine learning algorithm reveals that we can create multi-variate prediction models that can assign samples to treatments with very high accuracy. The results of the data analysis are summarised in In an effort to understand the mechanisms underlying the complex relationships between the host locust and parasite infection, we compared the fatty acid profile of brain from infected locusts to that for their control counterparts. Among the 37 fatty acids analyzed in this study, results were consistently obtained from all tested samples for only seven FAs and included: saturated fatty acids: C14:0, C16:0, C18:0; monounsaturated fatty acids: C16:1; polysaturated fatty acid-Omega (n)-3: C18:3n3. Omega (n)-6: C18:2n6, C18:1n9C. N. caninum on S. gregaria, we examined the parasite impact on host gene expression using Affymetrix DNA microarray and Cross Species Hybridisation (CSH) analysis. Since N. caninum infection induced neurological injuries in locusts, it was important to test whether N. caninum infection had altered gene expression within locust brains. The analysis was designed to gain further understanding of the mechanisms for N. caninum neuropathy, and in particular, of the genes responsible for the capacity of N. caninum to establish brain infection. We used an established between the uninfected and N. caninum-infected locusts. Of these transcripts, 10 increased >1.5-fold and 6 decreased >\u22121.5-fold was able to infect the invertebrate desert locust with induction of signs of illness and death. The progression to a lethal N. caninum infection in this non-natural host may result from the cumulative deleterious effects of both direct and indirect consequences of parasite infection that lead to accelerated destruction and compromised host immune regenerative capacity. N. caninum did not elicit a typical mortality dose response curve after intra-hemocoel infection and the reason for this is not known. However, the lack of dose-dependent mortality can be explained by the existence of a threshold effect that occurs with relatively moderate number of parasites (103 to 104), after which large differences in parasite number make little difference. It is also possible that immune response might have been elicited against this virulent strain to control parasite dissemination and interfered with the fatal outcome following increasing infectious challenge doses of the parasite. Doses from 105 to 106 parasites might seem high for the establishment of an infection for downstream analysis. On the other hand, infection with less number of parasites didn\u2019t seem to cause any effect on the locust\u2019s sickness, mortality, or body weight. For these reasons, a dose of 104 was chosen for subsequent experiments, due to the reasonably long mean time of death after infection, which allows more time for data collection but is pathogenic enough to determine the effect of infection.Previously, species only andN. caninum into locusts, N. caninum migrated to the brain within 24 h accompanied by induced histological alterations and infection-specific inflammatory responses, suggesting that the insect immune response is recognising and responding to the parasites. However, because S. gregaria is not a natural host of N. caninum in the field, it unlikely that locust is able to mount a N. caninum-specific response. It remains to be investigated if histopathological changes are N. caninum-specific or inflammatory response to the parasite invasion.Also, there was a reduction of body weight and fecal output in response to infection, but not in a dose-dependent manner. This is most likely due to the use of pooled rather than individual fecal samples. The possibility that this was caused by changes in the diet of locusts was ruled out by dietary standardization. Parasites disappeared from the hemolymph of the locusts after 2 day PI. Infected locusts showed reduced mobility and sickness. The possible risk due to the manipulation of the locusts during weighing or collection of hemolymph was ruled out because these handling issues didn\u2019t seem to affect the control groups. Mock-infected group and environmental control locusts group, apart from the death of two and one locusts, respectively, appeared normal. The study of locusts\u2019 behaviour highlighted the difficulties associated with the behavioural assay. Many of the tested variables are not independent of one another . It could have been more useful to analyze the amount of time locusts spent doing each activity, rather than the number of bouts. Also, the assay has not been standardized to control for the time of day. Locusts show a certain amount of diurnal variation in activity levels that may have contributed to the variability of the assay results. Following the intra-hemocoel inoculation of N. caninum infection in locusts. An alternative explanation is that the parasite is strictly neurotropic and its absence in other tissues is due to the lack of significant neuronal tissues in other organs in the locust, unlike in vertebrates. These findings are different to an earlier report where the protist Acanthamoeba organisms were able to invade different organs of the infected locusts and did not exhibit any tissue-specific preference host. However, this is a new in vivo approach to culturing N. caninum and the first report of an establishment of N. caninum infection in an invertebrate host. The availability of this experimental invertebrate host opens an avenue for N. caninum research in defining determinants of the parasite virulence, and host roles in disease pathogenesis along with a reduction of the number of vertebrate animals used in research. The use of the invertebrate locust has proven valuable to discriminate molecules participating from both sides of the host-parasite interaction stage, are known to occur during animals . In the eraction . For exaand CNF1 . AssuminN. caninum tachyzoites from the brain of experimentally infected S. gregaria for up to 17 days PI. The isolation of a viable N. caninum isolates from the brain of infected S. gregaria provided an opportunity to examine whether passage of the parasites through a totally non-natural host induces phenotypic and/or genotypic changes in these parasites. Thus, the phenotype, genetic and chemical profile of N. caninum isolate derived from S. gregaria and the original isolate were compared. S. gregaria-derived isolate exhibited similar growth pattern to the original isolate (unpublished data). The two isolates were subjected to nucleotide sequence analysis of the Nc5 gene and compared to each other and to previously described N. caninum sequences available in Genbank database. PCR-sequencing analysis revealed that both parasite isolates are identical at least with respect to this gene. Due to the multi-copy nature of Nc5 gene direct sequencing of PCR products of this gene may not be that informative. Alternatively, cloning prior to sequencing followed by careful alignments of cloned sequences is a more sensitive approach to distinguish between all variants of the gene, and can enable the recovery of information useful for estimation of genetic diversity between N. caninum isolates. Minor, non-significant differences were found between the two isolates by using Raman spectroscopy. A large amount of spectral data obtained from the two isolates analysed by multivariate analysis did not reveal two distinct clusters, identifying only a few chemical metabolites that were different between the two parasite isolates between the FA profiles of the infected and control samples species using a whole genome oligonucleotide microarray of the fruit fly Drosophila melanogaster. In line with previous studies in mammalian data from the locust CNS and its wild type (NC-1) isolate with different virulence and cystogenic capability should be utilized.Transcriptome profiling analyses during ublished . These sS. gregaria can be used as an alternative to mammalian models of infection; particularly to investigate host-pathogen relationship isolates; this short segment of the DNA is not enough to determine genotypic changes, more sensitive methods, such as SNP analysis should be pursued in subsequent studies. Finally, study of the possible change in the protein expression pattern could be more informative than the Raman spectrometry analysis.Invertebrates confer additional advantages compared to vertebrate models, since they are less expensive, easy to obtain, maintain and handle experimentally and can facilitate the development and testing of new treatment/preventive strategies. The importance of invertebrate animals for the study of fungal pathogenesis has been reviewed recently . Studiestionship . Our stu10.7717/peerj.674/supp-1Figure S1Click here for additional data file.10.7717/peerj.674/supp-2Figure S2Hemolymph was collected by insertion of a pipette tip through the locust arthrodial membrane at the base of the walking appendages.Click here for additional data file.10.7717/peerj.674/supp-3Figure S3N. caninum strain isolated from locust brain (A) or original strain (B). Infected HBMECs are immune-labelled with primary monoclonal mouse anti-NcSAG1 antibody that recognizes surface antigen of the parasite and secondary goat-anti-mouse IgG FITC conjugate (green) and nuclear DNA stained with propidium iodide (red). Scale bars, 10 \u00b5m.Immunofluorescence micrographs of human brain microvascular endothelial cells (HBMECs) 4 hrs after infection with Click here for additional data file.10.7717/peerj.674/supp-4Figure S4N. caninum strain isolated from locust brain (A) or original strain (B). Abbreviations: host cell nucleus (n) and rhoptries (rop). Arrows points at parasitophorous vacuole, which encloses a number of tachyzoites (Ta). Bars in A = 3 \u00b5m and b = 2 \u00b5m.TEM of human brain microvascular endothelial cells (HBMECs) 24 h after infection with Click here for additional data file.10.7717/peerj.674/supp-5Figure S5PCA resulted in two relatively distinct components of three infected (blue) and three uninfected samples (red).Click here for additional data file.10.7717/peerj.674/supp-6Table S1Click here for additional data file.10.7717/peerj.674/supp-7Supplemental Information 1Click here for additional data file."} +{"text": "However, the function of WRKY TFs in the regulation of fruit ripening is unclear. Here, 23 tomato SlWRKYs that are similar to ethylene-responsive WRKY genes from other plant species, or show up-regulation during fruit ripening in previous genome-wide study, were selected, and their function in fruit ripening was investigated. Twelve SlWRKYs were found to be responsive to ethylene (SlER-WRKYs), showing expression patterns similar to those of genes related to fruit ripening. Eight SlER-WRKYs\u2014SlWRKY16, 17, 22, 25, 31, 33, 53, and 54, detected in the nuclei\u2014interacted with and activated the promoters of 4 genes related to color change: Pheophytin Pheophorbide Hydrolase (SlPPH), Pheophorbide a Oxygenase (SlPAO), Phytoene Synthase 1 (SlPSY1) and Phytoene Desaturase (SlPDS). Yeast two-hybrid and bimolecular fluorescence complement (BiFC) assays in Arabidopsis protoplasts indicated that protein interactions occurred between SlWRKY17 and SlRIN, SlERF2b or SlERF7; SlWRKY33 and SlERF7; SlWRKY54 and SlERF2b; and SlWRKY16 and SlWRKY17. Suppression of SlWRKY 16, 17, 53 or 54 by virus-induced gene silencing (VIGS) retarded the red coloration of the fruit. Our study provides comprehensive molecular evidence that WRKY TFs function in fruit ripening, particularly in color change, and are linked to the intricate regulatory network of other ripening regulators.WRKY transcription factors (TFs) play important roles in stress responses The findings in tomato show that ripening is regulated by a number of transcription factors (TFs) in conjunction with ethylene signaling2.The ripening of climacteric fruits is a complex, genetically programmed process that involves dramatic changes in color, texture, flavor, and aroma of the fruits. These changes are initiated by the plant hormone ethylene and coordinated by the expression of a large set of ripening-related genes3. Three TFs\u2014the MADS-domain protein Ripening Inhibitor (RIN)4, the Squamosa Promoter Binding protein Colorless Non-Ripening (CNR)5, and a ripening regulator of the NAC family of TFs, Non-Ripening (NOR)7\u2014have been proposed to function early in the transcriptional activation cascade upstream of ethylene production2. Additional components, including Tomato Agamous-Like1 (TAGL1), Apetala2a (AP2a)8, Fruitfull (FUL1 and FUL2), the HD-ZIP protein gene (HB-1)9, Ethylene Response Factor6 (ERF6)10, and Golden2-Like (GLK)11, have also been reported to be associated with early regulators and play important regulatory functions in the fruit ripening process. However, the links between this highly connected regulatory network and downstream effectors that modulate color, texture, and flavor are still poorly understood.Characterization of a number of tomato mutations that display defective ripening has provided novel insights into the control of ripening and has revealed an intricate regulatory network underlying the process12. A protein designated SGR (STAY-GREEN) in rice and Arabidopsis has been identified as a positive player upstream of chlorophyll degradation that dismantles Chl-protein complexes16. The accumulation of lycopene in tomato fruits is correlated with the up-regulation of genes encoding the enzymes functioning in the biosynthesis of the pigment during fruit ripening19. The expression of two genes encoding the key lycopene biosynthesis enzymes, phytoene synthase (PSY) and phytoene desaturase (PDS), increases rapidly at the breaker stage20. Although the biological pathways functioning in color change during fruit ripening have been clearly outlined, knowledge regarding the regulation of the individual genes in the pathways is fragmentary. Most of the information has been obtained when searching for target genes of RIN or other key TFs functioning in early fruit ripening. Using high-throughput chromatin immunoprecipitation with subsequent microarray analysis (Chip-Chip) and transcriptome comparison of the fruit ripening between wild type and rin mutants, SGR1 and PSY1 were identified as direct target genes that are positively regulated by RIN22. In addition to PSY1 and SGR1, other genes related to chlorophyll degradation or lycopene biosynthesis are up-regulated during tomato fruit ripening, but the regulation of these genes is unclear.Color change is one of the most obvious traits that accompanies fruit ripening. The ripening stage of tomato fruits can be clearly characterized by the sequential color changing program\u2014green, breaker, turning, orange, light red, red\u2014which is carried out via chlorophyll degradation and lycopene biosynthesis. The chlorophyll (Chl) molecules degrade in a stepwise manner by the action of a series of enzymes, including Chl b reductase (NYC), PPH, PAO and red chlorophyll catabolite reductase (RCCR)25. WRKY TFs have been found to be involved in the acclimation to various plant stresses, including pathogen infection26, drought or cold stress28. Several stress-related hormone signals triggered by ABA, SA, and JA/MeJA are mediated by WRKY TFs32. Accumulating evidence also proves the involvement of WRKY TFs in various plant development processes, including seed development34, somatic embryogenesis35 and leaf senescence36. AtWRKY53 was demonstrated to play an important role in leaf senescence by integrating numerical senescence initiating cues and activating the expression of key senescence-associated genes, such as SAG12, CATALASE 1/2/3 and ORE937. Two SlWRKYs (SlWRKY31 and SlWRKY23) were found to increase at both the breaker and red ripe stages of tomato fruit ripening, indicating WRKY TFs may be involved in the regulation of fruit ripening38. However, the involvement of WRKY TFs in relation to fruit ripening and color development has not been systematically studied.WRKY TFs belong to one of the largest plant-specific TF family. By binding to the W-box [(T) TGACC (A/T)] promoter regions of their target genes, WRKY TFs play important biological functions in the modulation of a large set of genes involved in many plant processesSlWRKY TFs were selected from the whole gene family, based on their high sequence similarities to 25 ethylene-responsive WRKY genes from Arabidopsis thaliana, Oryza sativa, Gossypium (Gossypium hirsutum and Gossypium barbadense), and Brassica napus, or based on their up-regulation profile during fruit ripening in a genome-wide study39. Twelve of the SlWRKYs were found to be up-regulated by ethylene treatment during fruit ripening and thus were designated SlER-WRKYs. They showed overlapping expression patterns with 5 genes related to color change and 4 genes related to ripening: 3 genes related to ethylene biosynthesis and 1 gene coding polygalacturonase (SlPG). W-box elements were found in the promoters of these 9 genes related to fruit ripening. Eight of the SlER-WRKYs were selected to analyze their regulation of the genes related to color change. Furthermore, the interactions between the SlER-WRKYs and SlRIN or SlERFs and interaction among the SlER-WRKYs were analyzed.In the present study, in order to investigate the regulation of WRKY TFs involved in tomato fruit ripening, 23 SlWRKYs in the present study were issued according to the report of Huang et al.39 and the Solyc chromosome identifier (https://solgenomics.net)40. The relevance of others names as well as the functions proposed in the literature are also listed in Table\u00a0SlWRKYs and 25 senescence-related or ethylene-responsive WRKYs from Arabidopsis thaliana, Oryza sativa, Gossypium (Gossypium hirsutum and Gossypium barbadense), and Brassica napus were highly expressed after ethylene treatment and peaked at 5 d or 7 d. Compared with levels in the fruits of the ethylene treatment and control groups, the expression levels of SlPPH, SlPAO, SlSGR, SlPSY1, SlPDS and SlPG were markedly repressed by 1-MCP treatment throughout the whole ripening developmental process and RIN-binding sites . Among the selected 23 SlWRKY genes, EREs existed in the promoters of SlWRKY16, 17, 22, 23, 45, 47, 52, 54, 71, 80, and 81, and CArG boxes existed in the promoters of SlWRKY6, 17, 22, 25, 29, 31, 33, 39, 45, 46, 47, 52, 53, 54, 80, and 81 leaves, with 200-fold higher transcript levels of SlWRKY25, 33 and 54 than those observed in young leaves , which indicated that 6 of the SlER-WRKYs (SlWRKY16 and 17 were excluded) contain 1 to 2 putative nuclear localization signals in addition to the WRKY domains , indicating an interaction between SlWRKY16 and the promoter of SlACO1. Similarly, the growth of the Y1HGold cells after being transformed with the plasmids expressing the other SlER-WRKYs in the presence of ABA indicated that interaction was also observed for SlWRKY17 or 53 and the SlACO3 promoter (pSlACO3); for SlWRKY16, 33, 53 or 54 and pSlPPH; for SlWRKY17 and pSlPAO; for SlWRKY16, 17 or 31 and pSlPSY1; and for SlWRKY22, 25 or 54 and pSlPDS. No interaction was observed for any of the SlER-WRKYs and pSlSGR1 . After transforming pGADT7-GR1 Fig.\u00a0.in vivo dual luciferase assay via a tobacco transient co-transformation system and in vivo regulation assays51. Based on bioinformatics analyses, W-box elements were found in the promoter sequences of genes that involved in ethylene, cell wall, chlorophyll, carotenoid metabolisms, as well as several ripening regulatory TFs for tomato fruit , z-carotene isomerase (Z-ISO) and carotenoid isomerase (CRTISO)22. In the present study, interaction and strong activation of the SlPDS gene were detected for SlWRKY25 and 54. One of key synthesis genes, phytoene synthase (PSY1), was found to be a direct target gene of both RIN61 and FUL162. Recently, it was reported that RIN, FUL homologs, and tomato AGAMOUS-LIKE1 may form DNA-binding complexes, co-regulating fruit ripening41. In the present study, the interaction and activation of the SlPSY1 gene were detected for SlWRKY16, 17, and 31 42 and SlERF2b (TERF2/LeERF2)43, reportedly function in carotenoid accumulation and ethylene biosynthesis, respectively. SlERF2b (LeERF2/SlERF.E1) was recently confirmed to be markedly up-regulated during tomato fruit ripening and to be positively regulated by SlRIN64.The interaction of the SlER-WRKYs and SlRIN or SlERFs found in the present study further supports their direct regulatory roles in color change during fruit ripening. As described above, SlRIN is a key regulator of fruit ripening that functions in the early stage of the process and was observed to directly interact with several genes related to color change, including SlER-WRKY 16, 17, 53 or 54, which present high similarities to AtWRKY 6 and AtWRKY53, respectively , indicating the involvement of WRKYs in tomato fruit ripening. Eight of the 12SlER-WRKYs were found to have the potential to directly regulate 4 genes related to color change, SlPAO, SlPPH, SlPSY1 and SlPDS. These SlER-WRKYs may form complexes with each other or with other TFs and may connect to the intricate regulatory network that controls in tomato fruit ripening. Further genetic data by RNAi or CRISPR/Cas9 lines will help to confirm the function of the SlER-WRKY genes.In conclusion, 12 Solanum lycopersicum var. cerasiforme) were grown in the greenhouse under long-day conditions at a temperature of 26\u2009\u00b0C in the light and 18\u2009\u00b0C in the dark at the Vegetable Research Institute, Academy of Agriculture Sciences of Guangdong, Guangzhou, China. Green mature tomato fruits with uniform shape, color and size were selected and randomly divided into 3 groups of 90 fruits for each. These fruits were treated with 100\u2009\u00b5L L\u22121 ethylene, 1\u2009\u00b5L L\u22121 1-MCP (1-methylcyclopropene) or air (control) for 24\u2009h in airtight boxes at 25\u2009\u00b0C. Afterward, the fruits of each treatment were placed evenly into 3 baskets, which served as 3 replicates, and allowed to ripen at 25\u2009\u00b0C and 80\u201390% relative humidity (RH). Fruits from each replicate were randomly removed at 0, 3, 5, 7, and 9 d after treatment. Fruit respiration and ethylene production rates and firmness were measured at each time point. Fruit pericarp tissues at each time point were sampled, frozen in liquid N2 and stored at \u221280\u2009\u00b0C to measure the contents of chlorophyll and lycopene and for gene expression analysis. The leaves and fruits at different plant developmental stages were sampled from plants in the greenhouse. Samples from 3 different plants served as 3 replicates.Tomato plants 67. Five fruits from each treatment at each time point were measured. Fruit firmness was expressed as mean Newtons (N).Fruit firmness was determined using a digital force pressure tester equipped with a 2-mm-diameter round plunger with a flat surface 2 and extracted in 5 mL of dichloroethane for 3 h at 35\u2009\u00b0C68. The organic phase of both extracts was collected for detecting the absorbance at 484 and 652 nm using a spectrophotometer . The chlorophyll and lycopene content was expressed as \u03bcg g\u22121 fresh weight.Chlorophyll was extracted by grinding 1 g of fruit pericarp tissue in 5 mL of 80% (v/v) cold acetone and soaked for 30 min at 4\u2009\u00b0C. For measurement of the lycopene, 2 g of fruit pericarp tissue was ground in liquid NTM RT Reagent Kit) with gDNA Eraser . The cDNA products were extracted for use in RT-qPCR.Total RNA from the frozen pericarp tissues was extracted using a total RNA Extraction Kit . After obtaining pure RNA, the synthesis of cDNA was carried out using a cDNA synthesis kit (PrimeScripthttp://solgenomics.net/organism/Solanum lycopersicum/genome) or were adopted from those of the previous reports39. The primers of genes related to ripening and the SlWRKY genes used for qRT-PCR analyses are listed in Supplementary Table\u00a0EF-1\u03b1)69 as a reference gene according the 2\u2212\u0394\u0394Ct method70. The values represent the means of three biological replicates.The specific primers of the SlWRKY genes were designed based on the 3\u2032-untranslated region by searching the SGN database . The primers are listed in Supplementary Table\u00a0SlWRKYs were expressed in frame, with YFP encoding the yellow fluorescent protein under the control of the cauliflower mosaic virus (CaMV) 35\u2009S promoter. The resulting constructs were then introduced into Agrobacterium strain EHA105. Tobacco (Nicotiana benthamiana) infiltration was performed as described previously71. After 48 hours of infiltration, the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI), and the fluorescence signals of the DAPI-stained nuclei and the signals of the SlWRKY-YFP fusion proteins were imaged using an LSM710 confocal microscope .The open reading frames (ORFs) of SlACO1, SlACO3, SlPAO, SlPPH, SlSGR1, SlPSY1, and SlPDS genes were cloned by an enzyme restriction method into the pAbAi vector carrying the AUR1-C gene; the primers are listed in Supplementary Table\u00a0SlWRKY16, 17, 22, 25, 31, 33, 53 and 54 genes were cloned into pGADT7-AD vectors; the primers are listed in Supplementary Table\u00a072.Y1H assays were performed using a Matchmaker\u2122 Gold Y1H System . The promoters of the SlACO1, SlACO3, SlPAO, SlPPH, SlPSY1, and SlPDS genes into pGreenII 0800-LUC vectors using a One Step Cloning Kit . The primers are listed in Supplementary Table\u00a0SlWRKY16, 17, 22, 25, 31, 33, 53 and 54 were cloned into the pCambia35tleYFP vector using the One Step Cloning Kit; the primers are listed in Supplementary Table\u00a0Nicotiana benthamiana) leaves were co-infiltrated with Agrobacterium GV3101 containing the reporter and effector vectors as described above. The ratios of enzyme activities of firefly luciferase (Luc) to renilla luciferase (Ren) were analyzed using a dual luciferase reporter assay system and a Luminoskan Ascent Microplate Luminometer . At least six transient transformations were carried out for each assay, and the assays were repeated twice.Reporter constructs were generated by cloning the promoters of the SlWRKY16, SlWRKY17, SlMADS-RIN, SlERF2b, and SlERF7 were cloned into the pGBKT7 vector containing the GAL4 DNA-binding domain (DBD) to create different baits using a One Step Cloning Kit . The primers are listed in Supplementary Table\u00a0SlWRKY16, 17, 22, 25, 31, 33, 53, and 54 were cloned into the pGADT7 vector in-frame with the GAL4 activation domain (AD) to create relevant prey; the primers are listed in Supplementary Table\u00a0Y2H assays were performed using the Matchmaker\u2122 Gold Y2H System . The full-length coding regions of 73.The transcriptional activities of the selected 8 SlWRKY TFs were also detected in the Y2HGold yeast. The ORFs of the SlWRKY16, 17, 22, 25, 31, 33, 53 and 54 genes were cloned into the pGBKT7 vector. The primers for the cloning are listed in Supplementary Table\u00a0SlWRKY16, SlWRKY17, SlRIN, SlERF2b, and SlERF7 were cloned into the pSAT6-cEYFP-C1-B vector for in-frame expression of these genes with the C-terminus of YFP, and the ORFs of SlWRKY17, 33, 53 and 54 were cloned into the pSAT6-n (1\u2013174) EYFP-C1 vector for in-frame expression with the N-terminus of YFP. The primers are listed in Supplementary Table\u00a074. After obtaining the transfected cells, the results were imaged using a confocal microscope as described above.For the BiFC assay, the ORFs of et al.75, and the primers of SlWRKY genes were listed in Supplementary Table\u00a0et al.76, and Li et al.42, with little modifications. The Agrobacterium strain GV3101 containing pTRV1 and pTRV2-LIC-SlWRKYswere mixed in a 1:1 ratio and infiltrated into the mature green fruit tissue through the stylar apex with a 1-mL needle-less syringe. The fruit that infiltrated with pTRV1 and pTRV2-LIC without the gene fragments was used as control. Then tomato fruit were allowed to ripen at 25\u2009\u00b0C and 80\u201390% RH for 14 days after infiltration. Infiltration for invidious gene was performed 3 times with 10 fruits for each repeat. The red color a* values of each fruit were measured by 14 days after infiltration. RNA was isolated from the pericarp tissue of each fruit and the transcription levels of the SlWRKY genes were analyzed by real-time PCR as described above. The transcription levels and a* values of 6 representative fruit with efficient silencing were subjected for statistics analysis.For the VIGS experiment, pTRV1 and pTRV2-LIC (used for free cloning) vectors were employed. The plasmid construction was performed as described in Dong Supporting Information"} +{"text": "HDACs in this process has not been elucidated. In our study, an RNA interference (RNAi) expression vector targeting SlHDA1 was constructed and transformed into tomato plants. Shorter fruit ripening time and decreased storability were observed in SlHDA1 RNAi lines. The accumulation of carotenoid was increased through an alteration of the carotenoid pathway flux. Ethylene content, ethylene biosynthesis genes and ripening-associated genes were significantly up-regulated in SlHDA1 RNAi lines. In addition, the expression of fruit cell wall metabolism genes was enhanced compared with wild type. Furthermore, SlHDA1 RNAi seedlings displayed shorter hypocotyls and were more sensitive to ACC (1-aminocyclopropane-1-carboxylate) than the wild type. The results of our study indicate that SlHDA1 functions as a negative regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation.Histone deacetylation is one of the well characterized post-translational modifications related to transcriptional repression in eukaryotes. The process of histone deacetylation is achieved by histone deacetylases (HDACs). Over the last decade, substantial advances in our understanding of the mechanism of fruit ripening have been achieved, but the role of Ripening allows fruit to facilitate seed dispersal and provides essential nutrition in the human diet. In climacteric fruits , ethylene plays important roles in fruit development and ripening and is an essential factor for the ripening process5. Respiration is dramatically induced and the ripening of fruit is initiated by ethylene biosynthesis in climacteric fruits6, which is different from the case in non-climacteric fruits (e.g. grape and citrus). There are two key biosynthetic enzymes in the ethylene biosynthesis pathway: ACS (1-aminocyclopropane-1-carboxylate synthase), which transforms SAM (s-adenosyl-l-methionine) to ACC (aminocyclopropane-1-carboxylic acid)7 and ACO (1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE), which converts ACC to ethylene8. In SlACS2 RNAi transgenic tomato fruits, ethylene production and fruit ripening are obviously inhibited8. Previous studies also revealed that RNAi inhibition of SlACO1 delays ripening of climacteric fruits10. These findings indicated that normal function of ethylene biosynthesis is essential for the ripening process.Fruit ripening is a complex regulated process that involves numerous metabolic changes, such as changes in color, flavor, aroma and nutrition. The process is controlled by endogenous hormonalE4 in fruit is rapidly up-regulated following exogenous ethylene induction11. In fruit, E4 transcripts are suppressed by ethylene biosynthesis inhibition12. E8 is another a ripening-associated, fruit-specific expression gene in tomato that is regulated by ethylene13. Thus, illuminating regulation of these gene activities is important for us to understand the processes of ripening.In addition to ethylene synthesis, the ability to perception and response to ethylene is necessary for fruit ripening. The expression of rin, Nr, Cnr and TAGL1 have facilitated our understanding of the transcriptional control system underlying tomato ripening19. For example, the rin mutant displays inhibited fruit ripening and enlarged sepals, which have phenotypes ascribed to the function of two MADS-box transcriptional factors, SlMADS-RIN and SlMADS-MC. SlMADS-RIN regulates fruit ripening, and SlMADS-MC is involved in sepal development and the formation of abscission zones18.Tomato is usually considered to be an excellent model plant for studying climacteric fruit ripening. To date, the regulatory mechanisms controlling fruit ripening in tomato have been studied extensively. In these studies, a series of natural ripening-deficient mutants in tomato, such as HDAC genes in Arabidopsis, 18 HDAC genes in rice, 5 HDAC genes in maize20 and 14 HDAC genes in tomato21. HDACs have been grouped into subfamilies: RPD3/HDA1, HDT and SIR223. In tomatoes, nine HDACs belong to the RPD3/HDA1 subfamily(SlHDA1\u20139)24, three belong to the HDT subfamily and two belong to the SIR2 subfamily(SIR1 and SIR2). Based on domain organization and phylogenetic relationships the RPD3/HDA1 subfamily was subdivided into four groups: class I , class II , class III (SlHDA5) and class IV (SlHDA6). Until now, there have been some reports on HDACs in Arabidopsis and rice, but rarely in tomato.Histone acetylation is often associated with activation of transcription, whereas histone deacetylation is correlated with transcriptional repression. Histone acetylation levels are determined by the action of HATs (histone acetyltransferases) and HDACs (histone deacetylases). Over the past decades, an increasing number of HDACs have been identified in plants. There are 18 HDAC gene, SlHDA1, isolated from tomato fruits based on a cDNA clone. A previous report indicated SlHDA1 is mainly expressed in fruit and its transcript increases along with fruit development and ripening21. However, to date, SlHDA1 has not been studied for its functional attributes in tomato. In this study, RNAi repression of SlHDA1 was performed to investigate the exact role of SlHDA1 in tomato, and the results conformed our supposition that SlHDA1 acts as an inhibitor of fruit ripening.Here, we report the functional characterization of a SlHDA1 gene, five independent SlHDA1 silenced lines were obtained using RNAi. Total RNA was isolated from leaves, MG, B, B\u2009+\u20094 and B\u2009+\u20097 stage fruits of transgenic and wild type tomatoes. Real-time quantitative PCR (qPCR) results showed that the relative expression of SlHDA1 was significantly reduced in five transgenic lines compared with the wild type and \u03b2-carotene (orange)25. In this study, total Chl and carotenoids in the RNAi lines and wild type fruits at B, B\u2009+\u20094 and B\u2009+\u20097 stages were extracted and determined. As shown in Fig.\u00a0The wild type and transgenic tomato plants were grown under normal conditions. Flowers were tagged at anthesis, and the time from the anthesis to ripening stage was measured for the wild type and transgenic lines. Color changes were observed earlier in ype Fig.\u00a0. The ripSlHDA1 RNAi lines and wild type, the expression levels of carotenoid biosynthesis-related genes were measured in the fruit pericarp of SlHDA1 RNAi lines and wild type from MG to B\u2009+\u20097 stages by quantitative RT-PCR was up-regulated in RNAi fruits, while the expression of CYC-B, LCY-B and LCY-E was remarkably down-regulated in RNAi fruits compared with wild type. These results indicate that silencing SlHDA1 affects fruit ripening in tomato.To confirm the underlying causes of the differences in color and carotenoid accumulation between the PCR Fig.\u00a0. The res8. Carotenoid biosynthesis is also regulated by ethylene. To further investigate the relationship between SlHDA1 and ethylene26, ethylene production in SlHDA1 RNAi fruits and wild type was measured from stages B to B\u2009+\u20097. As shown in Fig.\u00a0SlHDA1 RNAi fruits.Ethylene biosynthesis, perception and signal transduction are essential for the initiation and completion of tomato fruit ripeningACO1, ACO3, ACS2 and ACS4) and ethylene response factor ERF127 were dramatically up-regulated in RNAi fruit pericarp from B to B\u2009+\u20097 stages medium supplemented with or without the ethylene precursor ACC, which can be taken up by the roots and rapidly converted to ethylene. The elongation of hypocotyls and roots was evaluated 7 days after sowing. The results showed that the average lengths of hypocotyl elongation in RNAi lines were slightly shorter than that of wild type in the absence (0\u2009\u03bcM) of ACC, but there were significantly shorter in the presence of ACC (5.0\u2009\u03bcM and 10.0\u2009\u03bcM) Fig.\u00a0. In addi\u03bcM) Fig.\u00a0.Figure 5ACS2, ACS4, ACO1, and ACO3 were all up-regulated significantly in RNAi seedlings in the presence of ACC(5.0\u2009\u03bcM) Fig.\u00a0. In addient Fig.\u00a0, which sSlHDA1 in fruit ripening, a set of ripening-related genes in wild type and transgenic tomato fruits were examined. Figure\u00a0RIN, E4, E8, Cnr and TAGL1 was markedly increased in the RNAi fruits. Additionally, LOXB, a fruit-specific lipoxygenase gene that is induced by ethylene28; PG, a ripening-related cell wall metabolism gene29; and Pti4, which is associated with defence responses; were also analyzed. Dramatic increases in the levels of these genes were also observed in transgenic fruits , whereas the other leads to \u03b1-carotene and lutein production 34. The relative ratio of lycopene and \u03b2-carotene in ripening tomato fruit is mediated by up-regulation of PSY1 and down-regulation of CYC-B, which are both regulated by ethylene36. In this study, PSY1 was notably increased in the pericarp of SlHDA1 RNAi fruits in ethylene biosynthesis have been cloned and characterized in many species.Ripening of tomato fruits is characterized by an autocatalytic increase in respiration and ethylene biosynthesis just prior to the initiation of ripening. Two modes of ethylene synthesis, system 1 and system 2, are well defined in higher plantsACS2, ACS4, ACO1, and ACO3 in wild type and SlHDA1 RNAi fruits and seedlings. The results showed that the transcript levels of ACS2, ACS4, ACO1 and ACO3 were noticeably higher in RNAi lines than wild type and transgenic plants were planted in a greenhouse under sodium lights for 16\u2009h days (25\u2009\u00b0C), 8\u2009h nights (18\u2009\u00b0C) and watered daily. Flowers were labeled at anthesis and fruit development was recorded as days post-anthesis (DPA). The ripening stages of tomato fruits were divided according to DPA and fruit color. The ripening stages of wild type tomato fruits were divided into IMG , MG , B , B\u2009+\u20094 (4 days after breaker) and B\u2009+\u20097 (7 days after breaker). For all plant samples, total RNA was prepared at the same time each day, and was immediately frozen with liquid nitrogen and stored at \u221280\u2009\u00b0C until required.In this study, wild type tomato (18 primer. A 1\u20132\u2009\u00b5L sample of cDNA was used to clone the full length SlHDA1 gene with primers FHDA1-F and FHDA1-R (Supplementary Table\u00a0Escherichia coli JM109 transformation and confirmed by sequencing (Invitrogen).Total RNA was isolated from all plant tissues including root, stem, leaf , flower, sepal and fruits of wild type tomato using Trizol according to the manufacturer\u2019s instructions. Then, 2\u2009\u00b5g total RNA was used to synthesis first-strand cDNA using the reverse transcription polymerase chain reaction with an Oligo(dT)SlHDA1 gene, an RNAi vector was constructed. A 309-bp specific DNA fragment of SlHDA1 was amplified with primers SlHDA1-RNAi-F and SlHDA1-RNAi-R by the freeze-thaw method. Transformed lines were selected for kanamycin (80\u2009mg\u2009l\u22121) resistance and then analyzed by PCR to determine the presence of T-DNA using the primers NPTII-F/R . All reactions were carried out using the SYBR\u00ae Premix Go Taq II kit in a 10\u2009\u00b5L total sample volume . For analysis of each gene, an NRT (no reverse transcription control) and NTC (no template control) were also performed. The tomato SlCAC gene and SlEF1a gene were also evaluated to be used as the internal standards for development studies43 and abiotic stress studies, respectively44. The relative gene expression levels were conducted using the 2\u2212\u25b3\u25b3CT method45. Primers used for quantitative RT-PCR are shown in Supplementary Table\u00a0The RNA extraction from all plant tissues including root, stem, leaf , flower, sepal and fruits of wild type and homozygous T2 transgenic plants, and cDNA synthesis were performed as described earlier. The synthesized cDNAs were diluted 2 times with RNase/DNase-free water. Quantitative real-time PCR analysis was performed using the CFX9646. Three biological replicates and three technical replicates were used for ethylene measurements.Fruits from the B, B\u2009+\u20094 and B\u2009+\u20097 stages were harvested and placed in open 100\u2009mL jars for 3\u2009h to minimize the effects of wound induced ethylene production caused by picking. Jars were then sealed and incubated at room temperate for 24\u2009h and 1\u2009mL of headspace gas was injected into a Hewlett-Packard 5890 series gas chromatograph equipped with a flame ionization detector. Samples were compared with standards of known concentration and normalized for fruit weight47. 1.0\u2009g sample were cut from pericarp in a 5 mm wide strip around the equator of MG, B, B\u2009+\u20094 and B\u2009+\u20097 of wild type and RNAi lines, respectively. Then grounded them with liquid nitrogen and 20\u2009ml of 60: 40% (v/v) hexane: acetone. The extract was centrifuged at 4000\u2009\u00d7\u2009g for 5\u2009min and the supernatant was carefully transferred to a new tube. The sediment were repeatedly extracted with fresh solvent until colorless and the absorbance of supernatant was measured at 450\u2009nm, 647\u2009nm and 663\u2009nm, respectively. The total Chl and carotenoid contents were calculated with the following equations: total Chl mg ml\u22121\u2009=\u20098.02(OD663)\u2009+\u200920.2(OD647) and total carotenoids mg ml\u22121\u2009=\u2009(OD450)/0.25. Individual tissue samples were taken from 3\u20134 fruits for each ripening stage in biological triplicate and three times for technical replicates.Tomato pigments were extracted from pericarp using a modified protocol from the previous reportFruits of wild type and RNAi lines were harvested at B stage, and placed on filter paper in greenhouse conditions. Phenotype was observed every two days.ACO1, ACO3, ACS2 and ACS4 in the wild type and transgenic lines were measured by qPCR. The expression of SlHDA1 was also detected in wild type seedlings treated with 0, 1.0, 2.0, 5.0, 10.0, and 20.0\u2009\u03bcM ACC.The seeds of wild type plants were sterilized and sown on MS medium supplemented with 0, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0\u2009\u03bcM ACC and then cultured in the dark at 25\u2009\u00b0C. Meanwhile, T1 seeds of RNAi lines were sterilized and sown on MS medium supplemented with 0, 5.0 and 10.0\u2009\u03bcM ACC and then cultured under the same conditions as the wild type plants. Hypocotyl and root elongation were measured 7 days after sowing, and at least 20 seedlings were measured for each culture. To further explore the molecular mechanism of the triple response of transgenic lines, the expression of t-test at P\u2009<\u20090.05.Data were analyzed by one-way analysis of variance (ANOVA) and different means were significant by a Supplementary Table S1 and S2"} +{"text": "Risk assessment for pulmonary embolism (PE) currently relies on physician judgment, clinical decision rules (CDR), and D-dimer testing. There is still controversy regarding the role of D-dimer testing in low or intermediate risk patients. The objective of the study was to define the role of clinical decision rules and D-dimer testing in patients suspected of having a PE. Records of 894 patients referred for computed tomography pulmonary angiography (CTPA) at a University medical center were analyzed. The clinical decision rules overall had an ROC of approximately 0.70, while signs of DVT had the highest ROC (0.80). A low probability CDR coupled with a negative age-adjusted D-dimer largely excluded PE. The negative predictive value (NPV) of an intermediate CDR was 86\u201389%, while the addition of a negative D-dimer resulted in NPVs of 94%. Thus, in patients suspected of having a PE, a low or intermediate CDR does not exclude PE; however, in patients with an intermediate CDR, a normal age-adjusted D-dimer increases the NPV. Diagnosis of pulmonary embolisms (PE) continues to challenge physicians largely because of the wide spectrum of presentations from vague symptoms to profound illness . Clinica ADJUST-PE trial, which showed that PE could be safely excluded in a larger number of patients using an age-adjusted cutoff for D-dimer (age \u00d710\u2009ug/L with a lower limit of 500\u2009ug/L) [Multiple studies have shown that combining D-dimer testing with either CDR improves their overall negative predictive value (NPV) and positive predictive value (PPV) . Le Gal 00\u2009ug/L) . Similar00\u2009ug/L) . Keeping00\u2009ug/L) .Thus, the exact role of D-dimer testing in the cohort of patients with an intermediate probability CDR remains still in question. In order to help clarify this issue, we performed a retrospective analysis of patients referred for CTPA to (a) confirm the predictive value of the Wells rule, Geneva score, and their individual elements in a University hospital population referred for CTPA to diagnose or rule out PE and (b) determine the utility of D-dimer testing in the population of patients with low and intermediate CDR scores.n = 4756) were screened for inclusion/exclusion criteria and 894 charts were reviewed by the authors. Patients were referred by either an emergency room physician or a hospital inpatient physician. Charts were reviewed for individual variables of the both CDR, D-dimer, and other commonly referenced signs of PE, which were analyzed in a database after subtracting all patient identifiers.This study was approved by the Institutional Review Board of the University of California Davis. Imaging reports of all patients referred for CTPA to evaluate for PE between 2012 and 2015 and chest imaging was collected from stored diagnostics from that admission. Blood gas data were employed only if results were collected within 4 hours from onset of symptoms. The original Wells rule and reviP value < 0.05.Logistic regression was used to model the probability of PE by each score, score element, and other clinical characteristic. Sensitivity, specificity, negative predictive value, and positive predictive value were calculated from 2 \u00d7 2 tables of predicted and actual outcomes. Binomial confidence intervals were calculated using the Agresti-Coull method . Data arThe final cohort assembled in this study included patients spanning an age range of 18\u201398, mean age 55.4, with a representative diverse population . The oveThe Wells rule and revised Geneva score, as well as individual components of both CDR and signs of poor oxygenation, were analyzed and depicted on receiver operating characteristic (ROC) curves (sensitivity against 1 \u2013 specificity). The predictive power of the Wells rule and Geneva score are shown in Tables n = 173) showed that-adjusted D-dimer (age \u00d710\u2009ug/L with a lower limit of 500\u2009ug/L) [P < 0.001). There was no correlation between age-adjusted D-dimer and either the Wells rule or Geneva score . Overall, the addition of D-dimer to high-value elements did not incrementally increase the predictive power .Subgroup analysis of patients whose D-dimer was measured (00\u2009ug/L) had a NPn = 678 according to Wells and n = 841 according to Geneva).The distribution of positive and negative CTPA and D-dimer by CDR risk is shown in low CDR indicated by Wells rule <2, overall 166 of 176 (94.4%) patients had a negative CTPA , yielding a NPV of 94.3%. In those patients with an intermediate Geneva score (119 patients scoring 4\u201310 on the CDR with a measured D-dimer), only 4 of the 67 (6.0%) patients with a negative age-adjusted D-dimer had a positive CTPA, with a similar NPV of 94.1%. Thus, the addition of age-adjusted D-dimer increased the NPV of an intermediate value from either CDR alone by ~6%.With either CDR,This study has several key findings: First, this analysis validates the modest performance of the Wells rule and Geneva score in predicting pulmonary embolism by CTPA as seen in the original studies , 10 and Recently, several studies have attempted to enhance CDR guiding PE diagnostic workup; these studies have isolated high-value elements such as signs of DVT and expaA unique feature of this paper is effort to remove the vague item \u201cPE as the most likely diagnosis\u201d by creating firm clinical criteria that could be uniformly applied. This method was slightly less predictive compareIn comparison with prior studies , fewer pThe NPV of a low Wells rule is approximately 94%, and the addition of a negative D-dimer test effectively excludes PE. The NPV of an intermediate Wells or Geneva score alone was less than that of a low score 89% and 86%, resp.), while the addition of a negative age-adjusted D-dimer improved the NPV similarly to that of a low score (~94%). Likely due to the small number of patients with D-dimer testing, this increase was not statistically significant, with overlapping 95% confidence intervals. Importantly, a negative age-adjusted D-dimer did not rule out a +CTPA, as, overall, 4 patients with a negative age-adjusted D-dimer had a +CTPA . Several studies have examined the role of D-dimer testing in improving the predictive value of a low or intermediate CDR. For example, Wells %, resp., and GuptThis retrospective study did not prospectively enroll patients. However, all patients who had a CTPA over the enrollment period were included in the analysis, yielding a robust sample size that was diverse in age, gender, and race. In addition, only a subset of patients received D-dimer testing, limiting the conclusions regarding the utility of D-dimer testing. Finally, as the patients receiving D-dimer testing were selected by the diagnosing physician, they may have had different perceived risks for PE not reflected in the data. Thus, the conclusions may not be broadly applicable to all patients presenting with possible PE.This study confirms the modest PPV and NPV of the Wells rule and Geneva score but affirms the value of clinical judgment (\u201cPE most likely\u201d) and the presence of signs of DVT as predictors of PE as diagnosed by CTPA. A normal age-adjusted D-dimer coupled with a low risk CDR effectively ruled out PE, while a negative D-dimer increased the NPV of an intermediate CDR to approximately 94%. Thus, the value of D-dimer testing, vis-\u00e0-vis clinical judgment, should be carefully evaluated in light of these findings and should not be used to \u201crule out\u201d PE."} +{"text": "The subject of the presented work was an attempt at optimization of the methods used for verification of the candidates for medical voluntary workers in a hospice and decreasing the danger of a negative influence of an incompetent volunteer on a person in a terminal stage of a disease and his or her relatives. The study was carried out in St. Lazarus Hospice in Krakow, Poland, and included 154 adult participants in four consecutive editions of \u201cA course for volunteers \u2013 a guardian of the sick\u201d organized by the hospice. In order to improve the recruitment of these workers, the hitherto methods of selection (an interview with the coordinator of volunteering and no less than 50% of attendance in classes of a preparatory course for volunteers\u201d) were expanded by additional instruments\u2014the tests whose usefulness was examined in practice. Knowledge of candidates was tested with the use of a written examination which consisted of four open questions and an MCQ test comprising 31 questions. Practical abilities were checked by the Objective Structured Clinical Examination (OSCE). A reference point for the results of these tests was a hidden standardized long-term observation carried out during the subsequent work of the volunteers in the stationary ward in the hospice using the Amsterdam Attitude and Communication Scale (AACS). Among the tests used, the greatest value in predicting how a given person would cope with practical tasks and in contact with the sick and their relatives had a practical test of the OSCE type. Involving volunteers in the care for patients and relatives of patients in the final stage of terminal illness constitutes a common standard in palliative care in Poland as well as in many other countries of Europe and the world. Hospice volunteers are people who perform various jobs and who are of a different background; what they have in common is their will to provide unpaid aid for those who need it .Volunteering brings, above all, great benefits both for patients and volunteers themselves , 6. Therstatus quo is a common standard in Poland.In order to reduce the risk of incompetent volunteers\u2019 actions on patients or their families, most Polish hospices provide special preparatory courses. However, a standardized system of preparing medical volunteers for hospice care has not been worked out. Presently, a lot of facilities have worked out clear principles of recruitment of volunteers. These principles look similar: a candidate who wants to undertake some actions involving a direct contact with the patient usually completes a preparatory course and is interviewed by the volunteer coordinator. Those who reveal behavioral disorders or unethical attitudes during the interview should not be approved. However, this criterion is subjective as it depends merely on the coordinator\u2019s experience and sensitivity. In turn, individual palliative or hospice care facilities work out their own training for candidates for hospice volunteers, and such training programs prepared in individual facilities vary considerably from one to another. They are usually courses lasting a few to a few dozen hours of classes. They do not usually end with examinations. So far, a standardized selection method has not been developed. Such The fact that introduction of courses for candidates for volunteers and trainings for volunteer coordinators has become more common recently proves that the issue of selection while recruiting hospice volunteers has become an increasing problem .The introduction of additional tests for candidates for medical volunteers in a hospice has not been intended to create formal barriers preventing individuals from undertaking this role. Its purpose was to protect the good of the patient and his family being under palliative care and also to protect the candidates for the volunteers themselves. Candidates who do not meet specific criteria may be suggested taking up some other forms of volunteer work. Thus, they may become volunteers of the administrative, maintenance, or fundraising staff, but not medical volunteers.The methods of assessment of the candidates which were the subjects of this study were not new. They have been extensively used, described, and tested in many countries as effective methods employed at the recruitment of medical workers , 12 and The study was carried out in the years 2010\u20132012 in St. Lazarus Hospice in Krakow, Poland.The subject of the presented work was the attempt at optimization of the methods used for verification of the candidates for medical voluntary workers in a hospice through the application of appropriate tools.Are there any methods for prediction of functioning volunteers in practice?Is there any possibility of application of methods for the assessment of volunteers, which is used for the evaluation of health care professionals?Which from the investigated methods of evaluation is the most appropriate for volunteers\u2019 verification?Research questions:For the study, a positive opinion of the Bioethics Committee of the Jagiellonian University had been obtained.In order to improve the recruitment of volunteers to work with patients in the hospice, the hitherto methods of selection (\u201can interview with the coordinator of volunteering and no less than 50% of attendance in classes of a preparatory course for volunteers\u201d) were supplemented by additional instruments\u2014tests whose usefulness was examined in practice.The knowledge of candidates was tested with the use of a written examination in the form of open questions (essay-type questions) and a multiple choice test (MCQ). Practical abilities were checked by the Objective Structured Clinical Examination (OSCE). A reference point for the results of these tests was a hidden standardized long-term observation carried out during the subsequent work of the volunteers in the stationary ward in the hospice.The written exam comprising four essay-type questions was aimed at revealing attitudes of the future hospice volunteers. In order to obtain the possibly most objective opinion about a candidate, for each question, four evaluation criteria had been prepared. For each of the criteria from 1 to 5, points could be obtained . For every question, volunteer could get a total of 4\u201320 points and for the whole test 16\u201380 points.The MCQ consisted of 31 items; for each item, one correct answer should be chosen from among four possible. It was a test of knowledge acquired during the course. The questions were focused on the knowledge of principles of interpersonal communication, propagation of the idea of hospice care, dilemmas associated with patient care, and patients\u2019 everyday problems.The practical exam consisted of three 5-min OSCE stations with standardized patients (SPs), who displayed the same behavior and expressed the same needs, and each time responding in the same way towards each test taker. Next, on the basis of the strictly defined criteria, the SPs assessed the future volunteers\u2019 abilities in regard to getting in touch, proper communication, attitudes towards the patient, respecting his or her dignity as well as the ability to recognize the patient\u2019s needs, and providing aid relevant to these needs.Stations 1 and 2 checked simple practical skills: assisting a weakened patient in walking and helping to change the underwear involving a bedridden patient. The last station checked the ability to communicate, with a psychologist acting as an SP.Before the examination, all the test takers received exactly the same initial information about the objectives and general principles of the exam and a briefing sheet comprising a detailed description of the organization of the examination with the requirements for test takers.patient and his family (ability to communicate),hospice workers and other volunteers (ability to cooperate),volunteering ,own person .The research tool used during the long-term observation was the Amsterdam Attitude and Communication Scale (AACS) modifiedFor greater validity of the prepared examinations, both the questions and the tasks for the volunteers as well as the evaluation criteria had been consulted with a clinical psychologist and with an experienced volunteer coordinator. In 2009, we conducted a pilot study covering 27 volunteers. As a result of these studies, the MCQ was extended by six additional questions, two questions were changed, and a more detailed checklist for the OSCE station no. 3 was created.N\u00a0=\u00a03) or practical (N\u00a0=\u00a02) exam.The study involved 154 people who had completed the course and decided to take the final tests. From among all the participants, 141 people (91.5%) were made subject to a hidden standardized long-term observation in the inpatient ward of the hospice. Within this group, 136 people went through all the phases of the study. The remaining five participated only either in the theoretical .The results of the essay-type questions were expressed in a number of obtained points. The MCQ was presented as a percentage of the correct answers. In the case of the OSCE, a subjective impression of the observer was presented next to objective assessment expressed in a number of obtained points. The relationship between objective and subjective evaluation in the context of this test was examined with the use of Spearman\u2019s rank correlation . The duration was divided into two categories: volunteer work being longer or shorter than 4\u00a0months.Due to the fact that a surprisingly high turnover of volunteers was observed during the study, the authors also decided to find out whether the test results could help to predict how long a given person will contribute to hospice volunteering (Mann-Whitney p\u00a0<\u00a00.0001) with the results of a hidden standardized long-term observation (Table sp\u00a0=\u00a00.67) and subjective impressions of the observer-simulated patient (rsp\u00a0=\u00a00.62) correlated higher with the observation result than the result of the written examination with open questions (p\u00a0=\u00a00.48) or the results of the MCQ (rsp\u00a0=\u00a00.44) cf. Table The results of all the used forms of examinations correlated significantly positively than any particular tests separately. However, this correlation was on a level comparable with the one obtained by the practical exam OSCE alone.The total results of all three forms of tests indicated higher correlation with the result of prolonged observation (rsp\u00a0=\u00a00.48) with standardized observation as compared to the first station (rsp\u00a0=\u00a00.46), but lower than the remaining stations.Assessment of each of the used stations of the OSCE type separately also correlated higher with practical coping of the volunteer with patient\u2019s care than a multiple choice test. The written examination with open questions obtained slightly higher coefficient of correlation and weaker with a practical test of the OSCE type . Open questions showed positive correlation on a level similar to all employed forms of assessment .Particular forms of examinations correlated positively with each other, and each of these correlations was statistically significant Table According to the performed qualitative analysis, the practical test of the OSCE type turned out to have significantly higher prognostic value than both theoretical tests. Moreover, in two cases, it revealed the candidates\u2019 features which precluded their undertaking of the work with the sick-fresh, not suppressed emotions after the loss of a close person in one of the female candidates and an aggressive attitude towards simulated patients in one of the male candidates. Carrying out this type of an exam allowed, therefore, to avoid unnecessary trauma by both potential candidates for volunteers and by the persons whom they would take care of while working in the stationary ward in the hospiceThe scores obtained in the theoretical tests, both in the MCQ and the open questions tests, did not reflect in any way the duration of the subsequent involvement in hospice volunteer work. People who did better in the OSCE and obtained higher marks were involved in volunteer work for a long time significantly more often with the results of the clinical performance observation (0.64) was also at a level similar to the level of the correlation of OSCE only (0.63) with the observation.The results of individual exams obtained in this study correlated significantly positively not only with the result of the long-term observation but also with each other. Thanks to this, on the basis of the mark obtained as a result of using one of the methods of testing, the result of a subsequent test taken by a given person can be predicted with great probability.In the study published in 2005, Auewarakul et al. [Based on the similarities between the results of the forms of testing the candidates for hospice volunteers used in this study and the results of similar exams used with medical students, a conclusion can be drawn that the empirically tested methods used for selecting medical staff may be used successfully, and after having been modified, they can be applied for selection of volunteers.On the basis of the above shown analysis of usefulness of the particular forms of testing, the subsequent conclusion can be drawn: OSCE seems to be the most valuable tool for selecting volunteers from among the instruments used in the study. Its correlation with the subsequent clinical performance, despite using a shortened form of examination (three stations), was at a similar or even higher level than that obtained in the studies in which its extended version was used. Naturally, the authors of the study would not be tempted to claim that this exam is the best possible tool for verification of volunteers, or that it could be used as a completely independent tool. For this reason, they suggest that the practical OSCE could be a valuable complement of methods used so far and not the only selection tool.Should carrying out OSCE in the form used in the study prove to be too challenging (the most difficult exam to carry out), it will be better, as the correlations between individual test results show, to use any other verification method than to resign completely from the assessment ending the preparatory training for future volunteers. In addition, the studies by Ramreje et al. prove thIt seems to be more reasonable, however, to introduce an examination resembling just one OSCE station than carrying out theoretical tests. Assuming that interpersonal skills and psychological preparation play the key role in preparing a volunteer to work with patients while selecting a single OSCE station, examiners should choose one that checks, above all, the ability to communicate with the patient and/or his family and not, e.g., manual skills. At such a station wrong stereotypical thinking, bias, religious fanaticism, lack of empathy are revealed most frequently, which were confirmed by the qualitative analysis of the OSCE type exam.It must be remembered that volunteers\u2019 future performance cannot be predicted with absolute certainty and clarity.Setting up the optimal cutoff value for the volunteers might be a big problem if something that clearly discriminates them, which was observed in the case of two individuals during the study, namely, aggression manifested in relation to the simulated patient in the case of one man and a strong experience of fresh mourning after the loss of a husband in the case of one woman, does not take place.The procedure of verification of volunteers expanded with a standardized tool such as OSCE, apart from benefits for patients and volunteers themselves, may have a positive influence on the organization of work in a ward, which could be attributed to the fact that volunteers of higher competences would work with the terminally ill and because of a lower turnover of volunteers. OSCE was the best predictor of both volunteers\u2019 clinical performance and the duration of their subsequent work as a hospice volunteer.Setting up the optimal cutoff value requires further research, so that exam results could be recognized in the categories of \u201cpass\u201d or \u201cfail\u201d.The empirically tested methods used for selecting medical staff may be used successfully for selection of hospice volunteers.A practical test of the OSCE type has a greater value for prediction of functioning volunteers in practice than open questions and MCQ.Even introducing an exam, resembling the single OSCE station could be a valuable instrument significantly increasing the value of the procedure of verification of volunteers for work in a hospice."} +{"text": "During the Ebola virus disease (EVD) epidemic in Liberia, contact tracing was implemented to rapidly detect new cases and prevent further transmission. We describe the scope and characteristics of contact tracing in Liberia and assess its performance during the 2014\u20132015 EVD epidemic.We performed a retrospective descriptive analysis of data collection forms for contact tracing conducted in six counties during June 2014\u2013July 2015. EVD case counts from situation reports in the same counties were used to assess contact tracing coverage and sensitivity. Contacts who presented with symptoms and/or died, and monitoring was stopped, were classified as \u201cpotential cases\u201d. Positive predictive value (PPV) was defined as the proportion of traced contacts who were identified as potential cases. Bivariate and multivariate logistic regression models were used to identify characteristics among potential cases.We analyzed 25,830 contact tracing records for contacts who had monitoring initiated or were last exposed between June 4, 2014 and July 13, 2015. Contact tracing was initiated for 26.7% of total EVD cases and detected 3.6% of all new cases during this period. Eighty-eight percent of contacts completed monitoring, and 334 contacts were identified as potential cases (PPV = 1.4%). Potential cases were more likely to be detected early in the outbreak; hail from rural areas; report multiple exposures and symptoms; have household contact or direct bodily or fluid contact; and report nausea, fever, or weakness compared to contacts who completed monitoring.Contact tracing was a critical intervention in Liberia and represented one of the largest contact tracing efforts during an epidemic in history. While there were notable improvements in implementation over time, these data suggest there were limitations to its performance\u2014particularly in urban districts and during peak transmission. Recommendations for improving performance include integrated surveillance, decentralized management of multidisciplinary teams, comprehensive protocols, and community-led strategies. Contact tracing is comprised of three main steps: identifying, listing, and monitoring persons who have been exposed to infected individuals, with the goal of rapidly diagnosing and treating new cases and preventing further spread of infection. This approach has been used to control transmission of infectious diseases including smallpox, tuberculosis, HIV, and syphilis, and while contact tracing has been used in prior outbreaks of hemorrhagic fever, these outbreaks were small in scale. During the 2014\u20132015 Ebola virus disease (EVD) epidemic in Liberia, contact tracing was implemented in all 15 counties on a scale that was unprecedented, particularly within both rural and crowded urban settings. This work provides insight into the magnitude that which contact tracing was implemented, its characteristics, as well as an assessment on its performance. Given that contract tracing is a critical tool for controlling disease spread, these findings aid in informing future planning and decision making for its implementation. In March 2014, Liberia detected its first cases of Ebola virus disease (EVD) in Lofa, a northern county bordering Guinea and Sierra Leone , 4, 5. CContact tracing is comprised of three main steps: identifying, listing, and monitoring persons who have been exposed to infected individuals, with the goal of rapidly diagnosing and treating new cases and preventing further spread of infection. This approach has been used to control transmission of infectious diseases including smallpox, tuberculosis, HIV, and syphilis , 11, 12.The 2014 EVD epidemic was the largest EVD outbreak in history and the first known EVD outbreak in West Africa [We performed a retrospective descriptive analysis of data collection forms for contact tracing that was conducted for the EVD epidemic in six of Liberia\u2019s 15 counties during June 2014\u2013July 2015. The six counties consisted of both rural and urban areas and represented 72% of the population of Liberia . Three oA contact was defined as a person who had direct or indirect exposure to any confirmed, probable, or suspect EVD case, or bodily fluids of a case, within the past 21 days , 23. ThiNational contact tracing guidelines and forms, which were initially adapted from existing WHO and CDC materials and finalized during the waning days of the epidemic, were used as the foundation for implementing the three steps of contact tracing: contact identification, listing, and monitoring. Once a case was detected, contact identification and listing were conducted by interviewing the case and/or family members to gather an initial list of potentially exposed persons. In most instances, this process was conducted by case investigation teams, which were distinct from contact tracing teams, and any of the following six types of exposure were added: (1) sleeping or eating in the same household; (2) direct physical contact with the body; (3) touching bodily fluids; (4) manipulating clothes or other objects; (5) through breastfeeding; and (6) attending a case\u2019s funeral.Contact tracers, chosen from within the community, located the listed contacts and identified any additional contacts missed in the initial investigation. Contact tracers transferred the information collected by case investigation teams to paper forms, including the contact\u2019s name, county, district, town, and exposure(s). The name, age, location, and unique case identifier of the case for which contacts were listed, i.e. the \u201csource case\u201d, were also recorded.During contact monitoring, contact tracers were expected to visit contacts twice daily (morning and afternoon) for 21 days post-exposure in order to identify and record whether the contact had EVD symptoms. This was determined initially through self-reports and physical observation, and eventually temperature readings were added for more objective monitoring. Contacts were monitored for nine symptoms: joint pain, fever (>38\u00b0 Celsius), weakness, nausea, diarrhea, headache, throat pain, red eyes, and mucosal bleeding.Following the outbreak, paper contact tracing forms were requested from all County Health Teams. Forms were received from six counties and the data were entered into a Microsoft Access database. Data were analyzed using Microsoft Access and Epi Info. Each form was considered a unique contact record and unit of analysis, though it was possible for individual contacts to be monitored more than once if re-exposed.Source cases were identified using unique case identifiers, name, age, county, and district. To assess the coverage of contact tracing, or the percentage of cases for which contacts were monitored, we calculated the ratio of source cases in the database to the total number of suspected, probable, and confirmed EVD cases in MOH situation reports for the same counties using the closest approximate dates , 27, 28.We analyzed records by county and district. Urban or rural classifications were assigned based on districts; districts that hold the county headquarters or that have settlements with a population of 5,000 or more persons were classified as urban . Two disWe divided the timeframe into four phases based on the observed epidemic trends of cases within Liberia , per epiEach record was assigned one of seven outcomes of monitoring, either designated on the form or imputed using supplemental information: (1) \u201ccompleted\u201d the monitoring period of 21 days post-exposure; (2) \u201cdropped\u201d if the source was determined to be not a case; (3) \u201clost to follow-up\u201d if the contact could not be located after three consecutive days; (4) \u201cpotential cases\u201d if the contact presented with symptoms and/or died and monitoring was stopped; (5) \u201crestarted\u201d if monitoring was reinitiated due to a new exposure; (6) \u201ctransferred\u201d if the contact moved to another jurisdiction; or (7) \u201cunknown\u201d for all remaining contacts with no outcome information. Contacts who presented with symptoms could be referred for medical evaluation without meeting EVD case definitions; hence, we use the terminology \u201cpotential cases\u201d.We calculated the positive predictive value (PPV) defined as the proportion of traced contacts\u2014excluding those with dropped and unknown outcomes\u2014who were potential cases. Sensitivity was defined as the ratio of potential cases identified during monitoring to the number of new cases in situation reports in the same counties , 27, 28.We used odds ratios and 95% confidence intervals to examine exposure types, symptom types, phases, and urban-rural amongst potential cases compared with contacts who completed monitoring; for ordinal variables, the lowest category was used as a reference group. Chi-square tests with p-values <0.05 were statistically significant. Two multivariate logistic regression models were used: (1) urban-rural, phase, and exposure type covariates, limited to records with \u22651 exposures, and (2) urban-rural, phase, and symptom type covariates, limited to records with \u22651 symptoms. Only statistically significant variables in bivariate analysis were included in the models. Nonparametric tests were used for continuous variables.This assessment is included under Johns Hopkins School of Public Health Institutional Review Board no. 6296 with DHP as principal investigator. A letter of agreement was signed with the Liberia MOH concerning the publication of contact tracing analyses. This assessment used retrospective data collected for public health surveillance purposes so informed consent was deemed unnecessary according to the U.S. Common Rule. We followed the Declaration of Helsinki, aiming to provide assurance that the rights, integrity, and confidentiality of participants were protected.We analyzed 25,830 records for contacts who had monitoring initiated or were last exposed between June 4, 2014 and July 13, 2015 in the six counties. Of these, 25,651 contacts were listed for 2,465 source cases; an additional 179 contacts had no source case provided. The overall contact-to-case ratio was 10:1 . The contact-to-case ratio increased with each subsequent phase and was higher in urban than rural districts. There were 9,241 EVD cases in situation reports in the six counties. The upper limit estimate of coverage, or the maximum percentage of cases for which contacts were monitored, was 26.7%, and was lowest during Phase 1. In the six counties providing data, 89.0% of the records were identified in Montserrado County, 8.6% in Margibi, 1.6% in Bong, 0.4% in Lofa, 0.4% in Sinoe, and 0.1% in Nimba Table 2Table 2. Temporal trends for contact tracing aligned with disease transmission trends . For 25,Of 25,830 total contacts, 17,876 (69.2%) contacts reported 34,284 exposure types, and 7,954 (30.8%) had zero exposures recorded. Among the 17,876 contacts reporting any exposure, direct physical contact with the body was the most common (73.1%), while funeral attendance (2.1%) and breastfeeding (0.2%) were the least common . Two or Of 25,569 contacts with an assigned outcome, 22,680 (87.8%) completed monitoring, 1,768 (6.8%) were dropped, 637 (2.5%) restarted, 334 (1.3%) were potential cases, 136 (0.5%) were lost to follow-up, and 14 (0.1%) were transferred. Most contacts completed monitoring during each phase except during Phase 4, when 53.6% of contacts were dropped . More coThe PPV was 1.4% overall, and was higher in rural (3.0%) than urban (1.1%) districts and highest during Phase 1 (4.7%), after which it decreased for subsequent phases. The sensitivity of monitoring, or the maximum proportion of new cases detected, was 3.6%, and was highest during phases 1 and 2. During 2014\u20132015, more than 25,000 persons in six of Liberia\u2019s 15 counties were identified, listed, and monitored for EVD, representing one of the largest contact tracing efforts during an epidemic in history. Nationwide, these efforts were even more substantial and required the dedication of responders, including the Government of Liberia, counties, and contact tracing teams. As a result, 334 contacts were identified as potential cases with the intention of providing earlier treatment and preventing hundreds of new infections.Relative to the scale of these efforts, however, these data suggest there were limitations to the performance of contact tracing within Liberia. Overall, there was a small proportion of monitored contacts that were identified as potential cases, and more than 97% of reported EVD cases from the six counties were not detected through contact monitoring. This is greater than expected, especially compared to other examples in West Africa where approximately 69% to 78% of cases were not being traced prior to case identification , 30. ThiPotential cases were more likely to be identified in rural districts and early in the epidemic, despite intensified efforts as the epidemic progressed. Possible explanations for why contact tracing was less effective in urban areas could include the following: higher population density and complex social networks making it more difficult to identify all contacts; less cooperation within urban settings; higher burden and strained resources; or a combination of these factors. These results support the concept that contact tracing is most successful when transmission is low, and models have shown that expanding implementation of contact tracing yields diminishing reductions in disease prevalence , 16. TheThere were, however, notable improvements in implementation over time; specifically, greater coverage, fewer contacts lost to follow-up, and higher contact-to-case ratios. During Phase 4, Liberia was able to focus more resources on eliminating the last transmission chains, including expanding the inclusion criteria to ensure no new cases went undetected . This woThe dynamics of contact tracing are complex, and its success is related to characteristics of the disease and etiologic agent, resources, and socio-political factors that influence its acceptability and implementation. Additionally, the approaches to contact tracing may differ depending on whether there is a vaccine or therapy available. Given that contact tracing remains one of the critical public health tools during outbreaks involving person-person transmission, optimizing its performance is paramount. While not exhaustive, we focus on four key challenges that may have limited the performance of contact tracing for EVD within Liberia, and propose recommendations for future efforts.First, an integrated surveillance and data management system was lacking and had to be established for reporting between the national laboratory, healthcare facilities and ETUs, and contact tracing and case investigation field teams . ConsequSecondly, the organizational structure for contact tracing likely led to inefficiencies in its implementation and management, particularly in urban districts. For instance, case investigation teams, who conducted contact listing, were often distinct from contact tracing teams who conducted contact monitoring. In some rural areas, teams responded in tandem thereby reducing gaps, yet this was more difficult in dense urban areas such as in Montserrado County. Additionally, the county level coordinated all aspects of the response\u2014not just contact tracing. In January 2015, Montserrado created decentralized sub-county sectors to oversee and synchronize all operations\u2014a change previously recognized as a critical step for halting transmission . ParticuThirdly, there were challenges with adapting and implementing contact tracing protocols, which had to be used by novice teams during the epidemic. For instance, the number of contacts per source case ranged widely in our analysis, from 1 to 424, and nearly one-third of contacts had no exposure documented, indicating that some contacts may not have met the inclusion criteria, thereby straining resources. Also, written guidance for identifying potential cases during monitoring did not specify how contact tracers should determine when to refer a contact for medical evaluation . EighteeFinally, community perceptions, stigma, and mistrust reportedly led to challenges in obtaining complete and reliable information, to delays or an inability to trace contacts due to evasion, and even to violence , 33. UndThis analysis represented both urban and rural settings, and Montserrado specifically, where the response was most intense. However, we were unable to collect forms from all 15 counties nor all forms from the six inclusive counties; for example, no forms were available for the EVD cluster that occurred in Margibi in July 2015. Despite commendable efforts, counties reported that paper forms were lost or destroyed due to perceived contamination risks. Using paper forms also led to variability in data quality, including illegible writing, misspellings, inversed source case and contact information, and difficulty in interpreting marks for visits and the presence of symptoms. Falsifying information on forms was a concern , such asThese factors, combined with the lack of information to ascertain the final status of EVD infection amongst source cases and potential cases, constrained our analysis. Likewise, we could not conclude whether symptoms reported amongst potential cases evidenced EVD infection. Our data primarily represented contact monitoring, as we did not have a comprehensive contact listing. Finally, EVD case counts from situation reports were unavailable to stratify coverage and sensitivity by urban-rural districts and/or phase, and these aggregated totals could not be linked to our individual-level database.Our findings suggest that despite the unprecedented scale of contact tracing for EVD in Liberia, there were limitations in its ability to detect new cases, especially in urban areas and during the peak case load. Since contact tracing remains a critical intervention for controlling outbreaks, we suggest rigorous implementation early in the outbreak and focusing on four key areas to optimize its performance within similar contexts: (1) strengthening integrated surveillance and electronic data systems, (2) decentralizing management of multidisciplinary teams for improved coordination and oversight, (3) instituting and reinforcing clear and comprehensive protocols, and (4) adapting community-led strategies to foster cooperation, trust, and ownership.S1 DatasetDe-identified listing of contact tracing records used for analysis .(XLSX)Click here for additional data file."} +{"text": "Appendicitis is a global disease affecting roughly 1 in every 12 people in the world, with the highest incidence between ages 10 and 19 years. To date, a wide variety of health outcomes have been reported in randomised controlled trials and meta-analyses evaluating treatments for appendicitis. This is especially the case in studies comparing non-operative treatment with operative treatment. A set of standard outcomes, to be reported in all future trials, is needed to allow for adequate comparison and interpretation of clinical trial results and to make data pooling possible. This protocol describes the development of such a global core outcome set (COS) to allow unified reporting of treatment interventions in children with acute uncomplicated appendicitis.We use current international standard methodology for the development and reporting of this COS. Its development consists of three phases: (1) an update of the most recent systematic review on outcomes reported in uncomplicated paediatric appendicitis research to identify additional outcomes, (2) a three-step global Delphi study to identify a set of core outcomes for which there is consensus between parents and (paediatric) surgeons and (3) an expert meeting to finalise the COS and its definitions. Children and young people will be involved through their parents during phase 2 and will be engaged directly using a customised face-to-face approach.The medical research ethics committee of the Academic Medical Center Amsterdam has approved the study. Each participating country/research group will ascertain ethics board approval. Electronic informed consent will be obtained from all participants. Results will be presented in peer-reviewed academic journals and at conferences.COMET registration: 1119 This protocol describes an international online Delhi study that should result in a globally relevant set of core outcomes for paediatric uncomplicated appendicitis.The protocol was developed in conjunction with an international steering committee\u00a0and patient representation, and follows all relevant core outcome set (COS)\u00a0development guidelines and standards.This study involves parents and patients in deciding what to measure in future uncomplicated appendicitis research.The involvement of young people in COS\u00a0development requires a customised approach. This protocol addresses this issue and describes a direct face-to-face involvement.Because of the global and multilingual aspect of the study, there will be a limited consensus discussion with only selected individuals. Also, due to feasibility, the direct face-to-face engagement of young people will take place only\u00a0in selected countries.et al,Appendicitis is a common gastrointestinal disease affecting roughly 1\u00a0in every 12 people in the world, with the highest incidence between ages 10 and 19 years.Inconsistent selection and reporting of outcomes limit the ability to adequately compare and interpret clinical trial results. Furthermore, it hampers data pooling and subsequent meta-analysis. It also increases the risk of selective outcome reporting, a form of publication bias. This, in turn, jeopardises the validity of results from individual trials, which feeds into subsequent systematic reviewset al in 2015, a wide variety of outcomes has been reported in randomised controlled trials (RCTs) and meta-analyses reporting on the treatment of appendicitis in children.et al proposed the development of a core outcome set (COS), which is a standardised collection of outcomes that should be measured and reported in all future trials.As demonstrated by Hall Outcomes considered important by patients and families are essential to a meaningful and complete COS.We aim to reach a global consensus among patients, parents, researchers and physicians on a minimal set of core outcomes that should be measured and reported in all future clinical trials investigating any type of treatment for acute uncomplicated appendicitis in children, including surgical treatment, NOT\u00a0or other treatments aimed at curing appendicitis.In the development of this protocol, we adhere to the Core Outcome Set-STAndards for Development\u00a0(COS-STAD) recommendations10.1136/bmjopen-2018-028861.supp1Supplementary dataet alThe paediatric appendicitis COS (PA-COS) development will consist of three phases: (1) an update of the 2015 systematic review on outcomes reported in uncomplicated paediatric appendicitis research,An international steering committee has been established and consists of the following; the authors, a parent/patient representative of the Dutch Foundation: \u2018Children and Hospital\u2019 and the lead local investigator of each participating centre (PA-COS development group). The steering committee will agree on the final version of the protocol at the start of the project and will provide input throughout the duration of the project. The steering committee members will also be involved in the development of the final COS. Within the steering committee, a smaller study management group has been appointed which will convene during regular (videoconference) meetings.et al performed a systematic review of RCTs and meta-analyses reporting treatment outcomes of children with appendicitis up to April 2014.Hall 10.1136/bmjopen-2018-028861.supp2Supplementary dataet al.et alAfter data extraction, a meeting of the study management group will be held to discuss potential similarities between the outcomes from the 2015 systematic review from Hall This study includes children and young people (5\u201318 years) who have been treated for acute uncomplicated appendicitis in the preceding 24 months, either with initial NOT or with surgery. Children less than 5 years old are excluded as different outcomes might be appropriate in this very young age group. Also, uncomplicated appendicitis is much less common in young children than in older children. Furthermore, there are no studies in which children below the age of 5 are treated non-operatively. Children will be engaged indirectly as we will urge parents to discuss the answers they provide with their child while filling out the Delphi questionnaire. Young people will be engaged directly through a customised face-to-face approach in selected countries. For the invited children, considering the complexity of the subject and methodology, age is limited to 12\u201318 years.Parents of children and young people (5\u201318 years) treated for acute uncomplicated appendicitis either with initial NOT or with surgery in the preceding 24 months or during the initial phase of the study were included. Parents will be asked to discuss the answers they provide with their child while filling out the Delphi questionnaire. Parents will be invited to participate by their child\u2019s treating physician or their designate in each participating country/hospital. Participants will be identified retrospectively by contacting patients who were treated in the past 24 months or prospectively by inviting parents to participate after their child has completed\u00a0the treatment.General and/or paediatric surgeons who care for children in the specified age group will be asked to participate. Surgeons will be identified and invited by the local coordinators in each participating country. These local coordinators are research groups that have previously registered a clinical trial on uncomplicated appendicitis in children. This should allow for inclusion of physicians who\u00a0also have experience in research on the treatment of appendicitis.www.clinicaltrials.gov by searching (January 2017) for \u2018appendicitis\u2019 with an age limitation of 5\u201318 years. Studies with a mixed population (children and adults) were excluded. Studies that had been completed before 2014, had not been updated since 2015 or with incomplete registrations were excluded. We found 111 trials, of which 12 trials assessed the treatment of uncomplicated appendicitis in children. Groups from the Netherlands, USA, Canada, Australia, Sweden, Finland, UK, France, Italy, Israel, Japan, Singapore and Malaysia were identified. Some trials included hospitals from multiple countries.It was decided to invite research groups that are currently conducting clinical trials on the treatment of acute uncomplicated\u00a0appendicitis in children. Groups were identified through There is no rationale for determining the number of respondents to invite for a Delphi study.The Delphi method is an effective tool for reaching consensus in a large group without the need for face-to-face contact.The list of outcomes from the systematic review will be formatted into questions, accompanied by an extensive plain language summary per outcome, including figures if appropriate. The Delphi questionnaire will originally be formulated in English and will be translated if required. Translation will only be performed by native-speaking professionals.Participants will be asked to score the importance of each outcome using a 1-point to 9-point Likert scale as recommended by the Grading of Recommendations Assessment, Development and Evaluation working groupParticipants will be divided into two stakeholder groups: parents (with their children) and surgeons. Parents will be asked to discuss the answers they provide with their child while filling out the Delphi questionnaire. Baseline characteristics (age\u00a0and country) will be ascertained. Parents will be asked if their child was treated with non-operative or operative treatment, time between registration and the first diagnosis of appendicitis, and if their treatment was with or without complications. They will also be asked whether they will be answering the Delphi together with their child. Surgeons will be asked their specialty , workplace , experience with NOT\u00a0and experience in research regarding appendicitis in children.All participants will be asked to score all previously identified outcomes according to their perceived importance for assessing the treatment effect. In the first round, there will be an option to suggest additional outcomes not yet listed.Participants will have between 4\u00a0and 8\u2009weeks to complete each round, depending on the response rate. During\u00a0that time, they will receive a reminder email every 2\u2009weeks as long as they have not replied to the questionnaire.by stakeholder group and for all participants using descriptive statistics. Outcomes will be analysed separately for each stakeholder group, as there is evidence that patients are likely to assign importance to outcomes differently from\u00a0surgeons,Results will be analysed both stakeholder groups scoring the outcome as 7\u20139 and less than 15% in both stakeholder groups scoring the outcome as 1\u20133.Greater than 70% of participants in one stakeholder group scoring the outcome as 7\u20139. This implies that these outcomes are highly regarded by an individual stakeholder group and should also be included.Greater than 90% of participants within \u2018Consensus-in\u2019 will be defined asboth stakeholder groups scoring the outcomes as 1\u20133 and less than 15% of participants in both stakeholder groups scoring the outcome as 7\u20139. Consensus-out can only be reached when there is consensus across both stakeholder groups.Greater than 70% of participants in \u2018Consensus-out\u2019 will be defined asOutcomes that do not meet any of these criteria will be defined as \u2018no consensus\u2019. A stratified analysis will be performed to check for skewing as a result of divergent opinions from a single country, or surgeons with or without research experience.At the end of round 1, there will be a meeting of the study management group to assess whether an alteration in the Delphi study is appropriate. If additional outcomes are suggested by Delphi participants, each outcome will be assessed by the study management group to determine whether it is indeed new and to which category it should be classified. Wording of the Delphi questionnaire will be adjusted if misinterpretation is suspected.all participants. Outcomes for which there was only consensus-in within a single stakeholder group will still be presented to the other stakeholder group to evaluate whether consensus can be achieved in both stakeholder groups. An overview of included and excluded outcomes will be available. The outcomes for which there is no consensus and the newly suggested outcomes from the previous rounds will be presented with the participants\u2019 individual scores and the median scores from each stakeholder group combined with a histogram showing the scoring distribution. Participants will be asked to score all remaining outcomes in the same manner as in round 1.All participants who\u00a0complete the previous round will be asked to participate in the next round. Only outcomes that have not yet been defined as consensus-in or consensus-out during the previous round will be presented in the following rounds to both stakeholder groups on more than 80% of the outcomes and more than five outcomes with consensus in. To give an estimate of the degree of agreement between respondents, the width of the IQR of the median ranking score will be calculated, potentially ranging from 0, meaning complete agreement, to 8, meaning least possible agreement. This will be calculated for both the individual stakeholder groups and\u00a0the entire group of respondents after the final round.Results will be analysed per stakeholder group and for all participants using descriptive statistics, including a stratified analysis. The same definitions for consensus in/out as in the first Delphi round are upheld. After the second round, there will be a meeting of the study management group to assess the need for alterations in the Delphi study and to decide whether or not to proceed with a third Delphi round, assuming consensus between We wish to check for discrepancies of opinion between parents answering the Delphi together with their child and children who are interviewed directly. For this, a form of in-person interaction will be organised with young people (12\u201318 years) who have been treated for appendicitis. They will be asked to comment on the preliminary COS selection established at the end of the Delphi study and to suggest additional outcomes and comment on outcomes that did not make the preliminary COS selection. This will either be done by a short, face-to-face, one-round questionnaire involving only outcomes relevant to children/young people, or in the form of a small consensus meeting (prioritisation meeting) before finalising the definitive COS. Doing this type of research requires experienced interviewers and resources. That is why the face-to-face engagement will take place only\u00a0in selected countries; however, we will aim to involve as many countries as feasible. Separate ethical board approval will be obtained as appropriate.If adequate consensus (we aim to achieve consensus on at least one outcome per OMERACT core area) is reached in the Delphi study, we will organise a face-to-face expert panel meeting with selected individuals with the purpose to ratify a pragmatic and well-defined set of outcomes. A secondary aim of this meeting is to enhance support and implementation of the final COS.The meeting will be held at an international conference for paediatric surgery. Through purposive sampling, approximately 30 \u2018experts\u2019 from across all stakeholder groups, including physicians, researchers and children/parent representatives, will be invited to participate in a face-to-face meeting with the steering committee. Journal editors and healthcare commissioners will also be invited to attend in an observational capacity with the purpose of promoting implementation and to provide comments on the final list of outcomes.In the event that adequate consensus cannot be reached in the Delphi process, we will organise a formal face-to-face consensus meeting or teleconference. In that case, we will select an appropriate representation of all stakeholder groups from the panel members who\u00a0participated in the Delphi study.The goal is to achieve a pragmatic COS that is applicable and feasible for all future trials that evaluate the treatment of uncomplicated appendicitis in children. There is no recommended maximum number of outcomes that should be included in a COS. However, if the final COS includes too many outcomes, the COS would not be feasible to use in practice. To achieve the goal of a pragmatic COS, we aim to arrive at a maximum of 10 outcomes, the same maximum number as the UK COS protocol specifies.Only outcomes for which consensus is reached internationally will be selected. To test for country bias, stratified analyses of the Delphi results will be performed. The results from the face-to-face engagement of young people will be taken into account for the final COS selection and will be reported separately. If there is no consensus between patients, parents and healthcare professionals, an outcome can still be selected if there is clear consensus within a single stakeholder group. These will be reported separately. The final COS will be categorised according to the four core areas of the OMERACT filter.Patient involvement is at the core of this study design. As\u00a0we will directly be be asking parents and patients, with experience in having uncomplicated appendicitis, what outcomes they feel should be part of future research. To ensure our design is appropriate for parents and children, we have involved the Dutch child and parents representation group as part of the steering committee. In that capacity, they provide input on the protocol and the study. To make sure the Delphi questionnaire is understandable and has no ambiguities, it is checked by a group of laypersons before the start of the Delphi study. Part of the Delphi study is giving feedback to all its participants after each round; this will also be done with the final study results.The medical research ethics committee of the Academic Medical Center Amsterdam confirmed that the Dutch Medical Research Involving Human Subjects Act does not apply to this study and that complete approval of this study by the committee is not required. Each participating country/research group will be asked to obtain ethics board approval or confirm that ethics board approval is not required. Electronic informed consent will be obtained from all participants. The face-to-face engagement of young people (12\u201318 years) will take place in selected countries, and separate ethics board approval will be obtained, as appropriate.All data will be handled confidentially and in accordance with the Dutch Personal Data Protection Act and the European General Data Protection Regulation. DelphiManagerIn the first quarter (Q1) of 2018, the following 13 countries were invited to participate in the project: the\u00a0Netherlands, USA, Canada, Australia, Sweden, Finland, UK, France, Italy, Israel, Japan, Singapore and Malaysia. Ten countries replied; Italy, Israel and Japan did not. In Q1 of\u00a02018, the systematic review was finished. In the second quarter of 2018, the Delphi questionnaire was developed and piloted. In the third quarter of 2018, all materials were translated. Between the fourth quarter of 2018 and Q1 of\u00a02019, institutional review board\u00a0applications were submitted in 10 countries and 15 participating centres. The anticipated start of the online Delphi study is May 2019. We anticipate to have the final COS ready by Q1 of\u00a02020. Dissemination of the results will be accomplished by publication in an international peer-reviewed scientific journal and by presentations at conferences. By involving the majority of the principal investigators who are currently involved in research on uncomplicated appendicitis in children, we aim to optimise uptake of the final COS. By involving journal editors and healthcare commissioners in the face-to-face consensus discussion, we aim to ultimately have the COS introduced as a requirement in future outcome reporting on the treatment of uncomplicated appendicitis in children. We will also actively send out the final COS to relevant journal editors and funding bodies to promote uptake in future research.The selection of potential outcomes will be done systematically and will provide a selection for the first Delphi questionnaire that reflects most issues pertinent to the treatment of uncomplicated appendicitis. By including systematic reviews/meta-analyses that also report on non-comparative studies, we expect to identify all reported treatment outcomes, including those from the relatively new field of NOT for uncomplicated appendicitis.To be able to arrive at a manageable list of outcomes that is appropriate for a Delphi study, the number of outcome terms needs to be somewhat limited. In order to achieve this, the outcomes derived from our systematic review will be merged in case of similarity. If outcomes are not generalisable and are\u00a0reported only\u00a0once, they will be excluded. This will be proposed and prepared by two independent reviewers and discussed in the study management group. However, the merging of outcomes will inevitably lead to some loss of detail.In order to reflect the views of different stakeholders, a variety of groups will be part of the development of this COS. This is the case not only on a national level but also on an international level, related to, for example, differences between countries in resources, treatment practices for acute uncomplicated appendicitis and cultural differences. For example, there is a large difference with regard to the standard length of hospital stay after an appendectomy for uncomplicated appendicitis. In the USA, much effort is devoted to reduce the number of admission days; in the UK, there is only limited attention for the duration of admission, and for instance, in Japan, an admission for 5 days is not uncommon. We can expect that these kinds of differences result in different opinions regarding the COS. By also involving patients and parents from the participating countries, we hope to correct for these differences.If consensus is reached in the Delphi study, we will not be organising a formal consensus meeting. The Delphi method can be used for reaching consensus in a group of respondents without the need for face-to-face contact. There is a risk of bias if a face-to-face consensus meeting leads to selection of only participants who are able to attend the meeting, which is especially a problem in a global consensus procedure. There are also problems regarding language barriers in an international consensus meeting. To check for interpretation errors in the Delphi method and to ensure a pragmatic and well-defined set of outcomes, the results of the Delphi study will be discussed in an expert meeting. However, the influence of this meeting on which outcomes are selected for the final COS is very limited, as this selection is primarily made in the Delphi study.Involving patients in COS development has recently become common practice with 88% (n=112 as of 12 April 2016) of ongoing COS development studies doing so.A limitation is that due to the international nature of our study, it will not be feasible to engage children directly in all the participating countries. That is why the face-to-face engagement will take place in selected countries.After careful consideration and consultation with the participating countries, it was decided not to include paediatricians, general practitioners, nurses or emergency medicine physicians. Although all these specialists play an intricate role in the diagnosis and care for children with appendicitis, they do not make the final decision regarding treatment or its provision. However, we will however, depending on the organisation of the healthcare system in each country, ask these stakeholders to comment on the final COS in order to ensure that essential outcomes are not missed. Since almost all research regarding treatment of paediatric uncomplicated appendicitis is initiated by (paediatric) surgeons, it was decided that researchers will not be included as a separate individual stakeholder group. However, involvement in research will be registered. While their opinion is vital to the development of a COS, it is likely researchers will be well represented in the (paediatric) surgeon stakeholder group. A stratified analysis will be performed to check for skewing of the results by surgeons involved in research. It was also decided not to include journal editors or healthcare commissioners. Even though their opinion is of great importance especially regarding implementation, it was determined that their opinion is not essential in establishing the outcomes selected for the COS. Also, there is much variability between countries regarding the role of these stakeholders, which would lead to major challenges regarding Delphi analyses of such a small stakeholder group. However, to enhance implementation and because of their expertise on the use of COSs, representatives of these stakeholder groups will be asked to attend the final consensus discussion.This study will not answer the question on how to measure the outcomes that are included in the final COS or at what time point the outcomes should be measured. However, we will attempt to come to a clear definition of each outcome. We expect that further research will be necessary to answer the question of timing and how to measure the outcomes. We will advise on this subject in the final report."} +{"text": "Urinary tract infections (UTIs) are the second most common infection presenting in the community. Clinical guidelines and decision aids assist health practitioners to treat a UTI; however, treatment practices vary due to patient needs and context of presentation. Numerous trials have evaluated the effectiveness of treatment interventions for UTI; however, it is difficult to compare the results between trials due to inconsistencies between reported outcomes. Poor choice of outcome measures can lead to impairment of evidence synthesis due to the inability to compare outcomes between trials with similar aims. Transparency in selecting and reporting outcomes can be mitigated through the development of an agreed minimum set of outcomes that should be reported in clinical trials, referred to as a core outcome set (COS). This paper presents the protocol for the development of a COS for interventions in the treatment of uncomplicated UTI in adults.This COS development consists of three phases. Phase 1 is a systematic review, which aims to identify the core outcomes that have been reported in trials and systematic reviews of interventions treating uncomplicated UTI in adults. Phase 2 consists of a three-round online Delphi survey with stakeholders in the area of treatment interventions for UTI. The aim of this online Delphi survey is to achieve consensus on the importance of the outcomes emerging from Phase 1 of this research. Phase 3 is a consensus meeting to finalise the COS that should be reported in trials evaluating the effectiveness of interventions for the treatment of UTI.It is hoped that the development of a COS for interventions for the treatment of uncomplicated UTI in adults will be adopted as a minimum set of outcomes that should be reported and measured within this context. If the findings from clinical trials related to treatment interventions for UTI are to impact on policy and practice, it is important that the findings from different treatment interventions are comparable across trials.The online version of this article (10.1186/s13063-019-3194-x) contains supplementary material, which is available to authorized users. Escherichia coli [Urinary tract infections (UTIs), sometimes referred to as cystitis or lower UTI, are the second most common infection presenting in primary care . In the hia coli . Treatmehia coli . Debate hia coli .Clinical trials are robust designs used to evaluate the effectiveness of healthcare interventions. However, the impact of their results on policy and practice may be limited by a lack of consistency in outcomes measured and reported across trials. This heterogeneity in outcomes makes it difficult to synthesise findings across trials and limits the ability of evidence to inform healthcare decisions . In addiTransparency in selecting and reporting outcomes can be mitigated through the development of an agreed minimum set of outcomes that should be reported in clinical trials. This is known as a core outcome set (COS) . ReportiThis paper presents the protocol for the development of a COS for interventions in the treatment of uncomplicated UTI in adults.The objectives are: 1) to conduct a systematic review to identify a comprehensive list of outcomes reported in trials examining the effectiveness of interventions for the treatments for uncomplicated UTI in adults; and 2) to develop consensus on a COS for evaluation of interventions for the treatment of uncomplicated UTI through a modified Delphi survey and consensus group meeting.This protocol has been developed using recommendations outlined in the Core Outcome Set Handbook , Core OuThe COS development will encompass three phases: Phase 1 is a systematic review identifying the core outcomes that have been reported in randomised trials and systematic reviews of randomised trials of interventions for the treatment of uncomplicated UTI in adults; Phase 2 is an online, three-round Delphi survey with stakeholders; and Phase 3 is a consensus meeting.Studies are all randomised trials and systematic reviews of randomised trials comparing the effectiveness of any interventions for the treatment of uncomplicated UTI in adults.All trials investigate the effectiveness of treatment interventions for uncomplicated adult UTI. For the purpose of this research, uncomplicated UTI is defined as the acute onset of dysuria, frequency, or urgency in healthy male and non-pregnant woman without known functional or anatomical abnormalities of the urinary tract . Papers Treatment interventions are defined as \u201canything that aims to make a change to someone\u2019s health. For example, providing a counselling service, giving a drug, or giving people information and training are all described as interventions\u201d . ReflectParticipants are otherwise healthy adults (over 18\u2009years old), male and non-pregnant female, who received treatment for an uncomplicated UTI. Pregnant women have been excluded as the treatment regime for a UTI in pregnancy may be different as highlighted by NICE guidelinThis systematic review will review the literature over the last 10\u2009years 2007\u20132017). Citation databases that will be searched are the Cochrane Database of Systematic Reviews , PubMed, and Embase. The PRISMA [07\u20132017. Titles and abstracts will be screened independently by at least two reviewers. Covidence, a web-based citation screening tool, will be used to manage the screening process. The full text of studies deemed potentially eligible will be obtained and screened independently for eligibility by two reviewers. Where there is uncertainty, these papers will be assessed by an additional reviewer. If there is a disagreement, these papers will be discussed with the advisory group.Author details, year, and journal titleInterventions under investigationAll intervention outcomes reported within the trial Outcomes will be extracted verbatim. For papers judged eligible for inclusion, the following data will be extracted to a purposefully designed database within Microsoft Excel:Extracted outcomes will be grouped into outcome domains by one reviewer, checked by a second reviewer, and approved by the COS advisory group. Outcome domains will be a broad term or phrase that will be used to categorise outcomes that are deemed similar; for example, all outcomes relating to \u2018time to cure\u2019 will be categorised under the domain name \u2018time to cure\u2019.The Delphi survey is a consensus building methodology. It involves a panel of stakeholders anonymously participating in sequential questionnaires (survey rounds) rating the importance of the reported outcomes. This survey will be administered online, which means the participants do not interact with one another thus removing group thinking.The aim of the online Delphi survey is to offer stakeholders the opportunity to rate the importance of outcomes identified from the systematic review process (Phase 1) for inclusion in the final COS and to identify additional outcomes of importance that were not captured within the review process.Stakeholders who have expertise in UTI treatment interventions will be invited to participate in the Delphi panel. Invitations to participate in the online Delphi survey will be made to three stakeholder groups: 1) members of the public who have experience in being treated for UTI; 2) healthcare professionals who have experience treating people with UTI and policy makers; and 3) researchers with expertise related to the treatment of UTI.A snowball sampling strategy will be used to identify experts in each of the stakeholder groups who will be sent an invitation to participate via electronic means . A list of researcher/academic stakeholder representatives will be identified through published papers related to the treatment of UTI. Healthcare professionals will be approached through relevant national and international professional organisations. Policy makers will be identified through published policies and briefs related to healthcare and UTI. Public representatives will be identified and invited to participate through established patient advocacy groups and social media activities. This process will ensure that the list of emerging outcomes is relevant and important to all stakeholder groups.Where appropriate, gatekeepers within relevant stakeholder organisations will be asked to distribute the COS invitation through their stakeholder mailing list on behalf of the research team. The research team will provide these organisations with an email invitation. This invitation email will explain the aims of the study, what it is about, what participants are asked to do, and why their participation is important.The invitation email will also contain an electronic link which will allow stakeholders who are willing to participate to register for the survey and provide their consent for completing all three rounds of the online Delphi survey. Registered participants will receive an email containing a link to round 1 of the survey only after they have consented to participate. The participants will then be given 2\u20133\u2009weeks to complete round 1.We aim to recruit similar numbers of participants to each stakeholder group. While there is an absence of evidence on optimal sample size for each group participating in a Delphi survey, our sample size overall, and within groups, will be guided by the COMET handbook feasibility considerations and recommendations . TherefoThe three-round online Delphi survey will be administered using an electronic web-based system distributed via email. Each round will be open for 3 weeks to give the stakeholders an opportunity to complete the Delphi. Participants who have not responded to the survey will be sent periodic reminders via email and a final reminder will be sent the day before the survey is closed. Participants who do not complete a round will not be invited to the next round. As per recommendations outlined by the GRADE group, each round will use the same nine-point Likert scale to rate the importance of the outcome for inclusion in the COS. A rating of \u2018limited importance\u2019 (rating of 1\u20133), \u2018not crucial importance\u2019 (rating of 4\u20136), or \u2018crucial importance\u2019 (rating 7\u20139) will be used . ParticiIn the first round of the Delphi survey, each stakeholder will be asked to provide some demographic information and then rate the importance of each outcome that emerged from Phase 1. One open-ended question will be included at the end of round 1 to give participants the opportunity to suggest outcomes they feel are important but have not been included in the survey. New outcomes that have been suggested by two or more participants will be considered for inclusion in round 2.All participants who completed round 1 will be invited to round 2. All outcomes included in round 1 will be carried forward to round 2. Any new outcomes suggested by two or more respondents in round 1 will also be included. For round 2 of the survey, the participants will receive their individual score for each outcome, the aggregated scores of their stakeholder group, as well as the other stakeholder groups from the previous round to consider when they are completing the survey. Based on this feedback, each stakeholder will be asked to rate each outcome again. In addition, participants in round 2 will be invited to consider if they are willing to attend a face-to-face consensus meeting to discuss the final set of outcomes.Participants who completed round 2 will be invited to the third and final round. Round 3 of the Delphi survey will contain the list of the outcomes that are rated as critical (rated 7\u20139) by at least 70% of respondents and rated as of limited importance by 15% or less of all respondents in round 2. In addition, to ensure that public prioritised outcomes are not overwhelmed by other groups, any outcome that had an average public score of 7 or more was also re-proposed for voting in round 3. Each participant will then be asked to rate each outcome for a final time using the same rating scale used in rounds 1 and 2.The aim of the third and final phase of this COS development will be to finalise the COS that should be reported when evaluating interventions for the treatment of uncomplicated UTI in adults.Participants will include representatives of the three stakeholder groups who completed Phase 2 of this research and who indicated they would be willing to participate. A convenience sampling strategy will be adopted to ensure that meeting participants will be composed of a mixture of representatives from each of the three stakeholder groups . It is also desirable to include national and international participants within this research. Attendees will be sent information about the results of the Delphi survey prior to attending the consensus meeting.If feasible, the 1-day consensus meeting will be scheduled to coincide with an academic conference of relevance to the treatment of uncomplicated UTIs in adults. Participants may attend in person or virtually by video conference. The chair will be independent, and the facilitator will encourage all stakeholders to have equal input during the meeting, adopting a collaborative approach to achieving consensus. Outcomes rated as 7\u20139, crucial, by \u2265\u200970% of participants and 1\u20133, of limited importance, by fewer than 15% of participants in round 3 will be considered to meet the definition of consensus. These outcomes will be brought forward to the consensus meeting. Outcomes that are rated 1\u20133 by \u2265\u200970% of participants and rated 7\u20139 by less than 15% of participants in round 3 will be excluded and not discussed at the meeting. Any outcomes that do not meet either definition will be classified as no consensus and brought forward to the meeting , 23.To our knowledge, there is currently no COS for interventions in the treatment of UTI. It is hoped that this COS will be adopted as a minimum set of outcomes that should be reported and measured within this context. The researchers propose a rigorous approach to the development of this COS, which adheres to best practice guidance from the COMET handbook, COS reporting guidelines, and other protocols which have adopted COS methodologies for other health conditions . The COSIn addition, use of the COS will assist in synthesising evidence from individual studies. To further broaden the transparency of this research, the final COS will be classified within a broader outcome taxonomy developed in 2017. The purpose of this taxonomy is to classify outcomes to increase efficiencies when searching for them in clinical trial registries .Additional file 1:SPIRIT 2013 checklist: recommended items to address in a clinical trial protocol and related documents. (DOC 113 kb)Additional file 2:PubMed search queries for the systematic review. (DOCX 17 kb)"} +{"text": "Staphylococcus pseudintermedius is a common bacterial pathogen in companion animal medicine and has demonstrated zoonotic potential. Here, we report six new Staphylococcus pseudintermedius prophage genomes of the Siphoviridae family, identified in isolates recovered from human and canine clinical specimens. Staphylococcus pseudintermedius is a common bacterial pathogen in companion animal medicine and has demonstrated zoonotic potential. Here, we report six new Staphylococcus pseudintermedius prophage genomes of the Siphoviridae family, identified in isolates recovered from human and canine clinical specimens. Staphylococcus pseudintermedius is a frequently isolated opportunistic pathogen of dogs and other animals, mainly causing pyoderma, wound infections, and otitis media and de novo assembled using the Geneious assembler on Geneious version 10.0 set to medium to low sensitivity (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the Swiss Institute of Bioinformatics Prosite database (http://prosite.expasy.org/), with priority given to Prosite predictions. Over 150 hypothetical proteins were identified in the novel prophages.The mobile elements involved in antibiotic resistance development and host adaptation of tigation , 12\u201316. rocesses \u201320. The ocesses \u2013, 17, 21.prophage . Here, w strains . Strains strains . Genomic strains , 20. LibZealand) . The PHAZealand) . The genZealand) . The phiZealand) . HypotheS. aureus, it has been shown that sequencing of exDNA can identify plasmidial or episomal prophages that would otherwise remain undetected (S. aureus, where prophages harboring virulence factors are commonly associated with clinical strains, no known virulence factors were detected in the identified S. pseudintermedius prophages (In detected , 20. Therophages , 28. FurMK075001, MK075002, MK075003, MK075004, MK075005, and MK075006 (SRR8957054, SRR8957055, SRR8957056, SRR8957057, SRR8957058, SRR8957059, SRR8957060, and SRR8957061.The prophage sequences of this study have been deposited in NCBI GenBank under the accession numbers MK075006 . The raw"} +{"text": "The subsequent L5-S1 disc degeneration associated with long fusion arthrodesis terminating at L5 in patients with adult scoliosis has been a common concern. However, few studies paid attention to its preoperative predictors, especially in spinopelvic parameters. The purpose of the present study was to clarify the preoperative predictors of subsequent L5-S1 disc degeneration after long fusion arthrodesis terminating at L5 in patients with adult scoliosis on spinopelvic parameters.In this retrospective study, we enrolled 67 patients with adult scoliosis, and the patients were divided into disc degeneration group (DD) and no disc degeneration group (NDD), based on the presence or absence of subsequent L5-S1 disc degeneration. The status of L5-S1 disc was evaluated by a modified version of radiographic classification. Characteristics and spinopelvic parameters of preoperative patients were collected as potential predictors for subsequent lumbosacral disc degeneration after long fusion arthrodesis terminating at L5 in patients with adult scoliosis. Multivariate logistic regression analysis and the receiver operating characteristic curve were used to identify the preoperative predictors, with an adjusted odds ratio (OR) and 95% confidence intervals (CI).P\u2009<\u2009\u00a00.001), and the other 31 patients were divided into group NDD (preoperative and last follow-up score 0.87\u2009\u00b1\u20090.49). There was no statistical difference in preoperative score (P\u2009=\u20090.583) of lumbosacral disc between two groups; however, significant statistical difference showed in last follow-up score (P\u2009<\u2009\u00a00.001). Multivariate logistic regression identified three preoperative predictors: pelvic incidence (PI) (P\u2009=\u20090.018), sagittal vertical axis (SVA) (P\u2009=\u20090.024), and sacrum-femoral distance (SFD) (P\u2009=\u00a00.023). PI <\u200948.5\u00b0 , SVA >\u20094.43\u00a0cm , and SFD >\u20095.65\u00a0cm showed satisfied accuracy for predicting subsequent L5-S1 disc degeneration.Thirty-six patients (53.73%) with subsequent L5-S1 disc degeneration were divided into group DD . PI <\u200948.5\u00b0, SVA >\u20094.43\u00a0cm, and SFD >\u20095.65\u00a0cm were preoperative predictors for the subsequent L5-S1 disc degeneration. More attention should be paid to prevent the L5-S1 disc from degeneration when these preoperative predictors exist, especially with two or more. Adult scoliosis, a spinal deformity with Cobb angle greater than 10\u00b0 after skeletal maturation, occurs in two types: idiopathic adolescent scoliosis in adulthood and de novo scoliosis \u20134.The distal fusion segment extending to the sacrum is undisputed for patients with adult scoliosis with presented severe lumbosacral disc degeneration, instability of lumbosacral segment, L5 spondylolysis, and nerve compression at L5-S1 needing decompression . Long fuTherefore, the purpose of our study was to clarify the preoperative predictors of subsequent degeneration in L5-S1 disc after long fusion arthrodesis terminating at L5 in patients with adult scoliosis and to provide evidence for surgical strategy. Besides, spinopelvic parameters have been addressed in the current study because of their important role in subsequent lumbosacral disc degeneration.This retrospective study was approved by the Institutional Ethics Board of the Third Hospital of Hebei Medical University. The retrospective study included a consecutive series of 67 patients with adult scoliosis who underwent surgical treatment at our institution from May 2004 and March 2016. The inclusion criteria were as follows: (1) presence of adult scoliosis with Cobb angle greater than 10\u00b0, (2) posterior-only surgical instrumented procedure terminating at L5, (3) no history of any spinal surgery, (4) fixed segments greater than or equal to four, and (5) follow-up period more than 2\u00a0years. The exclusion criteria were as follows: (1) lack of completed clinical data, (2) disability of lower limb, and (3) tumor or inflammation involving the spine. Finally, 49 women and 18 men with mean age at surgery of 59.24\u00a0years were reviewed in the study. Forty-one patients (61.19%) had de novo scoliosis in adulthood and 26 patients (38.81%) had a history of idiopathic adolescent scoliosis without treatment. The mean follow-up period was 4.85\u00a0years . Clinical data, including age, gender, body mass index (BMI), number of instrumented vertebrae, and spinopelvic parameters, were collected as potential predictors for subsequent lumbosacral disc degeneration.Spinopelvic parameters were measured on anteroposterior and lateral radiographs of the entire spine Fig.\u00a0. The sagt test; if the data was not in accord with normal distribution or homoscedasticity, Mann-Whitney U test would be used. Categorical data including gender and number of instrumented vertebrae was analyzed by chi-square test. The statistical significance was set as P value less than 0.05. The potential predictors which showed P\u2009<\u2009\u00a00.05 in univariate regression analysis were analyzed by multivariate logistic regression analysis with adjusted odds ratio (OR) and 95% confidence intervals (CI). PI, SVA, and SFD were entered into the logistic regression again to identify the correlation between subsequent disc degeneration and the predictors after adjusting for potential confounding factors. The sensitivity and specificity in PI, SVA, and SFD were evaluated by the receiver operating characteristic curve, and, at the same time, cutoff value in the three parameters was obtained to achieve good predicting.The data was analyzed by statistical software, the SPSS 21.0 software . Results are presented as the mean\u2009\u00b1\u2009standard deviation (SD). Quantitative data including age, BMI, degenerative degree, and spinopelvic parameters was analyzed by independent P\u2009<\u2009\u00a00.001). However, no significant statistical difference was found in preoperative average score between the two groups (P\u2009=\u20090.583). The number of instrumented vertebrae was four in three patients, five in four patients, six in 11 patients, seven in eight patients, eight in 28 patients, nine in six patients, ten in four patients, and 11 in three patients. There was no significant statistical difference in instrumented vertebrae number between group DD (7.56\u2009\u00b1\u20091.64) and group NDD (7.52\u2009\u00b1\u20091.54) (P\u2009=\u20090.927). Significant statistical difference was not found in average follow-up period (P\u2009=\u20090.377), BMI (P\u2009=\u20090.207), and gender (P\u2009=\u20090.355) , SVA and SFD showed significant difference. In multivariate logistic regression, PI , SVA , and SFD were identified to be predictors for subsequent L5-S1 disc degeneration (Table\u00a0P\u2009=\u20090.008), SVA , and SFD were shown in receiver operating characteristic curve analysis . It may be caused by the reason that the number of patients with instrumented vertebrae >\u200910 (three patients with fusion from T7 to L5) in our study was limited. Unexpectedly, all the three patients developed into subsequent L5-S1 disc degeneration. The relationship between number of instrumented vertebrae and subsequent L5-S1 disc degeneration should be further studied.The preoperative SVA >\u20094.43\u00a0cm was identified as a preoperative predictor for the subsequent L5-S1 disc degeneration. Many previous studies proved that the subsequent L5-S1 disc degeneration was highly correlated with sagittal imbalance , 22\u201324. PI, an anatomical parameter, being not affected by lumbar degeneration diseases after the end of bone growth, has been mentioned in previous studies and played a significant role in disc degeneration , 22, 25.Several limitations in the current study needed to be pointed out. First, the study was limited by its retrospective nature. Second, follow-up period in the study was medium-term. However, medium-term follow-up was not enough to evaluate the status of disc degeneration because the process was gradual. Third, the radiographic classification of the disc is divided into only four grades, so a more detailed classification method would be proposed and applied in this study. What is more, the number of patients with adult scoliosis after long fusion arthrodesis terminating at L5 in this study was small. Thus, further multicenter studies with a large sample would be preformed to clarify the correlation between the preoperative predictors and the subsequent L5-S1 disc degeneration.The prevalence of the subsequent L5-S1 disc degeneration in this study was 57.3% (36 of 67 patients). The PI <\u200948.5\u00b0, SVA >\u20094.43\u00a0cm, and SFD >\u20095.65\u00a0cm were identified as the preoperative predictors for the subsequent L5-S1 disc degeneration. Spine surgeons should pay more attention in choosing the surgical strategy when the preoperative predictors exist in patients, especially with two or more."} +{"text": "A modern genomics ecosystem has emerged. This commentary describes recent trends in clinical genomics that enable its successful integration in mainstream medicine. The rapid expansion of clinical genomics will have a positive impact on the healthcare of individuals worldwide. Time magazine cover proclaimed \u201cGenetics\u2014the future is now.\u201d Though it was prescient and certainly exciting to contemplate how genomics would transform healthcare and save lives, the proclamation was premature. A decade later, the completion of the Human Genome Project provided another occasion to expound on the merits and promises of genomic medicine. Despite this landmark scientific achievement and the subsequent exponential growth in our understanding of the genomic basis for human disease, the integration of genomics into healthcare has been slow because of the scarcity of access to appropriate genetic testing across all clinical specialties in which it matters. In this commentary, we describe the latest trends in clinical genomics as it relates to hereditary disorders and argue that we have finally reached a tipping point at which genomics has a realistic chance to radically transform medicine. This transformation will occur in key interrelated ways: by improving our understanding of the phenotypic spectrum of germline genetic disorders, enabling accurate molecular diagnoses in a greater number of individuals, connecting patients to therapies and clinical trials, and empowering individuals to comprehensively understand their genetic heritage, participate in patient advocacy, and consider informed proactive genetic screening for clinically actionable hereditary disorders. Together, these developments will stimulate a paradigm shift through the evidence\u2010based integration of genetics in various clinical specialties, thereby having a lasting impact on the healthcare of the global population.In January 1994, a Our optimism stems from a recent convergence of several essential components in a strengthening genomics ecosystem: rapidly expanding medical genetics knowledge, advances in genetic testing technologies and informatics solutions, the development of public genomics databases, large\u2010scale studies of clinical and research cohorts, and novel methods to improve phenotyping Figure\u00a0. Recent These advances in laboratory technologies and bioinformatics solutions have in turn encouraged the establishment and investigation of massive sets of genomic data from public and private efforts, dramatically enhancing our understanding of the variation that exists in both clinical cohorts and healthy individuals, and most importantly, across a variety of populations worldwide. Large\u2010scale initiatives such as OMIM, ClinGen, the 1,000 Genomes Project, gnomAD, DECIPHER, and the Database of Genomic Variants have been critical to the refinement of clinical genetic interpretations of sequence and copy number variants . However, at least two challenges will modulate the pace of this evolution: the high cost of whole\u2010genome sequencing and the limited understanding of the clinical relevance of noncoding regions of the genome. These challenges will surely be addressed as technologies improve and additional studies using whole\u2010genome sequencing are conducted around the world. In the meantime, targeted multigene NGS panels are having a significant impact on the accessibility of genetic testing owing to their versatility and low cost, and they are therefore increasing testing across clinical specialties and facilitating the deeper integration of genetics in medicine.Among the advantages of such panels, superior analytic sensitivity and wide availability make them excellent options as first tests for many disorders that can be diagnosed in the clinic with a moderate to high degree of confidence. Moreover, these panels contain all known genes associated with a specific disorder and are updated periodically to add newly discovered genes. As a result, well\u2010designed panels have a useful diagnostic yield: a recent review reported a positive diagnostic yield of 10% to 60% , on the contrary, provide unbiased assessments of the genome or exome, respectively, and are appropriate for patients with less well\u2010defined phenotypes or undiagnosed disease, particularly children with developmental delay. The diagnostic yields of CMA and WES are 15\u201320% and 25%,\u201340%, respectively . More hereditary disorders have been discovered and characterized in the last 10\u00a0years than in the previous 30. Recent discoveries of novel disorders have occurred in studies of large clinical cohorts and through individual case studies connected through forums such as Matchmaker Exchange and GenomeConnect, which allow clinicians and researchers worldwide to communicate about novel genotypes and phenotypes and recognize patients with the same disorders are emerging to provide deeper insight into variation in the human genome, in turn improving the interpretation of variants . Additional platforms will be developed during the next decade, essentially making AI tools standard requirements for genomics analysis. Clinical apps using facial recognition technology are also being developed to support facial dysmorphology assessments in pediatric disorders . The last few years have witnessed the successful development of life\u2010altering rare disease therapies . Genomics will need to demonstrate its value if it is to have its anticipated impact on global healthcare. Nonetheless, with the momentum of the twin forces of the falling cost of testing and the expanding knowledge of how to interpret the impact of genetic information on health, we anticipate that these challenges will be overcome. We are now witnessing the start of this exciting future.All of these advances portend a bright future for genomics\u2010enabled medicine, although formidable challenges remain and should not be underestimated. We need to improve the accuracy of penetrance rates for all disorders, recognize the influence of population\u2010specific genomic backgrounds, aggregate useful phenotype data to uncover meaningful genotype\u2013phenotype correlations, collect health outcomes data in both diagnostic and preventive genetics, and of course, develop safeguards for privacy of and access to genomic information. Medical education around the world should also provide more exposure to genetics. Even when these knowledge gaps in clinical genomics are filled and the challenges to integrating genomics into mainstream medicine are overcome, serious and daunting economic issues will remain due to healthcare inequities across the globe. Per capita annual healthcare expenditures range from more than $9,600 in developed countries in Western Europe to less than $20 in some countries in Africa and Southeast Asia (WHO Global Health Expenditure Database; The authors are medical team leaders and executives at Invitae, a genetic testing laboratory."} +{"text": "A promising approach to improve the poor antibacterial properties of dental composite resins has been the addition of metal oxide nanoparticles into the resin matrix. This systematic review aimed to determine whether the addition of zinc oxide nanoparticles (ZnO-NPs) improves the antibacterial properties of direct dental composite resins. This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and registered with the PROSPERO database: CRD42019131383. A systematic literature search was conducted using the following databases: Medline (Ovid), the Cochrane Library, SCOPUS, CINAHL, Web of Science, Trove, Google Scholar, World Cat, and OpenGrey. The initial search retrieved 3178 results, which were then screened against inclusion and exclusion criteria, resulting in a total of four studies that were eligible for qualitative synthesis within this review. All the included studies were in vitro non-randomized post-test design experimental studies. A lack of congruity in the results obtained from these studies that used different tests to evaluate antibacterial activity was evident. Although some studies demonstrated a significant improvement of antibacterial properties in composites containing at least 1% ZnO-NPs (wt %), they are unlikely to present any clear clinical advantage due to the short lifetime of observed antibacterial properties. Dental caries is a widespread infectious disease, in which the hard tooth structure is demineralized as a result of the acid produced by the bacterial fermentation of carbohydrates. Various direct restorative materials have been used to restore (fill) these carious defects ranging from metallic fillings to cements and polymer resin/inorganic filler-based composite resins ,2. CompoOne promising approach has been the addition of metal oxide nanoparticles into the resin matrix . As the Traditionally, nanofilled composites are known to allow for desired polishing results and are hence widely used for direct anterior restorations with an acceptable clinical longevity ,24. FurtThis systematic review was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and was The articles considered for inclusion were in vitro, in vivo, and ex vivo experimental studies. The inclusion criteria were as follows: (a) includes zinc oxide nanoparticles incorporated into direct dental composite resins; (b) investigates the antibacterial properties of direct dental composite resins incorporating zinc oxide nanoparticles; and (c) does not investigate only the synergistic antibacterial effects of multiple agents when zinc oxide nanoparticles are included along with other antibacterial agent(s) in direct dental composite resins.Exclusion criteria were as follows: (a) articles not published in the English language; (b) articles for which the full text was inaccessible; (c) case series, case reports, conference articles/proceedings, book chapters, theses, dissertations, reviews, ideas, editorials, and opinions.An electronic search was carried out into the following bibliographic databases and gray literature databases: Medline (Ovid); The Cochrane Library ; Scopus; CINAHL; Web of Science; Trove; Google Scholar; World Cat; and OpenGrey. There were no restrictions on publication date, language, or study type in the search.The following focus question was based on the PICO schema and usedThe articles were searched and retrieved using different combinations of medical subject headings (MeSH) terms/subject headings and natural language terms/phrases which in all instances encompassed the following integral concepts of the focus question: (a) antibacterial activity, (b) direct dental composite resin, and (c) zinc oxide nanoparticles. No limits on year of publication were placed for the database searched, and the last search was conducted in May 2020. The combinations and permutations of search terms used were optimized for each database according to the functionality of each database. The following limits determined the optimized search strategy for each database: (a) the number of retrieved articles after completion of the initial search must be less than the maximum number of articles retrievable by the database; (b) search syntax variations for each database; and (c) character limits of the database .The titles and/or abstracts of the articles retrieved were independently screened by two reviewers to identify articles that potentially met the eligibility criteria. Articles that definitively did not meet inclusion criteria and/or fulfilled exclusion criteria at this stage were eliminated. Any disagreements between the two review authors with regard to the eligibility of any particular article were resolved by consultation with a third reviewer until an agreement was reached.For articles that passed this process, the full-text manuscripts were catalogued and independently assessed by four reviewers for eligibility as per the eligibility criteria.Data from the full-text manuscripts of the articles identified as eligible for the review were extracted independently by two reviewers according to the data items listed in below. Any discrepancies or uncertainties in the data were resolved through discussion with a third review author until an agreement was reached.Author (year)\u2014presents the author(s) of the article and the year of publicationSample\u2014describes the study sample Treatment group\u2014describes the treatment group(s), including the average size of zinc oxide nanoparticles usedExperiment\u2014names the test conducted Methodology\u2014describes pertinent information on the corresponding test and the variable(s) measuredOutcome\u2014describes the outcome(s) of the experiment(s). All outcomes presented are statistically significant unless specified otherwise.The data relevant to the research question was extracted from the included studies and tabulated into the following fields for qualitative synthesis:The quality and risk of bias assessments were performed independently by two review authors using a modified version of the Methodological Index for Non-Randomized Studies (MINORS) tool 29]. An. An29]. The initial electronic search of the various databases yielded 3178 results. After the removal of duplicates, independent screening of titles and/or abstracts, five studies remained for assessment of eligibility based on full-text review. One study by Shen et al. (2016) was excluded from the review as nanoparticulate zinc oxide was not used in this study . A totalAll of the four studies included in this review were non-randomized in vitro experimental studies. The data extracted from the included studies are presented in S. oralis, S. gordonii, and A. naeslundii was used with a 10 wt % ZnO-NP composite resin. The authors found no statistically significant reduction in the number of CFUs between the 10 wt % ZnO-NP composite resin and the control [Three of the studies included in this review investigated the antibacterial properties of ZnO-NP composite resins via a direct contact test where the variable of viable colony-forming units (CFUs) was measured ,25. In tThe quality and risk of bias assessments for the four articles included in the review were carried out using the modified version of the Methodological Index for Non-Randomized Studies (MINORS) tool and are Much research has been carried out on the addition of antibacterial agents into direct dental composite resins to improve their antibacterial properties ,15,16,17Overall, there was a lack of congruity in the findings of the studies between the different tests/methodologies used to evaluate the antibacterial activity of ZnO-NP composite resins at lower weight percentage ZnO-NP loadings. Two studies included in this systematic review reported a significant improvement of antibacterial activity in composite resins containing at least 1 wt % ZnO-NPs, compared to the controls, in short-term direct contact tests utilizing a single species biofilm ,31; anotIn all of the three studies that assessed samples under electron microscopy, a qualitative reduction in the number of bacterial colonies was found with increasing ZnO-NP concentrations; however, the concentrations at which these changes were observed differed considerably amongst the three studies\u20141 wt % being the lowest effective loading depending on the study ,25,32. OS. sobrinus biofilm, whereas the other two studies used a S. mutans or Lactobacillus biofilm [S. sobrinus biofilm of Sevin\u00e7 et al. being at least 48 h more mature than those used in the other two studies.Three studies that investigated the antibacterial properties used a direct contact test followed by viable colony-forming units (CFUs) measurement reported variable results, specifically in relation to composite resins containing a lower concentration of ZnO-NPs ,25,31. NIn the literature, some studies suggest that ZnO-NP composites may have little antibacterial effect against multi-species biofilms . In regaBased on experiments that attempted to simulate the aging of composite restorations, the literature suggests that the antibacterial effect of the ZnO-NP composites may not be long-lasting ,25. Of tS. sobrinus attachment and biofilm coverage and density were all qualitatively lower on the 10 wt % ZnO-NP composites compared to unmodified control composites in both SEM and CLSM [Three of the four studies included in this review visualized their samples of bacterial colonization of composite resin under a scanning electron microscope (SEM) and/or a confocal laser scanning microscope (CLSM) ,25,32. Sand CLSM . Finallyand CLSM ,25,32.S. sobrinus; no statistically significant difference was found between these composites [S. mutans biofilm compared to the control. The 0.5 wt % ZnO-NP composite did not show a statistically significant difference compared to the control [S. mutans biofilm [In addition to the above-mentioned tests, two of the studies performed tests unique to their respective studies ,32. Thesmposites . In the Two of the studies included in this review also assessed changes in the mechanical and physiochemical properties of the composite resins as a result of the addition of ZnO-NP into the resin matrix. Hojati et al. concluded that the addition of up to 1 wt % ZnO-NPs into composite resins does not adversely affect the mechanical properties of the composite . No adveThe studies included in this systematic review display three primary limitations; study design, short term evaluation of antibacterial activity, and limited bacterial species against which the antibacterial effects were evaluated. All the studies included in this review are in vitro studies; therefore, the results may not translate to clinically relevant conclusions. Conditions in an oral cavity differ substantially from the in vitro conditions used in the included studies. The biochemical nature of the oral cavity is constantly changing as it is being flushed with saliva, food, and fluid drinks. This dynamic chemical environment has the potential to alter the results/conclusions drawn from these studies of the antibacterial agents in dental composites in controlled in vitro settings . FurtherMost of the tests used to assess antibacterial activity were short-term (1\u20133 days). Long-term studies of the antibacterial effect of ZnO-NP are yet to be conducted. A short-term exposure of zinc nanoparticle incorporated composite against bacteria does not represent how well the restorations will perform in the oral cavity over an extended period of time. Around 60% of composite resin restorations are expected to last for more than 10 years, if sound restorative principles and techniques are employed during placement . TherefoFinally, most of the studies included in this review used single species biofilms to test the antibacterial activity of ZnO-NP dental composites except for Sevin\u00e7 et al., who used a three-species biofilm for a single test, and in this test, they found no statistically significant difference between the ZnO-NP composites compared to the control . Due to There is a lack of congruity in the data from these studies due to the variation in assessment methods used to evaluate the antibacterial activity of ZnO-NP direct dental composite resins. Some tests such as the single species direct contact tests, metabolic assay tests, and visualization under electron microscopy demonstrated a significant improvement of antibacterial properties in composites containing at least 1 or 2 wt % ZnO-NPs compared to controls. Other tests such as aged direct contact test, multi-species direct contact tests, and the lactic acid production test showed little to no difference compared to the controls, particularly at lower ZnO-NP concentrations, or over extended periods of time. Current evidence suggests that ZnO-NP composites are unlikely to present any clear clinical advantage due to the short lifetime of observed anti-bacterial properties, and the poor results against multi-species biofilms in the in vitro studies examined in this systematic review. Further research is warranted in developing ZnO-NP with enhanced long-term bioactivity and enhanced antibacterial efficacy against multi-species biofilms. Efforts to improve and develop standardized study designs to mimic the oral environment in vivo and methods for the optimization of materials including the ZnO-NP are paramount."} +{"text": "Advancing Maternal and Newborn Health in Afghanistan has supported education of midwives since 2002, in accordance with the national plan for midwifery education.Afghanistan has one of the world\u2019s highest maternal mortality ratios, with more than 60% of women having no access to a skilled birth attendant in some areas. The main challenges for childbearing Afghan women are access to skilled birth attendance, emergency obstetric care and reliable contraception. The NGO-based project The aim of this study is to explore women\u2019s experiences of professional midwifery care in four villages in Afghanistan covered by the project, so as to reveal challenges and improve services in rural and conflict-affected areas of the country.An exploratory case-study approach was adopted. Fourteen in-depth interviews and four focus-group discussions were conducted. A total of 39 women participated \u2013 25 who had given birth during the last six months, 11 mothers-in-law and three community midwives in the provinces of Kunar and Laghman. Data generated by the interviews and observations was analysed using thematic content analysis.Many of the women greatly valued the trained midwives\u2019 life-saving experience, skills and care, and the latter were important reasons for choosing to give birth in a clinic. Women further appreciated midwives\u2019 promotion of immediate skin-to-skin contact and breastfeeding. However, some women experienced rudeness, discrimination and negligence on the part of the midwives. Moreover, relatives\u2019 disapproval, shame and problems with transport and security were important obstacles to women giving birth in the clinics.Local recruitment and professional education of midwives as promoted by Afghan authorities and applied in the project seem successful in promoting utilisation and satisfaction with maternal and neonatal health services in rural Afghanistan. Nevertheless, the quality of the services is still lacking, with some women complaining of disrespectful care. There seems to be a need to focus more on communication issues during the education of midwives. An increased focus on in-service training and factors promoting quality care and respectful communication is necessary and should be prioritised. After decades of war and conflict, the situation for the people of Afghanistan continues to be difficult. The country has one of the highest maternal mortality ratios (MMR) in the world , with considerable variation between urban and rural areas \u2013 253/100,000 and 620/100,000 respectively , and theThe health sector in Afghanistan has three levels of service delivery: Health posts at community or village level; Basic and Comprehensive Health Centres (BCHC) in larger communities at district level; District, Provincial and Regional Hospitals providing secondary and tertiary services at provincial level Fig.\u00a01)1).Fig. Health posts offer antenatal care provided by community health workers, but no delivery care. Both Basic and Comprehensive Health Centres offer antenatal, intrapartum and postnatal care provided by midwives. A Comprehensive Health Centre offers services for a larger population than a Basic Health Centre, and also provides blood transfusions. A District Hospital should offer Emergency Obstetric Care including anaesthesia and surgery, e.g. caesarean section .The Basic Package of Health Services (BPHS) was introduced in 2003, to address the country\u2019s poor health indicators. The maternal mortality ratio in Afghanistan was by then 941/100,000 . After iAfghanistan has committed itself to the UN Sustainable Development Goals . In ordeThe profession of midwifery in Afghanistan was established early in the twentieth century, when 12 women from King Amanullah\u2019s family were sent abroad to receive education as midwives. During the following years, midwives were educated and recognised as respected health-care providers. After the Soviet invasion of 1979, some girls got free nurse-midwife training in Russia and in towns such as Kabul and Jalalabad, as experienced by one of the authors (KS). Yet after the onset of civil war involving the Mujahidin and the Taliban, and particularly after the Taliban took control in 1996, women were denied schooling, and the country\u2019s well-established health-care system was devastated .An educated, professional midwife is, according to The International Confederation of Midwives, \u2018 \u2026 a person who has successfully completed a midwifery education programme that is based on the ICM Essential Competencies for Basic Midwifery Practice and the framework of the ICM Global Standards for Midwifery Education, and is recognised in the country where it is located; who has acquired the requisite qualifications to be registered and/or legally licensed to practice midwifery and use the title \u2018midwife\u2019; and who demonstrates competency in the practice of midwifery\u2019 .Since 2002 the Afghan government and the international community have focused on development of the midwifery profession through diploma-level educational programmes of various lengths . MidwiveAdvancing Maternal and Newborn Health in Afghanistan educates midwives and nurses at different levels, including through HME, CME and Community Health Nurse Education (CHNE). In the NAC-supported midwifery programmes, subjects such as domestic violence, anti-corruption and peace work are included in the curriculum. Currently NAC is running health-education programmes in six provinces in Afghanistan. The project is funded by The Norwegian Agency for Development Cooperation (Norad), and approximately 1000 midwives have graduated from the programme since 2002.The NGO Norwegian Afghanistan Committee (NAC) has supported the education of midwives in Afghanistan since 2002, and since 2009 has had the professional cooperation of The Norwegian Association of Midwives (DNJ). The NAC programme Although this project has been successful in terms of deploying midwives in rural communities, it is not clear how women value current access to midwifery care during pregnancy and childbirth. There is in any case little research into how such interventions have been received by Afghan women. Both the security situation and the geography \u2013 with difficult transport and lack of infrastructure \u2013 have limited such studies.The aim of this case study is to explore women\u2019s experiences, perceptions and utilisation of local professional midwifery services at the time of pregnancy and childbirth in rural Afghanistan. It is intended to provide important insights into issues that need attention in order to further improve the education of workers in the field of reproductive health care and the organisation of services, and to increase women\u2019s access to quality care at birth. In order to attain the Sustainable Development Goals of reducing maternal and neonatal morbidity and mortality, access to health care and barriers limiting it are particularly important for people in fragile and conflict settings and must be improved .An explorative case-study approach was adopted for this research . This tyThe study was conducted in the provinces of Kunar and Laghman, two of the provinces where midwives educated through the NAC and DNJ programmes are working Fig.\u00a0.Fig. 2SKunar is in the northeast of the country. The province is predominantly rural, most of the area being mountainous or semi-mountainous, and the geography makes movement difficult, necessitating movement on foot, as well as transportation using pack animals or motorised vehicles. The population is approximately 428,800, the majority belonging to the Pashtun ethnic group . The majThe neighbouring province of Laghman is located further west, in the hilly areas of the Hindu Kush mountains, and has approximately 435,000 inhabitants. Here, too, the Pashtuns are the major ethnic group, other groups including the Tajik and the Pashai. The province is known for its lushness, and people make a living from farming fruit, vegetables and crops such as rice, wheat and cotton. The literacy rate in Laghman is currently 26%, representing an increase from 14% in 2005, and the percentage of births attended by a skilled birth attendant increased from 3% in 2005 to 36% in 2011 . This maA list of villages in the two provinces was developed, and four villages (two in Kunar and two in Laghman) were selected, based on what was feasible for the research team.The two villages in Kunar are located close to the river, while the two communities in Laghman are close to the mountain. Most of the people living in these villages are very poor. Many of the houses, which are well kept and clean, are made from mud. They contain little furniture, and the beds are placed outside the houses and serve as both beds and chairs. Often, several houses share an open oven in the courtyard, where women meet for cooking, cleaning and socialing. The girls usually take care of their younger siblings. Many of the children are poorly dressed, wearing no pants or shoes, even though boys seem better dressed than girls. In the rocky Laghman villages, cultivation is difficult, and many men periodically leave their family to try to get a job in Pakistan or Iran. This means that many women, for long periods, live alone with their children, under the protection of older male family members.A total of 39 women from these four villages participated in the research. Women who had given birth within the past 6 months, either at home, with help from another woman, or in a facility, were selected for the individual interviews. Mothers-in-law, women attending those in labour and childbirths in the community as well as other local women participated in the Focus Group Discussions (FGDs). Educated Community Midwives working in the same villages were also selected as informants Table\u00a0.Table 1A total of 14 women participated in in-depth interviews, and four FGDs were conducted. Their community leaders recruited the 14 women who were interviewed individually. These informants included three community midwives. Apart from the midwives, all women participating in the research were unable to read and write. All informants participating in the interviews were given Afghan pseudonyms. The age of these women ranges from 17 to 30. Table\u00a0The data from Kunar and Laghman were collected from July to September 2017 by Afghan research assistants, two from the NAC office in Kabul and two midwives from Laghman province. Fourteen interviews and four FGDs were conducted. The two interviewers from Kabul travelled to Kunar and stayed there for 2 weeks during the data-collection period. In addition to having detailed knowledge of Afghan culture, the interviewers closely observed the local context and traditions during their stay.The four interviewers had previously participated in a qualitative-research-method course conducted by NAC during spring 2017. An interview guide was developed and was tested prior to data collection\u00a0 into English. The interviews and FGDs each lasted about an hour and took place in private houses and local Basic Health Centres.Availability, Accessibility, Acceptability and Quality I can help myself during childbirth. I don\u2019t need help from others.\u201d (Afrooz)However, during their pregnancies and after talking with other women, some women had changed their minds about where to give birth, especially after having trouble. One woman explained:\u201cI had three children at home with a lot of problems. My neighbour advised me to go to the clinic. When I went there the midwives behaved so well and I was very happy and satisfied.\u201d (Khandan)Thus some women, regardless of their age, felt confident about coping with childbirth on their own. As has been observed elsewhere in Afghanistan, some viewed pregnancy and childbirth as natural processes that should not require external help wanted to help me when it was time for the baby to come. They wanted me to go to the clinic. I refused. I don\u2019t like women to see me \u2026 there \u2026 during the birth of the baby. I am ashamed during childbirth \u2026 So, I did it alone.\u201d (Afrooz)Several women confirmed this with similar statements about a lack of privacy on clinic premises and the potential of shameful exposure to other women: \u201cThe windows in the delivery room do not have curtains. The clinic has few beds \u2026 one woman was delivering on the floor with other women present. We don\u2019t feel relaxed there \u2026\u201d (Bahar).The privacy and intimacy of labour and childbirth apparently influence the choice of place of birth, and many of the women had decided to give birth at home because they felt it was disgraceful to expose themselves to a health professional. Some felt this to be so intimidating and embarrassing that they themselves refused to go to a facility when in labour:Thus it seems that women face many obstacles and considerations when it comes to choosing a place for birth, owing to a combination of individual, cultural, religious and practical considerations. In a study in which Afghan women were asked about their customs and traditions during the perinatal period, the women emphasised that being a good Muslim is particularly important during this period, when, for example, undressing in front of a stranger and exposing oneself to a provider are seen as being inappropriate . This inThe villages included in the study are situated in relatively remote areas of Kunar and Laghman provinces, and the walking distances to the nearest Basic Health Centres varied from 20\u2009min to 4 h. In the summer it is usually hot, and people seek shade from the sun. During fieldwork, for instance, discussions had to take place in shady areas between people\u2019s houses. In wintertime there is often a lot of snow in these areas, making transportation difficult. The only routes into most of these villages are by roads that in most cases can only can be accessed by donkey or bicycle. When discussing transport, the women explained that very few people in their areas have access to a car. Some car owners would be willing to lend their cars out, but suspicion and security issues affected their trust and willingness to do so. Some families had a donkey they could use for transportation.\u201cToo often I gave birth to a dead baby ... [...] When I was full-term pregnant my labour pains did not start. In all my pregnancies my labour pain did not start ... So, I went to the clinic ... The clinic is two hours\u2019 walk away, far from my home. When I came to the clinic the midwife gave me an injection to start the labour, but the baby was already dead in my womb ... The midwife advised me to come [early] to the clinic the next time. If not, my baby might die, again.\u201d (Bahar)Families and communities would often help the women to reach a facility, when judged necessary. One woman who lived 1 h\u2019s walk from the clinic explained that it was hard to get there, but each time she was brought to the facility by her spouse. \u201cMy first five children were born in the clinic. My husband carried me to the clinic.\u201d (Delara).Most of the women in the study lived far away from a health facility Some women explicitly planned to give birth in the clinic, and prepared themselves as best they could: \u201cI got advice from the midwife to give birth in the clinic. We are poor ... So, when I was pregnant, I sold a sheep and saved money for the birth of my baby.\u201d (Afrooz).The people in the villages represented in the research were generally very poor, and during the FGDs poverty was often mentioned as a general problem that prevented them from having access to transport. Nevertheless, it seemed that many women and their families were trying to plan transport when their labour started. Many women explained about various efforts on the part of relatives to facilitate transport in case of need.In spite of being prepared for the financial costs of safe birth care, it is difficult for most women and their communities to prepare for the lack of security during travel to clinics.Every mother should have antenatal care. However, many women don\u2019t come because of the poor security situation.\u201d .The insecurity situation in the study area, particularly at night, posed a risk to women who wanted and needed to go to the clinic, as explained by a community midwife: \u201c\u201cThe most challenging thing is safety. This problem with lack of security is very, very difficult and challenging for us, both for me as a midwife to go to work, and also for the women in labour. When going to the clinic we use the same road as the bombers \u2026 we are on the roads where something happens every day \u2026 Something ... an explosion, a suicide \u2026 or something else \u2026 It makes moving from one place to another very dangerous and difficult.\u201d The ongoing conflict and insecurity situation in Afghanistan is well known, is currently worsening and is an international concern. This pervasive situation reduces the availability of healthcare and limits access to essential health-care services in the long run . It is very important and good to give birth in the clinic rather than at home. If a problem happens such as bleeding or the woman collapses, it is treated well in the clinic. At home nothing will be done.\u201d (Maheen)The fact that the midwives explained about what was happening and why created trust among the women:\u201cMy last baby was born in the clinic. The midwife assisted me, she was kind and friendly. I had severe bleeding. The midwife said that a few pieces of placenta were left in the womb. She helped me, took the pieces out and stopped the bleeding.\u201d (Bahar)Moreover, the mothers seemed to appreciate the way their newborns were put in close bodily contact with them after delivery, as well as the prompt initiation of breastfeeding. \u201cThe midwife was so kind. She put my baby on my chest after he came out.\u201d (Afrooz).Women expressed genuine gratitude:\u201cThe midwife put my baby on my belly. After two hours I started breastfeeding.\u201d (Bahar).Another woman explained: The midwives in the village clinics were taught and pursued \u201cBaby-friendly environment\u201d guidelines \u2013 a programme launched by the WHO in 1991 with the aim of improving maternal health care. This includes, for example, immediate skin-to-skin contact and initiation of breastfeeding immediately after birth. These practices seemed to be particularly appreciated by the women, even though there is no tradition of immediate skin-to skin-contact and early breastfeeding in Afghanistan , 27.\u201cMy last childbirth was in the clinic ... The midwife assisted me, but she was not a kind woman. I did not feel comfortable. I had bleeding, and she had to send me to the hospital. Giving birth at home is risky \u2026 The clinic is better ... However, the midwife was not kind. I did not feel good.\u201d (Farzana)Some women even felt harassed and intimidated:I had severe pain, I was crying, I was moving around, I couldn\u2019t be calm. The midwife became angry and said \u201cYou behave like a donkey, you are not a human being!\u201d [ \u2026 ] Sometimes midwives become so angry with mothers, I don\u2019t know why. [...] I am happy that she helped me, but I feel so sad because she called me a donkey.\u201d (Camila)\u201cOther women explained that the physical environment and conditions in the clinic were additional reasons for not giving birth there. They complained about the poor equipment and the staff\u2019s behaviour.\u201cI went to the clinic to give birth. In our clinic there are midwives, but no female doctor [...] There was no light or fan \u2026 They did not turn on the generator ... it was so hot! [...] The midwife examined me, and afterwards she went to sleep. When I called for her, she became angry and did not behave well [...] They don\u2019t pay attention to the patients.\u201d In order to understand such poor caring behaviours in midwives, it is important to mention the professionals\u2019 workload, involving a poor shift system for the midwives. Sometimes they had to work for 24 or 32\u2009h consecutively. Moreover, it is suggested that some midwives suffer domestic violence in their family because of their work, and this may also result in disrespectful care of their patients .In spite of the many positive experiences in the various communities, a few women shared their negative experiences regarding the midwives and the care they received.In spite of these negative experiences, women expressed that midwifery services were appreciated by many, and that midwifery seemed to be an increasingly valued career path for women in the communities.\u201cI am really enthusiastic about this education, and I wish I could be a midwife in order to help my family and the villagers. But unfortunately I got engaged during my school period, and after marriage my husband didn\u2019t want me to continue my schooling, he didn\u2019t understand the value of education.\u201d Some older women expressed how the value of education had changed over time, as had their own ideas about it.\u201cBefore, we did not allow our children to learn and to get an education, I wanted my children to work in the field instead. Now I know and understand and meet educated people, I see their attitude and value in society. I want my granddaughters to learn and get an education.\u201d Another woman said something similar, and intended to involve her husband in promoting their daughter\u2019s education:\u201cSome people agree about women getting an education. I want my daughter to learn and get an education. I want to ask my husband to allow her to start [on the midwifery programme], if not I will make him allow her.\u201d Some even stated the importance of the profession at national level: \u201cI want my granddaughter to learn and to get [midwifery] education, to serve the people of Afghanistan.\u201d .The discussion during the FGDs and interviews usually began with talking about the trained midwives operating in the localities. In some of the villages the midwife was the only educated health-care provider in the community, and the profession as such represented a rare opportunity for education and increased status for women. This was discussed during some of the conversations.Thus training in midwifery seemed to provide some status and recognition, and was apparently considered to be an important pathway in terms of increasing education for women in the country.The aim of this case study was to explore women\u2019s experiences, perceptions and utilisation of local professional midwifery services at the time of pregnancy and childbirth in rural Afghanistan. Afghan women\u2019s utilisation and experience of health-care services during pregnancy and childbirth are affected by several factors, such as accessibility of services and lack of transport, particularly in rural areas of the country. In addition, financial and cultural barriers, women\u2019s level of education, their knowledge of danger signs and poor health-care provision are important factors promoting non-utilisation of professional health services \u201330.Women\u2019s experiences and utilisation of services in the four villages represented in the research, in Kunar and Laghman provinces, were predominantly positive. Most of the women were content with the services received, and most of them did attend the recommended antenatal care, and apparently appreciated the value of a skilled midwife in terms of health and survival for mothers and infants. Many told stories of serious consequences when not attended by a professional midwife, or when referred in time to a facility with emergency obstetric care.However, women\u2019s utilisation of midwifery services was still influenced by sociocultural values and attitudes that were not always favourable for women seeking skilled care. Value and attitudinal barriers included a lack of permission from relatives/in-laws, in addition to women\u2019s own, sometimes negative, perceptions of midwifery care. Women also felt shame about exposing themselves to others during birth, and for this reason some did not use midwifery services. Others were not satisfied with the service and care that midwives provided. Despite these experiences and barriers, most of the women interviewed in our study preferred to give birth in a health facility with help from a midwife, and did so. This indicates that the national midwifery education programmes, such as the NAC project, which focuses on recruiting women from the community into midwifery, have had a positive effect on women\u2019s access to midwifery services.Advancing Maternal and Newborn Health in Afghanistan, community midwives are educated and deployed in rural parts of Afghanistan. Women\u2019s access to health-care services in rural parts of Afghanistan is still limited. In the areas where this study was conducted, women generally do have access to midwifery health care during pregnancy and childbirth [Through the programme ildbirth . As showildbirth . One musildbirth .All the women in our study were familiar with the midwifery services. The fact that the midwives were known women recruited from their own communities could contribute momentum in terms of increasing awareness and furthering utilisation of midwifery services . In a stSeveral studies show that awareness and knowledge of midwifery services increase the utilisation of such services during labour and childbirth. Our findings are consistent with these studies. A cross-sectional study from Ethiopia showed that raised awareness and inclusion of both husbands and other family members in maternal health care seem to have increased the use of maternal health-care services . A studyWhen my husband comes home, the baby is born and I have washed and cleaned up everything\u201d, thus expressing the importance of removing birth dirt, this \u201cmatter out of place\u201d which according to Mary Douglas often symbolizes danger and power [Despite the fact that the women in our study had access to a health facility, some of them still preferred to give birth at home, claiming that they could manage labour and delivery alone, without help from others. There may be many reasons for this, such as experience-based self-confidence in handling a childbirth alone, as well as families\u2019 opinions and women\u2019s shame in exposing themselves to strangers, as expressed by a number of women interviewed during the research. In fact, shame may also be associated with the exposure of loss of blood and bodily fluids during birth. During a NAC meeting in the province of Kapisa, several women told the team that they preferred to give birth at home, alone. A woman explained: \u201cnd power .A cross-sectional study from all 33 provinces of Afghanistan shows that older women use a skilled birth attendant less often than younger ones do, and that factors such as distance to facilities and availability of female health-care providers are important determining factors in this decision . Other rGeography and climate in Afghanistan are factors that clearly have an impact on utilisation of health-care services in general and maternal health care in particular. Long winters, often with a lot of snow, as well as a poor infrastructure limit people\u2019s ability to travel. It can be difficult to get out of villages, whether on foot or on a donkey. In many villages few, if any, people have access to a car, and driving conditions can be difficult or impossible for long periods of the winter, and when the snow is melting in the spring.The women in our study generally described both the midwifery services and health facilities as being accessible, even though most of them did not live close to health centres. Despite this, the women explained that long distances, difficulties with means of transport and security problems were obstacles to their using these services. Although the intention of the BPHS is to ensure access to health services for people in rural areas, these services are not accessible to everyone. Walking to a health facility with painful contractions can be a challenge for any woman, even when the distance is short. Mayhew et al. showed that in Afghanistan utilisation of health facilities for birth is related to the walking distance to the clinic \u2013 the greater the distance the less the use of a health facility . Yet it After 40\u2009years of war, internal conflicts as well as the presence of the Taliban and IS make the security situation for people in Afghanistan extremely difficult . The midIn Afghanistan\u2019s sociocultural and religious context, pregnancy and childbirth are seen as particularly delicate and private issues, and health services aimed at women and children must accordingly be sensitive . In manyIn a war-afflicted society like that of Afghanistan, confidence in health workers as well as health workers\u2019 own safety are especially important. The way midwives are recruited to the NAC project seems to have informed the local communities and made them conscious of the importance of a professional service, and of the midwife\u2019s knowledge and skills and the difference they make for mothers and infants. However, some communities may in general have insufficient knowledge of midwives\u2019 life-saving skills. Some women expressed having had a brutal experience of midwifery services, or had heard about other women who had lost their lives and/or babies in clinics.A systematic literature review from 2014 describes barriers to childbirth in health facilities in 17 low- and middle-income countries. These barriers are similar to those found in our study, e.g. local traditions and influence of the family, availability, access and quality of care. Women in our study talked about family members who forbade them to give birth in a facility because this is not the tradition and it is perceived as being shameful. They talked about long distances to the facility and a lack of both money and means of transport. Many women in our study also experienced a lack of respect, privacy and intimacy in the clinic, and therefore decided to give birth at home. Families with social connections to skilled providers may be more accepting of the biomedical approach to maternity care and thus more willing to seek a facility-based delivery . This hiA study from 36 low- and middle- income countries, including Afghanistan , describMost of the women in our study appreciated the quality of the midwifery services, as regards both the professional and the interpersonal care they received. They expressed that the midwives\u2019 expertise, in particular in emergency situations, their resultant ability to save lives and their care and kindness combined to create important reasons why they decided to give birth in clinics. This stance was supported by the women who were initially sceptical of birth in a health facility. A study from Ethiopia shows that women value giving birth in a clinic, since this includes medical interventions and the ability to deal with complications . Also, iand care promoting early closeness between mother and neonate.\u2026 A study from five different provinces in Afghanistan representing the country\u2019s major ethnic, religious and language groups showed that a common tradition is for the baby to be given a bath immediately after birth, and for the mother to have a bath after 3 days. In this tradition a woman should not start breastfeeding before her breasts are clean, thus delaying the first breastfeeding by 3 days . AnotherIn cases such as in our study, where midwives are recruited from the local environment, and among local women, this might increase women\u2019s and their families\u2019 experience of safety and security. Midwives who operate within communities are furthermore able to provide a continuum of care through pregnancy and childbirth, as well as during the postnatal period, which is known to improve the utilisation of such services . In factHowever, other women in our study were not satisfied with the care and treatment they received, and preferred to give birth at home even though they knew this could be risky. They were reserved and excessively shy when it came to exposing themselves to someone outside their intimate family circle, even in medically related contexts. They complained of lack of privacy and poor facilities in the clinic, such as lack of beds and curtains, and said that modesty and the shame of exposing themselves prevented them from giving birth in clinics. Lack of privacy during childbirth is described in two different studies from Nepal, in which women expressed the view that shame and shyness about showing their genitals were the main reason for not delivering in a health facility , 44. TheIn our study, a lack of delivery beds meant that some women had to give birth on the floor, adding to their discomfort. A study from various maternity facilities in Kabul showed tSome women experienced disrespect on the part of the midwives, involving neglect and verbal harassment, and stated that the midwives did not provide good care. Disrespectful care and even abuse during labour and childbirth are documented in various studies. Women in Afghanistan frequently report disrespectful care, lack of compassion and abuse on the part of health-care providers , e.g. inHowever, the challenges facing professional health workers and how this may influence their ability to provide care need attention. As suggested in a report about maternal care in Afghanistan, gender norms and increasing insecurity may be factors underlying health workers\u2019 performance . For exaNevertheless, in our study women\u2019s overall perception and experience of midwives and services were positive, and for most women the advantages seemed to outweigh obstacles such as long distances, transport difficulties and security problems.This study provided recent and original qualitative data material in a context in which it is difficult to do research for security reasons and owing to remoteness. Triangulation of three methods of data collection \u2013 in-depth interviews, FGDs and observation as applied by the local data collectors and the main researcher over time \u2013 strengthened the validity of the findings.The interviews and discussions were run by four Afghan women and conducted in Dari/Pashto. These women are very knowledgeable about the local culture, traditions and rituals around the time of childbirth, in addition to having a deep understanding of the project and the training of community midwives. The joint critical analysis of the data by the local data collectors and the principal investigator (TT) is considered to be a strength of the research.Owing to the difficult security situation in Afghanistan in general, and for foreigners in particular, it was impossible for the main researcher (TT) to run the interviews herself. Use of local research assistants with limited experience of qualitative research was a major challenge. There is a risk that women selected for the study were more in favour of the programme than others, and that as a matter of courtesy the informants emphasised the positive aspects of the midwifery project\u2019s interventions. The data collectors might have been biased in their choices of interview respondents and interviewing processes, creating a more positive picture than warranted, since they all have a link to the NAC, either as employees or by virtue of having been educated through the NAC programme. In the process of translation from Dari/Pashto into English, important details might have been lost. Finally, the female data collectors had to overcome challenges such as transport, security and a limited time frame, which may have affected the data-collection process. The research team have been aware of and have critically discussed possible weaknesses, but we believe that the female research assistants\u2019 background and skills were significant in making the study possible and relevant.The burden of maternal and child ill health in Afghanistan is one of the highest in the world, and it is being worsened by a long war. Our study explored women\u2019s experiences in two rural provinces, where a midwifery-education project recruiting local women to the profession has been running, in line with the national midwifery-education programme. Our findings suggest that the midwife often becomes a central figure in local society, leading more women to choose to give birth in a health facility, when available and accessible. Most informants stated that the service was relatively satisfactory, and increased awareness of the importance of skilled care during pregnancy and childbirth was voiced by women, their relatives and the communities as a whole.Nevertheless, issues of privacy and shame as well as the experience of disrespectful care affected the acceptability of midwifery services for some. An increased focus on respectful care and attitudes, and on communication in both pre-service and in-service training of midwives, is necessary in order to improve the quality of services. As regards research, it would seem important that one understand how to better prepare and support midwives operating in such demanding working conditions, and in this way strengthen the quality of care.Additional file 1. Interview guide: In-depth interviews for women."} +{"text": "The purpose of this study is to explore undocumented immigrant women\u2019s experiences of, as well as their access to, maternity care services during pregnancy in Denmark. Recruiting through the two branches of a non-governmental organization (NGO)-driven health clinic in Denmark, we conducted 21 semi-structured interviews with undocumented immigrant women in Denmark from January 2018 to January 2019. The undocumented immigrant women experienced barriers such as fear of deportation, concerns about payment for services, and uncertainties about rules for access. Many of them described depending on NGO-driven initiatives to access maternity care services and found these as providing a safe environment for care. Our findings contribute insights towards understanding the health behavior of undocumented immigrant women and highlight the need for inclusive care to safeguard the health of the women and their children. Undocumented migration is defined by the International Organization for Migration as the \u201cmovement of persons that takes place outside the laws, regulations, or international agreements governing the entry into or exit from the state of origin, transit, or destination\u201d . The patIn Denmark, undocumented immigrants have restricted access to health care including maternity care . Women wThe Danish Red Cross provides primary health care, including diagnostics for common symptoms and infections, vaccination of children, acute dental care, and maternity care, to undocumented immigrants through a healthcare clinic established in collaboration with the Danish Medical Association and The Danish Refugee Council . The HeaAccording to the World Health Organization, being an immigrant in itself serves as a risk factor for having poorer general health than the background population . In SwedThe study was a part of an overall research project entailinA qualitative research design was chosen for this study, to gain in-depth knowledge of the women\u2019s experiences . Data foThe recruitment site of the study informants was the aforementioned Health Clinic for Undocumented Immigrants. We recruited 21 undocumented immigrant women through purposive sampling [n = 14) were 20\u201339 weeks pregnant at the time of the interview, and the mothers of newborns (n = 7) were interviewed 4\u20138 months postpartum. The women constituted a heterogeneous group. They were aged between 20 and 42 years old with a mean age of 29.4 and originated from The Philippines, Sudan, Morocco, Pakistan, Kenya, Tanzania, Uganda, and Bosnia. The majority were nulliparous (n = 15) and the remaining had 1\u20133 children. Most of the women lived with their partner, and the partner could be a Danish citizen, have obtained a Danish residence permit, or be undocumented like themselves. The women were employed in the informal sector predominantly as domestic workers, or not be employed at all. Some women were in the process of applying for residency, whereas some of them were neither applying nor planning to apply.The women interviewed during pregnancy 2016/679). During the process of systematic text condensation, four overarching themes with eleven subthemes emerged see .This theme describes which public maternity care services the women used. It describes their expectations about them, as well as their experience of gaining access to maternity care services. The informants were in contact with public maternity care providers before, during, and after giving birth. When needing care in the early stages of labor, or in relation to the induction of labor, some women experienced that they had to negotiate their entitlement to care with health professionals, making the women feel neither welcomed nor acknowledged. In some cases, they felt they had to argue their case to secure the safety and health of their unborn child.During the actual birth, the women expressed having felt well treated during encounters with public maternity care providers and that they had received both adequate care and support. They did not feel that their legal status had affected the care they received, nor the staff\u2019s perception of them. Marisol tells: \u201c\u2026They were really nice, and the midwife was really accommodating me well, and the midwife even knew that the hospital asked us to pay, but she said that she is just doing her job. And her job is to take care of me and my baby. She was really, really nice\u201d .For post-natal care, the women experienced being questioned about entitlement to care and about their living conditions. Rahima reports that she felt alienated by these questions: \u201cThey asked funny questions, like do we have a home. It\u2019s like they don\u2019t know anything about people who are different from them. We are normal people, just without the normal papers.\u201d . At discharge, they were given no appointments for follow-up, apart from one being recommended to seek follow-up for a neonatal infection at the Health Clinic. This caused them to worry and feel insecure. Fear of deportation was by far the dominant factor affecting the access to maternity care, as this made the informants hesitant to seek maternity care at public hospitals. Eventually, they all did seek public hospitals, but not until it felt highly necessary, as described by Jessa who had an episode of severe pain during her pregnancy: \u201cI thought I was going to die. It didn\u2019t come abruptly. I felt more and more pain\u2026But I knew we could not go to the hospital. But when I reached the point of thinking it\u2019s hospital or death, we went. I couldn\u2019t be deported if I was dead, you know. Dead people don\u2019t have passports\u201d .Even though the women were aware of the hospital staff\u2019s obligation to confidentiality, all contact with a public hospital was seen as posing a risk, which was why the women wished to spend as little time as possible in the hospital during the birth; arriving late and leaving quickly. Despite this attitude, the women clearly expressed that if their child needed hospital admittance, they would adhere to it, no matter what the legal consequence. They feared consequences relating to their reason for staying in Denmark, either being separated from their spouse or losing their job. Applicable for all the women was a profound fear of being separated from their children, as Dina explains: \u201cI am afraid that the police will take me. What if I am not with my daughter at the time, would she have to stay here without me? Or go to Africa alone? It\u2019s horrible to think of\u201d .The possibility of being required to pay for maternity services served as a barrier for the women, in regards to pregnancy care, but mostly related to birth. Many had heard rumors of other women being charged for services, or the informants themselves had been faced with a payment claim when seeking maternal care. For example, Marisol, who went to the labor ward for an assessment relating to her overdue pregnancy: \u201c...then they checked me and then they checked my tummy. They had ultrasound and checked my water, and said baby is fine, but it\u2019s time to start the birth. What do they call it? Induce the labor. But first we have to pay for my birth\u201d . Others were faced with the payment claim at discharge or had bills sent to family members or tenants, while some were not. The women reported that the cost of giving birth could be between 20,000\u201325,000 Danish kroner (2600\u20133300\u20ac), which they saw as unmanageable. Most had low incomes, and some were financially dependent on their partner or family members, who also had low incomes, such as social benefits or salaries from low-skilled jobs. Generally, the women expressed that financial hardship was an ongoing concern in their life and that the worries of possibly having to pay for maternity care added to this concern. The women feared that the debt could increase their risk of deportation or affect their chances of attaining residence permits in the future. It also caused worries of whether they would be refused care during birth or be given inadequate care. Furthermore, they worried whether they would end up with unmanageable bills afterwards. However, the concern about payment did not deter any of the women from seeking care during birth, as this was considered unsafe for the baby, nor were any of the women rejected at the labor ward.This theme describes the women\u2019s health-seeking behavior when facing barriers to access of the public system. The services included maternity care at the Health Clinic and the use of private maternity care services. Furthermore, the theme describes the women\u2019s experiences of using these services and which barriers they faced.The undocumented immigrant women expressed feeling deeply grateful to the Health Clinic for providing a place to seek maternity care. They were aware that the clinic staff were volunteers and worked for altruistic reasons, and it was essential for the women to know that the purpose of the clinic was to provide healthcare for undocumented immigrants. The women felt safe in there, and they felt safe in knowing that they would not be reported to the authorities. The staff did not pay attention to the lack of residence permits during visits, and as Christina expresses below, it made her feel welcomed and safe: \u201cHonestly, they are so friendly here, and it\u2019s so nice to see that people really want to help you, even though you are foreign\u2026and even though you don\u2019t have residency. You don\u2019t have nothing, but you have the clinic. And know that they won\u2019t report you to the police. It feels very safe\u201d . The availability of free healthcare services for undocumented immigrants was limited to the two largest Danish cities during the study period. This gave some of the women long transportation hours to reach the clinics. Dina told that she had logistic obstacles to reach the clinic: \u201cI want to come as the midwife has told me, but sometimes it is just not possible. It is too far and costs too much. I can\u2019t do anything about it. I have to come some other time then\u201d .Another logistic obstacle was the limited opening hours at the clinics, as they only offered midwifery consultations once a week. It was challenging to get off work, both for the women and their partners or other networks whom they depended on for transport, translation, or emotional support. There were also women who either lived near the clinics or simply considered transport and waiting time at the clinic as a premise in their situation: \u201c...no, its fine. I take the train. Sometimes two. I know the staff are volunteers, and I am just grateful that they want to help pregnant women like me\u201d .Some of the women did not live close to a hospital, did not have any means of transportation, or lived where public transport was not very efficient, especially at odd hours. This posed a challenge when needing to reach a hospital for the birth. In these cases, the women had to get help from their network, as taking a taxi was considered too expensive. Some of the women used private healthcare services during their pregnancy. One of these private services was an ultrasound scan of the fetus in private clinics. The women perceived ultrasound scans as necessary, and having ultrasound scans was perceived as being a part of a normal pregnancy. It was important for them to both connect with the fetus to make sure that there were no problems with it. Therefore, most women went to see the gender of the baby, or in early pregnancy to see the baby for emotional reasons. A few women went because there was uncertainty regarding the due date and they wanted an assessment through the ultrasound scan. Some of the women perceived the price of ultrasound scans as high (approximately 70 euros), but despite their limited financial means they found the expenditure worth prioritizing. Others did not consider the cost as particularly high, as they were aware that costs were connected to it both for Danish citizens as well as for themselves in their countries of origin. In addition to paying for an ultrasound scan, some women were also seen by a general practitioner. One woman reported that she went to see her husband\u2019s general practitioner in early pregnancy because she did not know where else to go. She was asked to pay for the consultation, and therefore only made a single visit. Another woman was rejected completely due to her lack of a residence permit: \u201cWe asked my husband\u2019s doctor, but he said: No! No papers. No doctor\u201d .This theme describes the women\u2019s perception of their entitlements to care, exploring their wishes for fair treatment, and the uncertainty they experience regarding care.The limited access to maternity care was perceived as unfair. The pregnancy and a deep concern for the baby\u2019s health gave the women a strong perception of them having the right to care. \u201cWhen I don\u2019t have papers. I feel sad about that. Why should my child not get the same help as other children? A child is a child\u201d . As Dina expressed, the perception of entitlement was supported by a profound notion of fairness and a wish to be treated equally to others: \u201c\u2026but if the midwife in at the hospital says, \u201cYou are not Danish. you can go home or pay\u201d I will think: I am not Danish, but I need help with my baby. I am a human being\u201d . Even though being pregnant or having a small child played a role in the perceptions of entitlement, the women were aware that it would not improve their chances of being granted residency. They were also aware of the fact that their children would only obtain a residence permit based on the legal status of their parents. While they were aware that their stay in Denmark was not legal, they did not see themselves as criminals or as doing anything wrong. Several expressed that their stay in Denmark was based on an urgent need. This factor supported their perception of entitlement. However, in their encounters with the public healthcare system, both the limited access and questioning of the women\u2019s entitlement caused them to feel like criminals, which did not correspond with how they saw themselves.There was a shared notion among the informants that if a staff member liked women from a certain country, or was sympathetic to the woman\u2019s situation, the access to and quality of care would improve. Thereby, the granting of access was seen as being up to the individual healthcare professional, more so than rules and legal rights. For some women, the perception of entitlement was affected by comparison to the level of access in their country of origin. They stressed that in their countries of origin, there was a high level of co-payment and corruption within the healthcare system. This comparison made them feel that the rules in Denmark were fair, even though the rules did place hardship and worries on the women. Another factor affecting the women\u2019s perception of entitlement was the severity and urgency of their need for maternity care. Even though they were not all confident that they would receive care, they felt entitled to care both during birth and in cases such as severe pain or bleeding. In contrast, they did not always feel entitled to preventive maternity care.Uncertainty played a marked role in the women\u2019s lives and was rooted in several issues. There was uncertainty related to the possibilities of obtaining a residence permit and thereby gaining rights to maternity care. This issue was present in the life of the women who were either involved in an application process or had some sort of prospect of obtaining a residence permit. One woman described it as \u201can exhausting waiting game\u201d and talked about how she had been involved in the application process for years. This uncertainty was closely linked not only to the fear of never obtaining the permit, but also to whether they would have continually limited access to maternity care during birth and whether this would affect the health of their newborns.Another area where the women were affected by uncertainty was regarding care during birth. Some reported that they were uncertain about where to go, and more so talked about distress regarding whether they would be allowed care during birth and whether the quality of care would be affected by the lack of a residence permit. One woman expressed that she had never had previous contact with the hospital and had heard negative stories about other undocumented immigrants who had not been treated well. This made it feel like a gamble to plan to give birth at the hospital.The perception of a varying administration of access to public maternity care led to some uncertainty. Rosa expressed a feeling of unfairness: \u201cI think they should be more precise about it, because I know some people in the same situation like me, and they have access to hospital, and things like that, but in another region. But in my region, it\u2019s like they have other rules. So, it\u2019s like kind of unfair\u201d .This theme describes how the women depend on help, both from the Health Clinic and their social network. The theme also includes what sources of information they turn to for support and information regarding pregnancy and birth.The Health Clinic was the women\u2019s preferred place to seek maternity care, and for most it was the only place to seek care during pregnancy. When they did not have anywhere else to go, they felt dependent on the help they received at the clinic: \u201c...I solely depend on this place. If I could not go here, where would I go?\u201d . The informants not only felt dependent in regard to the care they received at the clinic, but also expressed that they needed the clinic to establish contact with the hospital at the time of the birth: \u201cThe clinic can assure me that nothing will happen to me when I go there. Because I remember before I got pregnant, I had to do some x-rays, and that was a bit risky, you know, going to the hospital, but the clinic assured me that nothing would happen. They made an appointment with the doctors , and therefore I got treatment and they were nice to me. I would not go without a referral from the clinic\u201d .This dependency was also expressed by worries about having a medically urgent need, such as bleeding or going into labor, outside the clinic\u2019s opening hours. The women did not have a strategy for such a situation, but simply expressed despair when asked about it. When Mabel was asked where she would seek care, if needed, outside the Health Clinic opening hours, she replied: \u201cThat\u2019s what I am afraid of! Where can I go? I simply don\u2019t know. I have nowhere else to go. I would just have to wait then\u201d .When having questions regarding pregnancy issues, none of the women turned to official sources such as maternity and emergency services for advice. Rather, they turned to friends and family who had either professional experience with maternity care or personal experience of their own. Asking family members was a way of obtaining both information and comfort. However, as these family members were living in the women\u2019s country of origin, the cost of telephone calls limited contact to some extent.Another used source of information was the internet, not only to get answers to immediately raised questions but also for obtaining knowledge regarding birth and breastfeeding: \u201cI attended antenatal class in YouTube. Only because I don\u2019t know how else I would prepare in Denmark. Especially if you don\u2019t have social security number, so it was like self-study\u201d . The internet was seen as an easily accessible source of information. However, the women were aware that the internet was not always a reliable source and that information should be read with precaution.The women who had partners with Danish residence permits experienced a deep dependence on these men. \u201cWell, it\u2019s a little bit hard when you come to a different county, different town, when you don\u2019t have nobody, so I only depend on my husband\u201d . For most informants, the partners were responsible for establishing the initial contact with the Health Clinic and hospitals. During consultations and hospital admittance, the partner, in some cases, served as a linguistic translator. The women also experienced their partners as their \u201cadvocates\u201d, making sure that their needs were met by healthcare professionals, but also in supporting them in dealing with the legal aspects. There were other aspects where the women expressed a need for protection. Mabel reported that she only left the house in the evenings because her husband could then go with her. In those instances, she felt safe, as he would be able to speak to the police if they were stopped, and be able to speak Danish to not draw unnecessary attention: \u201c\u2026I never leave the house without him (the husband). I need him for protection. What if the police stop me? Maybe they won\u2019t think about me, when he can answer in Danish\u201d .Even though most of the women expressed that they had a limited social network and that separation from family and other social networks in the country of origin was mentioned as a stressor, friends were the key to maternal health services. This was such because the friends knew about ways to obtain maternity care as an undocumented immigrant or they could offer support in terms of providing knowledge about health and provide practical aid such as transport, linguistic translation, or emotional support.This study examined how undocumented pregnant women experience maternity care services during pregnancy as well as their access to public maternity care in Denmark. Our analyses revealed that they encountered several barriers to access the public health system and thus relied on alternative strategies such as using NGO clinics and informal networks. We will discuss our findings of health-care seeking behavior in relation to groups of undocumented immigrant women in other countries, as well as the potential consequences for birth outcomes. In this study, we found that one of the main barriers to accessing public maternity care was the fear of being reported to the migration authorities when in contact with public health care providers. Another barrier was concern about payment and the consequences of not being able to pay. Scandinavian studies among undocumented immigrant women support these findings, and point to the continuous insecurities having both psychological and physiological impacts on their pregnancies ,26. MateThe analysis revealed that the undocumented pregnant women in this study felt safe at the Health Clinic, and that they established a trust-based relationship with the health providers volunteering there. Studies have shown, that a caring relationship between providers and the undocumented immigrant women is essential to facilitate access to quality maternity care during pregnancy ,25,30. WSeveral of the women in this study paid for ultrasound scans conducted by private providers. They perceived this as being part of what they called a \u201cnormal pregnancy\u201d, and the price was perceived as either manageable and/or inevitable. The WHO only recommends one ultrasound scan before gestational week 24 and the The analysis revealed that the undocumented immigrant women experienced several barriers such as fear of deportation and concern about payment when accessing maternity care services. These factors can lead to underutilization of maternity care services, or no utilization at all. Studies among immigrant groups in general show a tendency of underutilizing maternity care services during pregnancy , thus ouA strength of this study was the heterogeneity of the study population; we were able to recruit women of different ages, country of origin and living circumstances. Furthermore, given the difficulties in recruiting undocumented immigrants for research in general , it is cInformation regarding migration history, lifestyle, and general health including mental health would have added to the analysis and given valuable perspectives to the research question and possibly added to the heterogeneity of the participants. However, in respect of the Health Clinic\u2019s policy on safeguarding the safety of their patients and due to the time aspect of the data collection, these issues were not included in the data collection. The different entitlements to care for undocumented immigrants across Western countries affects the transferability of the study. However, a number of findings are confirmed in similar settings in other countries ,26. It cUndocumented pregnant women experience several barriers to accessing maternity care services in Denmark. Accessing public maternity care services is, apart from legal restrictions, dominated by fear of being reported to the immigration authorities and hence deportation, concerns about payment, and the management of rules from the health provider\u2019s perspective. Many of them depend on NGO-driven initiatives, the Health Clinic, and their personal networks for care and protection. The undocumented immigrant women experience feeling safe in accessing maternity care services at the Health Clinic. However, some also have substantial logistic barriers to attending. Our findings contribute insights towards understanding health-seeking behavior among undocumented pregnant women and highlight the need for accessible and quality maternity care services. To safeguard the health of the women and their children, inclusive policies for access to health care in Denmark for all must be implemented, as well as heightened awareness from public health care providers to provide care to this group. Further studies are needed on the health needs and health-seeking behavior of women not in contact with the Health Clinic, to fully understand the challenges of access to maternity care for undocumented pregnant women."} +{"text": "Epimedium and two species of Vancouveria was conducted. The 53 taxa were clustered, based on their karyotype similarity coefficients. Results showed that the 53 taxa studied were all diploid with 12 chromosomes (2n = 2x = 12). Each taxon had one pair of satellites located on pair I of homologous chromosomes. Moreover, the karyotype types of the 53 taxa studied were all type 1A or 2A of Stebbins. It can be concluded that the karyotypes between species are indeed very similar and the genome of Epimedium was conservative in evolution. The cluster analysis of karyotype similarity coefficients could provide valuable clues for the systematics and taxonomy of Epimedium. Results of the cluster analysis strongly supported the previous taxonomic division of E.subg.Rhizophyllum and E.subg.Epimedium. The results also showed that the interspecific relationship was closely correlated with geographical distribution in E.subg.Epimedium and the taxa native to east Asia had the highest genetic diversity in Epimedium. Finally, the origin of the modern geographical distribution of Epimedium was inferred. Results of the present study have significant scientific values in further studies on resource utilisation, taxonomy and phylogeny in Epimedium.In order to evaluate the genome evolution and systematics, karyotype analysis of mitotic metaphase chromosomes in 51 taxa of Epimedium L., Berberidaceae), is an important traditional medicinal plant in China. It is effective in strengthening kidney and curing rheumatism , E.sect.Macroceras , E.sect.Polyphyllon and E.sect.Epimedium . Epimediumsect.Diphyllon (native to China), the most complex group in the genus, was further divided into four series: E.ser.Campanulatae, E.ser.Davidianae, E.ser.Dolichocerae and E.ser.Brachycerae.Epimedium. For example: 1) the interspecific relationships are still unclear, especially in E.sect.Diphyllon (native to China); and 2) China is the diversity centre of this genus, but the process of diversification is still unclear, especially regarding the modern discontinuous distribution . All the karyotypes were symmetric to Stebbins\u2019 type 2A or 1A and similar to each other with one pair of middle satellite chromosomes. It can be seen that differences between homologous chromosomes were insufficient and karyotypes between species were very similar. Despite many relevant studies on the karyology of Epimedium were collected from China, Japan, Germany and the United States. The details, including collection location and voucher specimens of these experimental materials, are detailed in Table Fifty-three experimental taxa, including 51 TCL), relative length (%), arm ratio (L/S), arm index and location of the centromere were measured and calculated. RLR (the relative length ratio of the longest and shortest chromosome) and P (the proportion of chromosomes with arm ratio over 2:1) were also calculated. The classification of chromosome type was conducted according to The preparation method of chromosome slides referred to the method of The calculation formula of karyotype similarity coefficients referred to e\u00a8=a~\u00d7\u03b2 (1)a~ij=\u2211k=1nxik\u00b7xjk-\u2211k=1nxik\u2211k=1nxjk\u2211k=1nxik-x\u00af2\u00b7\u2211xjk-x\u00afj2 (2)\u03b2=1-dD (3)D is the sum of distances and d is the square root of the product of inner distance (id) and outer distance (nd):In formula (3), D=\u2211k=1nxik+\u2211k=1nxjk (4)d=di\u00b7dn (5)di=\u2211k=1nxik-xjk (6)dn=\u2211k=1nxik-\u2211k=1nxjk (7)According to the above formulae, the karyotype similarity coefficients of mitotic metaphase chromosomes of all the 53 experimental taxa were calculated. Furthermore, the 53 experimental taxa were clustered, based on the karyotype similarity coefficients by the UPGMA method . The cluEpimedium taxa are diploid with 12 chromosomes (2n = 2x = 12). Pair I of homologous chromosomes in each studied taxon has one pair of satellites. The mitotic metaphase chromosomes of the 51 Epimedium taxa studied were illustrated in Fig. RLR, P and karyotype are all detailed in Table Epimedium taxa studied, the majority of chromosomes were metacentric (m) chromosomes or submetacentric (sm) chromosomes. Subtelocentric (st) chromosomes were few and telocentric (t) chromosomes were not found. The average values of RLR and P of the 51 taxa studied were 1.41 and 0.33, respectively. According to Epimedium taxa tested were highly symmetrical with type 2A or 1A. It also can be seen that the karyotypes between species were very similar in Epimedium species.All 51 studied Vancouveria species studied also were diploid with 12 chromosomes (2n = 2x = 12). Furthermore, pair I of homologous chromosomes of each species also has one pair of satellites located. The mitotic metaphase chromosomes and the karyotype parameters of the two species studied are given in Fig. Vancouveria species studied, only two chromosomal types, i.e. m and sm types, were found. The average RLR of the two species studied was 1.41. The P values of the two species studied were 0.00 and 0.17, respectively. Moreover, the karyotypes of the two species studied were 1A and 2A, respectively. It also can be seen that the karyotypes of the two Vancouveria species were very similar.The two Epimedium, i.e. E.truncatum, E.leptorrhizum and E.dolichostemon were clustered into the group of genus Vancouveria.The karyotype similarity coefficients of the 53 taxa studied were calculated and each of them has one pair of satellites located on pair I of homologous chromosomes. These results are consistent with the previous research reported Mediterranean and Western Asian taxa and 2) East Asian taxa. The present results supported this classification, consistent with the previous studies on the morphology (Epimedium or the group of Vancouveria, indicating that the genetic diversity of East Asian taxa are the highest in the genus.Cluster analysis of karyotype similarity coefficients showed that, although the genome was conservative in evolution and the karyotypes between species were highly similar, the cluster analysis of karyotype similarity coefficients still provided some valuable clues for studies on phylogenetics and taxonomy in rphology , cytologrphology , moleculrphology and phytrphology , all of"} +{"text": "Complement deficiencies are rare and often underdiagnosed primary immunodeficiencies that may be associated with invasive bacterial diseases. Serious infections with encapsulated organisms are frequent in patients with a deficiency of the second component of complement (C2), but no data are available on long-term follow-up. This study aimed to evaluate the long-term clinical outcome and the importance of an early diagnosis and subsequent infection prophylaxis in C2 deficiency. Here, we report the 21-year follow-up of a whole family which was tested for complement parameters, genetic analysis and biochemical measurements, due to recurrent pneumococcal meningitis in the elder brother. The two sons were diagnosed with homozygous type 1 C2 deficiency, while their parents were heterozygous with normal complement parameters. For the two brothers, a recommended vaccination program and antibiotic prophylaxis were prescribed. During the long-term follow-up, no severe/invasive infections were observed in either patient. At the age of 16, the younger brother developed progressive hypogammaglobulinemia of all three classes, IgA, IgM and IgG. A next generation sequencing panel excluded the presence of gene defects related to primary antibody deficiencies. Our data show that early diagnosis, use of vaccinations and antibiotic prophylaxis may allow a normal life in hereditary C2 deficiency, which can be characterized using functional and genetic methods. Moreover, a periodical check of immunoglobulin serum levels could be useful to detect a possible hypogammaglobulinemia. The complement is a multi-functional complex system of the innate immunity comprising more than 30 proteins which are produced mainly by the liver and consist of activators and inhibitors interacting with each other to form three pathways of activation ,2,3. ThiBacterial infections and autoimmune diseases are clinical conditions most frequently associated with complement defects . HomozygPatients with hereditary C2 deficiency are at risk of developing serious infections with encapsulated organisms ,8 and shHere, we report the case and the 21-year follow-up of two brothers with type 1 C2 deficiency. Patient 1 was diagnosed with complement deficiency after the second episode of pneumococcal meningitis. Patient 2, the younger brother, benefitted from familiar profiling and avoided severe infections.Two Italian brothers born in 1997 and 2000 respectively from healthy, unrelated parents attended our University Hospital. Patient 1 was the first to come to our attention after the second episode of pneumococcal meningitis. Both patients were diagnosed with C2 deficiency, whereas in their parents C2 levels were near the lower limit of normal range. Forty healthy subjects served as normal controls and provided the ranges reported in \u00ae, Sanofi Pasteur Europe S.a.s, Lione, France), 7-13-23 valent absorbed pneumococcal polysaccharide capsular vaccines , quadrivalent meningococcal conjugate vaccine and with the conjugate vaccine against N. meningitides serogroup B as soon as it became available. They regularly carried out recalls for pneumococcal and quadrivalent meningococcal vaccines.Upon diagnosis, both brothers were immunized with anti-Haemophilus influenzae type B conjugated vaccine . This assay was done as previously described ,14. BrieC2 specific activity was calculated using hemolytic assay in which the recovery of complement activity of a serum from a patient with a well characterized deficiency of a single complement component is obtained by adding the patient serum, as previously described .The functional test for the classical complement pathway was performed using a Wieslab Complement System kit , following the manufacturer\u2019s instructions. The wells of the microtiter strips were coated with a specific activator of the classical complement pathway, and the serum was diluted in a buffer containing specific blockers of the other two complement pathways in order to ensure that only the classical pathway was activated during incubation. The wells were then washed, and C5b-9 was detected with a specific alkaline phosphatase-labelled antibody against the neoantigen expressed on C9 during C5b-9 formation. After a further washing step, the specific antibodies were detected by means of incubation with the alkaline phosphatase substrate solution. As the amount of complement activation correlates with color intensity and is measured in terms of absorbance, the results are expressed as percentages of the activity of a standard sample . This method allows evaluation of complement activity detected in serum through classical pathway activation using the terminal complement complex C5b-9 as detection system .Genomic DNA was amplified using PCR with primers designed to specifically amplify exon 6 (ex6F: 5\u2019-GCCTGGGCCGTAAAATCCAAATCCA-3\u2019 and Ex6R: 5\u2019-GCACAGGAAGGCCTCTGCTGCAGGC-3\u2019), including the most common Type I C2 deficiency mutation ,11,12 unPatient 1 is currently 24 years old; he was born to unrelated Italian parents at 40 weeks of gestation after an uneventful pregnancy. At 12 months of age, he suffered from pneumococcal meningitis with a delayed cerebrospinal fluid (CSF) sterilization (15th d), resulting in bilateral sensorineural hearing loss (SNHL). At 32 months of age, the patient suffered a second episode as meningo-encephalitis with isolation of S. pneumoniae from the CSF. Despite CSF sterilization after 5 d of antibiotic treatment, encephalic foci persisted causing a mild right cerebellar lesion.Possible risk factors for recurrent bacterial meningitis were investigated, such as anatomic defects of cribriform plate or paranasal sinus and asplenia, with negative results. In-depth investigations to rule out primary immunodeficiencies showed normal values of serum immunoglobulins, IgG subclasses, T and B cell number and activity. Detailed functional activity of the classical pathway of the complement system was measured, revealing a reduced activity due to C2 deficiency. Patient 2, current age 21 years, had the same diagnosis of C2 deficiency from the age of 6 months after the diagnosis in his brother. He was born at 40 weeks of gestational age after a physiologic pregnancy. The patient did not experience any severe or invasive infection before diagnosis of C2 deficiency was made.As reported in Further analysis performed with a recovery test of complement activity showed C2 deficiency in the siblings with levels of 0 and C2 levels near the lower limits of normal subjects in the parents. The amplified DNA visualized on agarose gel showed two bands in the parents, representing the wild type and the deleted allele of 174 bp and 156 bp, respectively. In the two children only a band of 156 bp was present .Sequence analyses of exon 6 of the C2 gene in all the family members revealed in the two parents the presence of the 28-base pair deletion at the heterozygous state. The two affected siblings were botDuring follow-up, Patient 1 did not suffer from severe/invasive infections. Due to recurrent episodes of rhinitis, allergological evaluation was performed at 6 years of age, which showed elevated IgE levels with specific IgE positivity for Alternaria and mites. A CT scan of the facial mass and the petrous rocks showed adenoiditis and chronic sinusitis. Although the symptoms were partially controlled by frequent nasal rinses and topic steroid cycles , at the age of 7 years the patient underwent adenoidectomy and enlargement of the sphenoid sinus ostium. Due to the severe SNHL following the first episode of pneumococcal meningitis, at the age of 14 he underwent cochlear implant surgery without complications. Brain MRI has remained stable over time. The patient has always shown normal levels of IgA, IgM, IgG, IgG subclasses and lymphocyte subpopulations as well as the development of protective specific antibody titers after vaccinations .Patient 2 has never experienced severe infections. Like his brother, he presented with recurrent episodes of rhinitis leading to the diagnosis of allergies to grass; nasal polyposis was diagnosed at the age of 13 years. The patient performed regular annual controls with immunological evaluation: at the age of 3 years, IgG levels were at the lower limit of normal values for his age with normal IgG subclasses, IgA and IgM. The antibody response after a tetanus vaccination was normal. At the age of 16 years, the patient showed low immunoglobulin serum levels for all classes and low IgG1 subclass , with an adequate antibody response to anti-pneumococcal vaccination, normal T cell distribution and functional evaluation, as well as normal peripheral B cell maturation with switched memory B cells being at the lower limit of normal values for his age . No evidence of autoimmunity was found. The patient did not show any further alteration of the immunological profile, maintaining a good health status.A next generation sequencing (NGS) panel of known genes associated with hypogammaglobulinemia, agammaglobulinemia and common variable immunodeficiency (CVID) was performed and did not reveal the presence of any pathogenetic variant. In particular the following genes were evaluated: AICDA, BLNK, BTK, CARD11, CD19, CD79A, CD79B, CD81, CECR1, CR2, CTLA4, CTPS2, CXCR4, DKC1, DNMT3B, FCN3, GATA2, ICOS, IGHM, IGKC, IGLL1, IL21R, INO80, LRBA, LRRC8A, MAGT1, MOGS, MRE11A, MS4A1, MSH6, NBN, NFKB1, NFKB2, PIK3CD, PIK3R1, PLCG2, PMS2, PRKCD, RAG1, RAG2, SH2D1A, STAT3, TCF3, TNFRSF1 3b(TACI), TNFRSF13C, CD40, TNFSF12, CD40LG, TPP2, TRNT1, TTC37, UNG2, XIAP.This study, in which we report the case and the 21-year follow-up of two brothers diagnosed with homozygous type 1 C2 deficiency and of their parents, shows that early diagnosis, use of vaccinations and antibiotic prophylaxis may allow a normal life in hereditary C2 deficiency. Moreover, hypogammaglobulinemia may complicate the clinical course of the disease. Deficiency of complement components represents approximately 1\u20136% of all primary immunodeficiencies, but these may increase up to 10% in certain communities ,18,19,20The mild hypogammaglobulinemia presented by Patient 2 has worsened over time, with deficiency in all the three classes of immunoglobulins (IgA-IgM-IgG) by the age of 16, without any further immunological alteration or severe infection, and thus not fulfilling the criteria of the European Society for Immunodeficiencies for CVID ,25,26. CAssociation between C2 deficiency and antibody deficiency has only been reported in a very limited number of cases ,28 and dIt is known that the classical complement pathway has a major role in B-cell development and differentiation: C2 seems to play a direct role in the production of IgG4 and IgD and the classical pathway per se contributes to the development of a normal lymphonode germinal center, as demonstrated by the observation that knockout mice for complement C3 receptor CD21/CD35 have an impaired B-cell differentiation . MoreoveOur report supports the utility of searching for complement deficiency as part of the immunological screening in all cases of invasive bacterial disease in which favoring anatomical causes are excluded. The long-term follow-up without any infection in our two patients with hereditary C2 deficiency indicates that an early diagnosis of complement deficiency allows a prompt intervention with vaccinations and antibiotic prophylaxis, which in turn avoids infections and thus improves the quality of life. Although hypogammaglobulinemia is not a classical immunological finding in hereditary C2 deficiency, our data along with those of others suggest the need of further longitudinal studies in patients with hereditary C2 deficiency to better define this possible association."} +{"text": "Candida tropicalis is a virulent fungal pathogen for which echinocandins are the primary therapy. Emergence of resistance to echinocandins of C. tropicalis carries potentially ominous therapeutic implications. Methods. We describe herein two patients with breakthrough C. tropicalis fungemia during echinocandin therapy, characterize their molecular mechanism of resistance, and systematically review 13 previously reported cases of echinocandin-resistant C. tropicalis bloodstream infections (BSIs) and other diseases. Results. Among these 15 patients with echinocandin-resistant C. tropicalis infections, the median age was 61 years (ages 28\u201384 years) and 13 (86%) were immunocompromised. Thirteen (86%) of all patients had a history of pervious or concurrent exposure to echinocandins. Isolates of C. tropicalis from 11 cases, including the two index cases, underwent DNA sequencing of the FKS1 gene for mutations known to confer echinocandin resistance. The amino acid substitution Ser654Pro was shown in four cases, while other FKS1 mutations encoded Ser80S/Pro, Phe641Leu, Phe641Ser, Ser80S/Pro substitutions. These mutational events were not associated with collateral increases in minimum inhibitory concentrations to antifungal triazoles and amphotericin B. Overall mortality in patients with echinocandin-resistant C. tropicalis infections was 40%. Among those six patients who died, two received monotherapy with voriconazole, one was treated with fluconazole, one remained on caspofungin, and two were switched to liposomal amphotericin B. Nine patients (60%) survived after being treated with an antifungal agent other than an echinocandin. Conclusions. Emergence of resistance to echinocandins by C. tropicalis, occurs during antifungal therapy, is associated with high mortality, is mediated by a diverse range of FKS1 mutations, retains in vitro susceptibility to triazoles and amphotericin B, and constitutes an emerging threat to patients with hematological malignancies.Background. Candida species to echinocandins in general, and specifically Candida tropicalis ..C. tropiFKS1 gene for mutations known to confer echinocandin resistance was performed in 11 cases, including our two index cases isolates had the amino acid substitution Ser80Pro; three cases (27%) showed the amino acid substitution S645P, and one each yielded the amino acid substitution F641L and F641S, respectively.DNA sequencing of the ex cases . Three cOverall mortality was 40%. Nine patients (60%) survived after being treated with an antifungal agent other than an echinocandin. Treatment was changed to liposomal amphotericin B or voriconazole in three cases respectively, and one each to voriconazole plus caspofungin, fluconazole, voriconazole plus liposomal amphotericin B, liposomal amphotericin B plus flucytosine and caspofungin plus voriconazole. Among the six patients who died, two were treated with voriconazole, two with liposomal amphotericin B, and one with fluconazole, while one continued to receive caspofungin . C. tropicalis to echinocandins in immunocompromised patients either during treatment or prophylaxis with an echinocandin. Candida tropicalis is a highly virulent pathogen in neutropenic hosts, where it causes increased mortality corresponding to greater degrees of tissue invasion in experimental models and immunocompromised patients with hematological malignancies [FKS1 gene resulting in a serine-to-proline substitution at position 654. FKS genes encode the (1\u21923)-\u03b2-d-glucan synthase, which is responsible for the synthesis of the structural polymer of the fungal cell wall and is the target enzyme of the echinocandins. Hot spot mutations are known to confer elevated MICs to the echinocandins. Other antifungal agents, particularly liposomal amphotericin B, are necessary to treat these echinocandin-resistant infections caused by C. tropicalis.This report describes the emergence of resistance of gnancies ,7,22. ThC. tropicalis with caspofungin and micafungin MICs of 2 \u00b5g/mL showing a L644W missense mutation in the hot spot 1 region of the FKS1 gene [Caspofungin was the first echinocandin approved by the Food and Drug Administration and the European Medicines Agency in 2001. Less than a decade later, Desnos-Ollivier et al. described the first case of echinocandin-resistant KS1 gene . This isCandida tropicalis isolate BL38734 showed a point mutation involving an amino acid substitution in one of the alleles of the FKS1 gene, while isolate 2 showed a point mutation in both alleles of the FKS1 gene. This amino acid substitution is located in the highly conserved hot spot 1 region of the FKS1 gene and has been associated with echinocandin resistance in Candida species [ species . C. tropicalis resistant to echinocandins have been reported to date [C. tropicalis fungemia is common among hematologic malignancies and especially in neutropenic patients [In addition to our institutional cases, 13 cases of fungemia caused by to date ,18,19,20Candida species infections was associated with caspofungin resistance [C. tropicalis, two were not exposed to echinocandin, but to voriconazole [C. tropicalis BSIs after receiving an echinocandin with two of those 8 cases receiving voriconazole and caspofungin before they developed resistant C. tropicalis strain [Candida glabrata, where echinocandin-resistant isolates may be also be resistant to antifungal triazoles [C. tropicalis to fluconazole, other triazoles, or amphotericin B has not been a prevalent problem in immunocompromised patients. Fluconazole or amphotericin B may be used in treatment of BSIs caused by echinocandin-resistant isolates of C. tropicalis. However, the delay in effective therapy of echinocandin-resistant C. tropicalis infections in patients receiving an echinocandin may result in worsened outcome. Thus, rapid molecular identification of echinocandin-resistant C. tropicalis and other Candida spp. from the blood stream may provide life-saving information before conventional blood cultures become positive. Exposure to antifungal agents has been described as a major risk factor for drug resistance . Prolongsistance ,16,23,24conazole , and thes strain ,17. In criazoles , resistaC. tropicalis that emerged while on micafungin (C. tropicalis emerged while on caspofungin [To our knowledge, our index cases represent the first described echinocandin-resistant cafungin . The antpofungin ,14,16 anpofungin . Nine papofungin ,19,20,22pofungin . Candida species have defined an MIC of \u22651 \u03bcg/mL as non-susceptible C. tropicalis, an MIC of 0.5 \u03bcg/mL as intermediately susceptible, and MIC \u2264 0.25 \u03bcg/mL as susceptible C. tropicalis [Candida species where the MIC distributions were remarkably distinct among the three echinocandins, C. tropicalis MIC distributions were similar for the three echinocandins [C. tropicalis, anidulafungin appears to have the lowest in vitro MIC compared to caspofungin and micafungin (C. tropicalis were in vitro intermediately susceptible to anidulafungin and micafungin but non-susceptible to caspofungin [C. tropicalis were also non-susceptible to fluconazole [The CLSI published species-specific antifungal interpretive breakpoints for resistance for echinocandins against each opicalis ,23,25,26cafungin . One isopofungin ,14,15,16conazole ,15 and oC. tropicalis is a highly virulent Candida species that is usually susceptible to echinocandins. However, emergence of resistance may develop following exposure to echinocandin treatment. The resistance is most commonly caused by a serine-to-proline substitution in the known hot spot 1 region of FKS1. Liposomal amphotericin B is an effective empirical alternative in patients with hematological malignancies pending susceptibility testing [C. tropicalis and other Candida species is highly recommended, as emerging trends in resistance may serve as a basis for changing patterns of practice in the use of echinocandins.In conclusion, testing . Finally"} +{"text": "The links between hearing impairment (HI) and dementia have been well documented, but factors mediating this relationship remain unknown. Major consequences of HI are social and emotional dysfunction, and as the risk of dementia increases linearly with the severity of HI, it is plausible that socio-emotional difficulties may play a role in this association.The aim of this study was to develop and validate a tool to analyse levels of hearing-related disability, to investigate ultimately whether subjective disability contributes to risk of cognitive impairment compared with hearing thresholds alone.Development and validation of the questionnaire, the Social and Emotional Impact of Hearing Impairment (SEI-HI), was conducted in four phases: (1) content; (2) scoring and outcomes; (3) validation; (4) feasibility in a sample of people with cognitive impairment.Considerable evidence was found for the internal and external reliability of the tool with high construct validity, concurrent validity and test-retest values of the SEI-HI questionnaire. A feasibility check on 31 patients with mild cognitive impairment or dementia showed the SEI-HI questionnaire was easy to administer and well-received.The SEI-HI questionnaire is a relevant instrument to assess hearing-related disability which can be used in people with cognitive decline to assess further impact on risk of developing dementia.The online version of this article (10.1007/s10072-020-04492-5) contains supplementary material, which is available to authorized users. Hearing impairment (HI) is one of the most common disabilities of the ageing population affecting over 466 million people worldwide . One of mechanism linking HI to dementia remains to be elucidated. One theory is HI may indirectly increase the risk via psychosocial pathways indicating a good test-retest reliability. There was not a statistically significant change in SEI-HI questionnaire scores between time 1 and time 2 .There was a strong positive correlation between participants scores on the SEI-HI questionnaire at time 1 and time 2 (4 to 8\u00a0weeks later), ICC\u2009=\u2009.905, rS\u2009=\u2009.890, p\u2009<\u2009.001, 95% CI , and Examiner 2, rS\u2009=\u2009.737, p\u2009=\u2009.002, 95% CI , was not significantly different.The presence of examiner bias was excluded, as the difference between test-retest correlations for Examiner 1, All participants with MCI and dementia were able to complete the SEI-HI questionnaire with no difficulty or reported issues, with support of the experimenter. Clinical characteristics, outlined in Table The SEI-HI questionnaire has demonstrated a high level of reliability and validity as a measure of current psychosocial impact of hearing-related disability in older adults.\u03b1 scores. Due to the spectrum of disability associated with HI, despite the high \u03b1, all items were included to ensure the breadth of relevant questions to maximise clinical potential.Internal consistency was demonstrated by strong Cronbach Due to individual differences in many factors including lifestyle, attitudes, comorbid health conditions, and available support networks, it is logical to assent that not any two people with the same levels of HI will be affected in the same way , 26. BecThe test-retest reliability of the SEI-HI questionnaire is very satisfactory at ICC\u2009=\u20090.905. As on average a 6-week timescale had passed, it can be assumed with reasonable certainty that participants would not remember their previous scores and thus the coefficient has not been inflated as a result of the retesting procedure. Any minor changes could be reflective of changes in circumstance or attitudes towards the disability or irrelevant temporal factors, such as mood, which may cause a fluctuation in scores over time .Altogether, this lends support for the use of the SEI-HI questionnaire, not only as a cross-sectional instrument to measure current subjective hearing disability, but to be also used for longitudinal purposes. Due to the strong correlation, small standard error and no evidence of experimenter bias, it can be expected that changes over time are as a result of intervention rather than experimental error . ContinuAlthough the development of the SEI-HI questionnaire is based upon the scenarios reported in the HHIE, it aims to measure slightly different aspects than the HHIE and SAC, and it is promising to see the significant correlation between scores on the SEI-HI questionnaire and these measures, supporting the specificity of the SEI-HI questionnaire. This high specificity means that it does not allow for cross-condition comparisons. For example, the socio-emotional difficulties measured are due to common problems in relation to HI, and not suitable for measuring quality of life affected by other conditions or disabilities. Similarly, although the SEI-HI questionnaire has shown high validity and reliability in our sample, it may have limited uses in other cultures and thus may not be generalisable to non-English speaking countries. However, using the principles upon which the scale was designed and validated would allow for translation and validation into other languages. Participants recruited for Phase 4, with MCI to mild dementia, were able to complete the SEI-HI questionnaire with support from the experimenter. This has not been designed as an informant questionnaire, due to the sensitive and subjective nature of hearing disability. In cases with a more severe cognitive impairment, it will be left to the clinicians\u2019 or researchers\u2019 judgement to include carer-responses to aid answering these questions.Given the clinical importance of investigating this association, it is essential to have a specific, valid and reliable questionnaire to compute current levels of hearing functioning. To the best of our knowledge, the SEI-HI questionnaire is the first validated instrument to measure current levels of subjective hearing disability in recent years. To conclude, this study has shown that the SEI-HI questionnaire is a favourable and relevant instrument to assess current levels of subjective hearing disability regardless of hearing threshold. It can be used with confidence to control for subjective levels of disability in people with varying levels of HI, to assess further the risk of cognitive decline. The use of the SEI-HI questionnaire would help to determine whether the social and emotional impacts of HI have more of an influence on the risk for dementia, in addition to hearing thresholds alone.ESM 1(DOCX 23\u00a0kb)"} +{"text": "Prostate cancer is a complex disease affecting millions of men globally. Radiotherapy (RT) is a common treatment modality although treatment efficacy is dependent upon several features within the tumour microenvironment (TME), especially hypoxia. A hypoxic TME heightens radioresistance and thus disease recurrence and treatment failure continues to pose important challenges. However, the TME evolves under the influence of factors in systemic circulation and cellular crosstalk, underscoring its potential to be acutely and therapeutically modified. Early preclinical evidence suggests exercise may affect tumour growth and some of the benefits drawn, could act to radiosensitise tumours to treatment. Intracellular perturbations in skeletal muscle reactive oxygen species (ROS) stimulate the production of numerous factors that can exert autocrine, paracrine, and endocrine effects on the prostate. However, findings supporting this notion are limited and the associated mechanisms are poorly understood. In light of this preclinical evidence, we propose systemic changes in redox signalling with exercise activate redox-sensitive factors within the TME and improve tumour hypoxia and treatment outcomes, when combined with RT. To this end, we suggest a connection between exercise, ROS and tumour growth kinetics, highlighting the potential of exercise to sensitise tumour cells to RT, and improve treatment efficacy. A lack of oxygen to the tumour (hypoxia) impairs radiotherapy treatment efficacy.Exercise may beneficially alter components of the TME and act as a sensitiser for treatment.Reactive oxygen species generated by exercise and redox-sensitive mechanisms within the tumour, alongside a host of other exercise-induced mechanisms, may prove pivotal in facilitating the treatment effects.Prostate cancer (PCa) is the most diagnosed male malignancy in the developed world, with an annual incidence of approximately 1.3 million cases . Indeed,Chemical radiosensitisers have proven to be effective complementary agents for RT treatment , 11, altMalignant tumours stimulate neovascularisation to sustain growth. Normally, angiogenesis is a highly regulated process; however inhospitable conditions within the TME and accumulating growth factors trigger an \u2018angiogenic switch\u2019, whereby the natural balance between proangiogenic and antiangiogenic factors is disturbed , 20. ThiCells exposed to cycling hypoxia exhibit a robust stabilisation and accumulation of HIF-1\u03b1, leading to a more aggressive phenotype and reduced responsiveness to RT , 27. MoeA recent meta-analysis confirmed higher levels of post-diagnosis physical activity can reduce PCa-specific mortality . This imSeveral reviews posit exercise as an anti-tumour therapy, alongside contemporary treatment methods, through regulating the systemic milieu and conceivably tumour intrinsic factors , 48\u201351. hormesis\u2019 [Given the potential of exercise to influence tumour vasculature, oxygenated blood perfusion, and thus hypoxia, it follows that exercise could act as a radiosensitiser, reducing radioresistance. Treatment response relies on well-oxygenated tissue (permitting oxygen fixation) and at lower partial pressures of oxygen, radiosensitivity is considerably reduced . Preclinormesis\u2019 or on thormesis\u2019 . Despite\u22121) and seemingly protective effects [Preclinical findings concerning exercise and the TME have provoked considerable research and clinical interest pertaining to the mechanisms of action surrounding tumour hypoxia and RT efficacy. The apparent intensity-dependent nature of the responses or gradual, chronic effects sensitise tumour tissue for RT would be highly advantageous, further to whether these mechanisms amplify treatment efficacy and tolerance. Exercise training may in some part, reprogramme the systemic milieu towards an anticancer phenotype, reducing tumour hypoxia and tumour proliferation through biological, epigenetic, metabolic, and inflammatory mechanisms. Redox signalling is integral in this beneficial response to exercise, though limited quantitative data exist pertaining to the influence of such species (modest and high levels) within the TME and their effect on human cancer cells ."} +{"text": "During glutamine sufficiency, both Notch\u2010positive and Notch\u2010negative T\u2010ALL cells promote glutamine catabolism, leading to mammalian target of rapamycin complex 1 (mTORC1) activation and cell growth and proliferation. However, during glutamine scarcity, only Notch\u2010negative T\u2010ALL cells can perform a metabolic adaptation by promoting glutamine synthesis. By contrast, Notch\u2010positive T\u2010ALL cells maintain glutamine catabolism during glutamine restriction, leading to glutamine addiction and mTORC1 dependency. in\u00a0vitro and in\u00a0vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1\u2010induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1\u2010driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1\u2010driven leukemia.The cellular receptor Notch1 is a central regulator of T\u2010cell development, and as a consequence, Notch1 pathway appears upregulated in >\u00a065% of the cases of T\u2010cell acute lymphoblastic leukemia (T\u2010ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T\u2010ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti\u2010Notch therapies in T\u2010ALL models. In this work, we report that Notch1 upregulation in T\u2010ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1\u2010driven T\u2010ALL both T\u2010ALL is frequently driven by the oncogenic receptor Notch1. However, treatments targeting Notch signaling result in resistant or relapsed disease, and still 20% of childhood patients and 40% of adult patients do not survive [Notch receptors (Notch1\u20104) are heterodimeric peptides, including an extracellular subunit and a transmembrane and intracellular subunit which interact through a heterodimerization domain present in both subunits. When a ligand of the DSL (Delta/Serrate/lag\u20102) family located in the surface of a neighbor cell binds to the extracellular domain of the Notch receptor, it induces sequential cleavages in Notch by an ADAM metalloprotease and by a \u03b3\u2010secretase, releasing the Notch intracellular domain (NICD) from the membrane .Although seminal works already showed the importance of glutamine in the control of metabolism of human leukemia long ago , 5, stilin\u00a0vivo study using T\u2010ALL mouse models reported that glutaminolysis plays a critical role in leukemia progression downstream of Notch1. Glutaminolysis is thus proposed to be a key determinant of the response to anti\u2010Notch1 therapies, as the inhibition of Notch1 blocks glutaminolysis [In the case of Notch1\u2010driven T\u2010ALL, very little has been described regarding the participation of glutamine in Notch1\u2010mediated T\u2010cell malignant transformation or even in other types of cancer. Further, the mechanistic relationship between Notch1 and glutamine in the control of cellular homeostasis is not clear, as contradictory conclusions have been obtained. For instance, an inolysis . Confirminolysis . Howeverinolysis . This stin\u00a0vitro and in\u00a0vivo. We observed that Notch1 upregulation leads to proteosomal degradation of GS, responsible for glutamine addiction in Notch1\u2010activated leukemic cells. Concomitantly, Notch1 also induces the upregulation of GLS and the subsequent activation of mTORC1 signaling pathway, leading to mTORC1 dependency in Notch\u2010driven T\u2010ALL. Beyond confirming the model by which Notch1 induces glutaminolysis, our results propose that Notch1 executes a program leading to the upregulation of glutamine catabolism and cell growth signaling, and blocking glutamine anabolism, which ultimately leads to glutamine addiction in Notch1\u2010driven leukemia. Our results also pointed at the potential therapeutic benefits of targeting glutamine availability and mTORC1 signaling specifically in Notch1\u2010positive T\u2010ALL patients.Herein, we show that Notch1 activation induces glutamine addiction in T\u2010ALL cells both 22.1l\u2010methionine sulfoximine (MSO), z\u2010VAD\u2010FMK caspase inhibitor, bis\u20102\u2010ethyl sulfide (BPTES),CB\u2010839, and diazo\u20105\u2010oxo\u2010l\u2010norleucine (DON) GLS inhibitors were obtained from Sigma\u2010Aldrich . DAPT and MG132 (proteasome inhibitor) were purchased from Santa Cruz. SMARTvector lentiviral human GS shRNA were obtained from Dharmacon . The plasmid GS human tagged ORF clone was purchased from Origene and the empty vector pJS27 MND\u2010DEST SV40\u2010Blasticidin is a gift from R. Iggo (Institute Bergoni\u00e9). The plasmid MND\u2010LUC\u2010IRES was obtained from the Vector Platform at the University of Bordeaux (France).Antibodies against Notch1 total , cleaved Notch1 , c\u2010myc , \u03b2\u2010actin , cleaved caspase 3 , cleaved caspase 8 , cleaved PARP , S6 , phospho\u2010S6 (Ser235/236) , S6K , and phospho\u2010S6K(T389) were obtained from Cell Signaling Technology . Antibody against GS was obtained from BD Biosciences . Antibody against GLS was purchased from Abcam . Antibody against hes1 is from Santa Cruz Biotechnology . The secondary antibodies anti\u2010mouse and anti\u2010rabbit were obtained from Cell Signaling Technology. The mTORC1 inhibitors rapamycin (RAP) and temsirolimus as well as the GS inhibitor 2.2\u22121) supplemented with 10% of FBS , glutamine (2\u00a0mm), penicillin , and streptomycin , at 37\u00a0\u00b0C, 5% CO2 in humidified atmosphere. Mycoplasma contamination check was carried out using the PCR Mycoplasma Test Kit . For glutamine withdrawal experiments, cells are incubated in RPMI without glutamine (GIBCO) with dialyzed serum (Dutscher) during indicated time. Glutamine was added at 2\u00a0mm final concentration. The different inhibitors were used as follows: BPTES (30\u00a0\u00b5m), DON (30\u00a0\u00b5m), CB\u2010839 (10\u00a0\u00b5m), zVAD (20\u00a0\u00b5m), DAPT (10\u00a0\u03bcm), MG132 (5\u00a0\u00b5m), MSO (1\u00a0mm), RAP (100\u00a0nm).CUTLL1 and H33HJ\u2010JA1 (CRL8163) were obtained from the collection of the laboratory of Marisa Toribio (Spain). HPB\u2010ALL, LOUCY, MOLT4 were purchased from DMSZ. All the cells lines were grown in RPMI high glucose at 3500 g for 20\u00a0s. Subsequently, 400\u00a0\u03bcL of the homogenate was transferred to a fresh tube and shaken at 200 g for 30\u00a0min at 4\u00a0\u00b0C. Next, the aliquots were centrifuged for 15\u00a0min at 18\u00a0000 g at 4\u00a0\u00b0C. Seventy\u2010five microliter of the supernatant was transferred to a fresh tube and placed at \u221280\u00a0\u00b0C for 20\u00a0min. The chilled supernatants were evaporated for 2\u00a0h. The resulting pellets were suspended in 100\u00a0\u03bcL water/acetonitrile/formic acid. Concentrations of all metabolites were determined with a semi\u2010quantitative method, using calibration curves with chemical standards. Samples were measured with an ultra\u2010high\u2010performance liquid chromatography (UPLC) system coupled to a time\u2010of\u2010flight mass spectrometer . A 2.1 x 100\u00a0mm, 1.7\u00a0\u00b5m BEH amide column (Waters), thermostated at 40\u00a0\u00b0C, was used to separate the analytes before entering the MS. Solvent A (aqueous phase) consisted of 99.5% water, 0.5% formic acid, and 20\u00a0mm ammonium formate while solvent B (organic phase) consisted of 29.5% water, 70% MeCN, 0.5% formic acid, and 1\u00a0mm ammonium formate. In order to obtain separation of the analytes, the following gradient was used: from 5% A to 50% A in 2.4\u00a0min in curved gradient (as defined by Waters), from 50% A to 99.9% A in 0.2\u00a0min constant at 99.9% A for 1.2\u00a0min, back to 5% A in 0.2\u00a0min. The flow rate was 0.250\u00a0mL\u00b7min\u22121, and the injection volume was 2\u00a0\u03bcL. All samples were injected randomly. After every 16 injections, a quality control sample was injected. The MS was operated in either positive or negative electrospray ionization . Moreover, two different scan modes were used namely full scan (50\u20131200\u00a0Da) and enhanced duty cycle . For all MS modes, the cone voltage was between 20 and 25 depending on the analyte. Capillary voltage in ESI+ was 250\u00a0V and in ESI\u2212 500\u00a0V. A 2\u00a0ng\u00b7mL\u22121 leucine\u2010enkephalin solution in water/acetonitrile/formic acid (49.9/50/0.1 %v/v/v) was infused at 10\u00a0\u03bcL\u00b7min\u22121 and used for a lock mass which was measured each 36\u00a0s for 0.5\u00a0s. Spectral peaks were automatically corrected for deviations in the lock mass. Data for the following set of metabolites were obtained from full scan in either ESI+ or ESI\u2212 . Extracted ion traces were obtained for threonine (m/z\u00a0=\u00a0+120.0661), serine (m/z\u00a0=\u00a0+106.0504), choline (m/z\u00a0=\u00a0+104.107), succinic acid (m/z\u00a0=\u00a0\u2212117.0188), fumaric acid (m/z 115.0031), pyruvic acid (m/z\u00a0=\u00a0\u221287.0082), malic acid (m/z\u00a0=\u00a0\u2212133.0137), citric acid (m/z\u00a0=\u00a0\u2212191.0192), and oxaloacetic acid in a 20 mDa window and subsequently smoothed and integrated with quanlynx software (Waters). Data for the following set of metabolites were obtained in EDC mode. EDC functions for MTA, glutamate, glutamine, methionine, SAMe, SAH, spermine, spermidine, and 2\u2010ketoglutaric acid were optimized for the mass of the analyte in question. The following metabolites were measured in ESI+ mode where MTA was measured in scan function 1 (EDC at 298), methionine in scan function 2 (EDC at 152), glutamate in scan function 3 (EDC at 148), glutamine in scan function 4 (EDC at 147), SAH in scan function 5 (EDC at 385), SAMe in scan function 6 (EDC at 399), spermidine in scan function 7 (EDC at 146), and spermine in scan function 8 (EDC at 203). 2\u2010Ketoglutaric acid was measured in ESI\u2212 in scan function 1 (EDC at 145). Extracted ion traces were obtained for MTA (m/z\u00a0=\u00a0+298.097), methionine (m/z\u00a0=\u00a0+150.0589), SAH (m/z\u00a0=\u00a0+385.1294), SAMe (m/z\u00a0=\u00a0+399.1451) , glutamate (m/z\u00a0=\u00a0+148.061), glutamine (m/z\u00a0=\u00a0+147.077), spermidine (m/z\u00a0=\u00a0+146.1657), spermine (m/z\u00a0=\u00a0+203.2236), and 2\u2010ketoglutaric acid (m/z\u00a0=\u00a0\u2212145.0137), in a 20\u00a0mDa window and subsequently smoothed and integrated with quanlynx software (Waters). Concentrations in the samples were calculated with the power\u2010fitted calibration curves. Concentrations were converted into amount of analyte (pmol) per million of cells, taking in account the analyte loss due to sample workup. All samples were injected randomly and in duplicate. Each measurement represents average of three independent experiments, each experiment performed in triplicates. Metabolite concentration was expressed as pmol per million of cells. Alternatively, glutamate levels were determined using a chromatography coupled to a QTRAP\u00ae 6500+ LC\u2010MS/MS System , with a LC column and a corresponding guard column. MRM data were processed using multiquant\u2122 3.0.2 Software (SCIEX).Cells were centrifuged at 300\u00a02.46 cells were seeded in Seahorse\u00ae WF RPMI medium in XFe24 cell culture microplate pretreated with poly\u2010d\u2010lysine and centrifuge 5\u00a0min at 300\u00a0g. To eliminate residues of carbonic acid from the medium, cells were incubated for at least 30\u00a0min at 37\u00a0\u00b0C with atmospheric CO2 in a nonhumidified incubator. Extracellular acidification rate (ECAR) was assayed in a Seahorse\u00ae XF\u201024 extracellular flux analyzer by the addition via port A of 100\u00a0mm of glucose , port B of 10\u00a0\u00b5m of oligomycin and port C of 500\u00a0mm of 2\u2010DG . Three measurement cycles of 2\u2010min mix, 2\u2010min wait, and 4\u2010min measure were carried out at basal condition and after each injection. At the end of the experiment, each well was washed twice with 50\u00a0\u00b5L of PBS and proteins were extracted with 50\u00a0\u00b5L of RIPA lysis buffer at room temperature. Protein concentration in each well was measured by a bicinchoninic acid assay according to the manufacturer's instructions . ECAR was normalized based on protein quantification. Glycolytic capacity represents the maximum ECAR rate reached by a cell following the addition of oligomycin, effectively shutting down oxidative phosphorylation and driving the cell to use glycolysis to its maximum capacity.CUTLL1 and H33HJ\u2010JA1 cells were incubated in the presence or absence of glutamine for 72\u00a0h. To measure the glycolytic capacity, 4\u00a0\u00d7\u00a0102.5The lentiviral production was carried out as described . Subsequ2.6g, 5\u00a0min then washed with phosphate\u2010buffered saline (PBS 1\u00d7), and lysed on ice using RIPA buffer, supplemented with protease inhibitors (Sigma), phosphatase inhibitors (Sigma), and PMSF 1\u00a0mm . Protein quantification was performed with bicinchoninic acid assay kit (Thermo Fisher). After the electrophoresis, the proteins were transferred to a nitrocellulose membrane with Trans\u2010Blot Turbo Transfer System (Bio\u2010Rad). The membranes were incubated for 30\u00a0min in PBS 1\u00d7 with 0.01% Tween\u201020 and 5% BSA. Incubation with primary antibodies is overnight at 4\u00a0\u00b0C and incubation with secondary antibodies is 2\u00a0h at room temperature. Finally, membranes were imaged using the Chemi Doc MP Imager (Bio\u2010Rad).All cell lines were seeded in 10\u2010cm plates. After the treatment, cells were centrifuged at 300\u00a02.7To assess cell proliferation, viability, and death, cells were seeded in 125\u00a0000 cells per mL in 24\u2010well plates for 7\u00a0days counting in triplicate. The number of total and alive cells was determined using the TC20 Automated Cell Counter (Bio\u2010Rad) according to the manufacturer's instructions. Cells were counted every day with the counter with trypan blue 5% solution (Bio\u2010Rad). After 7\u00a0days, proliferation curve has been established with the standard derivative. Cell viability was then calculated as an end\u2010point or for 7\u00a0days in triplicate.2.8For apoptotic cell death, after treatment, cells were stained with annexin V and propidium iodide (PI) or 7\u2010aminoactinomycin D (7\u2010AAD) following the manufacturer's protocol (BD Biosciences). Then, annexin V and PI/7\u2010AAD staining were analyzed using BD FACS Canto BD Biosciences flow cytometer. The analysis of the data was performed using the software facs diva (BD Biosciences).2.9mRNA extraction was performed with Trizol . One microgram of total mRNA was reverse\u2010transcribed using the GoScript Reverse Transcription system following the manufacturer's protocol. Quantitative real\u2010time PCR was performed using SSO Advanced Universal SYBR Green Supermix (Bio\u2010Rad). Expression levels of each gene were evaluated with normalization to RPL29 and GAPDH housekeeping genes. The sequence of the specific primers used for rtPCR is listed in the following table:2.103H\u2010labeled glutamine. Cells were seeded at the concentration of 500\u00a0000 cells per mL in RPMI without glutamine for 4\u00a0h. Labeled glutamine was added at the concentration of 2.5\u00a0\u00b5Ci and incubated for 15\u00a0min. Cells were collected and centrifuged at 1000\u00a0g in 4\u00a0\u00b0C for 5\u00a0min. Cell lysis was done after two washes in cold PBS with lysis solution followed by HCl 2N. Protein concentration was quantified then the radioactivity was quantified in 5\u00a0mL of scintillation solution. We obtained the values that are normalized to protein content.Glutamine uptake was assessed with 2.11scid Il2rgtm1WjlPrkdc/SzJ immunodeficient mice which were randomly assigned to the different treatment groups (20 mice/group). In the condition of glutamine withdrawal, the mice received glutamine\u2010free diet from 5\u2010week\u2010old and at 8\u2010week\u2010old mice received retro\u2010orbital injections of 0.5\u00a0\u00d7\u00a0106 EV or NICD H33HJ\u2010JA1 cells which are luciferase positive. Mice fed with a glutamine\u2010free diet did not show any morphological or behavioral difference with respect to their complete diet\u2010fed littermates during the duration of the whole treatment (4\u20135\u00a0weeks). We performed the first imaging 1\u00a0week after the injection and twice per week. We evaluated disease progression and therapy response by in\u00a0vivo luminescence bioimaging with the PhotonIMAGER . Temsirolimus (4\u00a0mg\u00b7kg\u22121) was injected intraperitoneally twice a week (every 3\u20134\u00a0days). When mice were sacrificed after 4\u00a0weeks of injection, blood and bone marrow samples were collected and analyzed for further analyses. Glutamine concentrations were determined in mice sera using the YSI 2950 Biochemistry Analyzer . Human CD3\u2010positive cells were analyzed by FACS using PE\u2010coupled anti\u2010human CD3 antibody (BD Biosciences).All animals are maintained in the Animal Facility A2 of the University of Bordeaux , led by B. Rousseau. The project has received the agreement of the Ethic Committee under the number APAFiS #94212017032614365349v7. We used 8\u2010week\u2010old male NOD.Cg\u20102.12post\u00a0hoc test was used to evaluate the statistical difference between more than two groups. t\u2010Test analysis was used to evaluate the statistical difference between two groups. Statistical significance was estimated when P\u00a0<\u00a00.05.The results are expressed as a mean\u00a0\u00b1\u00a0SEM of at least three independent experiments. One\u2010way or two\u2010way ANOVA followed by Bonferroni's comparison as a 33.1To better understand the role of glutamine in the metabolism of T\u2010ALL, we performed a metabolomic analysis in two different T\u2010ALL cell types (H33HJ\u2010JA1 and CUTLL1) in response to glutamine deprivation Fig.\u00a0a\u2010b. As e3.2hes1 and hey1 either with an empty vector (herein after referred as \u2018EV cells\u2019) or with a vector expressing NICD (\u2018NICD cells\u2019). The correct expression of NICD in infected cells was analyzed by qPCR Fig.\u00a0. To tracey1 Fig.\u00a0. The uprey1 Fig.\u00a0. Howeverey1 Fig.\u00a0. The losey1 Fig.\u00a0. As prevey1 Fig.\u00a0 and S2b.3.4in\u00a0vivo. For this purpose, we injected both EV and NICD H33HJ\u2010JA1 cells (luciferase positive) into mice receiving either a normal complete diet or a diet in which both glutamine and glutamate content was eliminated in complete diet\u2010fed mice similarly to EV cells , and we determined infiltration of leukemic cells in the bone marrow. As shown in Fig.\u00a0Glutamine metabolism is very closely connected to mTORC1, a master controller of cell growth and metabolism , 18, and4in\u00a0vivo using mouse models, as we observed that specifically Notch1\u2010positive leukemia was unable to progress in mice fed with a glutamine\u2010free diet. Finally, our results showed that Notch1\u2010induction increased GLS expression at the mRNA level. In parallel, Notch1 blocked the accumulation of GS in glutamine\u2010free conditions by enhancing the proteasomal degradation of GS, ultimately responsible for the addiction to glutamine.Aberrant Notch signaling has been reported to play an important role in the tumorigenesis of different types of cancer , 21, 22.Mechanistically, how Notch1 induces the proteasomal degradation of GS is still unclear. Recently, it was reported that glutamine induces the degradation of GS through the activity of the cullin\u2010RING ubiquitin ligase 4 (CRL4) complex. Thus, under glutamine\u2010rich conditions GS is acetylated at lysines 11 and 14 by p300, which allows its interaction with CRL4 (CRBN), and the subsequent ubiquitination and degradation of GS by the proteasome . How Notet al. [in\u00a0vivo. This work showed that, mechanistically, the inhibition of Notch1 induces glutaminolysis inhibition and triggers autophagy supporting leukemic survival and cell growth by recycling essential metabolites required for leukemic cell metabolism. Following a different approach, our results confirmed the important connection between Notch1 signaling and glutamine metabolism. However, and in contrast with their conclusions, our results indicated that GLS inhibition did not have the same impact than glutamine depletion on cell viability in Notch1\u2010driven leukemia, illustrating that glutamine is essential for Notch1\u2010positive T\u2010ALL cells for reasons that exceed just glutaminolysis. This conclusion was further supported by our observation that GS degradation (and not GLS) was responsible for the glutamine addiction phenotype.Although glutamine addiction has been reported in many cancer types , 24, 25,et al. , showed l\u2010asparaginase could be envisioned in this situation. l\u2010asparaginase is an enzyme which catalyzes the conversion of l\u2010asparagine to aspartic acid and ammonia. However, it has been reported that l\u2010asparaginase does not only degrade l\u2010asparagine, but also has a GLS activity although with lower affinity and lower maximal rate, leading to decreased levels of glutamine in blood [The results obtained in this study highlighted the potential involvement of glutamine restriction as a therapeutic approach for Notch1\u2010positive T\u2010ALL patients. In our model, we used a glutamine/glutamate\u2010free diet to fed mice\u2010bearing Notch1\u2010induced leukemia. Although the liver of mice can synthesize its own glutamine, our results showed that the blood glutamine levels in mice fed with glutamine\u2010free diet were actually decreased significantly compared to the normal diet. Thus, the establishment of glutamine\u2010free diets could be considered to apply for patients bearing Notch1\u2010driven leukemia. However, from the practical point of view, preparation of such a diet might be not achievable, or at least not affordable. Alternatively, the use of in blood , 27, 28.Finally, our results showed that a treatment combining glutamine depletion with the mTORC1 inhibitor reduces the viability specifically of Noch1\u2010driven leukemic cells. The mechanistic link observed between Notch1 and mTORC1 through glutaminolysis seems to operate as a mechanism to potentiate cell growth and leukemia proliferation, sustaining high glutamine catabolism and high mTORC1 activity. Targeting this axis by attacking to main points (glutamine availability and mTORC1 activation) could be envisioned as a potential therapy to treat Notch1\u2010positive T\u2010ALL patients.The authors declare no conflict of interest.RVD conceived the project. TLN, MJN, ST, and RVD designed experiments. TLN, MJN, ST, MT, CB, OG, JMP, BR, SvL, JMF, ER, HRR, MP, MB, IR, JC, BU, and PF performed experiments. TLN, MJN, ST, MT, CB, OG, and RVD analyzed data. RVD, PS, PSM, FP, MLT, and AMK secured funding. RVD, TLN, and MJN wrote the manuscript.Fig. S1. Glutamine sustains TCA cycle in T\u2010ALL cells.Fig. S2. Notch1 activation/upregulation correlated with glutamine addiction in T\u2010ALL cells.Fig. S3. mTORC1 inhibition synergizes with glutamine starvation to reduce cell proliferation in Notch1\u2010positive T\u2010ALL.Fig. S4. mTORC1 inhibition synergizes with glutamine starvation to induce cell death in Notch1\u2010positive T\u2010ALL.Click here for additional data file."} +{"text": "This study aims to observe the expression of NaSpecimens of 111 stage II\u2013IV CC patients were collected. We used western blotting, qPCR, and immunohistochemical staining to observe the distributions and expression levels of NCX isoforms in CC and distal normal tissues. Cox proportional hazards models were used to assess prognostic factors for patients.The expression of NCXs in most tumor specimens was lower than that in normal tissues. The NCX expression levels in tumor tissues from the primary tumor, local lymph node metastasis sites, and distant liver metastasis sites were increasingly significantly lower than those in normal tissues. The results of the Kaplan-Meier survival curves showed that the downregulation of any NCX isoform was closely related to the worse prognosis of advanced CC.NCXs can be used as independent prognostic factors for CC. Our research results are expected to provide new targets for the treatment of CC. NCX1 is distributed in several tissues and has been extensively studied. The NCX2 and NCX3 genes, however, were mainly found in the central nervous system \u201317. Theral cells . NCX1 anal cells . Upregulal cells . In the al cells , 27. How+]i) in HCT116 colon cancer cells, which suggested that NCX was positively involved in DMS-induced [Ca2+]i increase by using NCX inhibitors a priori and NiCl2 to block NCX\u2019s reverse transport of calcium ions into the cell to reduce [Ca2+]i i by downregulating NCX1/3, leading to the proliferation and migration of the HCT116 colon cancer cell line; moreover, the use of the NCX inhibitor SEA0400 significantly promoted the migration of CRC cells i, upregulate Ca2+ protein alpha 1D, and promote the migration of HCT116 colon cancer cells i of colorectal glandular epithelial tumors is increased, and the increase in [Ca2+]i activates calmodulin kinase II (CaMKII) and hypoxia-inducible factor-1a (HIF1a), which are involved in tumor progression [2+/calmodulin-dependent protein kinase II and HIF1a in CC cells promoting tumor cell invasion and metastasis is related to aerobic glycolysis, and the activation of downstream factors MMPs and EMT also participates in tumor cell migration [+/Ca2+/Li+ exchanger (NCLX) is significantly downregulated in patients with colorectal cancer. The reduction in NCLX led to transcriptional changes, and the expression of genes that regulate EMT and cancer stemness increased. Mesenchymal phenotype expression promoted colorectal cancer metastasis and drug resistance. In addition, the decrease or loss of NCLX expression can cause mtCa2+ overload, which leads to mitochondrial depolarization, increases mtROS production, initiates the mitochondrial Ca2+/ROS signaling axis to drive HIF1a activation and HIF1a-dependent glycolysis, promoting the migration, invasion, and metastasis of colorectal cancer cells [Research on NCXs has focused on the areas of the heart and brain for a long time; in recent years, articles on sporadic function have linked cancer with NCXs. For example, NCX1 was found to be highly expressed in esophageal cancer and considered to be involved in the pathogenesis of esophageal cancer . The ovegression . Inhibiter cells . The phy of NCXs . Therefogression , 27. Theigration , 54. Somer cells , 55.We preliminarily verified that NCX was a tumor suppressor in human colon tumor tissues. Certainly, the current research on the roles of NCXs in CC is still in an early stage, and there is no clarity on the mechanism of NCX regulation. As a next step, we will further verify the tumor suppressor mechanism of NCXs in a variety of in vitro cell lines. However, the three isoforms of NCXs can separately be used as independent prognostic factors of CC as long non-coding RNAs, autophagy-related genes, immune genes, and so on \u201358, whicThe expression of NCXs was decreased in CC tissues compared to adjacent normal tissues. The degree of downregulation of NCXs was positively correlated with the prognosis of CC. NCX1, NCX2, and NCX3 can be used as independent prognostic indicators of CC."} +{"text": "This paper proposes a new dynamic multi-objective optimization algorithm by integrating a new fitting-based prediction (FBP) mechanism with regularity model-based multi-objective estimation of distribution algorithm (RM-MEDA) for multi-objective optimization in changing environments. The prediction-based reaction mechanism aims to generate high-quality population when changes occur, which includes three subpopulations for tracking the moving Pareto-optimal set effectively. The first subpopulation is created by a simple linear prediction model with two different stepsizes. The second subpopulation consists of some new sampling individuals generated by the fitting-based prediction strategy. The third subpopulation is created by employing a recent sampling strategy, generating some effective search individuals for improving population convergence and diversity. Experimental results on a set of benchmark functions with a variety of different dynamic characteristics and difficulties illustrate that the proposed algorithm has competitive effectiveness compared with some state-of-the-art algorithms. F consists of m time-varying objective functions. x = defines the decision vector involving D variables, Li and Ui represent the lower and upper bounds of the ith variable xi, respectively. For two given decision vectors x and y, if \u2200j \u2208 , fi(x) \u2264 fi(y) and \u2203l \u2208 fl(x) < fl(y), then, x dominates y regarded as x \u227a y. If a vector x* can dominate any other solutions, x* is defined as Pareto optimal solution.The progress of optimizing multiple mutually conflicting objectives simultaneously and obtaining a set of tradeoff solutions is regarded as Multi-objective optimization problems (MOPs) , which it is the time instant of the problem.However, recent years, there exist an increasing number of multi-objective optimization problems recognised in various fields, such as scheduling , 6, planCompared with MOPs, dynamic dynamic multi-objective optimization problems have two important features: multiobjectivity and dynamism. It is generally known that multiobjectivity usually involves multiple conflicting objectives, which means the optimal solution of the problem will no longer be a single optimal value, but an optimal solution set containing tradeoff solutions. Dynamism in constraints and/or parameters causes the change of POF or POS and poses big difficulties to evolutionary algorithms. DMOPs are challenging due to the dynamic nature. They can be divided into a sequence of MOPs over the course of time. That is, the optimization goal is to obtain a sequence of approximations to the moving POS/POF.In recent years, much effort has been devoted to designing efficient and effective dynamic multi-objective evolutionary algorithms (DMOEAs). A widely used framework of DMOEAs in literature can be described as Algorithm 1. As shown in this framework, the whole procedure of solving DMOPs contains two main components: change detection and multi-objective algorithms including MOEAs and DMOEAs.Algorithm 1 The basic framework of DMOEAt = 1;1: Initialize time instance Popt;2: Generate an initial population 3: While the termination criterion is not satisfied4: Change Detection5: \u2003If change is not detected, evolve population using MOEA;6: \u2003Otherwise, evolve population using DMOEA;7: Return 3.As a significant component of DMOEAs, change detection is responsible for determining whether the environment has changed and in turn whether to implement a reaction mechanism. The existing dynamic extraction methods contain two categories: re-evaluating solutions \u201315 and cApart from the dynamic reaction mechanism, MOEAs are significant components of solvers for DMOPs, since DMOPs can be regards as a sequence of MOPs. That is, any MOEAs can be directly used to evolve the population during the (short) period of any static environments.As one of the most attractive and popular areas in intelligent computing field, the existing Multi-objective optimization algorithms can be classified into three categories as follows. The first class is Pareto ranking-based algorithms, which are designed based on the dominated relationships among population individuals. Some representative algorithms include the nond-ominated sorting genetic algorithm II (NSGA-II) , and strThe second class is indicator-Based algorithms, which are designed based on the performance indicators. The hypervolume , the epsDepending on the frequency or severity of change, changes may present various challenges, such as the finite computational time or resources to overcome the change, time-varying feasible region and constraint conditions. Therefore, effective and efficient dynamic multi-objective optimization algorithms are indeed important. Diversity and convergence are two important aspects in designing high-quality optimization methods, since the former aims to prevent the search from local optima whereas the latter helps algorithms to find promising solutions rapidly. Designing an effective strategy that is able to balance the diversity and convergence is one of the key topics in DMOPs. Existing Dynamic multi-objective optimization algorithms can be divided into four categories: diversity based algorithms, memory based algorithms, multi-population based algorithms, and prediction based algorithms.The main purpose of diversity based algorithms is to maintain the search population diversity for avoiding local optima when a change is detected. Recently, a increasing number of diversity maintenance methods have been proposed. A general framework proposed by Li maintainThe main idea of memory based algorithms is to record some historical information, which can be reused to accelerate the convergence of algorithms whenever a change occurs. Branke suggestsThe main idea of multi-population based algorithms is that multiple subpopulations can be advantageous at maintaining diversity. In , a self-et al. is used to predict the manifold of the whole search population by using the univariate auto-regression (AR) model. Besides that, many other prediction approach have been proposed in different ways, such as multi-directions Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0No**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The authors proposed a dynamic multi-objective optimization algorithm by integrating a new fitting based prediction (FBP) mechanism, good work; however, there are major comments to be addressed as follows:1) Introduction part is somehow poor, authors need to include a general description of Multi-objective optimization and the need for the multi-objective idea, then come to the dynamic multi-objective.2) The contribution should be clearly explained and this usually comes from the literature gaps.3) There is no section for related work. It is recommended to include a section named Related Work, in this section, the authors should critically review the recent studies on MO and DMO and find the limitations and drawbacks that drawn the problem they are trying to solve.4) Recent MO algorithms could be used for comparisons such as MOGWO, MOWOA, MOPSO, and others.5) No statistical test performed to find the significant difference between the proposed method and the state-of-the-art. It is recommended to use Anova test or t-test to find the p-value at a significant level less than 0.05.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article digital diagnostic tool,\u00a0 24 Jun 2021Revision Statement for \"Solving dynamic multi-objective problems with a new prediction-based optimization algorithm\"( No.PONE-D-21-11897)The referees' reviews are greatly appreciated and carefully considered in the revised version of the manuscript. The authors' responses to the reviewers and main changes in the revised version are summarized as follows.Academic EditorComment: Please submit your revised manuscript by Jun 18 2021 11:59PM.Response: Many thanks for the AE's time and assistance in dealing with our manuscript. We have carefully considered the reviewers' comments in the revision, and detailed responses to the comments are given as follows.Reviewer 1Comment 1: Introduction part is somehow poor, authors need to include a general description of Multi-objective optimization and the need for the multi-objective idea, then come to the dynamic multi-objective.Response: Many Thanks for the reviewer's valuable comments. We have revised the introduction section by a more rigorous literature review of the proposed area. The relevant description and concept of Multi-objective optimization have been added and marked in Introduction section in the 'Revised Manuscript with Track Changes'.Comment 2: The contribution should be clearly explained and this usually comes from the literature gaps.Response: We totally agree with the reviewer on this point. Therefore, we have cited the relevant literature gaps and added the relevant contents in the revised paper.\"...they neglect properties of decision variables, which is an important part of discovering.... Simultaneously, according to [54-55], curve fitting technique is a classic and popular technique, ... relationship between variables to a certain extent and predict possible regions or directions...\"Comment 3: There is no section for related work. It is recommended to include a section named Related Work, in this section, the authors should critically review the recent studies on MO and DMO and find the limitations and drawbacks that drawn the problem they are trying to solve.Response: Many thanks for your suggestion. In the revised paper, we have added the related work as Section 2 by increasing the review of on MO and DMO and providing their weaknesses. In detailed, the review of MO algorithms are divided into three categories, and some representative algorithms and limitations are also listed. For DMO, the existing algorithms were divided into four categories, and some representative algorithms and drawbacks are also presented.\"...As one of the most attractive and popular areas in intelligent computing field, ... optimization algorithms can be classified into three categories as follows...These algorithms can obtain good local optimal solutions, ...but it is difficult to achieve ideal global optimal solutions...Existing Dynamic multi-objective optimization algorithms can be divided into four categories: ..., and prediction-based algorithms....\"Comment 4: Recent MO algorithms could be used for comparisons such as MOGWO, MOWOA, MOPSO, and others.Response: We have enhanced experimental studies by carrying out more simulations according to this comment. We have compared with some well and recent MO algorithms to show the effectiveness of the proposed algorithm. In the revised manuscript, the relevant contents can be found in Section 4, and the simulation and comparison results are recorded in Table 10-12.Comment 5: No statistical test performed to find the significant difference between the proposed method and the state-of-the-art. It is recommended to use Anova test or t-test to find the p-value at a significant level less than 0.05.Response: Many thanks for pointing this out. We have added the t-test indicator in experimental studies, and summarized the comparison results at the bottom of each Table. This content can be found in all Tables in the revised manuscript.Many thanks for the supportive comments. We hope our effort address the reviewer's concerns with satisfaction. Kind RegardsAttachmentResponse to Reviewers.pdfSubmitted filename: Click here for additional data file. 5 Jul 2021Solving dynamic multi-objective problems with a new prediction-based optimization algorithmPONE-D-21-11897R1Dear Dr. zhang,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The authors did great efforts in response to the comments given in the first round of review. Most of the comments have been addressed. Therefore, the paper is acceptable for publication.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1:\u00a0No 21 Jul 2021PONE-D-21-11897R1 Solving dynamic multi-objective problems with a new prediction-based optimization algorithm Dear Dr. Zhang:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf. Seyedali Mirjalili Academic EditorPLOS ONE"} +{"text": "The UK prioritised delivery of the first dose of BNT162b2 (Pfizer/BioNTech) and AZD1222 (AstraZeneca) vaccines by extending the interval between doses up to 12 weeks. In 750 participants aged 50\u201389 years, we here compare serological responses after BNT162b2 and AZD1222 vaccination with varying dose intervals, and evaluate these against real-world national vaccine effectiveness (VE) estimates against COVID-19 in England.\u00a0We show that antibody levels 14\u201335 days after dose two are higher in BNT162b2 recipients with an extended vaccine interval (65\u201384 days) compared with those vaccinated with a standard (19\u201329 days) interval. Following the extended schedule, antibody levels were 6-fold higher at 14\u201335 days post dose 2 for BNT162b2 than AZD1222. For both vaccines, VE was higher across all age-groups from 14 days after dose two compared to one dose, but the magnitude varied with dose interval. Higher dose two VE was observed with >6 week interval between BNT162b2 doses compared to the standard schedule.\u00a0Our findings suggest higher effectiveness against infection using an extended vaccine schedule. Given global vaccine constraints these results are relevant to policymakers. The UK extended the interval until the second COVID-19 vaccine dose up to 12 weeks. Here, the authors show in a cohort of 750 participants aged 50\u201389 years that the extended schedule results in higher antibody titers and estimate a higher vaccine effectiveness for the extended schedule. In the United Kingdom, older adults were prioritised for vaccination at the start of the COVID-19 immunisation programme on 08 December 2020, initially with the Pfizer/BioNTech (BNT162b2) vaccine using the authorised 3-week interval between doses4. From 04/01/2021, the AstraZeneca (AZ) vaccine (AZD1222) was deployed and, with its more favourable storage and transport conditions, was used for vaccinating in care homes, community healthcare professionals and healthy adults aged 40\u201360 years. In January 2021, the UK Joint Committee on Vaccination and Immunisation (JCVI) recommended that with the emergence of a second wave as a result of the Alpha variant, the second dose of vaccine should be extended for up to 12 weeks to prioritise the first dose for those at highest risk of severe COVID-19 and death5. The decision to extend the second dose was based on early clinical trial data indicating nearly 90% effectiveness against SARS-CoV-2 within 3 weeks of the first dose of BNT162b2 vaccine compared to 95% from two weeks after the second dose6. Vaccinating more at-risk individuals quickly with a single dose was predicted to prevent more cases, hospitalisations and deaths than two doses at a 3-week interval7. This unique approach against authorised use and without formal clinical trials resulted in considerable international debate and prompted the need to evaluate immune responses and vaccine effectiveness following extended schedules.Older adults have been disproportionately affected by the COVID-19 pandemic, with age being the single most important risk factor for hospitalisations and deaths5. Real-world effectiveness studies indicate 50\u201370% protection against infection or mild disease for \u22658 weeks after one BNT162b2 dose and \u22656 weeks after AZD1222, with 75\u201385% protection against hospitalisation or death8.The COVID-19 vaccine responses after extended immunisation schedules (CONSENUS) evaluation aimed to assess immune responses in \u226550 year-olds receiving a COVID-19 vaccine as part of the UK extended immunisation schedule. Early analysis indicated that a single dose of BNT162b2 vaccine was associated with >94% seropositivity after 3 weeks in previously uninfected older adults, while two doses produced very high antibody levels, significantly higher than convalescent sera from adults with mild-to-moderate PCR-confirmed COVID-19We now report serological responses in 750 adults aged 50\u201389 years given two doses of BNT162b2 or AZD1222 at different intervals, comparing serological responses. These findings are evaluated against real-world vaccine effectiveness estimates against COVID-19 using similar dosing intervals in the same age group in England.2(1)\u2009=\u200940.3, p\u2009<\u20090.001).We recruited 750 participants aged 50\u201389 years - 421 received at least one BNT162b2 dose and 329 at least one AZD1222 dose seroconverting by 17\u201334 days and 35\u201355 days post-vaccination. S-antibody GMTs peaked by 35\u201355 days after vaccination at 29.8 (95%CI: 24.9\u201335.6) for 64\u201379 year-olds and 41.7 (95%CI: 28.5\u201360.8) for 80\u201389 year-olds, with levels sustained to 77\u201397 days post-dose 1 of 50\u201364 year-olds and 81.7% (49/60) of 65\u201379 year-olds seroconverted at 17\u201324 days, which increased to 95.6% (198/207) overall at 35\u201355 days. GMTs continued to increase from 13.7 (95%CI: 7.5\u201324.9) at 17\u201334 days to 38.2 (95%CI: 24.9\u201358.7) at 35\u201355 days in 50\u201364 year-olds, whilst, in older adults, the GMT peak was delayed until 56\u201376 days. The GMR for S-antibody was 62.01 (95%CI: 47.69\u201380.64) for BNT162b2 vaccine recipients aged 65\u201379 years at 17\u201334 days compared to pre-vaccine, followed by a GMR of 1.2 (95%CI: 1.08\u20131.34) from 17\u201334 days to 35\u201355 days after dose 1. For the AZD1222 vaccine in the same age group, GMRs were 14.68 (95%CI: 9.68\u201322.26) and 4.17 (95%CI: 3.28\u20135.3), respectively. In previously-uninfected individuals, GMTs remained lower in the 13 weeks post-dose 1 for both vaccines compared to convalescent sera from mild-to-moderate PCR-confirmed cases at 17\u201334 days after dose 1, this ratio declined with time and was no longer statistically significant at 56\u201376 days post-vaccination and 1.19 (VE: \u221219%) for AZD1222 and BNT162b2 in 65\u201379 year-olds, respectively 19\u201329, (ii) 45\u201364, (iii) 65\u201384, and (iv) \u226585 days. Sampling timepoints post-dose 2 were divided into 7\u201313 and 14\u201334 days. The vaccine used and dose intervals varied by age group depending on national recommendations and vaccine supply. Those receiving BNT162b2 at 19\u201329 day intervals and those reaching 85+ days for either vaccine were older whilst those receiving AZD1222 at 45\u201365 and 65\u201384 intervals were younger because initial vaccine rollout prioritised older adults and recommended a 3-week interval between doses.N\u2009=\u2009200; BNT162b2 extended: N\u2009=\u2009282 and BNT162b2 control: N\u2009=\u200987). BNT162b2 dose 2 responses were quick, peaking at 7\u201313 days followed by a 23% decline at 14\u201334 days. Amongst BNT162b2 recipients, GMTs were tenfold higher at 14\u201334 days post-dose 2 following a 65\u201384 day interval compared with a 19\u201329 day interval compared to AZD1222 at 14\u201334 days post-dose 2 , but not by AZD1222, where the GMT was 9633.2 (95%CI: 6233.9\u201314886.3) at 14\u201334 days post-dose 2.VE was higher across all age groups for both vaccines from 14 days after dose 2 compared to dose 1 but the magnitude depended on the interval between doses Fig.\u00a0. AmongstAmongst AZD1222 recipients aged \u226580 years, two-dose VE after 14 days was 96%\u00a0(95%CI:\u00a068\u201399) and 82% (95%CI: 68\u201389) following 45\u201364 and 65\u201384 days intervals, respectively . Additionally, it was anticipated that delaying the second dose would enhance boosting, extending the longevity of protection following a second dose. Lengthening intervals between vaccine doses to enhance boosting is well-recognised from studies of other vaccines, including hepatitis B13. Whilst other studies have reported higher GMTs following vaccination, direct comparison is not possible given the titres have been reported in different units using different serological assays15. The current study, which includes a wider age range, found no evidence of a difference in antibody decline with age after either dose, although a recent study reported lower serum neutralisation and binding IgG/IgA after the first dose with increasing age and lower potency against SARS-CoV-2 variants of concern (VOC) among \u226580 year-olds16. Following the second dose, neutralisation against VOC was detectable regardless of age.With BNT162b2, we found 8\u201310-fold higher GMTs after the second dose with a 6\u20139 or 10\u201313 week interval compared with the authorised 3-week interval, which was also associated with the more rapid waning of up to 50% between 1.5\u20133 weeks and 19 weeks after dose 2. These findings are consistent with our other as-yet unpublished study in \u226580 year-olds, where peak antibody responses were 3.5-fold higher following extended-schedule BNT162b2 immunisation although, interestingly, cellular responses were 3.6-fold lower with the extended interval schedule17. In our cohort, too, which focused on older adults, AZD1222 recipients had twofold higher antibodies after a 10\u201313 compared to 6\u20139 week interval.Among AZD1222, the initial clinical trials allowed permissive intervals between vaccine doses which showed minimal waning of antibodies or protection against symptomatic COVID-19 for up to 3 months after the first dose in healthy working-age adults, with better boosting after the second dose after a longer intervalIn previously-infected adults, we observed significant boosting of antibody responses after the first dose of both vaccines and after the second dose of BNT162b2, but not AZD1222. Notably, though, antibody GMTs following two doses of either vaccine were higher than those observed following mild-to-moderate COVID-19 regardless of interval.18. At the same time, BNT162b2 was preferentially used for healthy healthcare workers in hospitals early in the national vaccine rollout and older age groups vaccinated in the community. Since BNT162b2 was deployed earlier than AZD1222, we have longer population follow-up for this vaccine. Whilst the analyses adjust for key confounding variables, such as period and risk groups, whilst excluding those with previous COVID, it is possible that residual confounding persists to some extent. Additionally, the Alpha variant was dominant during the majority of the study period, which will affect comparisons with other international VE estimates where other variants were circulating and the follow-up time after the second dose differed20. Since May 2021, this has been replaced by the Delta variant, and whilst differences in VE between Alpha and Delta have been reported, VE remains high against hospitalisations and mortality more than 20 weeks post-vaccination23. VE estimates suggest evidence of a decline in VE beyond 10 weeks in those aged 80+ years, which could indicate immune senescence within this age group. However, large confidence intervals make interpretation difficult and further work is required to better understand differences in VE over time across the age groups.We were also able to compare immunogenicity with real-world VE data in England, which show substantial protection against symptomatic disease from 14 days after dose 2. Higher two-dose VE was observed with > 6-week intervals between BNT162b2 doses compared to the authorised 3-week schedule, including \u226580 year-olds. Among AZD1222 recipients, two doses provided the highest protection among \u226580 year-olds regardless of interval. Surprisingly, two-dose VE among 50\u201364 year-olds was lower, even when compared with adults in the same age group receiving two BNT162b2 doses. This may be due to the differential use of the vaccines in the national immunisation programme-, because AZD1222 vaccines do not require ultra-low temperatures for storage or transport, they were preferred for vaccinating care home residents who a higher risk of natural exposure and pre-existing immunity prior to vaccination, and for clinical risk groups in the community8. As of 28/06/2021, the vaccination programme is estimated to have prevented nearly 8 million infections and 27,000 deaths in England alone25.Notwithstanding this, it is important to emphasise that a single dose of either vaccine remains highly effective against severe endpoints, which is the primary aim of the vaccination programme, with 75\u201385% protection against hospitalisation in the oldest cohorts27. Despite this, S-antibodies have been found to correlate well with neutralising antibodies29.The strength of this study is the combination of sero-surveillance with real-world national VE data for two different vaccines in different age groups, including older adults who were excluded from initial clinical trials, with variable, real-world dosing intervals. Serological assessments provide an objective measure of vaccine responses which are important for comparing vaccines and schedules, but interpretation of serological data is limited as the way in which it correlates with protection is unknown, and the recognition that neutralising activity of antibodies and cellular immunity also play an important role in protectionAs with any observational study, there are limitations to VE analysis. There may be confounding factors that could increase the risk of COVID-19 in vaccinated individuals, for example, if vaccinated individuals adopted riskier behaviours after vaccination or unvaccinated individuals isolated themselves to reduce their risk of viral exposures. In addition, VE could be attenuated if there are high levels of protection from previous infection in the population or if there is misclassification of cases and test negative controls due to low sensitivity or specificity of PCR testing.Our findings suggest higher effectiveness against infection using an extended vaccine schedule. Given the global vaccine constraints, these results are relevant to policymakers in low and middle income countries especially in the context of highly transmissible variants and rising incidence in many parts of the world. An additional yet undervalued benefit of extended schedules is higher boosting and better protection after two doses of either vaccine, which potentially confer better protection against variants and for a longer duration than short-interval schedules. Our data also confirm previous findings of high protection after a single vaccine dose in previously-infected individuals, which is also important in the context of limited vaccine supplies. Ongoing evaluation of the protection conferred against new variants using an extended schedule will be critical.CONSENSUS recruited immunocompetent adults aged The sample size was determined based on providing reasonable precision for estimates of geometric mean concentrations at each timepoint, age group, the interval between doses and vaccine type for those not previously infected. To calculate precision an estimate of the standard deviation of responses post-vaccination was required. In the absence of data post-vaccination, this was estimated to be 0.5 log10 units based on data on responses post-natural infection. Using this estimate, and considering that recruitment may be variable in different groups the 95% confidence interval \u00b1fold-widths were estimated to be \u00b154%, \u00b139% and \u00b126% for sample sizes of 30, 50 and 100.The final numbers of samples available for analysis are shown in Tables\u00a031. Samples were tested according to the manufacturer\u2019s instructions, with additional dilutions if necessary to get an endpoint S-antibody level.Serum samples were tested for nucleoprotein (N) antibodies as a marker of previous SARS-CoV-2 infection and spike (S) protein antibodies, which could be infection- or vaccine-derived /mL to assess vaccine response; ref: 09289275190)8. The sample size was based on all available pillar 2 symptomatic cases and controls.A test-negative case-control design was used to estimate odds ratios for testing SARS-CoV-2 positive to in vaccinated compared with unvaccinated people with COVID-19 compatible symptoms who were tested using polymerase chain reaction (PCR), as described previously32. Pillar 2 tests performed between 26/10/2020 and 18/06/2021 we extracted for those who reported being symptomatic.All healthy adults aged \u226550 years in England were eligible for inclusion. Testing for COVID-19 in the UK is done through the hospital and public health laboratories (pillar 1) and more widely through community (pillar 2) testing33.Testing data were linked to individual vaccination histories in the National Immunisation Management System (NIMS), using unique National Health Service numbers, date of birth, surname, first name and postcode. NIMS data were extracted on 21/06/2021 with immunisation records up to 20/06/2021Geometric mean antibody titres (GMTs) were calculated with 95% confidence intervals (CI). Geometric mean ratios (GMR) of responses between timepoints were estimated using a mixed regression model on log responses including a random effect for each participant, separate models were fitted for each vaccine group. The GMR of responses by vaccine type at each post-vaccination timepoint was estimated via regression on log RocheS responses and included age group and sex. Statistical analyses were performed using STATA v.14.2. Individuals testing positive on the Roche N assay were considered to have had prior SARS-CoV-2 infection. Infection status was changed if a seronegative participant seroconverted on the Roche N assay during the study and remained positive thereafter.Logistic regression was used to estimate the odds of vaccination in PCR-confirmed cases compared with those who tested negative for SARS-CoV-2. Only those swabbed within 0\u201310 days of symptom onset were included in the analysis because the sensitivity of PCR testing decreases beyond 10 days after symptom onset. Vaccine effectiveness was calculated as 1 minus odds ratio.8 Analyses were run separately for 50\u201364, 65\u201379, 80+ and 80+(early cohort) year-olds. For 50\u201364 year-olds, only unvaccinated or vaccinated individuals from 01/02/2021 were included because, before this, only health and social care workers were eligible in this age group. For age 65\u201379 and 80+ year-olds, a cut-off date of 04/01/2021 was used when the AZD1222 vaccine became available. The 80+ early cohort age group included only those unvaccinated or first dose vaccinated before 04/01/2021 with the BNT162b2 vaccine, mostly with a 3-week interval between doses.To estimate vaccine effectiveness in fully susceptible people, we excluded those with a previous positive PCR or antibody result prior to 8 December 2020. Estimates were adjusted for a week of onset, 5-year age bands, gender, NHS region, index of multiple deprivation (quintiles), ethnicity, health/social care worker, care home resident, flagged as clinically extremely vulnerable in NIMS, and flagged as being in the extended risk groups in NIMS among <65 year-olds onlyVE was estimated by vaccine manufacturer and according to intervals after the first dose as well as from 14 days after the second dose split by intervals between first and second doses of 19\u201329, 30\u201344, 45\u201364, 65\u201384 and 85+ days.Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"} +{"text": "We report that in a cohort of 45,965 adults, who were receiving either the ChAdOx1 or the BNT162b2 SARS-CoV-2 vaccines, in those who had no prior infection with SARS-CoV-2, seroconversion rates and quantitative antibody levels after a single dose were lower in older individuals, especially in those aged >60 years. Two vaccine doses achieved high responses across all ages. Antibody levels increased more slowly and to lower levels with a single dose of ChAdOx1 compared with a single dose of BNT162b2, but waned following a single dose of BNT162b2 in older individuals. In descriptive latent class models, we identified four responder subgroups, including a \u2018low responder\u2019 group that more commonly consisted of people aged >75 years, males and individuals with long-term health conditions. Given our findings, we propose that available vaccines should be prioritized for those not previously infected and that second doses should be prioritized for individuals aged >60 years. Further data are needed to better understand the extent to which quantitative antibody responses are associated with vaccine-mediated protection. Longitudinal tracing of antibody responses to the ChAdOx1 and the BNT162b2 COVID-19 vaccines in 45,965 adults from the United Kingdom give indications for vaccine prioritization. Both vaccines have been widely used in the UK.Multiple vaccines have been developed that offer protection against COVID-19 by generating immune responses against the spike antigen of SARS-CoV-2. On 8 December 2020, the United Kingdom (UK) started its national vaccination programme with the Pfizer\u2013BioNTech BNT162b2 vaccine3. To maximize initial coverage, in early January 2021, the dosing interval was extended to 12 weeks for all vaccines, regardless of the licensed dosing schedule. Up until 6 April 2021, 31.7 million people (60.2% of the population aged \u226518 years) have been given a first dose, and 5.7 million people (10.8%) have received two vaccine doses (https://coronavirus.data.gov.uk/details/vaccinations).Vaccines were initially administered to priority groups, including care home residents, people >80 years old, healthcare workers and those clinically vulnerable (\u226516 years), and then offered to the rest of the adult (\u226518 years) population in decreasing age order4), and 70% (95% CI\u2009=\u200955\u201381%) after the second dose of ChAdOx1 (ref. 5). Several studies have examined the immunogenicity of vaccines in healthcare workers, who were typically the earliest groups to be vaccinated. A study of 3,610 healthcare workers found that 99.5% and 97.1% seroconverted after a single dose of BNT162b2 or ChAdOx1, respectively, and that higher quantitative immunoglobulin G (IgG) levels were achieved in previously infected individuals6. Other studies have also found that single-dose BNT162b2 elicited higher antibody levels in previously seropositive individuals, levels that were comparable to those after two doses of vaccines in seronegative individuals9. Outside trials, there are limited data on post-vaccine antibody responses in other groups, especially older adults who were underrepresented in the ChAdOx1 trial5. A study of 185 individuals aged >70 years showed high seropositivity after one or two BNT162b2 doses10. Another study, of 100 individuals aged 80\u2013100 years, showed almost universal high antibody responses 3 weeks after a single dose of BNT162b2, with spike-specific cellular responses in 63% of participants11. However, the representativeness of these small cohorts is unclear.The efficacy of the ChAdOx1 and BNT162b2 vaccines against symptomatic laboratory-confirmed SARS-CoV-2 infection has been reported in large randomized controlled clinical trials as 52% \u2009=\u200930\u201386%) after the first dose and 95% (95% CI\u2009=\u200990\u201398%) after the second dose of BNT162b2 (ref. ISRCTN21086382), which includes a representative sample of households and has longitudinal follow-up, to study population-wide anti-trimeric spike IgG antibody responses after SARS-CoV-2 vaccination by time since vaccination, considering the vaccine type (BNT162b2 or ChAdOx1), the number of doses received, the presence or absence of prior SARS-CoV-2 infection and demographic factors. Our results build on the REACT-2 study, a serial cross-sectional UK study of antibody responses using a binary point-of-care lateral flow assay12. Specifically, we investigate longitudinal data in the same individuals with a validated quantitative laboratory antibody assay, which has previously been shown to correlate with neutralizing activity (correlation coefficient of 0.76)13, allowing the assay to act as a potential correlate of protection based on the strong association between quantitative neutralizing activity and protection from infection14. Supporting this, quantitative readings from the assay are associated with protection from infection in those previously infected15.Real-world data provide information on populations who may not participate in clinical trials and can be used to assess the efficacy of interventions as deployed. We used the UK\u2019s national COVID-19 Infection Survey post-vaccine antibody responses showed that positive anti-spike IgG results increased over the 2\u20134 weeks after the first vaccination and varied significantly by age Fig. \u20137. FewerIn participants with prior evidence of infection, before vaccination, younger participants were more likely to be seropositive. For example, on the day of vaccination 90% (95% CI\u2009=\u200982\u201395%) for 20 year olds, 85% (95% CI\u2009=\u200980\u201388%) for 40 year olds, 78% (95% CI\u2009=\u200975\u201382%) for 60 year olds and 70% (95% CI\u2009=\u200961\u201378%) for 80 year olds receiving ChAdOx1 were seropositive (Supplementary Table P\u2009=\u20090.02). Seroconversion by 60 days was less common following ChAdOx1 than after BNT162b2 vaccination (aOR\u2009=\u20090.47 [95% CI\u2009=\u20090.44\u20130.51]), while receiving two doses of BNT162b2 increased seroconversion compared with one BNT162b2 dose (aOR\u2009=\u20092.11 [1.69\u20132.66]). Patient-facing healthcare workers were more likely to be anti-spike IgG positive by 60 days post-vaccination (aOR\u2009=\u20091.63 [1.29\u20132.08]), and participants who had long-term health conditions were less likely (aOR\u2009=\u20090.64 [0.60\u20130.69]). There was evidence of greater seropositivity post-vaccination in participants from non-white ethnic groups (aOR\u2009=\u20091.54 [1.27\u20131.90]). A 10-unit increase in deprivation percentile resulted in higher seropositivity post-vaccination (aOR\u2009=\u20091.28 [1.13\u20131.46]). There was no evidence of independent associations between antibody positivity and household size or working in social care or long-term care facilities.A total of 28,144 participants had an anti-spike IgG measurement 14\u201360 days after their first ChAdOx1 or BNT162b2 vaccination, of whom 24,977 (88.7%) had no evidence of prior infection and were included in a separate logistic regression analysis to investigate associations with antibody positivity. In all, 20,505 (82.1%) had a positive post-vaccine anti-spike IgG result. Age, sex, vaccine type, ethnicity, social deprivation, healthcare roles and long-term health conditions were associated with seropositivity after vaccination Table . There w\u20131 equivalents, with 95% CIs in parentheses) were reported for ChAdOx1 and BNT162b2, respectively: 73 (65\u201381) and 113 (104\u2013123) for 80 year olds; 94 (87\u2013100) and 163 (153\u2013175) for 60 year olds; 113 (99\u2013129) and 236 (214\u2013261); for 40 year olds; and 127 (94\u2013171) and 334 (266\u2013420) for 20 year olds achieved at titres of 28\u2009ng\u2009ml\u20131. The rate of increase in antibody levels was also slightly slower following the ChAdOx1 vaccine. For example, the estimated mean time to reaching the threshold for antibody positivity after the first vaccine in 40 year olds was 10 days after receiving BNT162b2 but 14 days after receiving ChAdOx1 . Many health conditions were more common in low responders and asthma (1.25 [1.03\u20131.52]) independently associated with low responses.We used descriptive latent class mixed models to identify evidence for different subgroups of responses after vaccination. The best-fitting models identified four classes of antibody responses post-vaccination for both vaccines Fig. . In a \u2018p7. A single dose of ChAdOx1 resulted in lower absolute antibody levels and slower responses compared with a single dose of BNT162b2. However, antibody levels after a single dose of BNT162b2 waned over time, whereas levels remained approximately constant after a single dose of ChAdOx1. Importantly, we did not identify a group who did not respond at all to vaccination; however, ~6% of participants were low responders to both vaccines, with low responses independently associated with several long-term health conditions.In this study based on 45,965 vaccinated participants from a large random sample of the UK population, we showed that post-vaccine anti-spike IgG responses vary by prior infection status, age, sex, vaccine type and number of doses received. In those who were previously infected, all age groups achieved high antibody responses after the first vaccination. In those without evidence of prior infection, older participants had lower and slower responses after the first vaccine dose than younger participants. Two vaccine doses achieved high responses across all age groups, and particularly increased the number of older people seroconverting to similar levels to those receiving one dose after prior infection, as recently reported in a smaller number of younger individuals12 of binary point-of-care lateral flow assay results after a single dose of BNT162b2. However, our results showed a much higher antibody response than reported in REACT-2, especially in older people, despite studying the same UK population. These differences probably reflect the lower sensitivity of the assay used in REACT-2, despite efforts to adjust for this12. In our study, mean quantitative responses were not far from the positivity threshold, particularly for older age groups, which demonstrates the challenge in applying binary thresholds to what are essentially continuous data. This is particularly important given that the antibody levels required for protection are still unclear, with a study using the same assay as our study identifying a gradient of protection associated with quantitative antibody levels below the positivity threshold following previous infection15. Our study provides additional comparative data on antibody responses following the ChAdOx1 vaccine. Studies of healthcare workers also support an inverse association between antibody response and age in those receiving a single dose of the BNT162b2 vaccine17 or the ChAdOx1 vaccine6.The relative differences in vaccine response by age and previous infection status are similar to those reported by the REACT-2 study18. These findings are consistent with observations that females generate stronger humoral immunity and greater vaccine efficacy than males20. However, a UK study of 3,610 healthcare workers (median age of 41 years) did not find any association between sex and single-dose ChAdOx1 or BNT162b2 antibody responses6, which is possibly explained by our finding that sex differences in antibody responses become more marked above 60 years of age.We found in those without evidence of previous infection, at older ages, females had a higher probability of being IgG positive post-vaccination than males, and females were more likely to be in the high-response latent class. Sex differences in antibody levels have also been described following natural infection21, we found that in previously infected participants, a single dose of ChAdOx1 or BNT162b2 led to high anti-spike IgG antibody positivity and quantitative levels. Where vaccine supplies are limited, this supports prioritizing those without evidence of previous infection for vaccination and, in particular, delaying or omitting second doses in those with robust serological evidence of previous infection in these scenarios. We also find that receiving two vaccine doses significantly increased seropositivity and antibody levels in older participants, but the short-term incremental increase in 20\u201340 year olds with a second vaccine was much smaller, thereby suggesting that older age groups should be prioritized for a second vaccination. However, protection from infection following seroconversion is not absolute6, with seroconversion rates after the first dose vaccination exceeding the reduction in symptomatic infection. Therefore, vaccine efficacy against clinical outcomes as well as antibody responses should contribute to prioritization decisions. In a related UK-wide study22 and a study from Israel23, high levels of protection from infection following natural infection were observed that were comparable to those seen after two doses of vaccination without prior infection. In the latter study23, the authors question whether previously infected individuals require vaccination; our data show that vaccination does boost antibody responses after previous infection, although the impact on protection from infection over varying timescales requires further study.Consistent with several previous studies24. Similar short-term (6 weeks) protection against symptomatic infection, hospitalization and death with single doses of both vaccines was also seen in adults aged >70 years in England25.Our latent class analysis identified four distinct types of vaccine response. The low-response class was more common in older participants and those with long-term health conditions, but comprised a similar percentage receiving the different vaccines. Further follow-up is needed to identify whether the modest increases in antibody levels achieved still lead to some protection against key outcomes such as hospitalization, death or onward transmission, and to what extent second vaccine doses boost this initial suboptimal response. This low-responder group could be identified by a negative antibody result on our assay from day 28 post-vaccine. Similar underlying latent classes were identified following single doses of the two vaccines, with different mean responses overall due to different percentages estimated to fall into the high-response and medium-response classes for ChAdOx1 and BNT162b2. Further studies are also required to assess whether different degrees of response are associated with different rates of waning over time and protection against clinical outcomes. A recent study of 10,412 of long-term care residents showed 65% and 68% protection against laboratory-confirmed SARS-CoV-2 infection 28\u201342 days after vaccination with the ChAdOx1 and BNT162b2 vaccines, respectively, which suggests that differences in antibody responses may have a limited impact on outcomes, at least in the short term22, we found a 76% reduction in symptomatic SARS-CoV-2 infection following a first vaccine dose, rising to 95% after two doses, with no evidence of differences after BNT162b2 and ChAdOx1 vaccinations. A major outstanding question is the extent to which antibody titres are correlates of post-vaccination protection and its duration. Pooled data from clinical trials and a post-infection study show a strong relationship between mean neutralization levels and reported protection14, estimating that protection from infection is likely to fall over 250 days , but with largely preserved protection from severe infection. In this current study, antibodies were measured in only a subset of survey participants, so data are currently insufficient to directly estimate the relationship between post-vaccine antibody titres and protection from infection. However, previous studies provide some indication. For example, using the same assay, post-infection antibody titres of 28\u2009ng\u2009ml\u20131 mAb45 equivalents (or 7 million fluorescence units) were associated with 50% protection from any subsequent PCR-positive result in healthcare workers15. Most of those vaccinated in this study achieved levels of >28\u2009ng\u2009ml\u20131, including at older ages, for example, mean 73 and 113\u2009ng\u2009ml\u20131 for 80 year olds 28 days after the first ChAdOx1 or BNT162b2 dose, respectively. Only ~6% of individuals were in the low-responder latent class, with peak antibody levels reaching only ~10\u2009ng\u2009ml\u20131.Using data from all participants in the national survey26.Study limitations include currently insufficient data to analyse responses following two doses of ChAdOx1 (533 participants without and 66 with prior infection). Data on antibody responses between 8 and 12 weeks after the first dose without a second dose were also limited. Further follow-up will be required to assess the duration of responses to all vaccines and how variations in the interval between first and second doses affects this. Although our study is representative of those vaccinated to date in the UK, vaccination prioritization means that we have fewer data on healthy younger adults. We show that multiple long-term health conditions are associated with lower antibody responses, but additional studies are required to understand their significance for vaccine protection. Our study assesses responses using a single assay; however, responses were calibrated to a monoclonal antibody such that these can be readily compared with other studies. Neutralizing antibody and T-cell responses were not assayed in this study. However, a recent much smaller study of T-cell responses post-vaccination in healthcare workers found qualitatively similar findings27. Further data from this study and others will be needed to assess the extent to which quantitative antibody levels can be used as a correlate of vaccine-mediated protection.In summary, in this population-representative study of individuals vaccinated to date in the UK, vaccination results in detectable SARS-CoV-2 anti-spike IgG levels in the majority of individuals after first vaccination. High rates of seroconversion and high quantitative antibody levels following one dose of vaccine after previous infection and in younger previously uninfected individuals potentially supports single dose or delayed second dose vaccination in these groups if vaccine supplies are limited, although the potential for this to lead to antigenic evolution requires investigationISRCTN21086382) from 26 April 2020 to 6 April 2021. The survey randomly selects private households on a continuous basis from address lists and previous surveys conducted by the ONS or the Northern Ireland Statistics and Research Agency to provide a representative sample across the four countries constituting the UK . Following verbal agreement to participate, a study worker visited each household to take written informed consent. This consent was obtained from parents/carers for those aged 2\u201315 years, while those aged 10\u201315 years also provided written assent. Children aged <2 years were not eligible for the study. At the first visit, participants were asked for consent for follow-up visits every week for the next month, then monthly for 12 months from enrolment. For a random 10% of households, those aged \u226516 years were invited to provide blood monthly for serological testing from enrolment. Nose and throat swabs were taken from all consenting household members, according to the follow-up schedule they agreed to at enrolment. Any individual aged \u226516 years from a household where anyone tested positive on a nose and throat swab was also invited to provide blood at all subsequent monthly visits. Participants provided survey data on sociodemographic characteristics and vaccination status. Details on the sampling design are provided elsewhere28. The study protocol is available at https://www.ndm.ox.ac.uk/covid-19/covid-19-infection-survey/protocol-and-information-sheets. The study received ethical approval from the South Central Berkshire B Research Ethics Committee (20/SC/0195).We used data from the UK\u2019s Office for National Statistics (ONS) COVID-19 Infection Survey \u2009=\u20090.221738\u2009+\u20091.751889\u2009\u00d7\u200910\u22127\u2009\u00d7\u2009fluorescence units\u2009+\u20095.416675\u2009\u00d710\u22127\u2009\u00d7\u2009\u2009\u00d7\u2009.SARS-CoV-2 antibody levels were measured using an ELISA detecting anti-trimeric spike IgG developed by the University of Oxford\u20131 was used to identify IgG-positive samples, corresponding to the 8 million units with fluorescence detection. In this analysis, measurements <2\u2009ng\u2009ml\u20131 and >500\u2009ng\u2009ml\u20131 were truncated at 2 and 500\u2009ng\u2009ml\u20131, respectively.A threshold of \u226542\u2009ng\u2009ml28) and PCR traces exhibit an appropriate morphology.PCR assays of combined nose and throat swabs were undertaken using the Thermo Fisher TaqPath SARS-CoV-2 assay at high-throughput national \u2018Lighthouse\u2019 laboratories in Glasgow and Milton Keynes (up until 8 February 2021). PCR outputs were analysed using UgenTec FastFinder 3.300.5, with an assay-specific algorithm and decision mechanism that allows conversion of amplification assay raw data into test results with minimal manual intervention. Samples are called positive if at least a single N-gene and/or ORF1ab are detected values are determined, S-gene detection alone is not considered sufficient to call a sample positive10(mAb45 units)) anti-spike IgG antibody measurements after the first vaccination. Given the prior hypothesis that response to vaccination would vary differentially by age and time according to vaccine type and prior infection, we built separate models by vaccine type, for those receiving one or two vaccinations, and by prior infection status. For participants receiving one vaccine dose, four models were fitted, for each vaccine and in those with and without evidence of prior infection. Two-dose models were only fitted for those receiving BNT162b2 without evidence of prior infection, as the sample sizes were small for other groups: 315 participants with prior infection receiving two BNT162b2 doses; 533 participants without prior infection receiving two ChAdOx1 doses; 66 participants with prior infection receiving two ChAdOx1 doses and quantitative . We excluded measurements taken after the 90th percentile of observed time points for all models to avoid undue influence from outliers at late time points. Any participant receiving a second BNT162b2 dose after the 90th percentile for the single BNT162b2 dose group (61 days) was censored at this time point and included in the one-dose group participants were censored in this way). Age was truncated at 85 years in all analyses to avoid outlier influence.Models were adjusted for participant age using a tensor product of B-splines to allow for nonlinearity and interaction between age and time since vaccination. The smoothing penalty was selected using fast restricted maximum likelihood as implemented in the mcgv R package. We included a random intercept for each participant to account for repeated measurements using a random effect smoother with the number of basis functions equal to the number of participants. The 95% CIs were calculated using the following formula: prediction\u2009\u00b1\u20091.96\u2009\u00d7\u2009standard error of prediction. The date of the first vaccination was set as P value was <0.05.To investigate predictors of antibody response in those without prior evidence of infection, we considered the latest antibody measurement per participant between 14 and 60 days after the first vaccine. We used multivariable logistic regression to examine the association between antibody positivity and vaccine type and doses received by this measurement time, demographic factors , household size, deprivation ranking , whether the participant reported working in patient-facing healthcare or social care, whether they reported working in a care home (any role), and whether they reported having a long-term health condition. Nonlinearity in age was accounted for using restricted natural cubic splines with internal knots at the 20th, 40th, 60th and 80th percentiles of unique values, and boundary knots at 5th and 95th percentiles. We tested for and added interactions between age and other variables if the interaction For those without evidence of prior infection who received a single dose of vaccine, we also investigated whether we could identify distinct patterns of antibody responses, using latent class mixed models to identify subgroups with different antibody trajectories after the first vaccination. Natural cubic splines were used to model time since vaccination as a fixed effect and a random intercept was added to account for individual variability. Within-class between-individual heterogeneity may also be present in the trajectories; however, models accounting for random slopes failed to converge. Age with natural cubic splines (same as above), sex, reported long-term health conditions and whether the participant was a healthcare worker were included as covariates for class membership. The 95% CI of the estimation was calculated by a Monte Carlo approximation of the posterior distribution of the predicted values, using the 2.5% and 97.5% percentiles. The number of classes was determined by minimizing the Bayesian information criterion for each vaccine, and then fitting the maximum number of classes (four) to both groups for comparability.To compare prevalence of long-term health conditions across different subgroups identified, participants from England were linked to the General Practice Extraction Service Data for Pandemic Planning and Research via their NHS number ). A range of pre-existing conditions across organ systems were identified from diagnosis codes over the 10-year look-back period 1 January 2010 to 24 January 2020 (the date of the first known case of COVID-19 in the UK). Body\u2013mass index was the most recently recorded measurement over the look-back period, without imputation. Participants were recorded as being on antihypertensive medication, diabetes medication, corticosteroids or immunosuppressants if they were prescribed these treatments within 90 days of the end of the look-back period. All clinical variables were derived from primary care records only; hospital admissions data were not used.\u20131. For the logistic model for antibody response 14\u201360 days post-vaccination, the C-statistic showed modest discriminatory power (0.66), but there was no evidence of misspecification (Homer\u2013Lemeshow P\u2009=\u20091.00).Analyses were performed using the tidyverse (v.1.3.0), mgcv (v.1.8-31), splines (v.3.6.1), lcmm (v.1.9.2), ggeffects (v.0.14.3), sandwich (v.3.0-0), arsenal (v.3.4.0), emmeans (v.1.5.1), cowplot (v.1.1.0), gmodels (v.2.18.1) and mgcViz (v.0.1.6)) libraries in R (v.3.6). Model diagnostics used residual checks for generalized additive models, including distributions and quantile\u2013quantile plots using check.gamViz, which showed normally distributed residuals but with some skew due to the assay upper limit of 500\u2009ng\u2009mlFurther information on research design is available in the Supplementary Information\u20131) with 95% CI according to age, vaccine type and dose, and prior infection status by weeks after vaccination. Supplementary Table 4: Characteristics of classes identified from latent class mixed models for single dose ChAdOx1 and BNT162b2 vaccines in those without evidence of prior infection. Supplementary Table 5: Previous health conditions identified from linked primary care records in 29,575 participants from England without evidence of prior infection.Supplementary Tables 1\u20135. Supplementary Table 1: Participants\u2019 characteristics by cohort. Supplementary Table 2: Predicted percentage probability of anti-spike IgG seropositivity with 95% CI according to age, vaccine type and dose, and prior infection status by weeks after vaccination. Supplementary Table 3: Predicted anti-spike IgG levels (ng\u2009mlReporting Summary"} +{"text": "Cancer treatments often require intensive use of healthcare services and limit patients\u2019 ability to work, potentially causing them to become financially vulnerable. The present study is the first attempt to measure, on the German national level, the magnitude of absolute income loss after a cancer diagnosis.This study analyzes data from the Socio\u2010Economic Panel (SOEP) survey, one of the largest and most comprehensive household surveys in Germany, consisting of approximately 20,000 individuals, who are traced annually. The empirical strategy consists of ordinary least squares (OLS) and multinomial logistic estimators to measure changes in job income, work status, working hours, and pension as a result of reporting a cancer diagnosis for the period between 2009 and 2015. Sample consistency checks were conducted to limit measurement error biases.Our results show that job incomes dropped between 26% and 28% within the year a cancer diagnosis was reported. The effect persisted for two years after the diagnosis and was no longer observable after four years. The finding was linked to an increased likelihood of unemployment and a reduction of working hours by 24%. Pension levels, on the other hand, were not affected by a cancer diagnosis.These findings suggest that many cancer patients are exposed to financial hardship in Germany, particularly when the cancer diagnosis occurs during their working age and before requirements to obtain a pension are met. Further research seems warranted to identify particularly vulnerable patient groups. The present study is the first attempt to measure, on the German national level, the magnitude of income loss after a cancer diagnosis. This study analyzes data from the Socio\u2010Economic Panel (SOEP) survey, one of the largest and most comprehensive household surveys in Germany, consisting of approximately 20,000 individuals, who are traced annually. Our empirical results suggests that many cancer patients are exposed to financial hardship in Germany, particularly when the cancer diagnosis occurs during their active life and before requirements to obtain a pension are met. The World Health Organization (WHO) reported that around 9 million people died from cancer in 2016, positioning it as the second most common cause of death globally after cardiovascular diseases.Cancer patients might experience financial hardship primarily as a result of increasing out of pocket (OOP) expenses and reduced working hours and productivity after their diagnosis. Numerous studies have explored this issue; most of them originating from the United States, to a lesser extent from Europe, where evidence from Germany is limited.Certainly, the availability and extent of the German healthcare and social security systems defray a large proportion of the costs to patients for cancer treatment. Health insurance coverage encompasses virtually the entire population and benefits are generous in both scope and scale, constraining the spending amount incurred in healthcare for cancer patients.Nevertheless, existing social safety nets do not entirely protect cancer patients from financial vulnerability in Germany. For example, the Law for the Modernization of the Statutory Health Insurance (SHI) in 2004 led to a considerable increment in patient's contributions in medical treatment, medicine and transportation costs.Recent surveys indicate that many cancer patients still experience large expenditure increases and income losses in Germany. These studies reported substantial OOP payments associated with hospital stays, transportation and medication, as well as diminishing working time and significant reductions in income. For instance, surveys administrated to cancer patients in hospital settings suggest that a proportion between a third and a half of the interviewed did not return to work after cancer treatment.This evidence suggests that cancer patients may still face healthcare expenditures and, most notably, large income losses after their diagnosis, despite nearly full health insurance coverage of anti\u2010cancer treatments and medications as well as extensive social security programs in Germany.2This study analyzes data from the Socio Economic Panel (SOEP) household survey implemented by the German Institute of Economic Research (DIW). It is a longitudinal panel, which started in 1984, and interviews adult household members annually. It is the largest and most comprehensive household survey in Germany, consisting of around 20,000 individuals from 12,000 households.Two different sample consistency checks were performed to guarantee that only reliable cases were included in the analysis. The first of these tests ensured period consistency, meaning that an individual selecting any disease diagnosis and the no disease diagnosis option simultaneously for any given year is considered an inconsistent observation. Such cases were very rare: in the sample, on average, only four of those cases per year were identified. Given the wording of the question, the second check ensured time consistency by certifying that an individual who reports a cancer diagnosis in a given year also reports a cancer diagnosis in every subsequent year. Moreover, because the sample contains missing values a strict time consistency check, which excludes series with incomplete information, was conducted in addition to the aforementioned check. The final sample fulfilled the period and time consistency checks, and the strict time consistency check was further applied to test for the robustness of results. The percentage of observations that follows the time consistency condition is higher in 2009 than for the other years, as the absence of a previous period makes the condition less likely to be rejected. The empirical strategy addresses this bias with the inclusion of time year dummy variables.The analysis focused on four different outcomes: job income, work status, working hours, and pensions. Job income constitutes the sum of salary and wages from the main job, income from secondary employment and income from self\u2010employment for the individual in a given year; it does not comprise social benefits or other transfer payments. Work status options include full\u2010time, part\u2010time, or unemployment, while working hours refer to annual work hours of the individual in a given year. Pensions includes old\u2010age, disability and civil servant pensions, widow and orphan pensions, company pension and private pension for the individual in a given year. Except for work status, all of these items in the survey are open\u2010ended questions. A single analysis of each of the elements which compose the aggregates for job income and pensions was not feasible, as some of them are limited in the number of observations and might lead to inefficient estimators. Sample averages were calculated for these four outcomes and by cancer diagnosis to identify any patterns. This initial evidence was further explored with an empirical strategy that models the four different outcomes as a function of a cancer diagnosis. A first regression equation is as follows:it is the outcome variable to be estimated of individual i in year t. Except for the work status, which is a categorical variable, the outcome variables are all in logarithmic form. CDiagit is the main explanatory variable signaling the cancer diagnosis status. In a first version, it is a dummy variable that takes the value of 0 if individual i reports no cancer diagnosis in year t (labeled as \u201cno cancer diagnosis\u201d) and 1 otherwise (labeled as \u201ccancer diagnosis\u201d). In a second version, it takes the value of 0 if individual i reports no cancer diagnosis in year t and in any other year (labeled as \u201cnon\u2010cancer control\u201d), the value of 1 if individual i reports no cancer diagnosis in year t but reports a cancer diagnosis in any other year (labeled as \u201cbefore cancer diagnosis\u201d), and the value of 2 if individual i reports a cancer diagnosis in year t (labeled as \u201ccancer diagnosis\u201d). Comorbit, Genderi, HHMemit, Ageit, Eduit, and Workingit control for others characteristics of the individuals, namely the number of comorbidities, gender, household position, age, education level and working status of individual i in year t, respectively. Comorbit is a six\u2010level categorical variable that is introduced in the regression equation as dummy variables signaling each category the variable might take: 0 comorbidities, 1 comorbidity, 2 comorbidities, 3 comorbidities, 4 comorbidities and 5 comorbidities. Genderi is a dummy variable that assigns a value of 1 if the respondent is a woman and 0 otherwise. HHMemit is a five\u2010level categorical variable transformed into a set of dummy variables in the regression equation indicating each category: household head, partner, child, relative and non\u2010relative. Ageit is a continuous variable and Age2it its squared form allowing for a non\u2010linear relationship between the age of the respondent and the outcome. Eduit is a three\u2010level categorical variable addressed in the regression equation by dummy variables for each category: less than high school, high school and more than high school. Finally, Workingit is a dummy variable that takes the value of 1 if the respondent is currently working and 0 otherwise. The first level of the categorical variables is assumed as baseline category. The construction of these control variables is found in detailed in Appendix\u00a0i and \u03c4t, and \u03b5it is the error term. Lastly, \u03b2 coefficients in the models with continuous outcome variables were estimated with an ordinary least squares (OLS) estimator, likewise, a multinomial logistic estimator for the model with the categorical outcome variable.Outcomeit is thus in this equation conditional to individual i being actively working in year t. In this way, it could be observed if the effect of a cancer diagnosis on the outcomes held when censoring the unemployed population. The outcome variables, work status and pension, could not be introduced in this model, as variation is limited. While cases of individuals actively working and receiving a pension are rare, the working status outcome loses a category when restricted to the working population sample. For this reason, the control variable Workingit was also omitted, and a working sector fixed effects variable, denoted by \u03c9i,, was included. Outcome variables are all in logarithmic form and \u03b2 coefficients were to be estimated by means of an OLS model.The second regression equation below was executed across a sample containing only individuals that are actively working. Outcomep\u2010value of at least 10 percent is reached. p\u2010value levels of at least 10 percent are denoted in the tables with an asterisk (*).A detailed description of the variables included in the models is found in Appendix\u00a03Initial evidence was captured with sample averages for the four different outcomes and distinguishing by the cancer status of individuals. Job incomes were considerably lower for periods in which a cancer diagnosis is reported, as observed in Figure\u00a0x\u03b2)\u20141. Relative to working full\u2010time, reporting a cancer diagnosis did not significantly increase the likelihood of working part\u2010time, as observed in column 3. It did increase, however, the likelihood of not working significantly, as observed in column 4. The decrease in the number of working hours as a result of a cancer diagnosis was also statistically significant and of 24% in size, as exhibited in column 5. On the other hand, the level of pensions did not seem to be significantly affected after reporting a cancer diagnosis. This result held when the sample was restricted to the population 65\u00a0years of age and older, as shown in columns 6 and 7.Regression results in Table\u00a0Table\u00a0t\u2010x on the outcome in t is equivalent to the effect of the cancer diagnosis in t on the outcome variable in t+x. For this reason, we interpret the coefficient of the lagged variable in x periods, as the effect of a cancer diagnosis on the outcome x periods after the diagnosis. The contemporaneous impact of reporting a cancer diagnosis was negative and statistically significant, as exhibited in column 1. The size of the effect was similar to those previously estimated. The coefficient for the first time lag of the cancer diagnosis variable was negative as well and statically significant, being smaller in size in comparison to the contemporaneous effect, as seen in column 2. Conversely, the coefficient for the second time lag, shown in column 3, was not statically significant.The analysis presented in Table\u00a0Finally, in a first robustness test, the effects for men and women were analyzed separately. An interaction term between the CDiag and Gender variables was introduced in the regression equations for the four outcomes and executed among the full and the working population samples. Nonetheless, the interaction term usually failed to be statistically significant across all models specifications, therefore we cannot conclude that the effect on the various outcomes is different for men and women. In a second robustness test, regression coefficients were re\u2010estimated with the sample that fulfills the strict time consistency check, which excludes observations with incomplete information. Estimations remained stable in terms of sign and significance levels. These results are available upon request.4The present study provides evidence for a topic that is poorly understood Germany. The fact that anti\u2010cancer treatments and medications are commonly accessible and the extent of social security is ample, unlike in some other countries in Europe, reinforces the belief that financial hardship is not a major concern for cancer patients in Germany.Results showed that job incomes decrease between 26% and 28% within the year a cancer diagnosis was reported. The study also found that reporting a cancer diagnosis increased the likelihood of work inactivity and reduced the number of working hours by 24%. Lower levels in job income and working hours were also encountered when the analysis was restricted to the working population only. The effect in income persisted two years after the cancer diagnosis was reported, but was not observable four years thereafter. This result might be a consequence of differences in patient characteristics that are not addressed by the control variables. Patients that leave the sample quickly are more likely to be in a late cancer stage and face larger income loses. On the other hand, patients that remain in the sample over a long period of time are more likely to be in an early cancer stage and therefore in a healthier status that allow them to return to their job. For example, cancer patients that left the sample two years after a reported cancer diagnosis experienced a drop in income per year of 28% on average, while the same figure is of 18% for those leaving the sample four years after the cancer diagnosis is reported. In contrast, pension levels were not significantly affected by a cancer diagnosis. This may be due to those within the population who already receive a pension having been entitled to it before a cancer diagnosis. Importantly, this evidence illustrates that cancer patients are more exposed to financial hardship when the cancer diagnosis occurs before requirements to obtain a pension are met.The absolute fall in job income predicted by the empirical strategy in this study cannot be directly compared with the findings from previous studies because it consists of a point estimate while existing research provides information on the frequency of predefined income intervals. Nevertheless, amount sizes are relatable. With an average job income of 15,613 euros prior to reporting a cancer diagnosis, the econometric models in this study forecasted an absolute fall in job income per patient between 4059 euros and 4371 euros per year, or between 338 euros and 364 euros per month, during those years in which a cancer diagnosis was reported. Corresponding numbers were between 100 to 500 euros per month for 60% of cancer patients in Bikowski,Other countries being evaluated on the impact on income and founded on longitudinal national registries or household surveys is observed for Denmark,A major limitation of this study is the self\u2010reported nature of the cancer diagnosis variable, which is subject to measurement error. Although the survey does not verify confirmed cancer cases, the implementation of period and time consistency checks decreases the likelihood of false positive observations appearing in the sample. This way, biases due to measurement error are limited. In addition, the coefficient measuring the impact of a cancer diagnosis is robust to different identification strategies and sample sizes. Another limitation of this study is the omission of the healthcare expenditure side of financial hardship in the analysis. The SOEP survey does not record OOP costs and therefore these cannot be measured. Research in this topic at a national level is still missing. Lastly, this study cannot address differences across cancer sites or any other epidemiological characteristics. The SOEP survey does not provide such information which has been identified to affect the size of the financial burden.5This study measured the extent of the income loss component in financial hardship from cancer patients in Germany with a nationwide survey. In particular, it examined changes in job income, work status, working hours and pension as a result of a cancer diagnosis. Our results show that job incomes drop between 26% and 28% within the year a cancer diagnosis was reported. This effect persisted for two years after the diagnosis and vanished four years thereafter. Furthermore, analyses revealed increases in the likelihood of unemployment and the reduction of working hours after a cancer diagnosis. However, pension levels are not affected by a cancer diagnosis. This suggests that the exposure to financial hardship is more critical when the cancer diagnosis occurs during the active work life and before requirements to obtain a pension are met. Current social security schemes protect cancer patients of certain work backgrounds only, and when they do, they offset income partially and only for a limited period of time. Self\u2010employed workers, students, and persons insured by their families are particularly at risk, as well as anyone with a work incapacity for a duration longer than one and a half years. This set of circumstances still needs to be acknowledged by the authorities and be called to the attention of policy makers to design a more inclusive and prolonged compensation mechanism to prevent cancer patients and their families from reaching poverty as well as to identify vulnerable groups.Given the nature of secondary data analysis, the need for an ethics approval was waived. The study was performed in accordance with the Declaration of Helsinki and follows the principles of Good Practice in Secondary Data Analysis.The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest , or non\u2010financial interest in the subject matter or materials discussed in this manuscript."} +{"text": "Medication adherence among older adults (aged 60 and above), particularly those with chronic conditions who take several medications, is critical, and tele-pharmacy services are a way to improve medication adherence. This study sought to determine the factors influencing medication adherence (MA) in older adults using tele-pharmacy services.The Joana Briggs Institute scoping review methodology was implemented. Searches were conducted in databases PubMed, Scopus, ProQuest, Web of Science, and Embase from 2000 to the present day, to identify both qualitative and quantitative studies focusing on the use of tele-pharmacy by older people. Factors impacting MA were extracted and analyzed into themes using a qualitative approach. A concept map was also designed summarising these factors.Of 7495 articles obtained in the initial search, 52 articles met the inclusion criteria. The analysis resulted in 5 themes and 21 sub-themes representing factors that impacted MA with tele-pharmacy. These themes are divided broadly into technology and user related factors. Technology factors included design of the tele-pharmacy intervention, commercial aspects, and adherence measurement method. User factors included user-health constraints, behaviors and perceptions.Industry, policymakers, and stakeholders should consider using tele-pharmacy services for improving medication adherence among older adults; however, ensuring interventions facilitate communication between patients and health care teams, and are accompanied by user training and support, is essential for technology uptake and effectiveness.The online version contains supplementary material available at 10.1186/s13690-022-00960-w. Nonadherence to medications, particularly in older people, is associated with an increased risk of hospitalization and death and a siThe shortage of health professionals and the closure of pharmacies in remote areas is a growing problem worldwide that health systems must address to improve medication adherence among patients . These iThe term tele-pharmacy describes the use of information and communication technology to deliver the components of pharmacy practice, including support to clinical services, remote education, patient counselling and monitoring, dispensing prescriptions and reconciliation of drug therapies. Tele-pharmacy is not a new initiative. Following the closure of several rural pharmacies, the first practical implementation of technology facilities in pharmacy service began in the early 2000s in North Dakota (USA). In recent years, the uptake of tele-pharmacy has increased exponentially, with strong support from relevant associations like the American Association of Health-System Pharmacists (ASHP) [The present scoping review was conducted based on the JBI manual of evidence synthesis in 9 steps, as shown in Table st January 2000 to 10th August 2021 to capture any studies of the initial implementation of tele-pharmacy in the US.The Participants, Concept and Context (PCC) were defined as follows: the population was adults older than 60 years old. The concept included medication adherence in older adults, and the context included all tele-pharmacy services. All full-text articles and conference papers reporting a qualitative or quantitative study of tele-pharmacy services in older people were included. Review papers were excluded. The search strategy was designed around the three keywords: older people, medication adherence, and tele-pharmacy , and the final map was reviewed by three experts in public health and pharmacy. The results were presented and discussed via the virtual panel to ensure the comprehensiveness and robustness of the results and accuracy of the final map. Furthermore, a 10n et al. , to illuInitially, searches returned a total of 7495 articles for the title and abstract screening , the device data indicated high MA rates (95-99%) in the intervention group versus the usual care group. However, the self-report data showed no significant improvement in the telemonitoring group compared to the control group . SimilarThe use of tele-pharmacy to improve MA in older people was impacted by a wide range of individual health variables. One of the most important factor was found to be the disease itself. For example, older patients with chronic diseases such as diabetes, hypertension, hyperlipidemia, chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD) and heart diseases were more committed to taking their medications, as were those with cancer , 43, 52 Notably, disability was examined in a few studies, limiting the utilization of technology tools in geriatrics tracking adherence , 39. A fThe last main theme identified was user behavior and perceptions, which included four sub-themes: baseline MA, patient behavior, preference, and long-term use of interventions. Notably, participants who initially maintained a high level of adherence before the tele-pharmacy intervention reported no significant change in their adherence following the use of the tele-pharmacy services , 57, 58.Several studies have demonstrated the importance of individual behavior in using the technology tools to improve MA , 23, 51.In addition to individual behavior, patient preference was another sub-theme that emerged from included papers. In one study, patients were pleased with the smart pillbox bottle's usability. Still, they were dissatisfied with its size and functionality because they already used weekly pill organizers to manage their medications . In anotThe other important issue impacting MA was the long-term effect of the tele-pharmacy intervention. In several studies, MA improved in the short term; however, this improvement was not persistent over time, and people stopped using the tele-pharmacy intervention over time. Studies associated this decline with poor long-term commitment to the interventions , 31, 34.The idea map depicted in Fig. Figure This review identified several factors related to the technology and user that affects MA in older people using tele-pharmacy services. Overall, older patients, particularly those with chronic diseases, demonstrated good MA when using tele-pharmacy services. However, this effect might not be sustained. In several studies, the improvement in MA observed when using tele-pharmacy interventions was short-lived, and after a few months of using the service, MA declined. The one exception to this appeared to be medication dispensing systems (MDS), with a study showing that high MA was maintained for up to 26 weeks . MDS incAn exciting result from this review was the key role that health professionals play in MA among older people, regardless of the tele-pharmacy technology used . This is likely because health professionals are a trusted source for patients and are often the first point of call for older people with a medication-related question or concern . This suOur trend analysis showed that older patients are becoming more comfortable with using mobile applications over time.\u00a0In addition, the majority of the included studies were completed between 2019 and 2021, indicating a recent surge in interest in this topic. Although there is a common perception that older people will not use technology for health, our review showed that this is rapidly changing, and in some cases, MA was higher in older groups (over 65 years) than in younger groups (less than 65 years) when using tele-pharmacy interventions , 45.This review showed that many smartphone apps, particularly those that include interactive features, have been effective in improving MA for older patients. Although patients found text message reminders helpful in taking their medications, they preferred mobile applications because they were interactive and provided individualized health monitoring and personalized medication information . AlthougIn contrast to mobile apps, we found fewer papers focusing on other types of tele-pharmacy interventions. Although in recent years, e-prescriptions and remote dispensing by pharmacists are increasingly being used to improve medication adherence, only a small number of studies investigated the utilization of these tools in older people , 49, 50.There was no consistent approach used for measuring MA in studies, which made comparison difficult across the studies. Investigating medication adherence was not the primary purpose of some studies and MA was reported only as a secondary outcome. However, we included those studies as their outcomes aligned with the aim of the scoping review.In conclusion, tele-pharmacy can improve MA in older people, however, several factors impact the achievement of this goal. These factors relate to the intervention, including design, commercial aspects, and adherence measurement, and to the user, including health constraints behaviors, and perceptions. Communication with the health care team and sufficient user training appear necessary for tele-pharmacy interventions to enhance MA. In addition, intervention designers should focus on the design, size, storage capacity, and security of technologies. Government and policymakers should provide subsidies for such interventions to encourage uptake among older users. There is no one-size-fits-all strategy, and each patient's physical and mental characteristics should be considered when recommending the best treatment option, including tele-pharmacy interventions.Additional file 1: Table 3. Search Strategy.Additional file 2: Table 4. Included articles extracted information.Additional file 3: Table 5. Determinants of elders\u2019 medication adherence in telepharmacy services with references."} +{"text": "AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH Remarkably, NH4Cl rescued the [Ca2+]i response, following desensitisation of the P4/PGE1 response is postulated to be a phenotypic reflection of the CatSper activation ,36. Howesponsive , its effsponsive ,45. In tThere are numerous published AR measurement protocol variations, including differences in the methods of isolating sperm from semen (swim-up or density gradient), media formulations , incubation time in capacitating conditions (3\u201324 h), P4 concentration (3\u20131590 mM) and exposure times (5 min to 24 h), AR indicator , method of detection and inconsistent assessment of viability, which may contribute to the discrepancy in the spontaneous and P4-induced AR , 10 mM 17\u03b1-hydroxyprogesterone (P4OH) and 5 mM prostaglandin E2 (PGE2). The concentrations of the CatSper agonist are at EC100 values.All the chemicals were purchased from Sigma-Aldrich, UK, unless stated otherwise. The sperm preparation gradients (PureCeption 40% and 80%) and Quinn\u2019s Advantage Sperm Washing Medium (SWM) were purchased from CooperSurgical, Inc., US. The allophycocyanin (APC)-conjugated Mouse Anti-Human CD46 (Clone E4.3) was purchased from BD Biosciences, Berkshire UK. The AlexaFluor 488 PNA and propidium iodide dye were purchased from ThermoFisher, UK. The progesterone (P4) and prostaglandin E1 (PGE1) were dissolved in DMSO. The final concentration of the DMSO in an experiment is maximally 0.02% 4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 0.33 mM Na-pyruvate, 12.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, 25 mM NaHCO3 and 3 mg/mL BSA, adjusted to pH 7.4 with NaOH, and the osmolarity adjusted to ~275 mOsm using NaCl.The HTF was composed of 72.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO+ was prepared by increasing the concentration of the KCl to 30 mM, while the pH and osmolarity were adjusted using NaOH and reducing NaCl by 30 mM, respectively.Additionally, the HTF-High KFollowing informed consent, samples for research were obtained from patients undergoing investigation and treatment at the Assisted Conception Unit (ACU), Ninewells Hospital, Dundee, and that were surplus to clinical requirement. Samples from healthy volunteer research donors with normal sperm motility parameters in agreement with World Health Organization 2010 criteria were useHealthy volunteer donors meeting the WHO normal semen criteria adhered to an abstinence period of 2 to 5 days, prior to the sample collection by masturbation in a sterile plastic container. The sample was then allowed to liquefy for 30 min at 37 \u00b0C, prepared by density gradient centrifugation (DGC).g for 20 min. The pellet was removed and washed using 5 mL SWM at 300\u00d7 g for 10 min. After washing, the cells were resuspended in human tubal fluid medium and left to incubate in capacitating conditions for 3 h. For experiments using high concentration of K+, the cells were capacitated in normal HTF for 3 h , centrifuged and resuspended in HTF K+, with the appropriate agonists, for 1 h.For the DGC, 1 mL of liquefied semen was layered on top of percoll gradients (1 mL 40% PureCeption underlaid with 1 mL 80% PureCeption) and centrifuged at 300\u00d7 The patients\u2019 cells were prepared according to the standard operating procedures employed by the Assisted Conception Unit at Ninewells Hospital, Dundee . The pat4Cl. The motility was assessed using four-chamber, 20-\u03bcM deep slides . At least 200 sperm cells were analysed per chamber, per condition. The motility readings were recorded intermittently over 30 min . The parameters measured included the progressive motility (PM), total motility (TM), and hyperactivated motility (HA). Proprietary algorithms on the CASA determined the percentage of cells displaying the HA automatically. Specifically, a subpopulation of the sperm displaying a curvilinear velocity (VCL) \u2265150 \u03bcm\u00b7s\u22121, linearity <50%, and an amplitude of lateral head displacement \u22657 \u03bcm of algorithms were designated as hyperactive i response i were carried out using a FLUOstar Omega reader at 30 \u00b0C. The filters were set to 488 nm (excitation), and 520 nm (emission). The baseline fluorescence was recorded for 30 s prior to the addition of the compounds.In total, 3 \u00d7 10affinity dye Fluoresponse . The sup4Cl, the agonists were added after a 1-min recording of the baseline fluorescence, followed 5 min later by the addition of NH4Cl, or vice versa. In all the experiments, the readings from an additional time-control well were taken, as were readings from a well that was exposed to the agonist(s) at the time point that matched the time point of the addition of the second agonist(s) in the paired experiment.To demonstrate that the P4 and PGE1 responses do not cross-desensitise, the experiments were carried out in accordance with the published protocols ,106; the2+]i. The peak [Ca2+]i response was defined as the maximum [Ca2+]i occurring immediately after the agonist addition.The normalisation of the background-corrected fluorescence data was, as described previously , using \u0394\u22126 per mL) were incubated with 2 \u03bcM 2\u2032,7\u2032-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM) for 30 min at 37 \u00b0C. The cells were centrifuged for 3 min at 500\u00d7 g; the supernatant was removed; and the cells were then resuspended into the HTF, buffered at pH 7.4. For calibration, five aliquots of the spermatozoa were placed into the HTF and buffered at 6.0, 6.5, 7.0, 7.5 and 8.0, respectively. A FLUOstar Omega plate reader (BMG Labtech) was used to detect the emitted fluorescence . The cell calibration was achieved following the cell lysis by the addition of 1% Triton X-100; a reading was taken from each well; and a calibration curve was constructed. The fluorescence measurements for the control wells (cells + 1% DMSO) and those containing the compounds were recorded using the 490/440 ratios. The unknown pHi values were calculated from the five-point standard curve.The measurement of the pHi was based on the previous methods ,108. AftThe oocytes were considered normally fertilized when two pronuclei (2PN) and two distinct or fragmented polar bodies were observed. Following the IVF, the fertilization rate was calculated from the number of oocytes normally fertilized, divided by the total number of inseminated oocytes. The fertilization rate was calculated where four or more mature oocytes (metaphase II) were present.The data are presented as the mean \u00b1 SEM unless stated. Unless indicated, the statistical significance was determined using a one-way ANOVA, followed by Tukey\u2019s post-test, using the statistical package GraphPad Prism 5, and is indicated as * <0.05, ** <0.01, *** <0.001."} +{"text": "This review seeks to understand how scientific research on trace elements in beef cattle has been developed and how the interaction of this theme with topics related to animal, environmental and human health has been established. Given the duality of many of the trace elements, being known as nutrients in small amounts or toxic when they exceed small concentrations, we brought the One Health perspective to analyse how researchers approach this research theme. In this work, we propose a path through scientific production to promote innovation, sustainability of animal production, food safety and human health.The objective was to investigate the context, approach and research topics present in the papers that analysed trace elements in beef cattle to identify gaps and scientific perspectives for the sustainable management of trace elements in livestock. The main research groups came from the United States, Spain, Japan, Brazil, India and Slovakia, which represented 31% of the papers produced. Only 37% of studies addressed aspects that integrated animal, environmental and human health. The reviewed papers concerned 56 elements and 15 bovine tissues . The main gaps were (1) lack of research in developing countries, (2) the need to understand the impact of different environmental issues and their relationship to the conditions in which animals are raised, and (3) the need to understand the role of many trace elements in animal nutrition and their relationship to environmental and human health. Finally, we highlight possible ways to expand knowledge and provide innovations for broad emerging issues, primarily through expanding collaborative research networks. In this context, we suggest the adoption of the One Health approach for planning further research on trace elements in livestock. Moreover, the One Health approach should also be considered for managers and politicians for a sustainable environmental care and food safety. Trace e\u22121 . Althoug\u22121 .The concentrations of trace elements in the environment and foods vary widely. Thus, in the context of the global environmental change that is occurring in the so-called Anthropocene era and consWithin the agriculture sector, mineral supplementation in animal production is of great importance. It is well known that an inadequate supply of essential trace elements leads to poor animal condition, adversely affecting immunity and reproduction . HistoriTrace element exposure in livestock also has important consequences for humans. It is well known that meat and meat products are some of the main contributors of trace elements, including toxic elements, and the concentrations of these elements in meat are directly related to those in animal feed . High leThe triple challenge of trace element exposure in livestock\u2014to optimize animal health and productivity while ensuring environmental sustainability and consumer/food safety\u2014leads to the need to consider trace element exposure, in particular trace element supplementation, from a holistic perspective, which is nowadays referred to as One Health. The One Health approach aims to ensure the well-being of people, animals and the environment through collaborative problem solving, integrating these highly interconnected components and enabling change towards better public health outcomes . AlthougDuring the last few decades, a large body of research has addressed trace elements in farm animals. Most of this research has focused on using trace elements, even at concentrations above physiological requirements, to improve animal performance. However, many challenges remain in understanding the dynamics of these elements from the animal/farm to the consumer to produce profitable but sustainable farms and thus accurately determining the possible short, medium and long-term impacts. Some aspects of animal production, such as breed and physiological state ,20, antaThe aim of this review was to collect and globally summarize the research carried out in the last few decades concerning trace elements in beef cattle and analyse the approach of researchers through bibliometric information and topics presented in their respective papers. Beef cattle were selected from among other livestock species as they are possibly the type of livestock that best exemplifies the triple animal\u2013environment\u2013consumer interaction and in which the One Health approach to trace element exposure would thus be of greatest benefit. We hope that this paper will identify the main gaps and prospects for advancing knowledge about trace elements in beef cattle production. This would facilitate future collaborations and enable responsible use of trace elements in animal farming while protecting animals, consumers and the environment.We carried out a search in the Scopus, Web of Science and PubMed scientific databases. To retrieve the papers that analysed trace elements in beef cattle tissues, advanced search strings were created using five sets of keywords in English, which appeared in titles and abstracts of papers published between 2000 and 2022. The search interval from 2000 onwards was defined to cover the United Nations Millennium Summit initiative held that year when the Millennium Development Goals were created and which in 2015 culminated in the proposal of the 17 Sustainable Development Goals . To prepA total of 3399 papers were obtained at the identification step, and 1507 duplicate papers were excluded. In the next step, the titles, abstracts and complete papers (when necessary) were examined, leading to the removal of 1542 papers considered outside the scope of this research and finally leaving a total of 350 papers.At the document examination step, papers on the following topics were excluded: (1) research on dairy cattle; (2) research on young suckling calves; (3) research with emphasis on reproductive aspects; (4) research in which trace elements were only determined indirectly; and (5) research with emphasis on diseases resulting from deficiency or secondary toxicity by trace elements. Exceptions to exclusions were established for criteria 1, 2 and 4, when the environmental conditions were considered preponderant, e.g., clinical cases investigating trace element intoxication resulting from environmental contamination.The papers accepted were subjected to an extraction phase to obtain general information such as year of publication, country where the research was conducted, authors and their affiliations and journal of publication. In addition, specific information was also obtained concerning the trace elements and bovine tissues analysed, analytical techniques and scope of the research with the One Health approach. Some papers that could not be accessed in full were analysed in relation to the information available in the databases, among which 15 papers were not available in digital format and another 8 papers required payment of a subscription.Within the One Health approach, the papers were classified into animal health, environment health and human health, according to the aspects addressed in each. At first, we accepted all papers from this analysis in the animal health field in view of the search and selection prerequisites, which identified papers that analysed trace elements in bovine tissues. Although some papers did not directly address animal health, we felt that they all provided useful information on this topic. Within the topic of animal health, we highlighted those aspects related to animal handling, classified as animal category and animal feeding . Environmental health was identified as a topic in papers that investigated natural and anthropogenic aspects that can alter the nutritional status of animals. Finally, the human health topic was identified in papers that evaluated trace elements as nutrients or contaminants in human diets.To proceed with the analysis of the co-authorship network, the correct spelling of the authors\u2019 names was first verified and where necessary changed. A network map was then generated using VOSviewer software, version 1.6.16, which enables generation of network maps combining information from co-authorships to trace the graphical representation, in which the nodes represent the relevance of the authors and the links represent the papers published in co-authorship.Bar charts, location maps and sector charts were generated using the ggplot2, hcmap and moonBook packages in R software, version 4.0.4. To assess whether the number of publications has tended to increase over the years, we performed a linear regression using the geom_smooth function, method lm from the ggplot2 package. In addition, we used the Inkscape software, version 1.0.1, for graphic adjustments and production of Boolean graphs.The search for literature concerning trace elements in beef cattle production in the period studied (2000\u20132022) identified 350 papers that analysed trace elements in bovine tissue. Despite being an important research topic, the number of publications per year was quite variable , with a moderate trend towards an increase in the number of papers on this topic published annually A, althouThe searches also led to the retrieval of research carried out in 61 countries, covering all continents, as can be seen in the map in Beef cattle are reared in many parts of the world, providing an important source of nutrients in the human diet , particiThrough the analysis of co-authorship network, it was possible to identify six main co-authorship networks in research and the main authors in each network . In thisAltogether, 1186 authors were identified. However, considering the visual aspect of the graph , only auAmong the networks identified, a distinction is made by mainly regional aspects, i.e., by the countries in which the networks were established. This stems from the regional aspects common to the authors, which may be favored by geographic proximity, language and regional research problems of related interests. In addition, the networks were also distinguished by the methodological approaches used and the general scope of the research. Network 1 is characterized by research related to animal health, addressing management aspects, mainly nutrition and animal performance, and the publications mainly appear in journals focused on Animal Science. The main research topics in this network include animal feed and monitoring of essential trace elements, particularly Cu and Zn, to assess animal performance. On the other hand, themes related to environment aspects and studies of potentially toxic elements, such as Pb and Cd, did not appear in this network. The other groups identified (Networks 2 to 6) are characterized by research with a broader and interdisciplinary scope. In these networks, the authors addressed environmental, human and animal issues within the research topics, e.g., differences in the location of the animal breeding areas in relation to the proximity of industrial or mining activities, evaluation of the risk of meat contamination and animal poisoning case studies in addition to practices adopted in animal production systems, such as feeding or animal performance. These networks study a greater variety of trace elements, and potentially toxic elements are often included. Furthermore, also in relation to analysis of the co-authorship network , we obseIn total, 56 trace elements were studied in the papers reviewed, although only 13 of these elements were studied in more than 32 papers A. Among The elements Cu, Zn, Fe, Se, Mn, Mo, Co, Ni and Cr are considered essential for ruminants and are therefore required for various biochemical and physiological functions of animals. These elements usually need to be added to animal feed to supply nutritional requirements ,36. ReceThe large number of studies investigating Cu and Zn in bovine tissue, as shown in The elements Pb, Cd, As and Hg do not have established biological functions and are considered undesirable and potentially toxic contaminants of animal feed in additAmong the less studied trace elements B, 20 eleThe importance of trace elements is continuously discussed and revised, with the elements being classified as toxic, beneficial or essential for living organisms, especially humans ,47. It hDetermining the nutritional requirements of trace elements is also important for identifying nutritional status and the subclinical detection of deficient or toxic concentrations through chemical analysis of animal tissue . This caConsidering the bovine tissues analysed, liver, muscle and kidney predominated in the studies reviewed C. The liMuscle was analysed in 128 papers and was generally found to have lower levels of trace elements than liver or kidney. On the other hand, muscle is the most commonly consumed by humans, and it assumes great importance in relation to meeting dietary requirements of essential nutrients and examining the risk of exposure to contaminating elements , and it Analysis of blood tissues and hair is very useful to assess bovine nutritional status in vivo, and it was also very representative in the papers reviewed, being included in 78 (blood plasma), 66 (whole blood), 62 (blood serum) and 23 (hair) papers C. RegardAnalysis of bovine hair was proposed by Combs as a potOther bovine tissues have mainly been studied to assess compliance with dietary requirements and to identify patterns of metal accumulation due to breed, food and environmental conditions. These data are scarce, and discrepancies may therefore occur, as verified by Berata et al. , who obsIn terms of analytical techniques for trace element determination in bovine tissue, 30 different techniques were identified. The main techniques used were flame atomic absorption spectrometry (F-AAS) or graphite furnace atomic absorption spectrometry (GF-AAS) and multi-element techniques such as inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) D. In addAtomic absorption spectrometry (AAS) techniques are traditionally widely used. However, for analysis of complex matrices that require analysis of multiple elements, multi-element techniques such as ICP are more suitable, especially because they have enabled analysis of a wide range of elements including ultra-trace elements in the order of parts per trillion in addition to not requiring long sample pre-treatment steps, which is one of the main disadvantages of AAS .ICP-OES provides an excellent, highly sensitive means of determining trace elements and can detect many elements simultaneously. However, the detection limit is similar to that of F-AAS. On the other hand, ICP-MS provides the best characteristics of all atomic spectrometry in terms of sensitivity, detection limits, transfer rate and multi-element measurement, although it is very expensive, despite having been developed over several years ,64.Other multi-elementary techniques of great potential for trace element analysis in bovine tissues are techniques that employ X-ray or neutron activation. In the present review study, we identified only four papers in which neutron activation analysis was used and two papers that used X-ray, indicating that these techniques can still be regarded as incipient for this type of study. We also observed that 40 of the trace elements identified in this research were analysed exclusively by multi-element techniques (mainly ICP-MS), which have led to improvements in the understanding of the dynamics of a wide range of trace and ultra-trace elements in the context of beef cattle production.Identification of studies using the One Health approach followed three steps. First, we initially admitted 350 papers in this review within the animal health interface, considering that animal health was supported in the stages of search, identification and selection of papers. Next, we identified 201 papers in the field of environmental health. Finally, we identified 164 papers that addressed issues related to human health. The multiple issues involved in this research indicated the complexity of the challenges involved in animal health, conservation of the environment and safe production of food, which go beyond the limits of farming and are established on a large scale. Such issues often interact with each other and thus can be elucidated through the One Health approach A.Interactions between the research topics occurred in 34 papers in the animal\u2013human health interface, 71 papers in the animal\u2013environment health interface and 130 papers in the One Health interface . This demonstrates how closely the investigative approach to trace elements permeates natural aspects, human activity, animal feed, human nutrition and differences between animals (among others) in different ways.We also observed that only 7% of the research studies carried out in the US were categorized as concerning the One Health interface (6 out of 83 papers), while 78% exclusively addressed the animal health interface (65 out of 83 papers). In comparison, 46% of surveys carried out in other countries (124 out of 267 papers) involved the One Health interface. This suggests that there is a distinction between the main research objectives that may be related to the interdisciplinary approach. In this case, in the US, research focused on animal health predominated, evaluating performance and productivity, while few studies integrated environmental and human health.Journal of Animal Science, the Professional Animal Scientist and the Animal Feed Science and Technology. On the other hand, the One Health approach predominated among interdisciplinary journals such as The Science of The Total Environment and Food Additives & Contaminants\u2014Part A. This indicates that the main journals in which these studies are published also express the differences between the research approaches adopted.The papers analysed came from 157 journals and cover a broad scientific scope, with emphasis on journals in the fields of animal and veterinary science B. We alsAlthough the concept of One Health permeated just over one-third of the papers analysed (37%), we highlight its potential to be incorporated in future research on trace elements in beef cattle and in animal production in general, thus favoring new approaches to addressing emerging issues in the global scenario. Examples of issues related to trace element monitoring that could be enhanced by this approach include identification of exposure risk sites, health risk diagnosis, support for regulatory compliance and identification of safer and more effective agricultural practices, among many others . Taking The information presented here refers to the number of papers in relation to year of publication, country where the research was carried out, authors involved, trace elements studied, bovine tissues analysed, analytical techniques used and the field of research. Both qualitative and quantitative information was reported, thus demonstrating the scientific progress made in this area. However, some knowledge gaps were identified: (1) lack of research in developing countries; (2) need for answers to a wide variety of questions related to the environment and the conditions under which livestock is raised; and (3) need to address the functions and interactions of a wide range of trace elements in animal metabolism and their relationship to environmental and human health.Few changes have been made in the trace mineral requirements of beef cattle since the sixth edition of the National Research Council (NRC) in 1984 . HoweverDespite the greater complexity of the One Health approach, which involves multiple issues, this approach has become widely accepted. Research with inter-, multi- or transdisciplinary approaches has been necessary to provide greater integration between disciplines and experts, as well as a broader interpretation or application of the results obtained . In thisThere are complex interactions that occur in the production system, which combine environmental conditions in addition to social, economic and cultural dimensions, resulting in unique characteristics in each location, which need to be recognized in research planning. These interactions require specific approaches to obtaining and interpreting results. In view of this, there should be greater interaction and collaboration among researchers to develop this type of research, which should favor greater sharing and advancement of knowledge.In the One Health approach, knowledge of the basic disciplines is reinforced, as this approach plays a fundamental role in aggregating complementary disciplines and different areas of expertise ,68,69,70Advances in the development and improvement of analytical techniques for the knowledge of the content of trace elements in bovine tissue have been shown to be essential. However, it is also important to consider access to cutting-edge technologies in less developed countries, especially due to the associated costs, which may constitute an important barrier limiting research in these regions. For instance, in Africa, the collaboration between national and international research centers provide better approaches to promote One Health technologies in remote areas centers .Regarding the 17 Sustainable Development Goals (SDGs) , it is eNew research should observe in an increasingly integrated way the environmental aspects of livestock and food quality to enable the advance towards precision livestock and reach Sustainable Development Goals. Thus, trace elements cannot be investigated in isolation within a production system, and integration of data that combines information from micro and macro systems, covering sociocultural, economic and especially environmental dimensions is required.This review enabled us to summarize the information obtained between 2000 and 2021 about trace elements in the production of beef cattle and to identify the main advances and knowledge gaps. The main research groups are located in the US and Spain, although most publications (69%) were produced by emerging research groups investigating this topic. Among the studies carried out in the US, only 7% were categorized as the One Health approach, while in other countries the corresponding proportion was 46%. Overall, only 37% of studies addressed aspects that integrated animal, environmental and human health.We found that 56 trace elements and 15 bovine tissues were studied; however, the main research targets were the elements Cu, Zn and Pb and the bovine tissues liver, muscle and kidney. We also noted the importance of improving analytical techniques to increase the number of trace elements studied and improve understanding about these elements in different contexts of animal production.Finally, we highlight the potential for extending international scientific cooperation to promote the inclusion of developing countries and greater collaboration across research disciplines in order to produce more significant advances in knowledge and more innovative and accurate responses to emerging issues worldwide. In this context, we suggest adoption of the One Health approach in planning new research on trace elements in animal production."} +{"text": "We examined the influence of three major environmental variables at the place of residence as potential moderating variables for neurofunctional activation during a social-stress paradigm. Data from functional magnetic resonance imaging of 42 male participants were linked to publicly accessible governmental databases providing information on amount of green space, air pollution, and noise pollution. We hypothesized that stress-related brain activation in regions important for emotion regulation were associated positively with green space and associated negatively with air pollution and noise pollution. A higher percentage of green space was associated with stronger parietal and insular activation during stress compared with that in the control condition. More air pollution was associated with weaker activation in the same brain regions. These findings may serve as an important reference for future studies in the emerging field of \u201cneuro-urbanism\u201d and emphasize the importance of environmental factors in urban planning. Simultaneously, urban living comes with an increased risk of stress-related mental disorders, such as depression or anxiety disorders2.Urbanization is associated with several benefits, but also challenges. \u201cCity life\u201d is, amongst others, linked to improved access to education, culture, health institutions, and employment4. Several factors could mediate this association. Apart from social factors , environmental factors have been associated with negative impacts on physiological and psychological health6.Interestingly, urban living is associated with increased amygdala activity in a stress paradigm, which is a key region for emotional processing and threat detection10. Furthermore, GS has been reported to play a part in everyday coping with stress: more GS in residential areas has been associated with a steeper decrease in the cortisol level during the day and a lower level of self-reported stress11. Only one functional magnetic resonance imaging (fMRI) study has investigated the association between GS and emotional wellbeing. GS exposure was especially beneficial in terms of mood improvement for urban dwellers who depicted lower, possibly less regulatory activity in the dorsal prefrontal cortex during processing of aversive emotional cues. That study suggested that urban green space (UGS) exposure might be a compensating factor for reduced neuronal regulation12. It is important to note, that population subgroups might benefit in different ways, caused by factors like e.g. age, level of education and social support15.Greater exposure to green space (GS) has been shown to be associated with improved mood, perceived general health, and increased physical activity, whereas a negative association was reported for obesity and body mass index (BMI)21. Across different air pollutants, particulate matter (PM) has been identified to be among the most prevalent and harmful22. Though less often investigated than PM, ambient gaseous nitric oxides NOx and/or NO2 have been associated with an increased risk of dementia, cerebrovascular disease, neurodegenerative syndromes, and poorer cognitive development in children25.Whereas GS in cities seems to promote mental wellbeing, urban air pollution has been shown to be related to neurotoxicity, neurodegeneration, worse cognitive performance, an increased risk for the recurrence of depressive symptoms, and suicide26. Several studies have indicated that noise might contribute to the development of metabolic and cardiovascular diseases, as well as to autonomic imbalance and vascular dysfunction30. Hence, GS, air pollution, and noise pollution are environmental determinants that distinguish urban areas from more rural areas, and have been shown to influence mental health: salutogenic for GS, and pathogenic for air pollution and noise pollution. A fMRI study on how air pollution and noise pollution may endanger psychological wellbeing has not been undertaken. Here, we assessed brain activation during a social-stress paradigm in male urban dwellers. Due to the menstrual cycle and hormonal fluctuations in women, stress responses have been shown to differ from men, which is why we included only men for this study33. We hypothesized that a higher percentage of residential GS and lower values of air pollution and noise pollution are associated with enhanced processing of neural stress.A third environmental risk factor in urban settings is noise. For example, noise annoyance has been reported to lead to a chronic stress response associated with an increased release of stress hormones1 to t4 are depicted in Fig.\u00a0p\u2009=\u20090.455, \u03b72\u2009=\u20090.023), but post hoc t-tests demonstrated an increase from pre-stress to post-stress (t(41)\u2009=\u2009\u2009\u2212\u20099.346, p\u2009<\u20090.001, d\u2009=\u2009\u2212\u00a01.35) and a subsequent decrease to the next time point (t(41)\u2009=\u20097.454, p\u2009<\u20090.001, d\u2009=\u20090.989). The cortisol concentration changed over time \u2009=\u20093.158, p\u2009=\u20090.048, \u03b72\u2009=\u20090.079), but post hoc t-tests were not significant between any of the four sampling time points (p\u2009>\u20090.05). Heart rate changed across the three time points measured \u2009=\u20098.238, p\u2009=\u20090.005, \u03b72\u2009=\u20090.195), with a significant increase during stress (t(36)\u2009=\u2009\u2009\u2212\u20098.353, p\u2009<\u20090.001, d\u2009=\u2009\u2212\u00a01.116), followed by a decrease during the scan after the stress task (t(38)\u2009=\u200910.389, p\u2009<\u20090.001, d\u2009=\u20091.034) . That is, the higher the amount of residential GS in a buffer of 5000\u00a0m, the lower was the cortisol AUCi in response to the social stressor. All other correlations were not significant . PM2.5 and PM10 showed negative associations with GS buffers\u2009\u2265\u20091500\u00a0m .Descriptive statistics for the mean value of percentage GS, air pollution, and noise pollution are listed in the supplement Tables and S4. nt Table . When add) Table . For NO2p\u2009<\u20090.05, TFCE-corrected) Fig.\u00a0. These ip\u2009<\u20090.017, FWE-corrected for comparison of nine buffer sizes). These data indicated that participants with a higher percentage of GS in a buffer of 5000\u00a0m around their residence tended to have stronger activation in these regions during the stress condition compared with that in the control condition. At a more lenient FWE-corrected threshold of p\u2009<\u20090.05, additional associations appeared in the right ventromedial (vmPFC) and ventrolateral prefrontal cortex (vlPFC), and ventral striatum (VS), left and right amygdala, precuneus, ventral posterior cingulate cortex (PCC), hippocampus, and ventral anterior cingulate cortex (ACC), as well as in the left lateral occipital cortex, superior parietal cortex, fusiform gyrus and insular cortex. Using the more lenient FWE-corrected threshold, brain regions found for 4000\u00a0m . This association was more pronounced for PM2.5 than for PM10, and comprised several regions in both hemispheres: frontoinsular cortex, hippocampus, amygdala, VS, inferior parietal cortex, thalamus, precuneus, PCC, dACC, dorsolateral PFC (dlPFC), ventrolateral PFC (vlPFC), and vmPFC. That is, participants exposed to a higher PM in their residential area had weaker stress-related activation in those brain regions. An association for the concentration of NO2 and NOx in residential areas was not found.In contrast to GS, the concentration of PM2.5 Fig.\u00a0a and PM1\u00a010 Fig.\u00a0b in resiAn association was not found between residential noise during the day and night for a buffer of 50\u00a0m or for 100\u00a0m.34. In terms of environmental variables, we showed stronger activation in brain regions involved in emotion-based regulation of stress compared with that in the control condition for participants with higher availability of GS, particularly within a buffer of 5000\u00a0m, whereas the buffers of 1500\u00a0m, 2000\u00a0m, and 4000\u00a0m showed stronger activation at a more lenient threshold. In contrast, less activity in a more extended set of brain regions was found for participants with a higher amount of PM at their place of residence, whereas there was no association with NO or noise pollution.We wished to examine the association between urban environmental variables and brain activity during a social-stress paradigm. Successful stress induction was judged by an increase in verbal stress ratings and heart rate, as well as by a significant main effect for cortisol concentration. Overall, the analysis of the main effect of stress showed a similar activation and deactivation pattern to that stated in studies on stress processing36. However, until now there has been no distinct operationalization of what constitutes \u201crestorative\u201d GS37. Ekkel and de Vries stated that cumulative opportunity metrics show a more consistent association with health than metrics of residential proximity accessibility 38. Our results showed no association between stress-related brain activation and the amount of GS for the small, \u201cwalkable\u201d buffers of 250\u00a0m, 500\u00a0m, or 1000\u00a0m. However, we found a positive association with larger buffers, particularly 1500\u00a0m, 2000\u00a0m, and 4000\u00a0m (not corrected for the number of buffers), and 5000\u00a0m (corrected for the number of buffers), which illustrated a stronger and more widespread activity pattern with increasing buffer size. The smaller buffers of 1500\u00a0m and 2000\u00a0m were associated with stress-related activation in the right vlPFC, an area well known for cognitive control of emotional processing40. The larger buffers of 4000\u00a0m and 5000\u00a0m were associated with a more diffuse activation pattern, including the insular cortex , the vmPFC, vlPFC, dlPFC, and vACC (which are important for emotion regulation), amygdala and ventral striatum , the fusiform cortex as well as the precuneus and PCC 41. In general, it seems that a larger amount of GS was associated with stronger activation in brain regions that are important for regulating emotions when processing a stressful task. Surprisingly, the GS buffers of 2500\u00a0m and 3000\u00a0m did not show an association with these brain regions. However, when applying a more lenient, uncorrected threshold, we observed a similar association with activation of the right vlPFC for these buffers as that found for 1500\u00a0m and 2000\u00a0m. Interestingly, for the buffer of 3000\u00a0m, additional regions appeared to be in agreement with the regions reported for 4000\u00a0m and 5000\u00a0m, which may indicate a shift in neural associations from more rostral to dorsal stress-related brain activity with an increasing percentage of GS.For many people, nature provides an opportunity to escape the stress of daily life and to regenerate their cognitive resources42. This matters especially for effects found within the two largest buffers. As such, our results might partly reflect an indirect association between GS and processing of neural stress, which may be influenced by the role of GS in filtering air pollution43. This hypothesis is supported by two results. First, we found a negative association between the amount of GS and air pollution for buffers\u2009\u2265\u20091500\u00a0m. Second, weaker activation was found for increased air pollution in the bilateral frontoinsular cortex, vmPFC, vlPFC, dlPFC, amygdala, hippocampus, precuneus, and PCC, which mirrored the pattern of brain regions that showed a positive association with GS. However, deactivations associated with PM comprised additional and larger areas than activations associated with green space. Interestingly, most of these regions are central nodes of three well-studied functional-connectivity networks in relation to the acute stress response: the salience network , default mode network , and central executive network (dlPFC)34. Though fMRI research on the influence of air pollution on stress processing is still missing, a previous study employed the Trier Social Stress Test outside the scanner and reported that higher PM2.5 concentrations in the residing neighborhood were associated with a greater autonomic response as indicated by lower heart rate variability and higher skin conductance levels44. This suggests that air pollution (at least PM2.5) potentially inhibits an appropriate stress response, which coincides with our results in which PM may lead to a general attenuation of stress-related activity in these networks.The observed associations with activity in the right vlPFC , as well as in the insular cortex, vmPFC, vlPFC, dlPFC, vACC, precuneus, and PCC may suggest a supportive effect of GS on coping with stressful events. However, we cannot infer how the amount of GS would exert such effects. Interaction with nature becomes less likely with increasing distance for people to reach GS2.5 in considerably more brain regions than for the larger PM10. This observation could be explained by an easier passage of the blood\u2013brain barrier by smaller particles compared to larger ones, but also by a different chemical composition of the two particle sizes45. A previous study reported that PM2.5 accounted for 65% of PM10, suggesting reducing especially PM2.5 is crucial for improving air quality46. For the environmental variables we used, PM2.5 was also present in PM10 and, thus, may have driven the effect of PM10.An association could be localized for the smaller PM39. Interesting to note is that while for PM2.5 the significant area covered mostly anterior-lateral amygdala regions, whereas for GS only anterior parts appeared significant. The amygdala is a complex of several nuclei with different in- and output regions, which have been shown to not only correspond to negative, but also positive emotional stimuli48. However, segregating the amygdala into anatomically and functionally specific regions has been challenging and could be an interesting research question for future studies to consider49.Surprisingly, the activity changes of the amygdala, a region which is associated with emotional processing, were opposite to what we would have expected to see, based on the assumption that green space promotes adaptive stress processing and air pollution weakens adaptive stress processing 51. Likewise, it was reported that a certain increase of PM10 and NO2 were associated with reduced volumes of many subcortical regions, amongst others, the amygdala52. Though volume does not equal function, air pollution-related reduction of white matter volume might play a role in the unexpected deactivation of the amygdala. Nevertheless, this and the replication of our results would have to be tested in future studies.Additionally, PM has been discussed to impair the central nervous system by stimulation of pro-inflammatory cytokines and oxidative stress, which lead to neuronal lossX or NO2 with stress-related activity. Studies have reported an increased risk for cerebrovascular and neurodegenerative diseases with the concentration of NOX or NO223. Furthermore, we did not find an association with noise pollution. Research has suggested that, besides objective noise pollution, subjectively perceived noise annoyance may play a part in psychological wellbeing, which was not assessed in the current study53.We did not find an association of NO31. Nor can we make assumptions for children and people with older age55. Second, the data is cross-sectional which prohibits any statement concerning causality. Third, the time of scanning and collection of environmental data did not match exactly due to the retrospective nature of our analyses: environmental data for the city of Berlin have been published only in an interval of several years. As air pollution data was extracted from the year 2015, hence 1\u00a0year before the acquisition of stress-related task activity, it could be argued that the order of events is sensible as the exposure to air pollution precedes the experiment. A different method, in terms of chronological order, had to be used for noise pollution, whose values were derived from the year 2017, a year after scanning, which might explain why we did not find effects at all. Also, it has been reported that noise exposure tasks were not successful as a stress induction method, leaving it questionable to use objective noise data for future studies56. Additionally, previous research suggests that besides objective noise pollution, subjectively perceived noise annoyance might play a role for psychological well-being53. Noise annoyance represents a conscious judgment, which is usually based on repeated experience of disturbances, an affective reaction to the disturbances or noise, and the limited ability to do something about it, which is also experienced as a loss of control57. And lastly, amount of green spaces was deducted from current land use data, thereby showing the least chronological conformity. As discussed above, we did find associations for rather non-walkable buffer sizes, which could have resulted from a more indirect effect in means of attenuating air pollution. Especially for green space, we have to consider the fact that we do not have information on, e.g., purpose, frequency or duration of use, which can help understand the impact of green space on health58. Fourth, we only had a significant main effect of salivary cortisol concentration, but post-hoc tests did not show significant differences between the time points, which might be due to the low absolute cortisol concentrations59. Another study reported a non-significant main effect when analyzing the entire sample due to the occurrence of non-responder60. Also, as we did not include a non-stressed control group, unfortunately, we cannot compare if cortisol concentrations in the stress group would have differed at all from a control group. Heart rate, however, resulted in a significant increase during the stress task and a decrease during the following non-stress resting state, which indicates a response of the cardiovascular system61. Likewise, verbal ratings increased from pre- to post-stress and decreased again after the second resting-state, which reflects a subjective experience of stress62.Our study had four main limitations. First, the study cohort was relatively small, not representative, and includes only age and BMI as potential biasing factors. Thus, the results cannot be generalized to a general population or to females because stress responses differ between men and womenAlthough we sought to offer an interpretation of our findings , our results should be interpreted as preliminary. Future studies have to find out not only whether we can replicate our findings but also if they are related to processes of emotion regulation.We found greater activation with increasing amounts of GS in brain regions relevant for regulating emotions in a stressful task. With an increasing PM concentration in the residential area, less activation in numerous regions was related to general attenuation of stress-related activity. However, we do not know whether the presence of residential GS directly influences processing of neuronal stress, whether it has indirect effects by filtering air pollutants, or whether other mediating factors are involved. This was the first study to describe changes in neuronal activity associated with the PM concentration. Depending on the place of residence, urban dwellers are exposed differently to stress-decreasing and stress-promoting environmental factors. Our results may help to understand associations of environmental inequality within a city and stress vulnerability of the brain. Further studies with larger sample sizes, equal inclusion of women and men, and preferably longitudinal observations as well as integration of different areas of expertise are needed to investigate more specifically the underlying mechanisms and implications of our findings.64. The study protocol was approved by the Medical Ethics Committee of Charit\u00e9\u2013Universit\u00e4tsmedizin Berlin . Written informed consent was obtained from all study participants. All methods were performed in accordance with the relevant guidelines and regulations.Originally, 50 healthy male volunteers, capable of german language, were recruited via mailing lists, advertisements on a website and on flyers. Of these, four were excluded from analysis due to incidental findings in their anatomical scan in order to have minimal distortion when matching the individual brain to a standard brain. Four more men were excluded because their addresses were outside of the city borders of Berlin. Thus, the final study cohort for analyses comprised 42 healthy men (mean age (SD)\u2009=\u200930.12 (5.57) years; range, 20\u201348\u00a0years) with a mean BMI of 23.71 . All participants had a mean depression score below clinical significance (Beck Depression Inventory: mean (SD)\u2009=\u20094.26 (4.05); range, 0\u201315). 33 participants reported to drink alcohol and 23 participants reported to smoke. School education was distributed as follows: 33 participants completed a high-school diploma, six completed 10th grade, one completed a vocational baccalaureate diploma, and two completed other degrees than listed. Professional education was distributed as follows: 26 participants completed university, six completed an apprenticeship, five completed university of applied sciences, two completed other professional educations than listed and three did not complete any professional education. This experiment was part of a larger study, which took place in 2016, in which participants needed to be tested after a working day, therefore scanning always took place on a Wednesday (n\u2009=\u200922) or Thursday evening (n\u2009=\u200919) between 17:00 and 22:00. Participants refrained from taking caffeine 2\u00a0h before scanning and from strenuous physical activity for the entire day . Further inclusion criteria were: no shift-work, non-smoking, a late chronotype as defined by a midpoint of sleep later than 04:30 a.m. (based on Munich Chronotype Questionnaire), no current or past psychiatric disorders , no current or past physical disorders of the major organ systems65. Public UGS was calculated as a total sum and as a percentage of public GS with a minimum size of 0.5\u00a0ha, including urban parks, urban forests, allotment gardens, and cemeteries, in different buffer areas around street addresses. The buffers we used were 250, 500, 1000, 1500, 2000, 2500, 3000, 4000, and 5000\u00a0m. In addition, the closest distance to UGS\u2009\u2265\u20092\u00a0ha was calculated to indicate potential differences in accessibility of larger GS. For participants residing close to the Berlin border (n\u2009=\u200912), the larger buffers surpassed the land-use data of the city of Berlin. To ensure that values were not underestimated or overestimated for these participants, data from forest areas in Brandenburg (http://www.brandenburg-forst.de/LFB/client/) were additionally calculated and compared with values extracted from Berlin only by the Wilcoxon signed-rank test . Therefore, analysis for this buffer was done with GS values including forest areas in Brandenburg.Urban green space (UGS) data were based on land-use data extracted from the Urban and Environment Information System provided by Berlin\u2019s Senate Department for Urban Development and Housing for 201965. Air-quality data were provided as a modelled annual mean value for the year 2015 on a raster of 500\u00a0m\u2009\u00d7\u2009500\u00a0m for PM10, PM2.5, NO2, and NOx. Emissions were determined at different spatial resolutions and aggregation level depending on the source group , heating of buildings on building block level, biogenic sources and construction sites on borough level) and aggregated to an uniform raster grid66. Noise data were based on a continuation of the \u201cstrategic noise maps\u201d of Berlin created in 2017. These maps combine information on the mean noise pollution of the main sources of urban noise during the night (22:00\u201306:00) and day (06:00\u201322:00). Noise data were extracted within a buffer of 50\u00a0m and 100\u00a0m of the participant\u2019s street address to take into account different levels of noise exposure depending on the orientation of the place of residence. For more information on the residential area studied, see Data on air quality and noise were also obtained from the Berlin\u2019s Senate Department for Urban Development and Housing60. Time pressure was introduced by an algorithm that reduced the available time to solve the set tasks depending on performance during previous trials and by presenting the remaining time via a visualized countdown . In the case of a mistake, the comment \u201cIncorrect!\u201d was presented on the screen. In the case of a correct answer, but slow performance, the comment \u201cWork faster!\u201d was presented on the screen. During the control condition, participants merely had to match figures and numbers.The ScanSTRESS task was used to induce acute social stress by carrying out figure rotations and mathematical subtraction tasks while being observed by a two-person panel who provided verbal negative feedback http://www.neurobs.com/).The task started after instructions from the panel with a practice run that included a control condition for figure rotation and subtraction and a stress condition for figure rotation and subtraction. Each run took 30\u00a0s, with a 10-s break in-between. After the practice run, the panel provided negative verbal feedback to increase subjective stress. Afterwards, the experimental run started with a control block consisting of 60\u00a0s of figure rotation, a 20-s break, 60\u00a0s of subtraction, a 20-s break, followed by a stress block with 60\u00a0s of figure rotation, a 20-s break, and 60\u00a0s of subtraction. This order was repeated twice so that the total duration of the task, including practice and experimental runs, spanned\u2009~\u200915\u00a0min. The adapted ScanSTRESS paradigm was programmed and presented with Presentation 18.1 provided by Neurobehavioral Systems , a fieldmap, two resting state scans (pre- and post-stress) and a working-memory task with emotional distracter images Fig.\u00a067.Figure0, t1), one directly before ScanSTRESS (t2), three after ScanSTRESS in-between other scans , and two after scanning . During these timepoints, participants also rated their subjective feeling of stress on a scale from 1 (\u201cvery low\u201d) to 10 (\u201cvery high\u201d). Heart rate was recorded during ScanSTRESS, as well as during resting-state scans preceding and following the stress task.Eight saliva samples were taken throughout the entire scanning protocol: two before scanning , including age and BMI as covariates. Effect sizes for t-tests were calculated using the website http://www.psychometrica.de/effect_size.html. The cortisol concentration as well as the subjective stress rating were analyzed for four time points (t1\u2013t4). We excluded t0 (because it was outside the scanner environment) and t5 to t7 67. One participant was excluded from analyses for t1 and t4, and two participants for t2, due to missing values for the cortisol concentration. The cortisol area under the curve with respect to increase (AUCi) was calculated, which was used for Pearson correlations with environmental variables68. Two participants were excluded from the AUCi analysis due to missing values for the cortisol concentration at one or more of the sampling time points. Heart rate was not recorded for three participants during resting-state scan before the stress task (r1), for three participants during stress task and for one participant during resting-state scan after the stress task (r2), providing 37 values for the t-test including r1 and stress and 39 values for the t-test including stress and r2.The cortisol concentration in saliva, stress rating, and heart rate were tested with a repeated-measures ANCOVA, followed by post hoc one-tailed paired-sample 71. Subject-level analyses were carried out in FSL using the general linear model, in which stress blocks were compared with control blocks. Group-level differences in activity between stress blocks and control blocks were assessed with a one-sample t-test, as well as the association of these differences with geographical data, each time including age as a covariate. Then, the resulting t-statistical maps underwent threshold-free cluster enhancement using the default parameter settings , and significance testing was carried out with permutation testing (4000 iterations) using TFCE_mediation (https://github.com/trislett/TFCE_mediation)72. In the latter step, a null distribution of random results was generated against which empirical findings were tested. This strategy resulted in statistical images that were Family Wise Error (FEW)-corrected across the whole brain at p\u2009<\u20090.05 for the main effect of stress. Taking the mutual correlation between the nine buffer sizes of GS into account (average r\u2009=\u20090.50), the Bonferroni-corrected significance threshold was p\u2009=\u20090.017 . Taking the mutual correlation between the two PM and NO values into account , the Bonferroni-corrected significance threshold was p\u2009=\u20090.047 for PM and p\u2009=\u20090.05 for NO. Voxel-wise uncorrected (t) and corrected (TFCE p) statistical maps of our analyses are available on NeuroVault (http://neurovault.org/collections/9333).During the ScanSTRESS task, gradient-echo planar images were acquired on a 3-T scanner with a 32-channel head coil using the following parameters: 426 volumes; repetition time (TR)\u2009=\u20091560\u00a0ms; echo time (TE)\u2009=\u200925\u00a0ms; flip angle\u2009=\u200965\u00b0. Twenty-eight slices of 3-mm isotropic voxels were acquired sequentially in descending order, and auto-aligned parallel to the anterior commissure\u2013posterior commissure line. A high-resolution T1-weighted image and a fieldmap image were acquired for registration. Preprocessing was carried out using FMRIB Software Library (FSL), Advanced Normalization Tools (ANTs), and Independent Component-Analysis based Automatic Removal of Motion Artifacts (ICA-AROMA)p\u2009<\u20090.05 from significant buffers and correlated with BMI in a Pearson correlation.In order to test possible associations of GS-related changes in brain activity with Body Mass Index (BMI), mean time series from the whole brain activity were extracted at a lenient threshold of More detailed information on preprocessing and analyses of fMRI data are reported in the Supplementary Information."} +{"text": "Circular RNAs (circRNAs) are a type of noncoding RNA, which play a vital role in the occurrence and development of esophageal squamous cell carcinoma (ESCC). While the role of novel circADAMTS6 in ESCC remains unknown. We assessed circADAMTS6 expression in ESCC tissues and cells, and the relationship between circADAMTS6 expression and overall survival of ESCC patients. Functional experiments in vitro and xenograft in vivo assay were applied to explore the functions and mechanisms of circADAMTS6 in ESCC. Results found that up-regulation of circADAMTS6 was associated with poor overall survival and may acted as an independent risk factor for ESCC prognosis. Knockdown of circADAMTS6 significantly inhibited the proliferation, migration and invasion of ESCC cells and growth of xenograft tumors in vivo. Induced AGR2 expression was able to rescue the loss of function induced by si-circADAMTS6 in KYSE150 cell. CircADAMTS6 may acts as oncogene by activating AGR2 and the Hippo signaling pathway coactivator YAP in ESCC. It has a high incidence in China, especially in Shanxi and Hebei Province. As the main pathological type of esophageal cancer in China, ESCC accounts for more than 90% of the total number of esophageal cancers4. Despite rapid advances in treatments, including neoadjuvant chemotherapy or immunity therapy, the prognosis of ESCC patents remains poor, with 5-year overall survival (OS) rate is less than 20%5. Consequently, our immediate concern is to investigate the pathogenesis of ESCC and to seek early screening indicators.Esophageal cancer is the sixth dominant cause of cancer-related mortality worldwide that threatens human health seriously7, which makes them resistant to RNA exonuclease compared to linear RNA. In addition, the tissue expression specificity of circRNAs endows them stably exist in saliva, plasma and other peripheral tissue, making them potential prognostic markers10. Growing evidence suggests that circRNAs are abnormally expressed in various diseases including ESCC and may act as tumor suppressor genes or oncogenes during the occurrence and development of cancer12. Our previous studies showed that ciRS-7 was over-expressed in ESCC tissue and accelerated the proliferation, migration and invasion ability of ESCC cells by regulating the miR-7/KLF4 axis to activate the NF-\u03baB p65 signaling pathway13. Cao et al. found that the up-regulation of circRNA-100876 promoted ESCC cell invasion, migration and epithelial mesenchymal transition (EMT), and associated with poor prognosis14. For instance, circUBAP2 plays a role of oncogene by regulating mir-422a/rab10 axis and may be\u00a0a predictive marker for the\u00a0prognosis\u00a0of ESCC15. Above discovers reveal the vital role of circRNAs in ESCC. However, many valuable circRNAs related to ESCC need to be further explored and identified.As a group of endogenous noncoding RNAs, circRNAs have stable covalent closed loop structure16. Studies demonstrated that AGR2 promotes tumor growth by inducing dephosphorylation of Yes-associated protein (YAP) in lung adenocarcinoma17. Besides, AGR2\u00a0overexpression promoted cell proliferation and migration and inhibited TNF-induced intestinal epithelial barrier damage by activating YAP18. However, little is known about the relationship of AGR2 and circRNAs in ESCC.Anterior Gradient Homolog 2 (AGR2) is a member of protein disulfide isomerase (PDI) family, which is overexpressed in ESCC, lung cancer, breast cancer and other cancerIn our research, hsa_circ_0072688 was discovered and dramatically expressed in ESCC tissues and cells. qRT-PCR results showed that knockdown of circADAMTS6 significantly reduced the proliferation, migration and invasion of KYSE150 and KYSE30 cells. Base on recent investigations indicating the function of AGR2 to cancer progression, we aimed to confirm regulatory effect of circADAMTS6 on AGR2 expression. Mechanistically, circADAMTS6 positively regulates the expression of AGR2 to accelerate the proliferation and invasion of KYSE150 cell by activating the expression level of the Hippo signaling pathway co-activator YAP. In conclusion, our study demonstrated that circADAMTS6 may play a vital role as an oncogene and serve as a tumor marker to promote early diagnosis and treatment of ESCC.19 to explore the characteristics of circADAMTS6 in ESCC cell and tissue. The results showed that the circADAMTS6 was resistant to RNase R treatment, which was different from the linear control gene GAPDH was applied by using the circADAMTS6 probe. 114 ESCC patients with clinicopathological and follow-up dates were obtained on Tissue Microarrays (TMAs). CircADAMTS6 positive cells were stained red and the nuclei were stained with DAPI in blue. Representative images of H&E and FISH staining of circADAMTS6 expression in ESCC tissues and adjacent normal tissues were displayed Fig.\u00a0. The outP\u2009<\u20090.05), but not corrected with gender or age in ESCC tissues (P\u2009>\u20090.05). Multivariate\u00a0analysis\u00a0indicated\u00a0that patients with higher circADAMTS6 expression had shorter overall survival time than those with lower circADAMTS6 expression, suggesting\u00a0that circADAMTS6 was an independent prognostic factor in ESCC , while phosphorylated Yes-associated protein (pYAP) was significantly increased (P\u2009<\u20090.05) Fig.\u00a0B. Consis05) Fig.\u00a0C.We further explored the relationship between AGR2 and circADAMTS6 by detecting whether AGR2 overexpressed could rescue the effect of knockdown circADAMTS6 in KYSE150 cell. Transfection of AGR2 overexpression plasmid significantly increased the expression level of AGR2 in KYSE150 cell Fig. . For resThe above results demonstrated that circADAMTS6 positively regulates AGR2 expression to promotes the progression of ESCC by activating the expression level of the Hippo signaling pathway coactivator YAP.22. Traditional tumor markers are not sufficiently sensitive and effective for early ESCC detection, largely ESCC patients are diagnosed in the advanced stage and the overall\u00a05-year\u00a0survival\u00a0rate\u00a0is relatively poor25. Despite the characteristics and functions of many circRNAs have been studied in depth in recent years, the mechanism of circADAMTS6 in ESCC metastasis and prognosis remains unknown. Our research aim to investigate the potential role of novel circADAMTS6 in the development of ESCC and elucidate its underlying mechanism.Increasing evidence indicates that circRNAs play vital roles in various physiological and pathological processes, suggesting that they may serve as potential diagnostic and predictive biomarkers in numerous diseases, including ESCC. CircRNAs interact with tumor-related miRNAs, proteases or signaling pathways, play important regulatory effects in the occurrence and development process of multiple tumors26. Subsequently, circADAMTS6 participates in IL-1\u03b2-induced human chondrocyte dysfunction though competing miR-324-5p and PI3K/AKT/mTOR signaling pathway27. In the study, we identified circADAMTS6 was notably high expressed in ESCC cells and tissues for the first time. In terms of its function and mechanism, circADAMTS6 promoted migration, proliferation and invasion of ESCC cells though the AGR2-YAP axis. Besides, high expression of circADAMTS6 was closely associated with pathological grade, tumor size, lymph node metastasis and poor prognosis. Correspondingly, silencing of circADAMTS6 was confirmed to suppress tumor growth in our xenograft mouse models. Notably, the above results demonstrated that the vital role of circADAMTS6, which may provide a potential therapeutic target for ESCC. As a widely used prognostic marker, the TNM classification is also the most universal tumor staging system in the world29. Then we guessed that the simultaneous use of TNM classification and molecular markers may predict the prognosis of ESCC patients more accurately. This is consistent with the research results of some scholars30.As a novel circRNA, circADAMTS6 was firstly reported to inhibit apoptosis in human chondrocyte by sponging miR-431-5p31. Recently, AGR2 was described to promote tumor metastasis via activation of the\u00a0mTOR/AKT signaling pathway32. Notably, high AGR2 expression levels was found in esophageal squamous tissue and\u00a0ESCC\u00a0cell lines, and was correlated with a worse prognosis in ESCC patients33. The above indicate the direction for further study of the mechanism of circRNAs in ESCC.As an evolutionarily\u00a0conserved pathway, the Hippo/YAP signal transduction plays a vital role in organ size by regulating cell proliferation, apoptosis and metastasis in various diseases. Non-phosphorylation of YAP mediates the transcriptional function of cells, while phosphorylated YAP is retained in cytoplasm and cannot perform transcriptional functions. Furthermore, overexpression and nuclear localization of YAP has been detected in ESCC, lung cancer and breast cancer, and associated with a poor prognosis. AGR2 is an oncogene and involved in cell proliferation, invasion and tumour progression via microRNA, circRNAs and several pathways. It targets and regulates YAP and amphiregulin (AREG) to promotes tumor growth in lung adenocarcinomaIn conclusion, circADAMTS6 may play a vital role as an oncogene and serve as an independent molecular marker to promote early diagnosis and treatment of ESCC. However, article does not involve circADAMTS6 overexpression experiment due to the efficiency of circADAMTS6 formation is too low and the amplification fold of circADAMTS6 was much lower than that of the linear transcript. More works remain to be done for further explore to elucidate other mechanisms.www.arriveguidelines.org).The Ethics Committee of the Fourth Affiliated Hospital, Hebei Medical University approved the study\u2019s protocol and exempted informed consent . Our research was conducted in accordance with the\u00a0Declaration of Helsinki. All animal experiments have been approved by the Animal Care and Use Committee of the Fourth Hospital of Hebei Medical University, and all experiments were carried out accordance with the guidelines. The study is reported in accordance with ARRIVE guidelines (The ESCC tissue microarrays (TMAs: No. EsoS180Su08-XT18-031) were purchased from Shanghai Outdo Biotech Co., Ltd. , involving 114 cancer tissues and 66 adjacent normal tissues. None of the patients received any treatment before operation or diagnosed with other cancers. All patients underwent accurate surgery base on their clinical examinations combined with pathological diagnosis.According to the hospital standard follow-up system, the patients were followed up and evaluated every 6\u00a0months after convalescence. July 31, 2015 was the deadline for follow-up evaluations. All the participants completed 0\u2013107\u00a0months (average: 31.70\u00a0months) follow-up survey. The survival time was defined as the date of operation to the date of death or the deadline of follow-up.The clinical features of the 114 patients were obtained from their medical records, such as age, gender, histological type tumor, pathological grade, primary tumor size and lymph node metastasis. According to the seventh edition of the American Joint Committee on Cancer (AJCC), metastasis status was assessed by the pathologic stage of the disease.Esophageal cancer cell lines were provided by the Scientific Research Center of the\u00a0Fourth\u00a0Hospital\u00a0of\u00a0Hebei\u00a0Medical\u00a0University\u00a0, and cultured in RPMI1640 supplemented with 10% fetal bovine serum , 100U/ml penicillin and 100\u00a0\u00b5g/ml streptomycin at 37\u00a0\u00b0C in humidified air\u00a0containing 5% carbon dioxide.KYSE150 and KYSE30 cells were maintained in six-well plates to 70\u2009~\u200980% confluence, and transfected with circADAMTS6-siRNA (CTAGGTTAAAAAATGGCCA) or si-NC (negative control siRNA) using Lipofectmine 2000 . Measured the expression of circADAMTS6 by real-time RT-PCR at 48\u00a0h after transfection.Genome DNA was extracted from ESCC cells by using a simplified Proteinase K digestion method. According to the manufacturer\u2019s instructions, total RNA from cells was extracted using TRIzol Reagent after transfection. RNase R treatment was managed using RNase R 3\u03bc/mg for 15\u00a0min at 37\u00a0\u00b0C.\u2212\u2206\u2206CT method was applied to compute the fold changes of the target gene expression.The cDNA was served as a template for Nucleic acid electrophoresis and quantitative real-time RT-PCR. All the primer sequences in our experiment were displayed in Supplementary Table RNA FISH was applied to explore circADAMTS6 expression in 114 ESCC tissues and adjacent normal tissues. Cy5-labelled circADAMTS6 probe (5\u2019 biotin -ATGGCCATTTTTTAACCTAGTA- 3\u2019 biotin) was designed and synthesized directly by Guangzhou Geneseed Biotech Co., Ltd. The FISH experiment was performed according to the manufacturer\u2019s instructions. Images were obtained by using a fluorescence microscope at room temperature.5 cells per well and seeded in 6-well plates, incubated 48\u00a0h. After overnight incubation, cell mono-layer was scratched with a 20\u03bcL pipette tip and washed 3 times with PBS. Photos of the wound were taken at 0, 24\u00a0h under the microscope after scratched.Diluted the cells to a density of 3\u2009\u00d7\u2009103 cells per well) in the logarithmic growth phase were seeded in 96-well plates and incubated at 37\u00a0\u00b0C. After 0\u00a0h, 24\u00a0h, 48\u00a0h, 72\u00a0h and\u00a096\u00a0h, 10\u00a0\u03bcl CCK8 was added to each well and incubated for 1\u00a0h. Finally, the absorption value of the whole wells was directly detected at 450\u00a0nm.To evaluated the proliferation of KYSE150 and KYSE30 cell lines by using CCK-8. Cells in five randomly selected fields.To detect the capacity of migration and invasion of ESCC cells through using transwell chamber precoated with or without matrigel. Briefly, 5\u2009\u00d7\u2009106)\u00a0into the right flank of mice. After tumor formation, 5\u00a0nmol si-RNA or si-NC was intratumorally injected into the two groups twice per week for three weeks. Tumor size was monitored weekly and volume was analyzed according to the formula:Four-week-old female BALB/c nude mice were purchased from SPF Biotechnology Co., Ltd , and randomly divided into two groups (6 mice per group). To evaluate the function of circADAMTS6\u00a0in\u00a0vivo,\u00a0xenograft\u00a0nude\u00a0mice\u00a0model\u00a0was\u00a0established\u00a0by\u00a0subcutaneously\u00a0injecting\u00a0KYSE150 cells . Chi-square test was employed to assess the relevance between circADAMTS6 expression levels and the clinicopathological features of ESCC patients. Analyzed the survival outcomes relied on the Kaplan\u2013Meier method and log-rank test, and evaluate the potential prognostic factors of overall survival based upon the Cox proportional hazards regression model. The results were expressed as the mean\u2009\u00b1\u2009S.D., and analyzed by using Student's t-test. All statistical tests were two-sided, and dates in statistically significant were defined as Supplementary Information."} +{"text": "This study focuses on the financing difficulties of small and medium enterprises (SMEs) in China to study the application of blockchain technology in developing the real economy. Deep learning neural network is applied to the vulnerability analysis and detection of smart contracts in blockchain technology by analyzing the connotation of blockchain technology and deep learning. A multiparty joint financial service platform based on blockchain technology is established to help SMEs financing institutions reduce transaction costs, thereby helping them reduce loan interest rates. Finally, Jiangsu Province is studied as a pilot unit. The results show that the Recall and F-score of Bidirectional Neural Network for smart contract vulnerability detection are higher than those of the original neural network. The Recall rate and F-score value of the Wide and Deep model are up to 96.2% and 94.7%, which are higher than those of other vulnerability detection schemes. The Timestamp vulnerability has the highest Recall rate, 94.2%, which can rely on a large amount of valid data to improve detection efficiency. The distribution of financing needs of SMEs in Jiangsu Province from 2020 to 2021 shows that the loan number of SMEs is generally not high. Still, financial institutions and enterprises must spend the same transaction cost. After a technology company in Nanjing made a loan through a blockchain financial service platform, its financing cost decreased by 0.5331%. Blockchain technology has played a great role in the financing process of SMEs, reducing intermediate links and credit costs, and promoting the development of SMEs and the real economy. The world's major economies are deploying digital technologies, the competition in key core technology fields is becoming increasingly fierce, and the global competitive landscape is also profoundly evolving. With the development of real economy business, both business scale and asset securitization product types have ushered in rapid development. How to better help enterprises, especially small and medium enterprises (SMEs), to obtain lower-cost financing through asset securitization is still a problem that the market needs to explore and solve. With the further support of the policy and the reform of the financial market, asset securitization as a new financing method is increasingly favored by enterprises \u20133. Due tSmart contracts take advantage of the distributed structure of blockchain and are widely used in financial and economic fields by automating peer-to-peer transactions. As a new technology, blockchain can effectively integrate financial resources to analyze and process data, improve the financial system from the direction of data, rules, and applications, improve the efficiency and quality of financial operations and services, and generate new financial formats or services \u201316. BlocBased on these questions, literature studies and surveys are employed. By analyzing deep learning and blockchain technology, it is applied in the financial field. The innovation lies in the use of deep learning neural networks in smart contract vulnerability detection, which improves the Accuracy and Recall rate of vulnerability detection. On this basis, a financial service platform based on blockchain technology is established. The platform is used to solve the financing difficulties of SMEs and provides new ideas for the development of the real economy in Jiangsu Province.Blockchain is a technological revolution that commodifies trust. Smart contracts are the tools that help people realize this revolution. It uses smart contracts to develop and implement a new paradigm of cryptoeconomics. Cryptoeconomics is one of the most interesting ways to achieve trust. Combining cryptographic determinism with the design of economic mechanisms to incentivize specific behaviors allows for the realization of novel systems. Systems are not possible until blockchain technology is developed. Blockchain is a peer-to-peer (P2P) network built on the Internet. Under the current business model, continuous recording of transactions is a core function of every company. These records track past actions and performance and guide future planning.Blockchain technology is a revolutionary new protocol for sharing and updating information by linking ledgers or databases in a decentralized, P2P open-access network. Blockchain is designed to ensure that data is stored and updated in a secure, tamper-proof, and irreversible manner. Although the technology is in its infancy, blockchain research is developing rapidly in different fields. The circular economy also focuses on enhancing sustainability and social responsibility alongside economic growth. Upadhyay et al. (2021) reviewedRecently, blockchain has emerged as a research trend with potential applications in a wide range of industries and contexts. One particularly successful blockchain technology is smart contracts. It is widely used in commercial settings, for example, high-value financial transactions. However, this has security implications. This technology has the potential to gain financial benefits from security incidents, such as identifying and exploiting vulnerabilities in smart contracts or their implementations. Ethereum is the most active and high-profile platform and is cBlockchain technology applies a series of distributed computing, data structures, and encryption mechanisms to create a secure online transaction system. In this trading system, buyers and sellers can directly conduct secure transactions. The content of the transaction is completely recorded and cannot be tampered with. The system eliminates all intermediate links. All intermediary chains involved in digitized transactions will be affected. For example, a large part of banks and many financial businesses serve as an intermediary to provide services to buyers and sellers. If buyers and sellers can connect directly through a secure platform, then the existence of these services is unnecessary . As a neIn In In As a piece of code that runs automatically on the blockchain, a smart contract can complete tasks according to preset rules. Manual programming will inevitably lead to loopholes, especially in the financial field, and the losses caused by loopholes are unpredictable. Such vulnerabilities are classified into code layer vulnerabilities, virtual machine layer vulnerabilities, blockchain layer vulnerabilities, and denial of service vulnerabilities due to different causes. Solidity language, as the main programming language for smart contracts, may cause vulnerabilities due to improper use of the language, such as arithmetic overflow and underflow. Mutation errors in smart contracts can cause programs not to perform as expected. Different blockchain systems will cause differences in application fields and characteristics, and the blockchain network cannot be used normally due to denial-of-service attacks by attackers. Vulnerabilities caused by these reasons need to be modeled for detection. Aiming at the problems of high false-positive rate, long time, and low degree of automation in automatic auditing technology in smart contracts, a smart contract vulnerability detection scheme based on deep learning is established, as shown in In N, and the number of types of contract vulnerabilities is denoted as T. Each sample is marked by vulnerability type, set to t, 1 \u2264 t \u2264 T. The vector of samples marked with t-vulnerability is denoted as Xt. Then, in the Forward layer, starting from the first vulnerability, the hidden layer output of each vulnerability calculated forward is denoted as h. Then, the output obtained by the t-th vulnerability is expressed as ht:\u03c3 represents the sigmoid activation function, shown in the following equation:Wi represents the weight preset by the system. b represents the bias value preset by the system. In the Backward layer, the output result of each vulnerability backward hidden layer obtained from the backward calculation from the t-th to the first vulnerability is denoted as h. Then, the result obtained by the backward calculation of the t-th vulnerability is expressed as ht:Assume that the number of samples of known smart contracts is zt by the vulnerability type through the training of the Deep component. Its decision set is denoted as Z={z1, z2, z3,\u2026, zt,\u2026zT}. The determination process is shown as follows: g represents the ReLU activation function, shown in the following equation:The output is the word vector and the discriminant The Wide component is to input the training set into the model for training, as shown in the following equation:Y={y1, y2, y3,\u2026, yT}. Among them, Xwide represents the training set vector and B represents the preset bias. Then, the training results of the Deep component and the Wide component are weighted to obtain the final prediction result pt, as shown in the following equation:WdeepT and WwideT represent the weights of Deep components and Wide components, respectively. Then, the deviation of the current model is calculated by the deviation of the predicted value pt and the vulnerability label, as shown in the following equation:The obtained set of predictions is denoted as t represents the vulnerability annotation. t\u2009=\u20091 when there exists the vulnerability, and t\u2009=\u20090 when there is no such vulnerability. \u22111T(pt \u2212 t) represents the error of a single smart contract. Finally, iterative training is performed by gradient descent until the preset weight is corrected to be smaller than the preset value. Gradient descent is shown in the two following equations:In equation , t repreq stands for gradient. Finally, \u03b1 vulnerability detection module is used for detection.where Loss \u00d7 Common evaluation indicators in the field of machine learning are Accuracy, Recall, Precision, F-score, True Negative Rate (TNR), False Positive Rate (FPR), False Negative Rate (FNR), and True Positive Rate (TPR). The calculation method is as follows:TP means True Positive, where the predicted result is positive, and the actual result is positive. FN stands for False Negative, where the predicted result is negative, but the actual result is positive. FP means False Positive, where the predicted result is positive, but the actual result is negative. TN stands for True Negative, where the predicted result is negative, and the actual result is negative.Google's TensorFlow 2.0 deep learning framework is chosen for experimentation. The operating environment is set to Intel(R) Core(TM) i7-8550U 1.80\u2009GHz processor, 32G RAM memory, 8G video memory, and kernel Linux version 4.8.0\u201352-generic. The dataset comes from web platforms and smart contract vulnerabilities that have been discovered. There are 40135 smart contract samples in total. The samples are divided into the training set and test set with the ratio of 8\u2009:\u20092. The composition of the sample distribution is shown in The cross-border financing of SMEs has greater financing risks due to the problem of information asymmetry. Due to the small scale of SMEs, there is a problem of nonstandard financial management. It is difficult for enterprises to collect information, and it is difficult to calculate credit lines in banks. Traditional trade finance cannot meet the requirements of the digital economy era in terms of trust mechanism construction and risk control. The advantages of blockchain technology information, such as nontampering, distributed ledger management, and information sharing, can provide technical support for financial trade. Taking Jiangsu Province as an example, SMEs account for most of the real economy. Not only can a credible trading environment solve the cross-border financing problem of SMEs but also it improves the status quo of the overall real economy. Some problems need to be solved urgently in Jiangsu Province, such as the time-consuming process of applying for loans for SMEs and the cumbersome examination and approval procedures.Cross-border financial blockchain services are the product of blockchain technology and cross-border financial services. Blockchain can create a trusted information exchange platform between businesses and financial institutions. Blockchain can improve the audit efficiency of financial institutions and increase the trust in SMEs through data sharing and optimizing the financing process of financial institutions. Thus, financial institutions are willing to provide loan businesses for SMEs. The adopted cross-border financial blockchain service platforms include financial institutions, SMEs, and foreign exchange administrations. The composition and functions of platform roles are shown in In In the cross-border financial blockchain service platform, the credit information of SMEs' cross-border financing has undergone informatization processing and credit approval. This can better solve the problem of information asymmetry between banks and enterprises, thereby improving the availability of cross-border financing for SMEs. Blockchain technology solves cross-border financing problems for SMEs through cross-border financial blockchain service platforms. System credit can be formed based on the smart contract algorithm in blockchain technology. This kind of credit helps blockchain technology to establish advantages in the application of SMEs in cross-border financing. The problems faced by SMEs in cross-border financing are finally solved through the mitigation mechanism of blockchain technology to the cross-border financing dilemma of SMEs. The application model is shown in In Although blockchain technology can help SMEs reduce costs, it cannot provide SMEs with the credit they deserve. This technology can only be used as a technical means to ensure the stability of credit circulation. The credit in the cross-border financing business of SMEs ultimately depends on the ability of the enterprise itself to determine whether the loan can be successful. Therefore, if SMEs have irregular management systems and imperfect financial systems, even with the help of blockchain technology, the credit rating of SMEs is still insufficient, and they still cannot obtain mortgage loans from financial institutions.Jiangsu Province is used as a pilot unit. From 2020 to 2021, the loan needs of some SMEs in Jiangsu Province are investigated. The use of blockchain financial services platforms for lending is encouraged. A science and technology company in Nanjing, Jiangsu, is taken as an example, and a specific usage analysis is carried out. The loan cost situation is compared.Compare several neural networks commonly used in smart contract vulnerability detection: RNN, Bidirectional Recurrent Neural Network (BRNN), LSTM, Bidirectional Long Short-Term Memory (BLSTM), Gated Recurrent Unit (GRU), and Bidirectional Gated Recurrent Unit (BGRU); the Recall and F-score results of vulnerability detection are shown in In The number of hidden layers and the number of nodes in each layer will affect the detection results. Set the number of nodes in the experiment to 100, 200, 300, 400, and 500. The number of hidden layers is selected as 2, 3, 4, 6, 8, 10, 15, 20, and 30, and the changes of the results are observed. The comparison between Recall and F-score is shown in In In the vulnerability detection framework of the Wide and Deep model, the comparison of detection results of different vulnerability types is shown in In The Recall rate of all vulnerabilities is around 90%, the highest in the TOD, with a Recall of 94.7%.This method is compared with the effects of Oyente, Slither, and Mythril, as shown in In The distribution of financing needs of SMEs in Jiangsu Province from 2020 to 2021 is shown in In Compared with large enterprises, the loan amount of SMEs in Jiangsu Province is generally not high, but financial institutions and enterprises must spend the same transaction cost. Using blockchain financial service platforms in the credit business can effectively reduce these costs. The transaction cost of financial institutions is reduced, and the financing cost of SMEs that finance on the cross-border financial blockchain service platform will naturally follow.A comparison of the cost of loan financing by a technology company in Jiangsu Province using this service platform is shown in In The reason why the financing obtains lower loan interest rates is that the cost of lending to SMEs by financial institutions in the service platform is reduced. Then, the loan interest rate of SMEs will naturally drop. Therefore, the advantage of the cross-border financial blockchain service platform to alleviate the cross-border financing dilemma of SMEs is to combine all the data information of government departments, financial institutions, and SMEs. The transparency and sharing of information enable the platform to use automated processing procedures to achieve economies of scale and reduce transaction costs. Ultimately, financing costs are reduced for SMEs. The development of SMEs drives the further development of the real economy.SMEs are an important part of China's real economy. From the perspective of development history, they have made great contributions to tax payment, labor employment, technological innovation, and even social stability. However, the difficulty and high cost of financing have always restricted the healthy and sustainable development of SMEs. With the advancement of science and technology, the development of deep learning and blockchain technology provides new directions for financing. Through deep learning in smart contract algorithms in blockchain technology, neural networks can help smart contract algorithms perform vulnerability analysis. A financial service platform based on blockchain technology is established, which can help SMEs financing institutions save costs and reduce loan interest rates, and has been verified in some SMEs in Jiangsu Province. As a result, the Recall and F-score of Bidirectional Neural Network for smart contract vulnerability detection are higher than those of the original neural network. The Recall rate and F-score value of the Wide and Deep model are up to 96.2% and 94.7%, which are higher than those of other vulnerability detection schemes. Timestamp vulnerability Recall rate reaches 94.2%, which can improve detection efficiency by relying on a large amount of valid data. As an important part of the real economy of Jiangsu Province, SMEs in Jiangsu Province will also drive the development of the real economy in Jiangsu Province. The application of blockchain technology in the financial field is very effective. However, there are still some deficiencies to be improved. For example, only four common smart contract vulnerabilities are analyzed in the smart contract security vulnerability detection process, and more instances should be used for verification. The established financial service platform has only been piloted in Jiangsu Province. In future research, SMEs across China will verify the application of blockchain technology in the financial industry, which will bring positive changes to the real economy."} +{"text": "Summary. This review discusses the various factors that are related to treatment nonadherence such as ineffective therapy, adverse effects with topical pharmacotherapy such as skin irritation and erythema as well as patient-related factors such as lack of knowledge of disease and a poor patient-physician relationship. Various methods are being adopted to increase adherence to treatments. Increased adherence to acne therapy has been associated with the use of dermocosmetics, such as moisturizers and cleansers. Encouraging the use of dermocosmetics in synergy with pharmacological regimens could support improved treatment adherence resulting in better clinical outcomes for acne patients. Acne is the most common inflammatory skin disease in adolescence. It is also prevalent in adults, especially females. The disease has a considerable impact on health-related quality of life. Many studies have reported the negative impact of acne on patients due to skin disfigurement, ineffective treatment, and adverse effects of the treatment. Numerous factors contribute towards nonadherence to therapy. Dermocosmetics as an adjunct to pharmacological regimens has the potential to improve clinical outcomes by increasing treatment adherence in patients with acne. Acne is a multifactorial skin disorder affecting approximately 9% of the world's population . Though The intensity of acne is moderate to severe in 15\u201320% of the affected population \u201311.The primary goal of the treatment is to address the immediate clinical signs and symptoms and prevent sequelae such as pigmentation and physical and \u201cpsychological\u201d scarring. The selection of pharmacotherapy is based on the intensity and duration of acne, evidence of sequelae, characteristics of the patient, and patient's preferences and expectations from the treatment .Nonadherence to the prescribed treatment plan can impact treatment outcomes and lead to financial burden. A large-scale observational study among 3339 acne patients from America, Europe, and Asia showed a poor adherence rate of 50% .Dermocosmetics are being adopted for the management of acne to achieve optimal clinical outcomes, improve adherence, and minimize the side effects that can arise from pharmacotherapy \u201316. DermThe aim of this review is (i) to provide an overview of the synergy between pharmacological regimens and dermocosmetics, (ii) to review the role of dermocosmetics to improve treatment outcomes and increase patient adherence to the treatment, and (iii) to consider how dermocosmetics can target additional pathogenic factors to improve treatment outcomes. In addition, other measures used to enhance treatment adherence are discussed.This article results from a focus meeting on acne disease burden, treatments, and their shortcomings including lack of adherence to prescribed treatments. The meeting was held in April 2021 and was attended by all the authors to reach consensus on the role of dermocosmetics in the context of acne management, with a specific focus on the synergy between pharmacological regimens and dermocosmetics and its impact on adherence in acne treatment.The management of acne using pharmacological regimens depends on numerous factors, including disease severity, the site affected, duration of disease, presence of sequelae, age, and gender of the patient, as well as treatment preferences. Topical and oral retinoids, antibiotics , azelaic acid, and benzoyl peroxide (BPO) represent some of the available therapies for acne treatment \u201322.Topical monotherapies for acne include retinoids, azelaic acid, and antimicrobials. The European evidence-based (S3) guidelines recommend topical retinoids for the treatment of comedonal acne, while azelaic acid and BPO can also be considered . A meta-Topical fixed combination therapies are advocated as first-line treatment by the European evidence-based (S3) guideline for the treatment of acne , 20. TheC. acnes [BPO is associated with concentration-dependent adverse events such as irritation and dryness that lead to poor patient adherence , 26. HowC. acnes . CombinaC. acnes \u201330. PatiC. acnes , 30, 31.C. acnes . In anotC. acnes .Consensus guidelines from the Global Alliance and European Dermatology Forum recommend fixed combination topicals including a retinoid and an antimicrobial (which may be an antibiotic) as first-line therapy for mild to moderate forms of acne and then an oral antibiotic for more moderate to severe disease , 31. TheThe use of topical retinoids such as tretinoin, tazarotene, adapalene, and trifarotene may lead to acne flares during the first few weeks of treatment . The infFixed topical combination therapy with clindamycin 1% + tretinoin 0.025% (Clin-RA) showed better efficacy and safety compared to the individual components in mild to moderate cases of acne vulgaris , 38\u201341. Topical retinoids and BPO can cause increased transepidermal water loss (TEWL), impaired permeability of stratum corneum (SC), and increased sensitivity of the skin to UV radiations . In addiDermocosmetics are used as adjuncts to pharmacological therapies for acne and can be formulated to target the main pathogenic pathways in acne. Dermocosmetics may minimize the side effects of acne medications and provide a synergistic effect by targeting additional pathogenic factors causing acne and/or improving the efficacy of other treatments . EffectiC. acnes colonization after application of an emulsion containing licochalcone A, L-carnitine, and 1,2-decanediol [Excess sebum production is one of the most important pathogenic pathways in acne. Dermocosmetics targeting excess sebum production are sebo absorbents that cause direct seboregulation or affect sebum composition. Few topical products such as nicotinamide, fullerene, epigallocatechin-3-gallate, and triethyl citrate + ethyl linoleate + salicylic acid reduce the skin's surface sebum level , 48. Acrcanediol . From socanediol , 52.p < 0.001) in the size and number of microcomedones in women with acne [To stimulate the new epidermal growth, exfoliating agents such as alpha hydroxy acids (AHA) and salicylic acids (SA) are used for superficial peeling in acne patients. A randomized controlled study showed a significant decrease was conducted to evaluate the effect of nicotinamide (NAM) on biofilms of C. acnes. NAM decreased the formation of biofilms when used with suboptimal dosing of tetracycline [ effects . Studies effects , 55\u201360. acycline .The effects of various dermocosmetics used in the form of creams, gels, and lotions are described in The development and manufacturing of dermocosmetics follow stringent regulatory standards to ensure patient safety . MultiplSales in the United States alone for cosmeceutical products are expected to increase by 7.4% per year. An average consumer in the United States uses at least 25 products containing hundreds of ingredients on their skin daily . Considep < 0.05) in noninflammatory lesions in arm A [p < 0.0001) as compared to baseline after treatment for 8 weeks [Dermocosmetic agents are prescribed based on the disease severity and patient characteristics. Controlled studies evaluating dermocosmetics as an adjuvant to pharmacological treatment for better adherence and improved clinical outcomes in acne patients are limited. In a double-blind, placebo-controlled, randomized, multicentre trial, 140 patients with moderate acne received either adapalene and nicotinamide + antibacterial adhesive agents + zinc-pyrrolidone carboxylic acid (Arm A) or adapalene and a placebo cream (Arm B) twice daily for six weeks. There was a significant reduction containing active ingredients such as 1,2-decanediol, regimen . The stu regimen . A key r regimen .The use of dermocosmetics as part of an acne regimen could result in better clinical outcomes and adherence to acne therapy. Various dermocosmetics such as cleansers , sebum-controlling agents (containing nicotinamide or zinc acetate), and antimicrobial or anti-inflammatory cosmetics should be strongly recommended in all acne patients in addition to the pharmacological regimen . The prop < 0.0001) [n\u2009=\u2009944) of patients had poor adherence to at least one treatment, and among patients treated with topical therapy only, poor adherence occurred in 40% (n\u2009=\u2009356) of cases [Acne patients' adherence to treatment is poor . An inte 0.0001) . In addiof cases .Poor treatment adherence is associated with two main factors: lack of knowledge about the acne treatment and adverse effects of the treatment . It has Patients' health literacy is vital in ensuring medication adherence. However, other supportive interventions play a critical role. Some of the interventions include electronic messages and phone calls, virtual engagement with a wider team, audio-visual/internet-based interventions, and patient support programs, which are highly effective in improving treatment adherence , 79. AnoFrom the literature, we know that during the interview with the patient, it is useful to investigate and address the most frequent causes of primary and secondary nonadherence such as little knowledge about the severity of acne, influence from the media or other physicians, fear of adverse reactions, or confusion about how to use treatment . Other fDermocosmetics as an adjunct to pharmacological regimens improve clinical outcomes, increasing patient adherence. There are different types of dermocosmetic preparations, some of which can provide additional benefits, and others that can worsen the acne condition.Generally, the safety level of dermocosmetics specifically developed for the management of acne is good, thanks to the high quality of the ingredients that compose them, and the close surveillance carried out prelaunch and postlaunch.The strong impact of the disease, not only from a morphological but also a psychological point of view, pushes patients to face overall high costs for improving their quality of life. Dermocosmetics fall within the so-called intangible costs but have the potential to improve the condition of these patients in association with correct medical treatment. Thus, the use of dermocosmetics should be advised by the prescribing clinician as an important part of acne management. There is still an unmet need to better understand a patient's expectations and educate people regarding acne to achieve better treatment outcomes. Therefore, adopting dermocosmetics as part of routine acne care provides a positive step towards improved patient outcomes."} +{"text": "Systemic inflammatory response syndrome (SIRS) is a non-specific exaggerated defense response caused by infectious or non-infectious stressors such as trauma, burn, surgery, ischemia and reperfusion, and malignancy, which can eventually lead to an uncontrolled inflammatory response. In addition to the early mortality due to the \u201cfirst hits\u201d after trauma, the trauma-induced SIRS and multiple organ dysfunction syndrome (MODS) are the main reasons for the poor prognosis of trauma patients as \u201csecond hits\u201d. Unlike infection-induced SIRS caused by pathogen-associated molecular patterns (PAMPs), trauma-induced SIRS is mainly mediated by damage-associated molecular patterns (DAMPs) including mitochondrial DAMPs (mtDAMPs). MtDAMPs released after trauma-induced mitochondrial injury, including mitochondrial DNA (mtDNA) and mitochondrial formyl peptides (mtFPs), can activate inflammatory response through multiple inflammatory signaling pathways. This review summarizes the role and mechanism of mtDAMPs in the occurrence and development of trauma-induced SIRS. Systemic inflammatory response syndrome (SIRS) is a non-specific exaggerated defense response caused by infectious or non-infectious stressors such as trauma, burn, surgery, ischemia and reperfusion, and malignancy, which can eventually lead to an uncontrolled inflammatory response . In trauc, and mitochondrial RNA (mtRNA), which are also related to trauma-induced SIRS (Early studies indicate that trauma-induced tissue damage leads to pathogen invasion and the release of pathogen-associated molecular patterns (PAMPs). The binding of specific pattern recognition receptors (PRRs) by PAMPs activates the innate immune system, a prerequisite step for generating immunogenic signals that ultimately lead to infectious SIRS . Gradualced SIRS .Trauma causes mitochondrial damage and dysfunction, leading to mtDAMP release and the induction of an immune response similar to that against pathogen infection . This phHuman mtDNA is a closed-circular double-stranded molecule coding 37 genes. Unlike nuclear DNA, mtDNA can be easily damaged by the lack of repair systems and histone protection . SimilarAs a PRR, Toll-like receptor 9 (TLR9) can directly bind to CpG DNA. After the stimulation by CpG DNA, TLR9 and its intracellular adaptor protein myeloid differentiation factor 88 (MyD88) localize in the endoplasmic reticulum, rapidly redistribute to the CpG DNA accumulation site, and transfer to the endosomal membrane and lysosomal compartment .2+ influx and MAPK phosphorylation, mediating neutrophil degranulation and migration, and inducing inflammation and interferon gene stimulator (STING) are widely expressed in mammalian cells that mediate the expression of type I interferon and other cytokines in infections and inflammatory diseases . cGAS iscGAS binds to mtDNA and catalyzes the generation of 2\u2032-3\u2032 cyclic GMP-AMP (cGAMP) from its substrates GTP and AMP . The sec+ outflow, mitochondrial dysfunction, reactive oxygen species (ROS) and mtDNA release, and lysosomal destruction) are upstream signals for inflammasome assembly and activation inflammasome is a macromolecular complex composed of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) . It is utivation , 36. Howtivation .Unlike cGAS, which binds to both oxidized and non-oxidized mtDNAs, NLRP3 prefers oxidized mtDNA (ox-mtDNA) , 37. Ox-Zhong et\u00a0al. confirmed that the newly synthesized mtDNA is indispensable for NLRP3 inflammasome activation . TLR actN-formyl methionine is the first amino acid during translation initiation. Severe trauma causes mitochondrial damage and the release of mtFPs into the blood circulation, where they bind and activate formyl peptide receptor (FPR), and recruit immune cells to mediate inflammatory response ; promotes ROS and proinflammatory cytokine production; and enhances cytoskeletal rearrangement and network formation and MAPK signaling pathways . MtFP-stormation , 48. Liuormation . Lee et\u00a0ormation . Studiesormation , 52. TheTFAM is abundant in the mitochondria and plays a key role in stabilizing the mtDNA structure and protecting mitochondrial function . TFAM damtRNA, synthesized through mtDNA transcription, can also lead to inflammation because of abnormal accumulation and release. Mitochondrial ssRNA can activate TLR8/MyD88 signaling pathway . After rCL is a crucial phospholipid in the bacterial membrane and mitochondrial inner membrane and plays a pivotal role in maintaining the electron transport chain, mitophagy, and apoptosis . Chen etExtracellular ATP (eATP) induces the generation and release of IL-1\u03b2, IL-6, and TNF-\u03b1 by activating phospholipase A2/D, MAPK, NF-\u03baB, and other pathways . In the c is released from damaged cells into the extracellular space and acts as DAMP to trigger the inflammatory response. Wenzel et\u00a0al. discovered that cytochrome c induced the inflammatory activation of microglia through the TLR4 signaling pathway , can reduce circulating mtDAMP levels and reduce the severity of organ injury in rat hemorrhagic shock models , acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and traumatic brain injury (TBI) . The folARDS is an acute inflammatory lung injury. Clinical studies have discovered that plasma mtDNA levels correlate with ARDS severity in trauma patients and can also predict the risk of ARDS during distal injury , 78.In vivo experiments of burn-induced ALI mice revealed that elevated plasma mtDNA levels may enhance neutrophil infiltration and post-burn ALI through cGAS/STING and NLRP3 signaling pathways promotes mitochondrial degradation and mtDNA release, which activates the cGAS/STING signaling pathway and exacerbates inflammation and kidney injury after renal IR . Zhao etIn a cisplatin-induced AKI mouse model, mitochondrial-targeted therapy with SS31 peptide improved renal oxidative stress by inhibiting the mtROS/NLRP3 signaling pathway . In addiin vivo , which can lead to neuroinflammation. This inflammation can be caused by mtDAMPs, which trigger the release of proinflammatory cytokines and polarize microglia/macrophages toward an M1-like phenotype . A serie2+-activated K+ channels, big K+ (BK) channels, is important for K+ transport. MaxiK channels are important in a variety of physiological functions, including regulation of neuronal firing, endocrine cell secretion, smooth muscle tone, and cellular proliferation and migration , which was related to the increase of ROS content in hypoxic smooth muscle and the damage of membrane integrity, leading to graft failure (In smooth muscle, Thankam et\u00a0al. found that TFAM, cytochrome failure . In mice failure .In a retrospective study on patients undergoing a liver transplant, Nagakawa et\u00a0al. discovered that the measurement of plasma mtDAMPs might predict the post-transplantation recovery of the patients . In burnThe circulating mtDNA level after pancreaticoduodenectomy is associated with inflammatory responses and may be used as an early marker of the postoperative disease course . During Post-trauma tissue damage releases mtDAMPs from the mitochondria into the cytoplasm or the extracellular space, leading to proinflammatory cytokine release and immune cell activation through a series of signaling pathways. MtDAMP-induced inflammatory reaction protects the body; however, excessive inflammation damages organ function and causes SIRS and MODS, which are poor prognostic factors for trauma patients. TW and JY designed the project; JY and XH wrote the paper; ZW, RL, LG and MZ performed literature search and revision; and all authors participated in the data analysis and interpretation. All authors contributed to the article and approved the submitted version."} +{"text": "Interfaces at thenanoscale, also called nanointerfaces,play afundamental role in physics and chemistry. Probing the chemical andelectronic environment at nanointerfaces is essential in order toelucidate chemical processes relevant for applications in a varietyof fields. Many spectroscopic techniques have been applied for thispurpose, although some approaches are more appropriate than othersdepending on the type of the nanointerface and the physical propertiesof the different phases. In this Perspective, we introduce the majorconcepts to be considered when characterizing nanointerfaces. In particular,the interplay between the characteristic length of the nanointerfaces,and the probing and information depths of different spectroscopy techniquesis discussed. Differences between nano- and bulk interfaces are explainedand illustrated with chosen examples from optical and X-ray spectroscopies,focusing on solid\u2013liquid nanointerfaces. We hope that thisPerspective will help to prepare spectroscopic characterization ofnanointerfaces and stimulate interest in the development of new spectroscopictechniques adapted to the nanointerfaces. Increasing surface area through nanostructurationhas become a successful strategy to improve the chemical and catalyticreactivity3 or energy storage properties4 of nanomaterials. Nevertheless, properties which are notsimply related to a larger surface area also appear, such as (quantum)confinement effects or more reactive edge/defect states. Over thelast years, the controlled synthesis of model nanomaterials with uniformstructure and interfacial properties has enabled a better molecularunderstanding of interfacial effects on nanomaterials.5 The characterization of interfaces involving nanomaterials,referred to as nanointerfaces in the following, is therefore gainingmomentum.Many fundamental chemicalreactions and adsorption processes governingapplications in energy storage and energy conversion, catalysis, andbiology, among others, are taking place at interfaces.6 while \u201cinterfacial\u201dproperties associated with their surface chemistry may also be probedby classically \u201cbulk-sensitive\u201d techniques.7 It is therefore of high relevance to clarifythe interplay between the characteristic length of the nanointerfacesand the probing and information depths of spectroscopic techniquesused for their characterization.A variety of spectroscopic techniques have alreadybeen appliedto provide optical, chemical, or electronic information on nanointerfaces.Depending on the excitation wavelength, the probing depth of the techniquesmay affect the information depth, from where spectral informationis recorded. As a result, spectroscopic techniques with a short informationdepth are often referred to as \u201csurface-\u201dor \u201cinterface-sensitive\u201d while techniques with longerinformation depths (>100 nm) are labeled as \u201cbulk-sensitive\u201d.This classification may become misleading when considering nanointerfaceswith characteristic lengths of the same order of magnitude as theprobing and information depths. Indeed, for nanomaterials, some \u201cbulk\u201dproperties from their core region may be accessed through \u201csurface\u201dsensitive techniques,in situ/operando characterizationof interfaces, which can be found elsewhere for more specific fields.While we mostly focus on solid\u2013water nanointerfaces, the conceptsintroduced here hold for other types of nanointerfaces.In this Perspective, we wouldlike to introduce the main conceptsto be considered for the characterization of interfacial phenomenainvolving nanomaterials. First, a definition and taxonomy of nanointerfacesis proposed. The relevance of nanointerfaces is highlighted, takingthe specific field of electrochemical energy storage as an example.Then, the concepts of probing and information depths are explained,which are of uttermost importance for spectroscopy at nanointerfaces.Finally, these concepts are illustrated by selected examples fromthe literature based on infrared and X-ray spectroscopies. We do notattempt to give an exhaustive review of spectroscopic techniques developedfor 22.19 but a clear definitionhas been lacking so far. By analogy with the EU definition of nanomaterials,10 we propose to define a nanointerface as theboundary between two phases, with at least one of the phases havingone or more characteristic dimensions in the size range of 1\u2013100nm. The characteristic dimension, or length, is an external dimensionthat defines the scale of the phase and can be, for example, the diameterof a nanoparticle or the length of a nanowire. Each phase has threecharacteristic lengths for the three spatial coordinates, which definesits dimensionality: 0D when the three characteristic lengths are nanometric, 1D when two are nanometric, and 2D when only one dimensionis nanometric . Note that2D materials with a characteristic length smaller than 1 nm due tomono- or few atom-thin 2D planar arrangements are usually still consideredas nanomaterials.Theterm nanointerface has been regularly used in the nanomaterials\u2019community over the last years,Nanostructuredinterface: A solid materialwith macroscale dimensions and a nanostructured surface form a nanostructuredinterface when exposed to a liquid phase. In this case, both phaseshave non-nano and nanoscale components.Nanocolloidal interface: A colloidaldispersion of a solid nanomaterial in a liquid phase leads to a nanocolloidalinterface. Large interfacial areas are formed when the size of thenanomaterial shrinks down and the nanomaterial concentration is high.Nanoporous interface: Aporous solidmaterial which pores are filled with a liquid phase leads to a nanointerfacewhen the pores have nanoscale dimensions.Nanolayered interface: When both phaseshave nanoscale dimensions only, they form an ideal nanointerface.This is for example the case for layered 2D materials with a liquidphase confined in the interlayer spacing constituting nanoslits asshown in According to this definition, a taxonomy ofnanointerfaces can be derived. Four main classes of nanointerfacesbetween two media, A and B, are possible based on whether A and/orB have their respective characteristic lengths at the nanoscale, asillustrated in 11At the interface, boththe solid and the liquidphases will havean interfacial volume with properties that differ from the bulk volume.This interfacial volume, also known as \u201cinterphase\u201d,usually spans only a few nanometers on both sides of the nanointerface.Villevieille recently summarized the difference between the interfaceand interphase.2.212 Precisely60 years later, this revolutionary but simplistic model was improvedby Guoy and Chapman independently of each other by breaking the rigidorder of the Helmholtz double layer under the consideration of thermalmotion of the cationic and anionic species in the electrolyte followingthe Poisson\u2013Boltzmann theory and thereby defining the EDL asa diffuse layer and a diffuselayer as defined by Guoy and Chapman.15 The compact layer was further split into the inner (IHP) and outerHelmholtz layer (OHP) by Graham in 1947 to account for the specificionic species, which is today considered as the classical theory ofEDL to consist of atomistic layers of opposite charge witha linearly decreasing potential 2a.12 Prese layer 2b.13,14 y ofEDL 2c.16\u221218C is defined asr and\u03f50 the relative and vacuum permittivities, A the interfacial area, and d the distancebetween the opposite charges. Nanomaterials can reach much highercapacitance values than conventional capacitors due to the atomicscale of the EDL (d) and the large surface area .21Although there are manyparameters affecting theEDL ,none have had an impact as fundamental as the presence of nanoporesor nanoconfinement for application in electrochemical storage systems,more precisely supercapacitors, in the past few decades. Briefly,supercapacitors store energy through the formation of an EDL, whichcan be considered as a capacitor, for which the capacitance 23 Traditionally, nanopores were believed to hamper the electrochemicalperformance due to a limited ion accessibility that would hinder theEDL formation.25 Although it was shown as early as 1977 that EDL formation is possiblein nanopores as small as 0.377 nm through desolvation of the cationicspecies,26 it took until 2006 to provethat pores smaller than 1 nm can lead to increased capacitance values.27 These groundbreaking results are based on the unimodal pore designof a carbide-derived-carbon structure, which challenged the generalunderstanding of the contribution of pores smaller than the solvatedions to the overall capacitance of supercapacitors as the highestcapacitances are achieved with pore sizes comparable to the ion size.28 This phenomenon cannot be explained by employing \u201cclassical\u201dEDL models from which the adsorption of the ionic species at the porewalls would be expected to mesopores(2\u201350 nm) and macropores (>50 nm).expected 3a. But, expected 3b.21 SucIn situ/operando spectroscopyis essential for this purpose, and we discuss in the following sectionimportant considerations for probing the EDL and electrochemical reactionsat nanointerfaces.This short descriptionof nanointerfaces for electrochemicalstoragesystems emphasizes the importance of having advanced theoretical andexperimental methods to unravel the unique physical and chemical propertiesof nanointerfaces. 2.330 In the context of nanointerfaces,probing depth (volume) and information depth (volume) are particularlyrelevant. The probing depth relates to the depth of the sample, normalto the surface, which is exposed to the electromagnetic wave and dependson the excitation energy as well as the sample composition. On flatsurfaces, the probing depth is calculated from the attenuation lengthof the incident photons at a given energy and the angle of incidenceof the excitation beam.31 The attenuationlength depends on the material and the excitation energy. On nanomaterialswith a more complex morphology, considering a probing volume is moreappropriate than a probing depth because the presence of curvatureeffects or rough morphology will affect the angle of incidence ofX-rays and complicate the definition of a normal plane to the surface, on the other hand,refers to thedepth (volume) from where a specific percentage of the spectral information is recorded. The information volumedepends on the detection technique that is used to record spectralinformation. It can be equal or smaller than the probing volume. Atypical example of a spectroscopy technique with different probingand information depths is XPS, which is a photon-in electron-out technique,as shown in 2.4Matchingthe probing volume and the volume ofthe nanointerface (S1): Ideally, only the volume of the nanointerfaceshould be probed. When both phases A and B of the nanointerface haveonly nanoscale characteristic lengths (such as nanolayered interfaces),even spectroscopy techniques with large probing volumes will probeonly interfacial signal. When both macro- and nanoscale componentsare included in the nanointerface : if the probing depth ismuch larger than the characteristic length of the nanointerface, theinformation depth can be reduced. In the previous example, XPS wasshown to be highly surface sensitive because of the detection of electronshaving small attenuation length and thereby ensuring a small informationdepth even though a large volume is excited by X-rays. The bulk componentof the probed materials is hence not contributing to the spectralsignature. If one would instead detect the photons emitted as secondaryprocess following the X-ray absorption, the bulk component of theprobed materials would contribute more to the spectral signature dueto the longer attenuation length of X-ray photons.Ensuring selective sensitivity to phase A orB (S3): A spectroscopic technique with high selectivity toeither the phase A or B may allow the removal of bulk contributions,thereby increasing sensitivity to the nanointerface. When phases Aand B are constituted of different elements, the element sensitivityof X-ray spectroscopy can be used to probe both phases separately.This approach is particularly successful when either A or B has onlya nanoscale component .If not, probing and information volumes also have to be optimizedusing the two previous strategies.Ensuring selective sensitivity to the interface(S4): Local changes of optical and/or electronic propertiesin the interfacial region can also be used to probe selectively interfacialsignal. High selectivity may be achieved by specific selection rulesfor optical spectroscopy. A typical example is the change of the refractiveindex at the interface, which ensures a high selectivity to interfacialsignal with Sum Frequency Generation (SFG) techniques.33Now that the main concepts of nanointerfaces,probing, and information volumes have been introduced, the variousstrategies to detect spectral information from nanointerfaces canbe overviewed:in situ/operando characterizationof nanointerfaces for electrochemical energy conversion and storage,further constraints need to be considered. Following electrochemicalprocesses at nanointerfaces during operating conditions implies theuse of electrochemical cells as close as possible to real devices.In general, such cells are based on a 2- or 3-electrode system witha window transparent to the absorbed and emitted photons (or electrons).34 The active nanointerface is therefore buriedin a complex cell design, which requires spectroscopic techniqueswith relatively long probing depths.35 Onthe other hand, maintaining an information volume sensitive to interfacialsignal is also required. Having these constraints in mind is necessaryto carefully design experimental schemes relevant for practical in situ/operando electrochemical characterizations.The different strategies mentioned above will be illustrated withspecific examples of nanointerfaces characterized by optical and X-rayspectroscopies in the next sections.In general, the best strategy willdepend on the typeof nanointerfaces 1 as well33.1In optical spectroscopy, the absorption orthe reflectance of a sample is measured following its excitation byan electromagnetic wave. Depending on the wavelength, the incidentphotons have different energies and excite different transitions.IR spectroscopy probes the vibrational modes of chemical bonds andis particularly sensitive to the surface termination of a materialand its interaction with a solvent, for instance. Increasing the energy,the UV/visible range can probe the transition between two electronicstates of a molecule or the band structure of a material and its interbandstates such as surface and defect states. Applying optical spectroscopyto nanointerfaces is a great source of information. However, IR andUV/visible spectroscopy are bulk-sensitive (information length inthe range of micrometers or millimeters) by default, and one mustapply the aforementioned strategies to gather information from theinterface.36 may allow a significant reduction of the informationvolume (strategy S2). Surface plasmons are coherent electronic modesthat exist at the interface between two materials. The absorptionof the plasmon depends on the size and shape of the nanoparticles,as well as its interaction with the environment. For instance, thequantum size effects in silver nanoparticles are dominated by interfacialinteractions, and the surface plasmon resonance frequency is sensitiveto the local medium dielectric constant.37 It can be exploited for sensing chemicals, gases, and biologicalanalytes.39In the case of noble metal nanoparticles, localizedsurface plasmon resonance (LSPR) occurring in the visible rangeFourier transform infrared (FTIR)spectroscopy, or IR spectroscopyfor short, has been applied extensively to the characterization ofaqueous solutions. It is especially sensitive to the water H-bondingenvironments and benefits from relatively simple set-ups. IR spectroscopyperformed in transmission or diffuse reflectance geometry has a probingand information depth of several millimeters. Attenuated Total Reflectance(ATR) uses an evanescent wave which only penetrates a few micrometersinto the sample deposited on top. Nevertheless, in the context ofnanointerfaces, this geometry can still be considered bulk-sensitive,as the information depth is much larger than the characteristic lengthof the objects of interest. Therefore, in all three measurement geometries,the volume of the nanointerface has to be increased in order to matchthe volume probed with IR spectroscopy (strategy S1).40 In SEIRAS, local surface plasmons created bymetallic nanostructures enhance the electric field of the infraredlight up to a factor of 105,41 allowing the detection of as few as 500 molecules.42 SEIRAS has been used extensively in spectroelectrochemicalstudies of CO2 reduction44 and formicacid oxidation.46 Nevertheless, this techniqueis limited to adsorption on thin metallic films49 or, for best signal enhancement,very well-defined metallic nanostructures.53 It cannot be applied to the characterizationof most nanointerfaces. A similar strategy can be applied to reducethe information volume of Raman spectroscopy, and we refer to recentreviews for more details on surface- and tip-enhanced Raman Spectroscopy(SERS/TERS).54IR spectroscopycan also, in some cases, be surface-sensitive byreducing the information depth (strategy S2). For example, surface-enhancedinfrared absorption spectroscopy (SEIRAS) is a technique that canachieve sensitivity down to single molecular layers in liquid.33 These techniques have an extremely short probing depth. Theey havebeen mostly developed for flat interfaces, but have also been appliedto probe some nanointerfaces, such as the graphene\u2013water interface55 or buried perovskite layers.56 While developments in the characterization of colloidalnanoparticles have been made in recent years,57 the application of these techniques to nanointerfaces with a roughmorphology in electrochemical cells is not straightforward at thecurrent stage.Finally, the use ofinterface-sensitive selection rules is anothersuccessful approach to probe nanointerfaces with optical spectroscopies(strategy S4). In particular, SFG and Second Harmonic generation (SHG)are based on the detection of nonlinear optical processes occurringdue to the breaking of symmetry, such as at interfaces.58 By analyzingthe reflection at different polarizations of the incident light, onecan gain information on the orientation of adsorbed species.59 Also known as polarization modulation infraredreflection\u2013absorption spectroscopy (PM-IRRAS), this extensionof IRRAS overcomes the experimental difficulties caused by the lowerreflectivity of water compared to a metal surface and allows the investigationof thin films on air\u2013water interfaces.60 Again, IRRAS techniques are limited to smooth surfaces and are thereforenot applicable to the characterization of nanoparticles, nanoporousmaterials, or layered nanosheets.The use of surface selection rules at metallicsubstrates can alsobe applied to infrared spectroscopy. Infrared reflection\u2013absorptionspectroscopy (IRRAS) is based on the different intensities of p- ands-polarized light especially at grazing angles of incidence, allowingthe detection of monolayers.3.261 In this section, we highlightsome examples of how different bulk-sensitive IR methods have beenused to investigate nanointerfaces.Many solid\u2013waternanointerfaces have been investigated with FTIR while exposed to variousenvironmental conditions such as heating, cooling, or changing humidity.In addition to probing interfacial water layers, IR spectroscopy iscommonly used to monitor heterogeneous catalytic reactions in thegaseous phase.63 MOFs,65 oxides,68 and biologically relevant systems.70Transmission IR spectroscopyis optically the simplest way to record IR spectra, since the infraredlight goes through the sample at normal incidence. There is no needfor additional optical components, and the absorption of light isindependent of wavelength (unlike in ATR). However, this geometryplaces some restrictions on the sample. Powder samples must be pressedinto a pellet, and the thickness of highly absorbing samples suchas water must be controlled to avoid saturation of the absorptionbands. Any investigations of nanointerfacial water must thereforebe conducted in the absence of bulk water. Despite these limitations,transmission IR spectroscopy has proven to be a very useful techniqueto study interfacial and confined water in various materials suchas nanotubes,62 or imogolite63 nanotubes. At relatively high humidity, thenanotubes are filled with water, while no extensive liquid water filmis formed due to the enhanced water condensation on nanotubes. Inhydrophobic carbon nanotubes, even at fully hydrated state, danglingH-bonds dominate the IR spectrum allows the direct investigation of powders without the requirementto apply pressure, as often is the case for transmission measurementsusing a KBr pellet, or ATR measurements where good contact with theATR crystal is required. The benefits of this method are preservingthe structure of the nanomaterial and better transport of reactants.DRIFTS is therefore particularly well adapted to nanoparticle-gasnanointerfaces and it has been used extensively in catalysis as evidencedby numerous recent reviews in the field focusing on carbon dioxidemethanation on MOFs,78 The chromium terephthalate-based MIL-101-Cr MOF was investigatedat different pressures and the experimental spectra were comparedto MD simulations , which is not possiblewith optical spectroscopies. X-ray spectroscopy excites core electronsof the atoms to unoccupied surface states or to vacuum, providingelement specific information such as the oxidation state of the atomsor the chemical bonds it is involved with. They also offer a widevariety of detection techniques, which can provide information ondifferent electronic states but can also be used to modulate the informationdepth. For XPS, based on the analysis of the kinetic energy of photoelectrons,the information depth can be tuned in the range of \u223c0.1\u201310nm by changing the X-ray excitation energy. This information depthremains limited to a few nanometers due to the short attenuation lengthof electrons in matter31 The soft X-ray region, enabling the probingof light elements94 and of valence shellsof transition metals,95 is particularlyinteresting for nanointerfaces, with probing depth ranging from severalmicrometers for nonabsorbing phases to a few hundreds of nanometersfor absorbing phases. The downside to such a short attenuation lengthis the need for the measurement to be carried out under vacuum, whichimplies complex experimental set-ups. With probing depths of severalmillimeters, hard X-rays are well adapted for in situ/operando characterization in real devices but requirethe removal of bulk components for characterizing nanointerfaces (strategyS1).In X-ray absorption spectroscopy (XAS), the X-ray excitation energyis scanned over an energy range enabling transitions to partial unoccupiedelectronic states of the element of interest. Thanks to the fine-tuningof incoming X-rays, electronic transitions corresponding to a particularelement and bonding state can be selectively excited. The probingdepth will vary with the elements to be probed because it dependsstrongly on the elemental X-ray cross-section and the X-ray energy.96 All these techniqueshave different information volumes which are shown in 97 These spectroscopy methods, alsobased on X-ray photon-out detection, have similar probing and informationdepths as fluorescence yield XAS and will not be discussed further.In the following, we will illustrate with selected examples how thedifferent XAS detection techniques can be applied to characterizenanointerfaces.The true X-ray absorption can be detected in transmission,butthe detection of secondary processes following X-ray absorption isalso possible. The main detection modes are fluorescence yield andelectron yield, while ion yield has also been proposed in the gasand liquid phase.4.294 This ensures a significant probing volume for a nanophase with elementshaving absorption edges lying within the water window, such as carbon,nitrogen, titanium, or vanadium. Coupled with transmission and fluorescenceyield detections, which have information depths of a few tens to hundredsof nanometers within absorbing materials, both the solid and the waterphases can be selectively characterized by tuning the excitation energyas shown in 98 Since nanodiamonds have adiameter (characteristic length) of \u223c5 nm, the full volumeof the nanodiamonds is probed by XAS. Due to their small size, mostof atoms are contained within 1 nm from the surface, therefore XASis still mostly sensitive to the interfacial region on such smallnanoparticles. A similar approach was used to probe TiO2 nanoparticles in colloidal dispersions.100In thecontext of solid\u2013water nanointerfaces, the penetration depthin the range of 1 to 10 \u03bcm of X-rays in the so-called waterwindow (280\u2013535 eV) is a great asset.101 such as the aqueous phase in a nanocolloidal dispersion.98 As a result, the information volume of fluorescenceyield is smaller than the probing volume and the spectra may be distortedfor concentrated phases. On the other hand, transmission detectionenables the measurement of absolute absorption cross sections, assumingthat the sample can be made thin and homogeneous enough to allow reliableX-ray transmission.102 We applied previouslytransmission XAS to probe interfacial restructuring of water aroundnanodiamonds.103 In particular, a different H-bondingnetwork was observed for increasing nanodiamond concentration . Despite the large probing volume, interfacialinformation can still be obtained when probing small colloidal nanoparticles(<10 nm) because of the high ratio of surface atoms to core atoms.An example is shown in articles 11c.105107 The advantage of this techniquecompared to XPS is that only secondary processes leading to a photocurrentare detected. As a result, the photoelectrons do not have to be detecteddirectly and the current can be measured in the electrochemical cellusing a simple ammeter. On the other hand, the origin of the detectedphotocurrent is still subject to debate. This process was first demonstratedon a graphene layer exposed to an applied potential and then characterizedby XAS (106 By introducing a lock-in detection, this techniquewas also used to characterize solvent restructuring at gold- and platinum-waterinterfaces under applied potential.108 Interestingly,the pre-edge observed at the oxygen K-edge of water was found to behighly sensitive to the applied potential (The short informationdepth of electron yield detection limited the characterization ofsamples in vacuum due to the low mean free path of photoelectronsas discussed earlier. Nevertheless, by measuring the drain currentthrough a conductive sample exposed to a liquid, Velasco-Velez etal. showed the possibility to detect electron yield directly at solid\u2013liquidinterface.dby XAS 12a.106 Botential 12b. This110 Thisdetection scheme, called ion yield detection, relies on secondaryprocesses resulting from photoelectron emission in liquid and wasfound to have an information depth closer to fluorescence yield thanelectron yield.111 In fact, since the ionyield detection relies on the diffusion of ions to the counter electrode,it does not suffer from the self-absorption of emitted X-ray photonsand the information volume for concentrated species such as an electrolyteis larger than for fluorescence yield (109 X-ray excitation of \u03c0*C=C transitions, whichled to X-ray absorption for transmission detection, did not inducean ionic yield signal for amorphous carbon dots unlike for graphiticcarbon dots (Using a similar philosophy, Sch\u00f6n et al.showed that bymeasuring X-ray induced ionic current in solution instead of photocurrentflowing through a material in electrical contact with an electrode,XAS in solution can be measured.ce yield 9. We latbon dots 12c. As a112 and requires further research. The developmentof X-ray induced photocurrent techniques, especially to charged nanointerfaces,will certainly contribute to a better understanding of electrochemicalprocesses in the future.We have attempted to describe two differentmethods of detectingX-ray induced photocurrent signals as depicted in 5in situ/operando characterization of photo/electrochemical processes at nanointerfaces.Three further aspects were not discussed in this Perspective but arealso essential when designing an experiment focusing on nanointerfaces:The temporal resolution of the spectroscopytechniqueis a critical parameter that Mmust be taken into account. Molecularprocesses occurring in the EDL at ultrashort time scales will notdiffer between classical and nanointerfaces. However, diffusion processesare likely to be much faster due to the reduced volume of nanointerfaces.54The spatial resolution of the spectroscopytechniquemay be particularly important for some nanointerfaces. We assumedhere that the nanointerfaces to be probed are homogeneously distributedthrough a large volume. However, this is likely not the case for manynanointerfaces, due to local inhomogeneities that may lead to differentinterfacial processes. In this case, nanospectroscopy techniques,enabling spatial resolutions of the same order of magnitude than singlenanomaterials to be characterized, will be required.113 and/ordifferent information depths.114Multimodal spectroscopy, combiningdifferent spectroscopytechniques measured simultaneously, is a promising approach that mayoffer complementary information from the different phases of a nanointerface.This can be achieved using correlation analysis between techniqueswith different selection rulesMolecular processes atnanointerfaces are playing a major rolein many applications and require adapted spectroscopic tools to probethem. We proposed here a definition and a classification of nanointerfaces,illustrated with solid\u2013liquid nanointerfaces. We showed thatthe comparison of the characteristic lengths of the nanointerfaceof interest with the probing and information depth/volume of the spectroscopictechnique is necessary to properly assess the volume which can beaccessed. These considerations are particularly important when assessingthe possibility to perform 115 In addition,computational spectroscopy is generally of great help for the interpretationof spectroscopic results and must be included in the experimentalworkflow whenever possible.116 To conclude,we would like to emphasize that explicitly mentioning the probing,information, and characteristic lengths in further studies on nanointerfaceswould be highly beneficial to facilitate the understanding of theresults, especially when introducing new spectroscopic methods.While this Perspective was focused on experimentalapproaches,the coupling with theoretical calculations is often crucial to drawa full picture of chemical reactions at nanointerfaces. Moleculardynamics can provide crucial information on solvation and interfacialprocesses at the nanoscale, which are not always easily accessibleby spectroscopy techniques."} +{"text": "Materials with an extremely low thermal and high electrical conductivity that are easy to process, foldable, and nonflammable are required for sustainable applications, notably in energy converters, miniaturized electronics, and high-temperature fuel cells. Given the inherent correlation between high thermal and high electrical conductivity, innovative design concepts that decouple phonon and electron transport are necessary. We achieved this unique combination of thermal conductivity 19.8\u00a0\u00b1\u00a07.8 mW/m/K (cross-plane) and 31.8\u00a0\u00b1\u00a011.8 mW/m/K (in-plane); electrical conductivity 4.2 S/cm in-plane in electrospun nonwovens comprising carbon as the matrix and silicon-based ceramics as nano-sized inclusions with a sea-island nanostructure. The carbon phase modulates electronic transport for high electrical conductivity, and the ceramic phase induces phonon scattering for low thermal conductivity by excessive boundary scattering. Our strategy can be used to fabricate the unique nonwoven materials for real-world applications and will inspire the design of materials made from carbon and ceramic. Carbon/ceramic nonwovens with sea-island nanostructure perfectly balance extremely low thermal and high electrical conductivity. Meanwhile, thermal stability is another practical challenge. While carbons have excellent stability in inert environments, they degrade at around 400\u00b0C in air (2 nanowire aerogel (14 mW/m/K) (3N4 nanofelts (11 mW/m/K) (Ar atmosphere) , from 20\u00b0 to 250\u00b0C under the air atmosphere. Last, the stabilized nonwovens were carbonized and ceramized in an inert nitrogen atmosphere at 1000\u00b0C for 1 hour (fig. S2C), yielding the foldable C/SiCON composite nonwovens comparable to the printing paper . The pol2; fig. S2C), flexible carbon nonwovens were obtained. The carbonization of fibers caused a reduction of the fiber diameter down to 470\u00a0\u00b1\u00a050 nm (fig. S6) because of an increase in density and mass loss. Raman spectra show typical carbon material peaks such as the G-line and the disorder-induced D-line (fig. S7). Meanwhile, XRD (x-ray diffraction) was used to investigate the crystalline reflection of the carbon fibers. Typically, a broad peak at 2\u03b8 of approximately 25\u00b0 is assigned to the disordered graphitic (0002) plane, and a weak peak at 2\u03b8 of about 43\u00b0 belongs to the overlapping of the . The fiber shape and the diameter were retained after stabilization . The stabilization of PAN fibers by direct heating from 20\u00b0 to 250\u00b0C at a heating rate of 2 K/min (fig. S2B), followed by carbonization resulted in brittle fibers. Insufficient hardening of the fibers caused melting and sticking of the fibers during carbonization, which led to a molten and brittle fibrous microstructure (fig. S5). We found that the stabilization with a step-wise temperature program from 20\u00b0 to 250\u00b0C used to stabilize PAN fibers in this work was essential to avoid the melting of PAN and the fracturing of the fibers. Thus, after the carbonization of the stabilized nonwovens at 1000\u00b0C (NThe findings on PAN were transferred to the preparation of PAN/OSZ-X nonwovens. The fibers with an average diameter in the range of 500 to 600 nm were obtained using 10 to 30\u00a0wt % of OSZ in the electrospinning solution (fig. S6). The average fiber diameter increased to 1 to 1.6 \u03bcm when using higher amounts of OSZ (40 and 50\u00a0wt %) . OSZs were reported to form advanced Si-based ceramics through a polymer\u02d7to\u02d7ceramic transformation at high temperatures (~1000\u00b0C) in an inert gas atmosphere . In geneR/R0) of 1.01 (fig. S12). Moreover, we found that the electric conductivity increased slightly with increasing temperature from \u221250\u00b0 to 300\u00b0C, which is known for semiconducting graphitic materials derived from pyrolyzed PAN and suggests our materials have a broad range of working temperatures . The thermal diffusivity showed a clear falling trend with increasing SiCON content, particularly for the cross-plane thermal diffusivity . As a result, also the in-plane thermal conductivity decreased with increasing SiCON. We obtained the lowest in-plane thermal conductivity for C/SiCON-50 nonwovens (32\u00a0\u00b1\u00a012 mW/m/K), being well in the range of state-of-the-art polymer foams. The in-plane thermal transport measurements were performed in vacuum (<10\u22122 mbar). A similar decreasing trend of thermal conductivity was also observed in the cross-plane direction, which was all conducted under ambient conditions. First, the cross-plane thermal conductivity of C/SiCON-X nonwovens was measured via TPS. The minimum value was obtained for the C/SiCON-50 nonwovens with 10\u00a0\u00b1\u00a00.1 mW/m/K . Furthermore, LFA was used to confirm the cross-plane thermal conductivity. Although higher thermal conductivity values were recorded, like 19.8\u00a0\u00b1\u00a07.8 mW/m/K in the C/SiCON-50 nonwovens , it clearly showed the same trend as TPS: The higher content of SiCON, the lower cross-plane thermal conductivity. The lower thermal conductivity in the case of the TPS measurement can be attributed to an additional interfacial resistance between the measurement sensor and the nonwoven sample surface. Regardless, both cross-plane thermal conductivity values of C/SiCON-50 nonwovens are extremely low . Although the nonwovens constitute an open porous structure, heat transport via the gas phase apparently does not substantially add to the heat transport of these fibrous skeletons.To determine the effect of the ceramic phase on the thermal and electrical properties of the nonwovens, the thermal conductivity and electrical conductivity of the C/SiCON nonwovens with varying SiCON contents were investigated. The increase of the SiCON ceramic phase decreased the electrical conductivity gradually from 20.1 to 4.2 S/cm at room temperature (RT) measured in the in-plane direction but did eratures . The theWe highlight the unique combination of extremely low thermal conductivity and high electrical conductivity of our C/SiCON nonwovens by an Ashby plot compared\u22128 and 4.60 \u00d7 10\u22128 W\u03a9K\u22122 for graphitic materials belonging to the SiCON phase and the typical C\u2550C (1500 cm\u22121) carbon phase (29Si-NMR [Si-29 nuclear magnetic resonance (NMR)] spectroscopy, a distinct SiO4 signal at \u2212104 ppm (parts per million) was found. A broad peak in the \u221250 to \u221290 ppm region was characteristic for complex, mixed SiCxOy, SiNxOy, and SiCxNy environments (I(D)/I(G)] in the Raman spectra and Lc of graphitic carbon in C/SiCON\u02d750 fiber were smaller than that of pure carbon fibers (table S3). Further investigation from the selected area electron diffraction pattern with three Debye-Scherrer rings in the cross-sectional TEM (transmission electron microscope) image of C/SiCON-50 fiber tests . In addition, a methane burner burning experiment in air was conducted \u00a0=\u00a095,000 g/mol; registration no. 26658-88-8]. OSZ Durazane 1800 was obtained from Merck (Germany) and used as received. Dicumylperoxide (DCP) was obtained from Sigma-Aldrich (Germany) and used as received. N, N\u2032-dimethylformamide and 99.9% acetone were obtained from Thermo Fisher Scientific GmbH (Germany) and used as received. The normal printing paper was Orange Label print paper from Canon, A4, 80 g/m2 (https://staples.de/orange-label-performance-papier-a4-80-g-m2-weiss/364513/).PAN was obtained from Dolan GmbH (Germany) and used as received .A combined Raman Imaging/Scanning Force Microscope System with WiTec Control FIVE 5.3 software was used for RAMAN measurements. Laser is equipped with a UHTS 300 spectrometer combined with a back-illuminated Andor Newton 970 electron multiplying charge-coupled device camera .NMR experiments were performed with a Bruker Avance II 300 (magnetic field of 7.05 T) spectrometer in a 4-mm triple resonance sample head at a rotation speed of 10 kHz. The 2 flow rate was up to 70\u00a0liters/min.Pore size measurements were carried out on a capillary flow porometer PSM 165/H . The standard test liquid Topor (surface tension\u00a0=\u00a016.0 mN/m) was used. The specimens were cut into squares (length about 2 cm) and covered the sample holder hole. The sample holder diameter is 11 mm, and Nwww.mikrolabor.com, Germany). According to the company, the following methods were used: carbon in the polymer, oxidized, and ceramic state.The measurements were carried out at Mikroanalytisches Labor Pascher .The samples were burned with a combustion additive at about 1200\u00b0C in a stream of oxygen; the contained carbon burned to COThe samples were burned at 1050\u00b0C in an oxygen stream. The combustion water formed was determined by IR spectroscopy.2, or NOx formed were passed over copper oxide and reduced to N2 after switching the carrier gas to CO2, excess oxygen was bound. The nitrogen formed was purged into an azotometer. Acidic reaction gasses and the carrier gas CO2 were bound by potassium hydroxide solution. The nitrogen gas was measured volumetrically.The sample was melted at a temperature of 900\u00b0C in the oxygen stream with a catalyst. Ammonia, NThe sample was degassed in a heated graphite crucible. The released nitrogen was measured with helium as carrier gas with thermal conductivity detection.Samples were pyrolyzed at 1500\u00b0C in a glass carbon tube with carbon contact. Oxygen was detected as CO and measured by thermal conductivity detection.2 produced by the reaction of the released oxygen with the graphite was pumped off and detected by IR spectroscopy.The sample was degassed in a hot graphite crucible. The CO/COAfter pressure digestion with nitric acid, the silicon dioxide formed was digested with sodium hydroxide solution under pressure. Detection was carried out by ICP-AES (inductively coupled plasma\u2013atomic emission spectroscopy).The sample was digested under melting (with a soda-borax mixture in a Pt crucible) and then dissolved with water. The detection was done by ICP-AES.z-number and absorption effects on the evaluations of EDS spectrum, based on the chemical compositions of a C/SiCON-50 nonwoven measured in Laboratory Pascher.We performed HAADF-STEM imaging and chemical analyses on a field emission scanning transmission electron microscope , equipped with an EDS and electron energy-loss spectrometer to observe and analyze the chemical compositions and relative thicknesses of the samples against a chemical standard of a C/SiCON nonwoven, respectively. For the TEM sample preparation, we used ultramicrotomy and Ar-milling methods to make thin cross-sectional specimens using an ultramicrotomy machine and Jeol Ion Slice. The TEM thin foils were a thickness of 50 to 150 nm, but we selected approximately the same thickness areas for the EDS by using zero-loss spectra in the EELS. The EDS maps were taken at a resolution of 5 to 10 nm per pixel and a dwell time of 16 \u03bcs using a subnanometer-sized electron beam with less than 0.09-nA probe current at 200-kV acceleration voltage. To accumulate statistically enough characteristic x-ray counts in a quantitative EDS map, the total acquisition time was about 60\u00a0min. During the acquisition, an image drift correction function was always activated to prevent artifacts in the profile. To get quantitative compositions of the samples, we corrected 2, the flame was immediately extinguished, and an LOI value of 100 O2% was recorded (movie S4).The LOI experiments were carried out on an Fire Testing Technology Oxygen Index Apparatus ISO 4589-3\u2013NES 715 . The nonwoven samples were cut in shapes (width about 1 cm and length about 6 cm) and fixed in the device. The respective oxygen and nitrogen composition were adjusted before the measurement. Then, the samples were exposed to the oxygen and nitrogen composition atmosphere for 5 min and followed by burning with a methane flame. The composition of oxygen and nitrogen at which the sample burned down independently was recorded. When the methane flame was removed and even at 100% OThe burning test was conducted in a hood in the air atmosphere. For the burning tests, the nonwovens were cut in rectangular shapes (width: 4 cm and length: 6 cm) and fixed in a metal frame. The metal frame with the nonwoven was placed on a metal rack, with a methane burner installed beneath it. Then, the samples were burned with a methane flame (rate: 0.5\u00a0liter/min). After ignition, the samples were burned for 5 min, and the whole process was recorded by video (movie S5)."} +{"text": "Although stakeholder collaboration is key for sustainable development of tourism in small islands, research on its determinants is only emerging. The lack of empirical studies hampers an understanding of how effective stakeholder partnerships for sustainability in small tourism islands can be formed and sustained. To partially address this knowledge gap, this study explores stakeholder collaboration for solid waste management in the island of Koh Phayam, Thailand, from the perspective of stakeholder theory, social capital and proximity effect. Semi-structured interviews (n = 26) reveal a lack of understanding of collaboration benefits alongside leadership and reciprocity among stakeholders. However, due to geographical proximity, the level of stakeholder trust in each other is significant, thus indicating potential for future successful partnerships. For these partnerships to become effective, a system of financial incentives for stakeholders to separate and recycle solid waste in situ should be designed. To improve stakeholder communication and reciprocity, capacity building workshops and round tables can be organised. Municipal authorities should lead on solid waste management, and a steering committee comprising the representatives of all other stakeholders needs to be established to oversee the work of municipal authorities. Lastly, the feasibility of setting private public partnerships for solid waste management in Koh Phayam should be considered given the significant extent of knowledge and trust among local stakeholders. External stakeholders, such as farmers, can be involved in management of organic waste, thus extending the scope of partnerships for sustainability beyond the island. Tourism has significant environmental externalities that should be mitigated towards sustainable development goals . WithoutThe figures from UNEP highlighThe challenge of solid waste management in tourism is pronounced in small islands as remotResearch on solid waste management in small island destinations is evolving but significant knowledge gaps persist . One is However, remoteness and size also suggest that tourism stakeholders in small islands know each other better, thus increasing the likelihood of collaboration, especially in critical situations, such as under a threat of environmental degradation. This is explained by the concept of social capital suggestiThis study explores the determinants of stakeholder collaboration for solid waste management in a small island destination through the prism of stakeholder theory, social capital and proximity. Koh Phayam, a small tourism island in Thailand, is used as a case study. Prior to COVID-19, Koh Phayam has been growing in popularity with tourists. This has intensified the challenge of solid waste management calling for urgent mitigative interventions. Stakeholder collaboration is key for the success of these interventions in small tourism islands ,17,24 anAs a theory of organisational management, stakeholder theory posits that organisations have interconnected relationships with different actors of their value chain known as stakeholders . StakehoStakeholder theory emphasizes that stakeholders should first be identified and then carefully managed . For exaAlthough stakeholder theory does not explicitly integrate the concept of stakeholder collaboration, it communicates the need for stakeholders to shape formal or informal partnerships for common goals . StakehoStakeholder theory is underpinned by the concept of social capital . Social Stakeholder collaboration represents a common strategy of working towards sustainability . For exaStakeholder collaboration for sustainability is defined as the ability of individuals and organisations, representing private and public sectors, and operating in a specific locality, to work together towards a common, pro-sustainable goal . This goThe benefits of stakeholder collaboration for sustainability are tangible and intangible . StakehoStakeholder collaboration for sustainability is hindered by the lack of stakeholder interest, but also by the limited understanding of the goals and outcomes of collaboration . StakehoLastly, in tourism, size of a destination plays a role. Larger destinations have more stakeholders; it can therefore be more difficult to engage them in collaboration for sustainability due to varying interests . In thisThe proximity effect is key for inter-organisational networking and succEmpirical evidence on the effect of geographical proximity on stakeholder collaboration for sustainable tourism in small islands is fragmented. The study by Graci showcaseIn contrast, Canavan reports Literature reiterates that collaboration reinforces the social capital of organisations by extending and strengthening a network of their relationships with stakeholders. The effect of geographical proximity can determine the success of stakeholder collaboration. Stakeholder collaboration is critical for sustainable development of tourism, especially in small island destinations where resources are limited. By pooling together, stakeholders can benefit from each other to enable progress of tourism islands towards sustainability. Research on stakeholder collaboration for sustainable tourism is however limited, especially in developing countries . In part2, maximum length 10 km, maximum width 5 km, permanent population of 964) in the Andaman Sea located 33 km off the west coast of Thailand. In 2021, circa 150000 tourists visited the island; this figure was however significantly lower than in the peak year of 2019 when about 400000 tourist arrivals were registered , there is little sense. I pay 30 Baht per month [0.9 USD] for [waste] collection. Even if I don\u2019t pay, they [solid waste collectors] still come and collect the rubbish. I don\u2019t think it [collaboration] is that important given the money involved is ridiculous\u2026\u2019 (TB2)\u2018This quote highlights a lack of financial incentives as one of the barriers for more effective stakeholder collaboration . CollectEvidence from developed countries shows that carefully designed financial incentives can be instrumental in promoting recycling and, ultimately, stakeholder collaboration for solid waste management . Even orA lack of leadership and limited understanding of stakeholder roles and responsibilities was another barrier . TourismThey [municipal authorities] told us to separate food from other waste. OK, I said, but where to? They [municipal authorities] said, oh, get your own bin. I said, why should I if I can dump it?.. I tried to compost food waste. I got one of those composter things. It was my own idea, no one helped me, no one told me what to do and how to do it. But then I had to stop. There is no space, and the odour is horrendous\u2026\u2019 (TB7)\u2018\u2018responsible leadership\u2019 in that, for example, there is no corruption and unequal treatment involved with a proposal. I don\u2019t know what happened, but the contract was never signed. I think they should have told us about why they\u2019d decided to turn them down\u2019 (C1)\u2018Communication is key for stakeholder collaboration for sustainability, especially in developing and transition economies and shouReciprocity was another barrier . Some stI went to local villagers and said, hey, how about I bring you organic waste and you compost here as you have more space than I do. But they say, oh, no, we don\u2019t want that as it stinks. So, this is how it goes here in terms of reciprocity [laughter]\u2019 (TB4)\u2018Reciprocal engagement is key for success of stakeholder partnerships for sustainability . In Koh As for enablers of stakeholder collaboration, trust was frequently mentioned . AlthougIt\u2019s a small island and we know each other. How can I say that I don\u2019t trust XXX , if I see him every week in the market? It\u2019s not about trust, it\u2019s about other things\u2026\u2019 (N3)\u2018Power was discussed as an enabling factor from a neutral perspective . There w\u2018powerful as individuals but powerless when together\u2019 (TB5). To reduce the negative effect of this \u2018powerlessness in togetherness\u2019, capacity building events and round tables are warranted. Such events can empower stakeholders by reinforcing their beliefs in that a success is achievable when working in partnerships. Such events can also strengthen perceived reciprocity i.e., another determinant of stakeholder collaboration, as highlighted earlier.The literature emphasizes power as a determinant of stakeholder collaboration for sustainability. However, power is largely discussed from the perspective of its imbalance or even Environmental awareness was discussed as an enabler with theIt\u2019s a beautiful island, almost untouched, you know. But I was shocked when I saw so much garbage here. I understand this is very much from tourism, but I feel the locals don\u2019t try to protect the environment either. The bins are provided for plastics, but it is felt that only tourists are using these bins\u2019 (T3)\u2018Stakeholder collaboration for sustainability should be underpinned by an understanding of why this collaboration is necessary and what it aims to achieve . In Koh Importantly, the barriers and enablers of stakeholder collaboration for sustainability are inter-related. For example, by building and reinforcing trust, stakeholders can reduce such barriers as restricted leadership and limited communication . LikewisWhen comparing the findings of the current study with the literature on stakeholder collaboration for sustainability in small tourism islands, some similarities and differences can be established. Similar to Graci , the curThe study participants outlined three determinants of successful stakeholder collaboration for future management of solid waste in Koh Phayam . First, Private public partnerships (PPPs) for sustainability were discussed . AlthougLastly, potential engagement of external stakeholders in solid waste management in Koh Phayam was discussed . There iIdeally, I\u2019d want my food waste collected every day. Preferably, for free but I\u2019d even be happy to give it out for a small fee. If this waste is then delivered to a farmer, I think that is ideal. No dumping in the sea, no unpleasant smell, yeah, I\u2019d feel good about it [laughter]\u2019 (TB3)\u2018This study explored the opportunities and challenges of stakeholder collaboration for solid waste management in a small tourism island, thus reinforcing the emerging body of literature on partnerships for sustainability. Through the lens of stakeholder theory, social capital and proximity principle, the study revealed such impediments of stakeholder collaboration for solid waste management in Koh Phayam as a lack of perceived benefits, leadership, communication, and reciprocity. The study indicated that geographical proximity exerted a positive effect on social capital and trust between stakeholders. It also ensured that disbalance of power, a major barrier to partnerships for sustainability in developing countries, was less pronounced in the case studied island.This finding suggested sufficient grounds for future stakeholder collaboration for solid waste management in Koh Phayam, subject to addressing other barriers, as highlighted above. From the theoretical perspective, the study contributed to a better understanding of geographical proximity as a determinant of effective partnerships for sustainability. This finding is relevant for small tourism islands, but it can also be extended to other tourism contexts, such as remote mountain destinations, characterised by a small number of stakeholders concentrated in a single locality.From the management perspective, the study highlighted several areas for intervention which can facilitate stakeholder collaboration for solid waste management in Koh Phayam. Financial incentives need to be designed to encourage stakeholder engagement in solid waste management. Capacity building workshops and round tables can streamline stakeholder communication and facilitate reciprocity. One stakeholder, most likely municipal authorities, should take the lead in solid waste management and a steering committee should be established to oversee a leader\u2019s activities. The feasibility of setting PPPs for solid waste management in Koh Phayam should be considered given that local stakeholders know and trust each other. Lastly, external stakeholders can be involved in management of organic waste, thus extending partnerships for sustainability towards other localities.The study had limitations which outlined directions for future research. First, not all stakeholders in Koh Phayam participated in interviews. Future research should aim at engaging other stakeholders and examine their opinions either by a method of interviews, or by focus groups and surveys. Second, the study did not incorporate stakeholders external to Koh Phayam and Ranong, such as farmers and national decision-makers. As these stakeholders are critical for the design of financial incentives for solid waste management alongside PPPs and industrial symbiosis networks, their opinions should be sought in future research. Third, this study established that organic waste in Koh Phayam was severely mismanaged. Future research should focus on organic waste as a key challenge in small tourism islands and examine how it can be managed more effectively. Fourth, the idea of building PPPs for solid waste management in Koh Phayam emerged during interviews. PPPs can be instrumental to progress small tourism islands towards sustainability; however, research on how they can be designed and implemented remains limited, especially in countries of the Global South. Hence, more studies are necessitated to better understand how PPPs can be most effectively developed in Koh Phayam or in other island destinations of Thailand to account for local political and socio-economic contexts. Lastly, this study was only concerned with Koh Phayam. However, Thailand has numerous small tourism islands, including the world-famous destinations of Phuket, Koh Samui and Koh Phi Phi. Future research should focus on these islands as they represent the mainstay of tourist demand.S1 Appendix(DOCX)Click here for additional data file."} +{"text": "To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum\u2014a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlights the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease.Heterozygous HNRNPU, from the molecular to the whole organism level using a genetic mouse model. We generated disease-associated gene expression signatures and compared them to signatures obtained from cells treated with different drugs to pinpoint compounds with the potential to rescue abnormal gene expression.The brain relies on strict regulation of gene expression for proper functioning. Mutations in genes that influence the expression of other genes are linked to neurological conditions such as epilepsy, autism, intellectual disability, and neurodegenerative disorders. Identifying targeted therapies for these genetic causes of disease are increasingly difficult since they often lead to a wide array of molecular and cellular effects from the widespread disturbance in gene expression. One potential approach is to identify therapies that shift the abnormal gene expression signature to a more normal state. To explore this \u2018transcriptome-guided\u2019 approach, we characterized the consequences of the loss of one copy of the gene expression regulator, HNRNPU haploinsufficiency, we extend this transcriptome-guided precision medicine approach to a monogenic neurodevelopmental disease.Given their functional complexity, high metabolic demands, and extensive diversity, it is unsurprising that neurons rely particularly on the strict regulation of gene expression. In fact, mutations in genes that cause gene expression dysregulation, including chromatin modifiers ,2, transHNRNPU (heterogeneous nuclear ribonuclear protein U) encodes a ubiquitously-expressed, DNA- and RNA-binding protein, hnRNP U, that localizes to the nucleus [de novo loss-of-function variants [HNRNPU in pediatric patients with a severe, and often treatment refractory, developmental and epileptic encephalopathy (DEE) characterized by early-onset epilepsy, moderate to severe developmental delay, autistic features, structural brain abnormalities, hypotonia, short stature and variable renal and cardiac abnormalities. HNRNPU is essential for mammalian development as lethality results by embryonic day 11.5 in mice carrying homozygous hypomorphic mutations [Hnrnpu in mouse cardiomyocytes was also associated with a lethal dilated cardiomyopathy and widespread transcriptional and splicing dysregulation including known cardiomyopathy disease genes [Hnrnpu haploinsufficiency in the brain have yet to be characterized. nucleus ,13, wher nucleus \u201319, pre- nucleus ,21 and c nucleus ,23. We avariants \u201328 and mvariants ,30 encomutations . Furtherutations . ConditiHnrnpu mouse disease model using in vivo developmental, morphological, electrophysiological, and behavioral studies. We also perform a comprehensive cell-type- and brain region-specific characterization of reduced Hnrnpu levels on gene expression using scRNAseq at a single postnatal time point. Using these data, we generate cell-type-specific disease expression signatures and identify vulnerable cell types in the mutant mouse brain. We then compare these signatures to publicly available gene expression signatures of cells treated with small molecules to identify compounds that correct disease-associated transcriptomic changes towards a normal state. Overall, this work provides a framework for high-resolution phenotyping of models of transcriptome-mediated diseases and outlines important considerations for the future development of targeted therapies for HNRNPU DEE.Here we assess the neurophysiological consequences and face validity of an HnRNP U protein expression is widespread in the brain, yet particularly concentrated within the cerebellum, hippocampus and neocortex . In mousHNRNPU are loss-of-function (Hnrnpu\u2014a region in which at least five premature truncating mutations were identified in human patients\u2014to induce a constitutive out-of-frame deletion (Hnrnpu+/113DEL or HET) with resulting premature stop codon in exon 2 was identified and used to expand the line, which was subsequently maintained on an inbred C57BL/6NJ background . Furthermore, we did not detect a truncated form of hnNRP U when using an antibody that binds N-terminal to the deletion breakpoint . Furtheral width . AdditioHNRNPU-associated DEE frequently demonstrate axial hypotonia along with moderate to severe developmental delay, primarily manifesting as delayed motor skills and severe speech impairment [Patients with pairment \u201328. We t-3) . This gr-3) and S5B.-3) .Hnrnpu+/113DEL pups showed a subtle increase in latency to fall at PND6 in the vertical screen test . There wctively) .Hnrnpu+/113DEL pups, we evaluated separation-induced ultrasonic vocalizations (USVs). USVs are functionally important signals that elicit maternal retrieval and care [Hnrnpu+/113DEL pups showed clear deficits in USVs , with an atypical trajectory characterized by a slow increase in the number of calls that peaked around PND9 and overall higher frequency compared to control calls (-3) Hnrnpu+/1 in USVs , includiND9 Figs . Furtherol calls . Moreovells (-3) . OverallHNRNPU mutations [in vivo spontaneous and evoked excitability phenotypes using electroencephalography (EEG) and electroconvulsive threshold (ECT) studies, respectively. Despite over 300 total hours of video EEG recordings among Hnrnpu+/113DEL adults, there was no evidence of spontaneous generalized epileptiform activity .HNRNPU mutations [Hnrnpu+/113DEL and WT adult mice .Hnrnpu+/113DEL brain. We compared gene expression profiles from mutant and WT cells of each population using a linear mixed model (The most downregulated gene observed in the subiculum was x 10\u221237) . This efrneurons . Its exprneurons . Interes= -0.36) . This fiMef2c, we examined the number of hnRNP U binding sites for each gene expressed in the brain of mice. Using available hnRNP U CLIP-sequencing data derived from mouse cardiac tissue, we found that Mef2c contains more hnRNP U binding sites than 99% of the other genes assessed [Because heterozygous loss of p (CMAP) ,58 proviHnrnpu haploinsufficiency in disease-relevant brain regions, we expect that this approach will require scRNAseq-derived signatures.Transcriptomic reversal approaches that have leveraged the CMAP and other resources have not only successfully identified targeted therapeutics in cancer ,59,60, bTo examine the importance of cell-type-specific effects, we compared compounds predicted to reverse the subiculum gene expression signature versus compounds predicted to reverse a \u201cpseudo-bulk\u201d hippocampal signature . We specifically focused on the downregulated genes in the disease signatures, as these genes were enriched for biologically relevant pathways and disease genes. The CMAP uses a \u201cconnectivity score\u201d to assess each compound\u2019s ability to reverse the query signature . This scFor the subiculum-derived signature, 128 compounds received a Connectivity Score less than the CMAP\u2019s recommended cutoff of -90 . 98 compHNRNPU haploinsufficiency, bulk RNA-sequencing may not generate adequate signatures to identify the compounds most likely to target the relevant disease mechanisms. Experimental validation will be required to verify that these candidate compounds in fact reverse the transcriptomic signatures and rescue Hnrnpu disease phenotypes. Nonetheless, these results highlight the potential importance of deriving cell-type-specific disease expression signatures for transcriptomic signature reversal approaches.Therefore, if transcriptional dysregulation of a specific cell type, such as excitatory neurons in the subiculum, contributes substantially to the pathophysiology underlying Identifying and modeling germline mutations associated with developmental and epileptic encephalopathies provides the unique opportunity to develop targeted therapeutics . While mHnrnpu does not lead to 50% reduction in gene expression as predicted by the protein truncating nature of the frameshift mutation. Despite only this partial reduction in expression, heterozygous intercrosses failed to produce any homozygous progeny, consistent with embryonic lethality in mice. These findings are also consistent with the observation that mutant ES cells containing embryonically lethal homozygous hypomorphic Hnrnpu mutations showed only a 40\u201380% decrease in Hnrnpu transcript levels [Intriguingly, like the human cortical organoid models previously described , we obset levels .Hnrnpu expression remains unknown, potential explanations include the enhanced stability of Hnrnpu transcripts, such as through autoregulation via self 3\u2019UTR binding [Hnrnpu expression through mechanisms like transcriptional adaptation [Hnrnpu expression.While the mechanism underlying this partial loss of binding or the captation . DiffereHNRNPU neurodevelopmental disease model. Hnrnpu+/113DEL mice displayed several phenotypes that overlap in presentation with the human disorder [HNRNPU-mediated disease phenotypes. Our study also builds upon prior behavioral work conducted in an independent heterozygous Hnrnpu knockout model that demonstrated altered circadian-mediated locomotor and metabolic activity in mice carrying an out-of-frame deletion of exons 3 through 6 [Here we performed a comprehensive neurophysiological characterization of an disorder \u201328. Mutadisorder ,67, and hrough 6 .Mecp2 mouse models, as well as a mouse model of CHD8-mediated neurodevelopmental syndrome [in vivo neurological phenotype of mutant mice, and allude to an important role for hnRNP U in regulating the expression of many disease-relevant, neuronally expressed genes.Through single-cell RNA-sequencing of the neocortex and hippocampus, we identified widespread transcriptional dysregulation across all the neuronal cell types examined in mutant mice. The overall magnitude of these expression changes was generally modest\u2014an observation similar to that described in Rett Syndrome, both in post-mortem human tissue and syndrome \u201371. Recesyndrome . In the Hnrnpu occurred in pyramidal cells of the subiculum. Downregulated genes in this cell type were also enriched for known neurodevelopmental disease genes including those associated with autism, developmental delay, and epilepsy. Although the subiculum functions as the primary output of the hippocampus and is important for normal hippocampal-related functions such as learning and memory and stress response, this brain region has also been implicated in pathological conditions such as the generation and spread of temporal lobe seizures [HNRNPU mutations, two patients reportedly had temporal lobe epilepsy [Mef2c, is associated with a neurodevelopmental disorder showing significant phenotypic overlap to patients with HNRNPU mutations [Interestingly, at this perinatal time point, we found that the greatest burden of gene expression dysregulation upon heterozygous loss of seizures \u201376. Whilepilepsy ,28. Furtutations ,78. WhilWidespread and cell-type-specific transcriptomic defects present a challenge regarding pinpointing targeted therapeutics. However, transcriptomic signature reversal may provide one route forward. This approach has been successful in identifying compounds that ameliorate seizures in non-genetic models of epilepsy . ThroughHNRNPU developmental and epileptic encephalopathy. This model system demonstrated developmental impairment, seizure susceptibility and cognitive dysfunction, recapitulating aspects of the human condition. We further observed pervasive gene expression changes of modest effect that occurred in a cell-type-specific manner, including many neuronally-expressed genes that converged on important neuronal pathways. This was accompanied by a nearly 50% reduction in expression of a single neurodevelopmental disease gene, Mef2c, in pyramidal neurons of the subiculum\u2014a cell-type particularly vulnerable to heterozygous loss of Hnrnpu. Overall, these findings imply a complex role for Hnrnpu in gene expression regulation of the brain\u2014a notion supported by its multifaceted role in regulating multiple levels of gene expression. This complexity is likely further amplified by many factors including context-dependent interactions with other gene expression regulators, developmental timing, sex-specific effects and species-specific variation that were not examined as part of this work, but do warrant further consideration. These results also support the exploration of alternative therapeutic approaches, with transcriptomic signature reversal of key, vulnerable cell-types as a promising, novel strategy for transcriptome-mediated neurodevelopmental disease.Here we characterized the neurophysiological and cell-type-specific transcriptomic effects of a mouse model of All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University Irving Medical Center.Hnrnpu+/113DEL mice were generated through The Jackson Laboratory\u2019s Genome Engineering Technology core using a CRISPR-Cas9 strategy targeting exon 1 of mouse Hnrnpu. Of 15 founder mice that survived, 6 appeared most promising based on TIDE analysis and were further evaluated using TOPO-TA cloning to validate the corresponding mutant alleles. A founder containing an out-of-frame 113-bp deletion was further expanded and maintained on a C57BL/6NJ background. All experiments were performed on the inbred background except for ECT studies, which were performed on the F1 hybrid background C57BL/6NJ (005304 JAX stock) x FVB/NJ (001800 JAX stock), as mutants on an inbred C57BL/6NJ were significantly smaller than WT controls. WT littermates were used as controls in all experiments. All mice were maintained in ventilated cages with controlled humidity at ~60%, 12h:12h light:dark cycles and controlled temperature of 22\u201323\u00b0C. Mice had access to regular chow and water, ad libitum. Breeding cages were fed a high fat breeder chow. Mice were maintained and all procedures were performed within the Columbia University Institute of Comparative Medicine, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.DNA was extracted from tail or ear clippings using the Kapa Mouse Genotyping Standard kit (KAPA Biosystems) and stored at -20\u00b0C. PCR was performed with 2x MyTaq HS Mix (Bioline), using the following Hnrnpu primers: FWD = 5\u2019-GTCCGTTCTGCAGCAGCACT-3\u2019, REV = 5\u2019- TTACCTCCCGCCTGCTGTTG-3\u2019. This amplifies a 745-bp product from the WT allele and a 632-bp product from the mutant allele.Hnrnpu 113-bp deletion. Mutant and wildtype pups were decapitated, and cortex and hippocampus were separately dissected in cold Hibernate A (Thermo Fisher). Tissue was diced into smaller pieces and dissociated in a solution containing pre-warmed Hibernate A, papain, and DNase for 20 min at 37\u00b0C. Dissociated tissue was then centrifuged at 300 g for 5 min at room temperature (RT), resuspended in pre-warmed Hibernate A, and triturated to further dissociate. Undissociated tissue was allowed to settle to the bottom of the tube, and the single cell suspension was transferred to a new tube and centrifuged at 300 g for 5 min at RT. The cell pellet was resuspended in complete medium containing Neurobasal A (Thermo Fisher), B27 Plus (Thermo Fisher), 1% FBS, Hepes, Glutamax and Penn/Strep. Cell viability and counts were obtained using a trypan blue exclusion assay, then further resuspended to the desired cell concentration using complete medium supplemented with laminin (5 ug/ml). Both cortical and hippocampal cells were plated on PDL-coated 12 mm coverslips in a 24-well dish at a density of 200,000 cells. Complete medium was changed the following morning to Neurobasal A, B27 Plus, Hepes, Glutamax and Penn/Strep, and 50% medium changes were subsequently performed every other day.P0 pups were tail sampled, weighed and genotyped for the On day in vitro 9 (DIV9), mouse primary cortical and hippocampal cells were washed 2x with 1X PBS, fixed for 15 min in 4% paraformaldehyde at RT, and again washed 2x with 1X PBS. Cells were incubated in a staining solution comprised of 5% donkey serum, 1% BSA, 0.3% TritonX-100 in 1X PBS for 15 min at RT, then subsequently incubated in the primary antibody diluted in the staining solution for 2 hr. at RT. Cells were washed 4x with 1X PBS, 0.2% TritonX-100, incubated with the fluorophore conjugated secondary antibody in staining solution for 30 min at RT then washed 4x with 1X PBS, 0.2% TritonX-100. Coverslips were mounted using ProLong-Antifade with DAPI on Superfrost Plus Microscope slides and allowed to dry in the dark prior to imaging. Imaging was performed using the Zeiss Axio Observer.Z1 Fluorescence Motorized Microscope and associated Zen2 Pro imaging software. Downstream image processing was performed using Adobe Photoshop, using auto-brightness and contrast for each individual channel and merged image. Primary antibodies used include: Mouse anti-Map2 at 1:500 (Sigma M4403), mouse anti-GFAP at 1:100 (Abcam ab10062), mouse anti-Gad67 at 1:500 (Millipore MAB5406), mouse anti-Satb2 at 1:100 (Abcam ab51502), rat anti-Ctip2 at 1:250 (Abcam ab18465), rabbit anti-HNRNPU at 1:500 (Abcam 20666). Secondary antibodies include: 488 and 568 Alexa Fluorophore conjugated donkey anti-mouse, donkey anti-rabbit and donkey anti-rat (Invitrogen), at 1:1000 dilution.Dissected tissue was snap frozen in liquid nitrogen and stored at -80\u00b0C until time of extraction. Tissue was thawed on ice and homogenized using a motorized pestle in RIPA buffer containing both protease and phosphatase inhibitor cocktails (Roche). Lysis was completed for 15 minutes on ice. Samples were subsequently centrifuged at full speed for 20 minutes at 4\u00b0C. The resulting supernatant was collected, and protein was quantified using the BCA method (Pierce) with BSA as a standard. All western blots were performed using the Novex NuPAGE system (Invitrogen). Protein lysates were diluted in LDS sample buffer and reducing agent, and heated at 70\u00b0C for 10 min. Using the Xcell SureLock Mini Cell gel box, a total of 5 ug of reduced protein lysates were loaded onto a 4\u201312% gradient Bis-Tris gel in 1X SDS Running buffer (Invitrogen) supplemented with the NuPAGE antioxidant, and ran at 180 V for 1\u20131.5 hrs. Using the Xcell II Blot Module, proteins were subsequently transferred to a 0.2 um methanol activated PVDF membrane at 30 V for 1.25 hrs at 4\u00b0C in Transfer buffer containing 20% methanol. Membranes were blocked for 1 hr at RT in 5% milk, then incubated overnight in the hnRNP U primary antibody at 1:1000 diluted in 5% BSA. Blots were washed 3x for 10 min in PBST, incubated at RT for 1 hr in a secondary HRP-conjugated anti-rabbit diluted in 5% BSA, then further washed 3x for 10 min in PBST. Proteins were incubated for 5 min in a standard ECL substrate (Pierce) and developed with either a Kodak X-OMAT 2000A Processor or iBright FL1000 Imaging system (Invitrogen). For a loading control, blots were subsequently incubated in an HRP-conjugated b-Actin secondary at 1:1000 (Santa Cruz #sc-47778) diluted in 5% BSA for 1 hr at RT, then washed and developed as previously described. Densitometry analysis was performed using the iBright FL1000 imager. Specifically, the Local Background Corrected Density, LBCD, (background-corrected volume/area) of each hnRNP U-probed sample was first normalized to the corresponding LBCD of \u03b2-Actin to generate an hnRNP U/\u03b2-Actin ratio. WT and HET ratios were further divided by the average WT hnRNP U/\u03b2-Actin ratio and plotted individually.Mouse cerebral cortex: Cortical tissue was collected and immediately stored in RNALater Stabilization Solution (Qiagen) at 4\u00b0C. After 24 hrs., the RNALater was subsequently removed, and samples were stored long term at -80\u00b0C. For RNA extraction, tissue was first mechanically homogenized using a motorized pestle in RLT buffer (Qiagen) supplemented with b-mercaptoethanol, then further homogenized using a QIAshredder spin column (Qiagen). RNA was extracted using the RNeasy Mini kit (Qiagen) per protocol instructions, and the resulting RNA concentration and purity were assessed using a NanoDrop. A total of 2.5 ug of RNA was used for the reverse transcription reaction, which was performed using the SuperScript IV First-Strand Synthesis System (ThermoFisher) with random hexamer priming. Resulting cDNA was used as input into pre-validated TaqMan Gene Expression Assays (ThermoFisher) and run with the TaqMan Fast Universal PCR MasterMix 2x (Applied Biosystems). The following TaqMan probes were purchased from ThermoFisher: mHnrnpu Mm00469329_m1 (spans exons 1\u20132) and mCyc1 Mm00470540_m1 (spans exons 1\u20132). A total of six biological replicates were evaluated. TaqMan assays were run on a QuantStudio 5 Real-Time PCR System (Applied Biosystems) using the comparative Ct method.All qRT-PCR analyses were performed using QuantStudio Design and Analysis Software v1.2 and Microsoft Excel. To analyze for gene expression differences, raw Ct values were first averaged across technical replicates, which ranged from 2\u20134 for each sample. For samples with a Ct standard deviation >0.3, a technical replicate was filtered out if its Ct value was one standard deviation above or below that of the mean of the replicates. We required samples to have at least technical duplicates to be considered in the final analysis. Delta Ct values were determined by calculating the difference between mean Ct values for experimental samples and corresponding loading controls. Delta delta Ct values for both HET and WT samples were then calculated using the average WT delta Ct from the corresponding time point and experimental batch, and then were independently plotted along with SEM. Statistical analyses were performed using Welch\u2019s two-sample t-test.Brains were extracted from PND0 pups and fixed in Bouin\u2019s solution overnight at room temperature. Fixed brains were embedded in paraffin with service provided by Columbia University\u2019s Molecular Pathology Core Facility. Coronal sections in 5 \u03bcm thickness were obtained using a microtome (Leica RM2125RT) and subjected to hematoxylin and eosin (H&E) staining. Briefly, the slices were deparaffinized in xylene and rehydrated with ethanol and water. Slices were then stained in hematoxylin, counterstained with eosin and subsequently dehydrated with ethanol and xylene. Stained slices were mounted with coverslips using Permount . Images were acquired using a Nikon Eclipse E800 Microscope packaged with NIS-Elements DV.4.51.00 imaging software. For presentation purpose, full size images were subjected to automatic brightness and contrast adjustment using Adobe Photoshop. Brain measurements were collected using ImageJ. The measurements were normalized to respective pup body weight. Student\u2019s T-test was performed on each set of measurements and Bonferroni corrected for multiple comparisons.On PND2, pups were tattooed on the bottom of their paws for identification using a 25\u201330 G needle and Ketchum tattoo ink, following the AIMS tattoo chart. The following developmental milestone studies were completed on P4, P6, P8, P10 and P12 within a 3 min time window for each test subject.Righting reflex: the latency to flip over from supine to prone position on all fours. Pups were gently placed on their backs on a hard surface and released. A stopwatch was used to measure the total time for each pup to right itself. The cutoff latency was 30 s.Negative geotaxis: the latency to face upwards from a downward-facing start position on an inclined mesh screen. Pups were placed at a downward facing position on a 45\u00b0 inclined screen and released. A stopwatch was used to record the time it takes for each pup to turn 90\u00b0 then 180\u00b0. The cutoff latency was 30 s. If the pup failed the trial by falling while turning, they were scored the maximum cutoff latency time of 30 s.Vertical screen: the latency to fall from a vertically positioned wire mesh screen. Pups were positioned to grasp the screen. Using a stopwatch, the time until fall was recorded. The minimum and maximum latency allowed was 1 s and 30 s, respectively.For each developmental test, every pup underwent two successive trials that were subsequently averaged and used in the downstream analyses. Throughout the testing period, the experimenter was blinded to genotype. The mean and standard error of the mean (SEM) for both genotypes were plotted across all tested timepoints. Permuted MWU p-values were reported for each time point. Briefly, WT and HET data were pooled, randomly sampled and MWU p-values calculated 10,000 times. Permuted MWU p-values were calculated as the total number of randomly sampled MWU p-values that fell below the actual MWU p-value divided by the total number of permutations .USVs were assessed on P3, P5, P7, P9 and P11. Each pup was gently removed from the nest and placed in a small, plastic container containing a 0.5 cm layer of fresh bedding. The cage lid was immediately returned to avoid irritating the dam and remaining pups in the nest. The container holding the pup was placed immediately into a sound-attenuating environmental chamber . After a 3 min recording, each pup was marked and returned to the nest. Ultrasonic vocalizations were recorded with an Ultrasound Microphone sensitive to frequencies of 10\u2013180 kHz and using the Avisoft Recorder (Version 4.2) software. Sampling rate was 250 kHz, format 16 bit. Ultrasonic vocalizations were analyzed using Avisoft SASLab Pro software (Avisoft Bioacoustics). Spectrograms were generated for each 1 min audio file, with an FFT-length of 512 points and a time window overlap of 75% . The spectrogram was generated at a frequency resolution of 488 Hz and a time resolution of 1 ms. A lower cut-off frequency of 15 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. Calls were inspected visually and manually labelled. Summary statistics were generated by Avisoft SASLab Pro and analyzed using Prism. All calls emitted over the 3 min recordings were quantified. For the qualitative analysis, given the extensive manual review required to measure duration, peak frequency, and amplitude, we chose to limit this analysis to 2 time points, 1 near the beginning (P5) and the other near the end (P7) of the canonical inverted U-shaped curve. One 1 min file (out of three 1 min files) that included the most USVs were analyzed for each mouse. Mean and SEM were plotted, and permuted MWU p-values were reported for each time point as described for pup developmental milestone tests.Video EEGs were performed on 6- to 8-week-old adult mice. Mice were anesthetized with tribromoethanol , and three small burr holes were drilled through the skull 2 mm lateral to the midline . One hole was also drilled over the cerebellum as a reference. Four Teflon-coated silver wires soldered onto pins of a microconnector (Mouser electronics cat# 575\u2013501101) were placed in between the dura and brain. A dental cap was applied on top. Each mouse was provided the post- operative analgesic Carprofen (5 mg/kg subcutaneous Rimadyl) and allowed a recovery period of at least 48 hours prior to recording. Signal was obtained on either a Grael II EEG amplifier (Compumedics) or Natus Quantum amplifier (Natus Neuro). Data was analyzed with either Profusion 5 (Compumedics) or NeuroWorks (Natus Neuro). Differential amplification recordings were recorded pairwise between all three electrodes and the reference, resulting in 6 total channels for each subject. Mouse behavior was captured throughout the recording period through video using a Sony IPELA EP550 camera with infrared light for dark recordings. Each mouse was recorded for 24\u201348 hrs. continuously.EEG spectral analysis was carried out using fast Fourier transform (FFT) over a 5 s sliding window, sequentially shifted by 2 s increments (bins). Brain states were semi-automatically classified into wake and slow-wave sleep using a custom-written MATLAB program activity). Semi-automated classification was validated manually by trained experimenters. Note that REM sleep was not classified in this study due to lack of EMG signals. EEG power was normalized to the total power in each mouse. Then, low gamma power (30-50Hz) in different brain states across the entire 24-h recording session was calculated and compared between the mutant and wildtype animals.All tests were performed on 6- to 8-week-old mice. Transcorneal electrodes were used to deliver a predefined stimulus with the Ugo Basile Model 7801 electroconvulsive device. High frequency (HF) electroshock was performed with the following fixed settings: 1.6 ms pulse width, 0.2 s shock duration and 299 Hz pulse frequency with variable settings of 4\u201312 mA amplitude. The individual threshold for each mouse was determined by testing in 0.5 mA intervals on sequential days until the threshold was reached. The behavioral endpoint evaluated was a maximal tonic hindlimb extension seizure, which often start with tonic extension of the forelimbs that evolves into full tonic hindlimb extension. The overall stimulus is calculated as the iRMS using the following equation: sq. root frequency (Hz) x pulse width (ms) x duration (s) x amplitude (mA).Elevated Plus Maze test (EPM): This classic test for anxiety-like behavior is based on rodents\u2019 innate fear for height and open space. The elevated plus-maze consists of two open arms (30 cm x 5 cm) and two closed arms (30 x 5 x 15 cm) extending from a central area (5 x 5 cm). Photo beams embedded at arm entrances register movements. Room illumination was approximately 5 lux. The test begins by placing the subject mouse in the center, facing a closed arm. The mouse is allowed to freely explore the maze for 5 min. Time spent in the open arms and closed arms, the junction, and number of entries into the open arms and closed arms, are automatically scored by the MED-PC V 64bit Software (Med Associates). At the end of the test, the mouse is gently removed from the maze and returned to its home cage. The maze is cleaned with 70% ethanol and wiped dry between subjects.Open Field exploratory activity: The open field test is the most used general test for locomotor activity. Each mouse is gently placed in the center of a clear Plexiglass arena lit with dim light (~5 lux), and is allowed to ambulate freely for 60 min. Infrared (IR) beams embedded along the X, Y, Z axes of the arena automatically track distance moved, horizontal movement, vertical movement, stereotypies, and time spent in center zone. At the end of the test, the mouse is returned to the home cage and the arena is cleaned with 70% ethanol followed by water and wiped dry.Catwalk: Free-pace walking was evaluated using the Catwalk XT system (Noldus Information Technology) which consists of an illuminated walled glass walkway (130 cm x 10 cm) and a high-speed camera underneath. Light is reflected and illuminates the stimulus (footprint) when downward pressure is applied. Walking patterns are captured with a high-speed camera mounted underneath the walkway. The experiment was done with dim room illumination (30 lux). The mouse is allowed to traverse the walkway as many times as needed to obtain at least 3 compliant runs (runs with a speed variation under 80% in 20 seconds or less). Pilot experiments using a 60% speed variation limit (most common in the literature) proved to be too stringent for most heterozygous mice. Parameters automatically collected by the software include, but are not limited to, paw statistics, intensity measures, stride length, width, base of support, distance between ipsilateral prints, cadence, % limb support, regularity index, speed, and speed variation. A highly trained experimenter visually inspected all automatically scored runs, and manually classified any prints that were too ambiguous for the software to identify accurately. The walkway is cleaned with paper towel moistened with 70% ethanol and wiped dry between trials.Acoustic startle response: Acoustic startle response was tested using the SR-Laboratory System . Test sessions began by placing the mouse in the Plexiglass holding cylinder for a 5-min acclimation period. For the next 8 min, mice were presented with each of six trial types across six discrete blocks of trials, for a total of 36 trials. The inter-trial interval was 10\u201320 s. One trial type measured the response to no stimulus (baseline movement). The other five trial types measured startle responses to 40 ms sound bursts of 80, 90, 100, 110 or 120 dB. The six trial types were presented in pseudorandom order such that each trial type was presented once within a block of six trials. Startle amplitude was measured every 1 ms over a 65 ms period beginning at the onset of the startle stimulus. The maximum startle amplitude over this sampling period was taken as the dependent variable. Background noise level of 70 dB was maintained over the duration of the test session.Fear Conditioning: This is a classic test for conditioned learning. Training and conditioning tests are conducted in two identical chambers that were calibrated to deliver identical foot shocks. Each chamber was 30 cm \u00d7 24 cm \u00d7 21 cm with a clear polycarbonate front wall, two stainless side walls, and a white opaque back wall. The bottom of the chamber consisted of a removable grid floor with a waste pan underneath. When placed in the chamber, the grid floor connected with a circuit board for delivery of scrambled electric shock. Each conditioning chamber is placed inside a sound-attenuating environmental chamber (Med Associates). A camera mounted on the front door of the environmental chamber recorded test sessions which were later scored automatically, using the VideoFreeze software . For the training session, each chamber is illuminated with a white house light. An olfactory cue is added by dabbing a drop of imitation almond flavoring solution (1:100 dilution in water) on the metal tray beneath the grid floor. The mouse is placed in the test chamber and allowed to explore freely for 2 min. A pure tone which serves as the conditioned stimulus (CS) is played for 30 s. During the last 2 s of the tone, a foot shock (0.5 mA) is delivered as the unconditioned stimulus (US). Each mouse received three CS-US pairings, separated by 90 s intervals. After the last CS-US pairing, the mouse is left in the chamber for another 120 s, during which freezing behavior is scored by the Video Freeze software. The mouse is then returned to its home cage. Contextual conditioning is tested 24 h later in the same chamber, with the same illumination and olfactory cue present but without foot shock. Each mouse is placed in the chamber for 5 min, in the absence of CS and US, during which freezing is scored. The mouse is then returned to its home cage. Cued conditioning is conducted 48 h after training. Contextual cues are altered by covering the grid floor with a smooth white plastic sheet, inserting a piece of black plastic sheet bent to form a vaulted ceiling, using near infrared light instead of white light, and dabbing vanilla instead of banana odor on the floor. The session consisted of a 3 min free exploration period followed by 3 min of the identical CS tone . Freezing is scored during both 3 min segments. The mouse was then returned to its home cage. The chamber is thoroughly cleaned of odors between sessions, using 70% ethanol and water.Y maze: The Y-maze is a standard test for assessing short term memory in mice, based on the mouse\u2019s natural tendency to explore novel locations. Memory impairment is indicated by failing to spend more time exploring the novel arm than the familiar arms. The test is conducted in the Y maze (Maze Engineer) consisting of three arms of equal arm lengths (35 cm), arm lane width (5 cm), wall height (10 cm). One arm is the start arm, with a \u201c=\u201c sticker velcroed on the wall, to the end of the arm. The two stickers (bus and plane) are velcroed on the wall at the end of the other two arms. The placement of the stickers was counterbalanced across animals. The novel arm preference test consists of two trials. In trial 1, each mouse is placed in the designated start arm and allowed access the start arm and the one other arm for 10 min. The third arm is blocked with an opaque door. At the conclusion of trial 1, the mouse was placed in a temporary holding cage for 10 min. For trial 2, the subject mouse was returned to the start location, and allowed to explore all arms for 5 min. A camera mounted above the maze and interfaced with the Ethovision software (Noldus Information Technology) automatically records distance traveled, arm entries, and time spent in each arm. The maze was cleaned with 50% ethanol and allowed to dry between trials and between animals.Morris water maze: Spatial learning and reversal learning were assessed in the Morris water maze using procedures and equipment as previously described . The appCLIP-seq experiments were conducted with hearts from two-week-old WT mice using anti-hnRNP U . Sample preparation, crosslinked-RNA recovery, library preparation and sequencing were performed according to published protocols . After lNeocortical and hippocampal tissue was dissected from postnatal day 0 pups and subjected to a papain dissociation. Following papain dissociation and tissue trituration, all neocortical and hippocampal samples were filtered through a 40 \u03bcm cell strainer to enrich for single cells in the resulting suspension. Cell viability was subsequently assessed, with a cutoff of 70% or greater to be used for sequencing. Single cell RNA-seq libraries were constructed using the 10X Chromium Single Cell 3\u2032 Reagent Kits v2 according to manufacturer descriptions, and samples were sequenced on a NovaSeq 6000. Reads were aligned to the mm10 genome using the 10X CellRanger pipeline with default parameters to generate the feature-barcode matrix.We used Seurat v3 to perform downstream QC and analyses on feature-barcode matrices ,54. We rNext, we used linear regression to regress out the number of UMIs per cell and percentage of mitochondrial reads using the ScaleData function on the integrated expression matrices. We then performed dimensionality reduction using PCA. For each dataset, we selected the top 30 dimensions to compute a cellular distance matrix, which was used to generate a K-nearest neighbor graph. The KNN was used as input to the Louvain Clustering algorithm implemented in the FindClusters function. For clustering via Louvain, we chose a resolution parameter of 0.8. We visualized the cells using UMAP via the RunUMAP function.To annotate and merge clusters, we performed differential gene expression analysis on the integrated expression values between each cluster using the default parameters in the FindMarkers function, which implements a Wilcoxon test and corrects p-values via Bonferroni correction. Additionally, we visualized the expression of canonical marker genes aggregated from previous single-cell publications \u201385. ClusWe performed cell-type-specific differential gene expression analysis using MAST , as implWe corrected the p-values using the Benjamini-Hochberg FDR method. We considered genes with a log2(fold change) value of at least 0.14 (10% difference) and FDR < 0.05 as differentially expressed. We performed gene ontology analysis using g:Profiler , using aFor the burden analysis, we down sampled the data to include 300 cells per population. To increase power, we pooled cells from male and female samples. We ran the differential expression analysis as described above, removing gender as a latent variable.For disease gene enrichment analyses, human homologs for all tested mouse genes were obtained using biomaRt. Significant and nonsignificant mouse genes were annotated based on the respective human homolog disease gene status. Genes without human homologs were not used in the analysis. The epilepsy-associated gene list was based on a prior publication . Autism We queried the Connectivity Map (clue.io) to identify compounds most likely to reverse disease expression signatures ,58. Our S1 Figin vitro 9 showing hnRNP U (red) co-staining with nuclear marker Dapi (blue) and cell markers (green) (A) Map2 , (B) Gad67 (inhibitory), (C) Ctip2 , (D) Satb2 and (E) Gfap (astrocytes). Scale bar = 50\u03bcm.Primary neocortical cell cultures at day (TIF)Click here for additional data file.S2 Fig(A) Map2 , (B) Gad67 (inhibitory), (C) Ctip2 , (D) Satb2 and (E) Gfap (astrocytes). Scale bar = 50\u03bcm.hnRNP U (red) co-staining with nuclear marker Dapi and cell markers (green) (TIF)Click here for additional data file.S3 Fig(A) A representative genotyping agarose gel showing the 113-bp deletion. (B) Western blot image using antibody targeting hnRNP U C-terminus (C) Western blot image using an antibody targeting hnRNP U N-terminus.(TIF)Click here for additional data file.S4 Fig(A) Representative H&E-stained coronal sections of WT and HET brains at PND0. Black arrows indicate the corpus callosum. Level of sections indicated by the respective cartoons. Scale bar = 1 mm. (B) Brain width, height, cortical thickness and hippocampal width were measured from caudal sections and normalized to respective body weight . Bonferroni-corrected T-test p> 0.99 for each test . Error bars = SEM.(TIF)Click here for additional data file.S5 Fig(A) Body weight of PND0 pups, also stratified by sex. Unpaired t-test for all mice p = 3.9x10-3 , for males p = 0.01 and for females p = 0.02 . (B) Pup weights stratified by sex . Permuted MWU p-values for males: PND4 & 6 = 1x10-4, PND8 = 7x10-4, PND10 = 6.4x10-3, PND12 = 0.03, and for females: PND4-12 = <1x10-4. (C) Adult body weights (n = 9 per genotype). Permuted Mann-Whitney U (MWU) p-values for all mice: wk4 = 2x10-4, wk5 & 6< 1x10-4, wk7 = 4x10-4, wk8 = 3x10-4, wk9 = 1.1x10-3, wk10< 1x10-4. (D) Adult weight stratified by sex. Permuted MWU p-values for males: wk4 = 0.20, wk5 = 0.11, wk6 = 0.06, wk7 = 0.06, wk8 = 0.06, wk9 = 0.06, wk10 = 0.05, and for females: wk4 = 9.0x10-3, wk5-10 <1x10-4. (E) Representative image of 8 wk adult male WT and HET. (F) Vertical screen test. Permuted MWU p-values: PND4 = 0.31, PND6 = 0.05, PND8 = 0.07, PND10 = 0.07, PND12 = 0.96. (G) Surface righting test. Permuted MWU p-values: PND4 = 0.3, PND6 = 0.42, PND8 = 0.07, PND10 = 7x10-4, PND12 = 0.15. (H) 90\u00b0 negative geotaxis test. Permuted MWU p-values: PND4 = 0.96, PND6 = 0.91, PND8 = 0.14, PND10 = 0.16, PND12 = 4.2x10-3. (I) 180\u00b0 negative geotaxis test. Permuted MWU p-values: PND4 = 0.39, PND6 = 0.2, PND8 = 0.16, PND10 = 0.47, PND12 = 0.08. . (J) USV\u2019s stratified by sex . Permuted MWU p-values males: PND3 = 0.36, PND5&7< 1x10-4, PND9 = 0.01, PND11 = 1x10-3; females: PND3 = 0.40, PND5 = 0.57, PND7< 1x10-4, PND9 = 0.58, PND11 = 0.16. PND = postnatal day. Error bars = SEM.(TIF)Click here for additional data file.S6 Fig(A) Summary table of EEG data. Sz = seizure (B) Elevated plus maze (EPM) percent time spent in open arm . Welch\u2019s t-test p = 0.47 . (C) EPM total arm entries . T-test p = 0.12 . (D) Open field ambulatory distance divided into six 10 min bins . Two-way repeated-measure (RM) ANOVA p = 0.026 . (E) Time spent in the center of open field, divided into 10 min bins . Two-way RM ANOVA p = 0.09 . (F) Catwalk front-paw (FP) and hind-paw (HP) print area . Unpaired, two-tailed t-test FP p = 0.024 , HP p< 1x10-4 . (G) Catwalk max contact max intensity (MCMI) of FP and HP . Mann-Whitney U (MWU) FP p = 0.08, HP p< 1x10-4. (H) Representative images of FP and HP prints of WT and HET mice. (I) Catwalk base of support for FP and HP . MWU FP p = 0.08, HP p = 1.5x10-3. (J) Morris Water Maze (MWM) latency to platform (acquisition period) measured over 5 consecutive days . Two-way RM ANOVA p = 0.22 . (K) MWM swim distance measured over 5 days . Two-way RM ANOVA p = 0.1 . (L) MWM probe trial measured as time spent in each quadrant of the pool . One-way RM ANOVA on ranks: WT T (trained quadrant) vs L (left quadrant) p< 1x10-3, T vs R (right quadrant) p = 0.2, T vs opp (opposite quadrant) p< 1x10-3. HET T vs R p = 2x10-3, T vs L p = 0.64, T vs opp p = 0.42. (M) Y-Maze percent duration spent in novel vs familiar arm . One-way RM ANOVA WT p = 5x10-3 , HET p = 3x10-3 . (N) Y-Maze percent spontaneous alternation . Welch\u2019s t-test p = 0.86 . (O) Fear conditioning percent freezing . MWU post-train p = 7x10-4, context p< 1x10-4, pre-cue p = 0.02, cued p = 1.8x10-3. (P) Startle response (y-axis) across different decibels (x-axis) . T-test p = 0.96. Error bars = SEM.(TIF)Click here for additional data file.S7 Fig(A) A representative example of EEG signals in a Hnrnpu heterozygous mutant mouse (Het). From top to bottom: EEG spectrogram (20-50Hz), raw EEG traces , brain states , and EEG traces in wake and SWS . (B) A representative example of EEG signals in a wildtype (WT) mouse. EEG traces in the bottom are enlarged from b1 and b2 positions. (C) Spectral analysis of EEG during wake and SWD in a recording session in Hnrnpu mutant and wildtype mice. (D) Quantitation of EEG low gamma power in Hnrnpu mutant and wildtype mice .(TIF)Click here for additional data file.S8 Fig(A) UMAP plots of cells from each hippocampal sample. (B) UMAP plots of cells from each cortical sample. Het_F: neocortical cells from a female; Het_M: neocortical cells from a male. Het_F = Hnrnpu+/113DEL female; Het_M = Hnrnpu+/113DEL male; WT_F = wildtype female; WT_M = wildtype male.(TIF)Click here for additional data file.S9 Fig Number of cells detected in each cell population, colored by sample. (A) represents hippocampal clusters and (B) represents neocortical clusters. Het_F = Hnrnpu+/113DEL female; Het_M = Hnrnpu+/113DEL male; WT_F = wildtype female; WT_M = wildtype male.(TIF)Click here for additional data file.S10 Fig Distribution of Connectivity Scores for the subiculum-derived and pseudo-bulk derived disease expression signatures. Top 20 compounds predicted to reverse the subiculum and pseudo-bulk signatures. Compounds in red represent compounds prioritized for both signatures. (E) Venn Diagram depicting the overlap between all compounds achieving a Connectivity Score less than -90 for the subiculum and pseudo-bulk signatures.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file." \ No newline at end of file