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+{"text": "Some environmental factors of possible aetiological importance for primary liver carcinoma (PLC) in males were analysed in a case-control study including 83 cases of hepatocellular carcinoma (HCC), 15 cases of intrahepatic cholangiocellular carcinoma (CC), 3 cases of haemangiosarcoma and 1 case of unspecified sarcoma in the liver--102 cases in total. Two matched controls were used in each case. One case with haemangiosarcoma was exposed to polyvinyl chloride. The case with unspecified soft-tissue sarcoma was exposed to phenoxy acids. A 4-fold increase in the risk of HCC was seen in alcoholics, and regular drinking gave a 3-fold increase in the risk. Exposure to organic solvents gave a 2-fold increase in the risk of HCC. No increased risk was observed for cases exposed to various other chemicals. Three cases of HCC had a previous diagnosis of porphyria acuta intermittens (PAI), versus no control. Six cases of HCC had a previous diagnosis of porphyria acuta intermittens (PAI), versus no control. Six cases with PLC had polyphyria cutanea tarda (PCT) which in 4 cases was related to alcoholism and in one case to haemochromatosis."}
+{"text": "An earlier case-control study from Western Australia reported a protective effect of maternal folic acid supplementation during pregnancy on the risk of childhood acute lymphoblastic leukaemia (ALL). The present study tested that association.A national case-control study was conducted in New Zealand. The mothers of 97 children with ALL and of 303 controls were asked about vitamin and mineral supplements taken during pregnancy.There was no association between reported folate intake during pregnancy and childhood ALL (adjusted odds ratio (OR) 1.1, 95% confidence interval (CI) 0.5\u20132.7). Combining our results with the study from Western Australia and another study from Qu\u00e9bec in a meta-analysis gave a summary OR of 0.9 (95% CI 0.8\u20131.1).Our own study, of similar size to the Australian study, does not support the hypothesis of a protective effect of folate on childhood ALL. Neither do the findings of the meta-analysis. Despite decades of research, the aetiology of childhood leukaemia remains enigmatic. There have been clear increases in the incidence rates of childhood leukaemia in New Zealand and other countries, highlighting the aetiological importance of unknown environmental factors . There iA case-control study from northern California looked at maternal diet and vitamin supplements in the twelve months before pregnancy, and found no statistically significant association between childhood ALL and any vitamin or iron supplements . Total dIn this paper we have tested the hypothesis raised by the Australian study that folate supplementation in pregnancy reduces the risk of childhood ALL. We have also assessed the effects of iron and multivitamin supplements. In addition to looking at maternal pregnancy consumption, we have looked at supplement use by the child.In the 1990s, we interviewed families for a case-control study of childhood cancers in New Zealand to test hypotheses related to infections and vaccinations, electromagnetic fields, chemicals and other exposures ,6. The sThe detailed methods of this population-based national case-control study are described elsewhere ,6. BriefThe controls were selected at random from birth records, while matching 1:1 to cases on age and sex. Of 303 eligible first choice controls, the mothers of 209 69%) consented and took part. Replacement controls were selected for the rest ,6.9% consenDid you take any vitamins or mineral supplements during your pregnancy, in the 3 months before, or while breastfeeding? Include iron or folate and any others.\" Those who replied 'yes' were then asked \"What was the name of the vitamin or mineral (please be specific)?\" They were then asked to specify their usage of each vitamin/mineral in each of the periods of interest. Information was also collected on vitamin and mineral supplementation of the child. Mothers were asked: \"Did {child's name} take any vitamins or mineral supplements for five days or more (or on 5 or more occasions) at any time prior to ...../...../..... {reference date}?\" For each case, the reference date was their diagnosis date. For each control it was the date on which they were the same age (in days) as their matched case was at diagnosis. If the mother said 'yes' to the question about supplement use by the child, then she was asked to specify the name of each vitamin or mineral supplement taken, and to answer questions about the timing, duration and frequency of its use. We excluded supplements taken by the child within 6 months of the diagnosis/reference date because they may have been taken as a consequence of early disease and were not likely to be related to causation.Home interviews were conducted using structured questionnaires. Mothers were asked \"Folate could be taken on its own, or more usually in combination with iron or as part of a multivitamin preparation. Separate analyses were attempted for 'any folate' , and for 'folate only' if numbers permitted.The main analysis involved the cases of ALL and controls, and was by unconditional logistic regression. This unmatched analysis was based on the cases and all the available controls, to increase the statistical power . In breaking the matching, we always adjusted for the matching factors (age and sex). Possible confounders were identified on the basis of plausibility and a 10% change in estimate. Such a strategy was used for other aspects of the study ,6. FolloFolic acid supplements were taken in pregnancy by 9% of case mothers and 9% of control mothers. Among the controls, only one of the 27 women who took folate Table  did not The unmatched analyses showed no statistically significant association between the mother's use of folate  in pregnancy and the risk of childhood acute lymphoblastic leukaemia  or multivitamins Table . The oddSince the report by Thompson and colleagues , severalThe authors of the case-control study from California reported no association between maternal pre-pregnancy dietary folate and childhood ALL . They alShaw et al. have reported findings from their large Canadian case-control study of childhood ALL and use of medications during pregnancy . They inLimitations of our study include its small size , and the low prevalence of folate supplementation reported by the mothers of the cases and controls. On its own, our study cannot exclude a protective association like that indicated by the Australian study \u2013 this is shown quantitatively by the overlap in the confidence intervals of the two studies. As in the Australian study, it was not possible to look at the effect of folate alone (without iron). We also lacked information on dietary folate. Fortification of foods such as breakfast cereals began in the mid 1990s, after the data collection for our study . Folate 2 = 74% (p = 0.02) because the findings of the Thompson study differ from the other two.Conducting a fixed effects meta-analysis  to combiSeveral possible biological mechanisms have been suggested for an effect of folate in reducing the risk of cancers. These relate to alterations in DNA methylation, a role of folate in DNA repair, methylenetetrahydrofolate reductase polymorphisms, and other possible mechanisms . Our resWe did not confirm the association found in the Australian study of a lower risk of ALL related to maternal folic acid supplementation in pregnancy. Our meta-analysis of three relevant studies showed no statistically significant association.ALL, acute lymphoblastic leukaemia. OR, odds ratio. CI, confidence intervalThe author(s) declare that they have no competing interests.JD, ME and DS designed the case-control study. JD co-ordinated the data collection; and checked and cleaned the data with assistance from PH. PH and JD conducted the analyses, which were interpreted by all the authors. JD wrote the report, and all the authors contributed to revisions.The pre-publication history for this paper can be accessed here:"}
+{"text": "A retrospective population-based case-control interview study has been conducted in three distinct areas in the north of England where local excesses of children with leukaemia have been reported. A total of 109 cases of childhood (0-14 years at diagnosis) leukaemia and non-Hodgkin's lymphoma who were born in one of the study areas and diagnosed there between 1974 and 1988 were included in the study. One control per case was matched on sex, date-of-birth and health district of birth. The objective was to compare residential histories of cases and controls and in particular to determine whether case children had lived in the same place at the same time more often than controls. The residential distance between two children was taken to be the smallest geographical distance between homes they had 'occupied' simultaneously for a period of at least 6 months between conception and diagnosis. Case children were more likely than expected to have other cases as their nearest neighbours by residential distance . A detailed examination of the nearest neighbour pattern permits the generation of further specific hypotheses. These suggest that persistent infection established in utero or early infancy may be involved in the development of some cases of childhood leukaemia. Horizontal transmission of the agent(s) within small communities may occur but there is no evidence of direct contact between cases."}
+{"text": "DPD exerted growth inhibitory activities in vitro against three human colon cancer cell lines . DPD-treated cells were arrested at G0/G1 as analysed by flow cytometric analysis. The expression of cyclin D was decreased in DPD-treated cells. The differentiation markers of carcinoembryonic antigen and fibronectin were significantly increased in colon cancer cells after treatment with DPD. The epithelium-like brush borders on HT-29 cell surface were also demonstrated at 1 week after withdrawal from DPD treatment. The DPD-induced cell growth inhibition and differentiation were irreversible after removal of DPD. The in vivo effect of tumour growth suppression by DPD was also observed in mouse xenografts. No acute toxicity was observed after an intraperitoneal challenge of DPD in BALB/c-nude mice weekly. These results suggest that DPD appears to be a new potentially less toxic modality of cancer therapy.A diamantane derivative 1,6-Bis [4-(4-amino-3-hydroxyphenoxy) phenyl] diamantane (DPD) was found to inhibit the growth of several cancer cell lines in the National Cancer Institute (NCI) Anticancer Drug Screen system. In this study, we examined the  N-1-adamantylcitraconimide, N-1-adamantylmaleimide (AMI) and N-1-diamantylmaleimide (DMI) exhibited modest growth-inhibitory activity against four cancer cell lines , and AMI and DMI exhibited antimicrobial activity against Staphylococcus aureus and Trichophyton mentagrophytes  of 0.50 (>100), 0.85 (22.3), 1.31 (6.24), 0.62 (>100) and 0.75 (>100) \u03bcM, respectively (unpublished data).For example, very strong anticancer effects of DPD were observed against one leukaemia (HL-60), one non-small-cell lung cancer (HOP-92), one colon cancer (Colo 205), one ovarian cancer (OVCAR-8) and one breast cancer (T-47D) cell line with GIColon cancer is a major cause of mortality in the Western world . Althoug1 phase of the cell cycle is an important period where various signals interact to determine the proliferation, quiescence, differentiation or apoptosis of cells  are now used as a second-line chemotherapeutic agent for patients who have failed to respond to previous 5-FU-based chemotherapy, but the survival remains poor for patients with metastatic colorectal carcinoma . In this\u22121 gentamycin . HCT-15 cells were cultured in RPMI-1640 with 20% foetal bovine serum and 0.01\u2009mg\u2009ml\u22121 gentamycin. Cells were incubated in a humidified atmosphere of 5% CO2 in air at 37\u00b0C. DPD was dissolved in DMSO at a stock concentration of 10\u2009mM and added to culture media at a final concentration of 0.5\u20134\u2009\u03bcM. Cells were seeded at 6 \u00d7 105 cells per 60\u2009mm or 1 \u00d7 106 cells per 100\u2009mm dish in growth medium. The following day the cells were replenished with medium containing the DPD. Cells were harvested and counted by haemocytometer at 24, 48, and 72\u2009h after treatment with DPD and used for further analysis.Three colon cancer cell lines Colo 205 (ATCC: CCL-222), HT-29 (ATCC:HTB-38), and HCT-15 (ATCC: CCL-225) were used in this study. Colo 205 cells were cultured in RPMI-1640 with 10% foetal bovine serum . HT-29 cells were cultured in McCoys 5A with 10% foetal bovine serum and 0.01\u2009mg\u2009mlAt appropriate times after DPD exposure, attached cells were trypsinised and combined with nonadherent cells. After centrifugation, cells were resuspended in culture media and stained with 0.4% trypan blue, and viable cells were counted using a haemocytometer.6\u2009ml\u22121 and 0.5\u2009ml of cell suspension was centrifuged at 400\u2009g for 5\u2009min at room temperature (20\u201325\u00b0C). The cell pellet was added on 250\u2009\u03bcl of solution A (trypsin buffer) and gently mixed. After incubation at room temperature for 10\u2009min, 200\u2009\u03bcl of solution B (trypsin inhibitor and RNase buffer) was added to each tube, gently mixed and then incubated at room temperature for 10\u2009min. This was followed by with the addition of 200\u2009\u03bcl of solution C (propidium iodide (PI) stain solution) and incubated for 10\u2009min in the dark on ice (2\u20138\u00b0C). The sample was filtered through a 50-mm nylon mesh and used for flowcytometric analysis.Cycle TEST\u2122 PLUS DNA Reagent Kit  was used for DNA staining. After washing the cells twice with buffer solution, the cell concentration was adjusted to 1.0 \u00d7 103 (1%), heat-inactivated foetal bovine serum), and fixed in 5\u2009ml of 1% methanol-free formaldehyde in PBS for 15\u2009min on ice. Cells were then centrifuged at 400\u2009g for 5\u2009min and the supernatant was discarded. Cold 75% ethanol was added drop by drop to the cell pellet and incubated at \u221220\u00b0C for a minimum of 2\u2009h. Just prior to staining, ethanol was removed by centrifugation at 400\u2009g for 10\u2009min. Cold 0.25% Triton X-100 5\u2009ml wash buffer was added to the cell pellet, vortexed and incubated for 5\u2009min on ice. A measure of 20\u2009\u03bcl of FITC-conjugated mouse anti-human cyclin D1, D2, D3 monoclonal antibody   was added to 100\u2009\u03bcl cell suspension (1 \u00d7 106 cells) and incubated for 30\u2009min at room temperature in the dark. The cells were washed with wash buffer, and then stained with PI solution (10\u2009\u03bcg\u2009ml\u22121) for 10\u2009min. The sample was filtered through a 50-mm nylon mesh and used for flow cytometric analysis.Cells were washed with 5\u2009ml of wash buffer (PBS (0.1%), NaNCells (20\u2009000) were analysed on a FACSCalibur flow cytometer (Becton Dickinson) using an argon-ion laser (15\u2009mW) with the incident beam at 488\u2009nm. The red fluorescence (PI) was collected through a 585-nm filter and the green fluorescence (fluorescein isothiocyanate) was collected through a 530-nm filter. The data were analysed using ModFit and Cellquest softwares on Macintosh computer.6 per dish) were seeded on 10-cm dishes and allowed to attach overnight, and then the medium was discarded and replenished with medium containing DPD for incubation at 37\u00b0C for 3 days. The conditioned medium was collected and stored at \u221220\u00b0C before analysis. The levels of FN production were measured by a quantitative enzyme-linked immunosorbent assay , using primary rabbit anti-human FN antibody and goat anti-rabbit peroxidase conjugated secondary antibody. The levels of CEA production were measured by a radioimmunoassay (RIA) kit . Both FN and CEA concentration were normalised to nanograms per 1 \u00d7 106 cells. The results are expressed as an average of duplicate assays from one of two independent experiments.Cells (1 \u00d7 106 per dish) were seeded on 10-cm dishes and allowed to attach overnight and then the medium was discarded and replenished with medium containing the DPD for incubation at 37\u00b0C for 3 days. At the end of 3 days, the medium was discarded, and the cells were replenished with fresh medium. Cells were harvested and the cells viability were examined by haemocytometer at days 0, 3, 4 and 5 after withdrawal from 1, 2, or 4\u2009\u03bcM DPD treatment for 72\u2009h.Cells (1 \u00d7 106 per dish) were seeded in 10-cm dishes and allowed to attach overnight, and then the medium was discarded and replenished with medium containing DPD for incubation at 37\u00b0C for 3 days. At the end of 3 days the DPD was withdrawn, the cells were replenished with fresh medium. At 1 week after DPD withdrawal, cells were washed with PBS, then fixed with 2% glutaraldehyde in PBS then post-fixed with 1% OsO4 in PBS, dehydrated in ethanol, dried, coated with gold and examined in a field emission SEM .The method of in vivo experiments have been carried out with ethical committee approval, and meet the standards required by the UKCCCR Guidelines  was calculated according to the following formula: V (mm3)=0.4AB2, where A and B are the longest diameter and the shortest diameter, respectively , respectively. The control group received DMSO vehicle. Tumour size and body weight were monitored twice a week throughout the experiment. Tumour size was measured as described above. Drug efficacy was assessed as tumour growth index (TGI)=Vn/V0, where Vn is the tumour volume of treated group on day n and V0 is the initial tumour volume. At day 32, all mice were killed by CO2 gas. Tumours, livers, kidneys, and lungs were collected, fixed, embedded, and stained with haematoxylin and eosin for pathological analysis.Colo 205 cells were harvested and resuspended in serum-free RPMI-1640 medium. Cells were adjusted to 1 \u00d7 10\u03bcM) or vehicle (DMSO) for 24\u2009h. Then the cells were further treated with or without CPT-11 (25\u2009\u03bcg\u2009ml\u22121) for another 72\u2009h. The antitumoral activities were assayed by MTT method. The detailed procedure was reported in a previous study  with a decrease of cells in S phase (9.7%) after treatment with 1\u2009\u03bcM DPD for 72\u2009h. Colo205 and HT-29 cells were mainly in G0/G1 phase (88\u201393%), and only a few percent of cells in S phase (5\u20138%) and the G2/M phase (2.3\u20135%) after exposure to 2 or 4\u2009\u03bcM DPD for 72\u2009h. The moderately increased G0/G1, G2/M phase and decreased S phase of HCT-15 cell populations were observed after their exposure to 2 or 4\u2009\u03bcM DPD for 72\u2009h. These results showed that treatment of colon cancer cells with DPD resulted in increased G0/G1 phase with concomitant decrease of cells in S phase.The cell cycle progression of Colo205, HT-29 and HCT-15 cells was examined using flow cytometry after exposure to 1, 2, or 4\u2009\u03bc\u03bcM DPD treatment for 0\u201372\u2009h, the percentage of cells with expression of cyclin D in Colo 205 and HT-29 cells decreased from 80.68 to 26.46% and 79.84 to 24.49%, respectively   .\u03bcM DPD for 72\u2009h, then cells were replenished with fresh culture medium and cultured for another week. Figure 6\u03bcM DPD-treated cells but not in control cells.To further investigate whether DPD-induced epithelium like brush border formation in HT-29 cells. Cells were treated with 2\u2009\u03bcM DPD for 72\u2009h , and then transplanted into BALB/c nude mice. Figure 73 at day 32. The tumour of DPD (1\u2009\u03bcM)-pretreated Colo 205-implanted animals was marginally larger than control. However, tumours from 2 to 4\u2009\u03bcM DPD-pretreated Colo 205 implanted animals grew to an average size of only 17\u201320\u2009mm3. The tumour incidences were 10 out of 10 (vehicle control), nine out of 10 (1\u2009\u03bcM), four out of 10 (2\u2009\u03bcM) and two out of 10 (4\u2009\u03bcM), respectively. These results showed that the tumorigenicity of 2\u20134\u2009\u03bcM DPD-treated Colo 205 cells was significantly (P<0.01) reduced.To evaluate whether DPD-treated cells are still tumorigenic, Colo 205 cells were treated with 0, 1, 2, or 4\u2009in vivo after tumour formation. Cancer cells were transplanted into BALB/c nude mice, and when the tumours were palpable (3\u20135\u2009mm), the mice were treated either with vehicle control or DPD . Treatment of nude mice with DPD (37.5\u201375\u2009mg\u2009kg\u22121), the tumour growth index was significantly (P<0.05) decreased in mice as compared to control groups at the end of experiment. Figure 8A\u22121)-treated animals had an average of only 4.99. The tumour growth index of DPD (18.75\u2009mg\u2009kg\u22121)-treated animals was slightly larger than control but not statistically significant.We further examined whether DPD is also effective \u22121, i.p., once a week) in nude mice produced no obviously acute toxicity. No significant reduction in body weight was found in DPD-treated mice  as compared to DPD (17.1%) or CPT-11 (18.3%) alone. These results clearly showed that DPD enhanced the antitumoral activity of the chemotherapeutic agent CPT-11.To further investigate whether DPD could enhance the antitumoral activity of the chemotherapeutic agent CPT-11. Colo 205 cells were treated with DPD or DPD in combination with CPT-11 and their cell cycle analysed. As shown in Figure 9Ain vitro against several cancer cell lines in National Cancer Institute (NCI) Anticancer Drug Screen. Therefore, in the current study, we evaluated the effects of DPD in vitro and in vivo on the human colon cancer cells. To the best of our knowledge, the present study is the first report that refers to the in vitro cytostatic and differentiation promoting and in vivo antiproliferative effectiveness of DPD.We have previously found that diamantane derivatives exert strong growth inhibitory activities 0/G1-to-S phase transition in the cell cycle, and the cyclin D is a start cyclin in cell cycle. The decrease of cyclin D in specific cell types may signal a switch between proliferation and differentiation (1 phase (>90%), and the expression of cyclin D was dramatically decreased in DPD treated cells. The data suggest that the presence of DPD is promoting the loss of cyclin D from the cell in response to some as yet undefined process involved in DPD-induced G0/G1 arrest and differentiation.In the current study, three colon cancer cell lines with distinct biological properties  were used and treated with DPD. The Colo 205 is a poorly differentiated cell line, and HT-29 is a well differentiated cell line . The twontiation . In the trans retinoic acid (ATRA) and sodium butyrate (NaB) have also been shown to upregulate FN production in human colon cancer HT-29 cells (Differentiation-inducing chemicals often inhibit growth in conjunction with the induction of differentiation in cancer cells. In many transformed cell types, the induction of differentiation is associated with an increase of FN expression (In vivo, NaB has a half-life too short to produce any therapeutic effect. Our results show that DPD is more active than NaB in suppressing cell growth and concomitantly promoting differentiation of HT-29 colon cancer cells. Differential cellular responses were observed in these three cell lines after treatment with DPD. The differences in the cellular responses to DPD may be due to the differences in the biological properties of the three cell lines.Several studies have shown that the upregulation of CEA expression is also associated with a differentiation induction response in human colon cancer cells (The human colon cancer cell line HT-29 offers a favourable study system for the evaluation of various inducers involved in differentiation (Treatment of Colo 205 tumour-bearing nude mice with DPD significantly decreased the tumour growth index in mice as compared to control groups. Moreover, once a week i.p. challenge of DPD in nude mice produced no obvious acute toxicity. These results together with the data that combination therapy showed DPD could enhance the antitumoral activity of the chemotherapy agent CPT-11 in colon cancer Colo 205 cells suggest that a G1-targeting and differentiation-inducing agent DPD, has potential for combination with other antineoplastic agents in treatment of human colon cancer cells."}
+{"text": "Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis.Triacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT) with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight) compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold.Beneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, In silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering. Rhodococcus opacus PD630 which naturally stores large amounts of TAG, up to 76% of cellular dry matter [Triacylglycerols are the main components in vegetable oils. TAGs are used for various purposes including food applications, cosmetics, oleochemicals and biofuels. Current raw materials for biodiesel and renewable diesel include vegetable oils, animal fats or recycled greases. The production potential of current vegetable oil sources is limited to replace commodity products derived from fossil resources. One alternative to produce TAGs is to utilize heterotrophic organisms which produce lipids from organic molecules, such as sugars. In addition to crop sugars, different waste and residue streams can be used as a source of sugars for heterotrophic TAG production. In the field of bacterial lipid production, significant yields of TAG have been obtained with y matter ,2.et al. [Escherichia coli was genetically engineered to produce fatty esters, fatty alcohols and wax esters straight from plant biomass containing simple sugars. Furthermore, Kalscheuer et al. [E. coli. In this approach, lipid and ethanol formation was combined with subsequent esterification of fatty acids and ethanol, resulting in formation of fatty acid ethyl esters (FAEE). More recently, a pilot-scale process with FAEE producing E. coli was carried out [In order to improve economic feasibility of the process the production strains need to be engineered and optimized . Advanceet al.  publisher et al.  have lauried out .Acinetobacter baylyi is a strictly aerobic and widely spread soil bacterium with simple growth requirements and wide substrate range. The laboratory strain ADP1 has shown potential for metabolic studies and biotechnological applications, mainly for the compact easily-transformable genome and a unique metabolic network [E. coli, thus providing possibility to exploit the knowledge applied to this microorganism so far [Acinetobacter strains have been already studied to some extent and especially the bifunctional enzyme WS/DGAT have drawn a lot of interest [in silico  and in vitro  techniques, the production rates can be potentially improved by target-specific gene knock-outs. Thus, the strain ADP1 serves as an excellent model organism for TAG production and metabolic engineering. network . The genm so far . The genm so far ,10. Furtinterest -14. Combin silico with the help of a genome-wide metabolic model and gene annotation data [The objective of this work was to study the lipid metabolism of ADP1 and construct genetic tools for metabolic engineering. Potential single gene deletions affecting TAG metabolism were scanned ion data . The effin silico calculations, literature, and predictions based on gene annotation data, a list of beneficial single gene deletions was obtained  is incapable of fatty alcohol biosynthesis and therefore wax ester synthesis, which is the end-product of a competitive storage lipid pathway. ACIAD3381 (poxB) is associated to acetate production, and therefore the deletion redirects the carbon flux towards storage lipid synthesis. The fourth deletion, ACIAD2837 diacylglycerol kinase (dgkA), catalyses phospholipid synthesis consuming 1,2-diacylglycerol, an important precursor of TAG, as a substrate. In preliminary tests, the phenotypes and growth characteristics of the mutant strains were determined. Few observations were made: it was verified by thin layer chromatography (TLC) analysis, that the strain ACIAD3383 (designated as M1) is unable to produce wax esters , multiple cloning site (MCS), transcription termination loop (t lpp), selection marker (cam(r)), and homologous sequences from ADP1 (downstream of ACIAD3383 and upstream of ACIAD3381) to knock out selected target genes. The strain M3 was transformed with the gene cassette and selected on LA plate containing 50 \u03bcg/ml chloramphenicol. The strain was designated as MT. In addition to the target genes, the gene ACIAD3382 was deleted for practical reasons; the group of the three adjacent genes ACIAD3381 - ACIAD3383 could be deleted using single knock-out cassette. The gene ACIAD3382 encodes a homocysteine synthase, metY. The function is essential for methionine biosynthesis, but there is another gene present (ACIAD2314) in the genome possessing the same function, for which the deletion of the gene was not expected not to have an effect on the phenotype. The lipid synthesis pathway of ADP1 is presented in Figure In order to study the cumulative effects of gene deletions on TAG production, a knock-out strain with three gene deletions was constructed . The deletion ACIAD2837 was left out from the knock-out combination for the negative effect on growth properties. For strain construction, synthetic gene cassette was used  2.4 - 3.6 g/l with M4 possessing the lowest number and the strain M3 the highest.The profiles of acylglyceride lipid fraction Figure , and intin silico model predicted, the lipid synthesis was shifted towards the intended lipid fraction. The most drastic changes were observed for the MT strain , indicating that combining the deletions have cumulative effects on TAG production.For extracted lipids the amount of total lipids was determined gravimetrically. For visualization and quantitative analysis of specifically TAG, preparative thin layer chromatography (TLC) was carried out. The amount of total lipids (mg/g CDW) was found to be similar in all strains Table . On the 2, biomass, and storage compound production. Also, no significant differences were observed in carbon usage between the knock-out strains; within the first 24 hours of cultivation dedicated as biomass accumulation conditions (phase I), approximately 20-35 mM of gluconate was consumed. Within the next 24 hours , gluconate consumption decreased to approximately 13-25 mM. According to optical density measurements, after transferring the cells to low nitrogen medium (phase II) the amount of biomass did not increase significantly. Thus, the consumed carbon was directed to other functions than biomass formation, as intended. The consumption of glycerol did not correlate with the measured TAG content of the cells, indicating that the endogenous glycerol synthesis does not limit the TAG production in the studied conditions. Moreover, for all the strains, glycerol was only consumed within the first 24 hours and only to minor extent (1-10 mM).The utilization of gluconate and glycerol, and the production of end metabolites were determined by High Performance Liquid Chromatography (HPLC). The presence of common end-products such as acetate, lactate and succinate was studied in ADP1 cultivations. Detectable amounts of the above mentioned known metabolites were not seen in any of the samples, indicating that carbon is mainly directed to COAcinetobacter baylyi ADP1 by target specific gene knock-outs, the lipid production of the strain was studied. Potentially beneficial gene deletions were screened by computational means exploiting the constraint-based metabolic model available for ADP1. The whole genome sequence database enabled the construction of genetic tools for specific molecular engineering to take place in vivo.Modelling the biochemical pathways and studying the effects of competitive or beneficial key enzymes in specific routes increase the knowledge and understanding of microbial metabolism . Metabolin silico results and literature, the four most interesting gene deletions with different action mechanisms on TAG synthesis pathway were chosen to be studied more in detail both individually and concurrently. To study the cumulative effects of gene deletions, three of the deletions were combined to form a strain with four gene deletions . As the goal was to shift the lipid metabolism towards TAG synthesis, the fatty acyl-CoA reductase ACIAD3383 (acr1) was deleted, totally blocking the wax ester (WE) synthesis [According to ynthesis . The celThe gene ACIAD3309 is annotated as a TAG lipase, and the corresponding knock-out strain M3 was experimentally shown to possess good cellular fitness and slightly increased TAG accumulation in the studied conditions. However, the role of the lipase in context of intracellular lipid metabolism is unclear, since the gene sequence contains clear signal sequence, indicating subcellular location and potential involvement in periplasmic or membrane lipid metabolism. Thus, further studies are required to show the interplay of different lipases in storage lipid metabolism.E. coli, the gene poxB (corresponding to ACIAD3381) is associated to acetate production and carbon flux [E. coli, acetate accumulation inhibits growth and bioprocess control systems are required to neglect the negative effects of acetate [poxB deletion and carbon fate is not straightforward. The deletion did not shift the carbon metabolism towards TAG production in M2 strain but rather towards WE formation in studied conditions, as shown in Figure poxB deletion for the wax ester deficient MT strain was that the carbon flow could be shifted from WE production towards TAG production. Since there is only one enzyme, WS/DGAT, responsible for the final step of producing both WE and TAG, such modulation in theory is possible.In bon flux ,18. In E acetate . The delThe strain MT with four gene deletions  was constructed using a synthetic gene cassette. For practical reasons (see: strain construction) ACIAD3382 was also deleted, although the gene is not known to be involved in lipid metabolism. The TAG content of the cells relatively increased compared to the single gene knock-out mutants and the wild type strain. Also, the lipid metabolism was shifted towards producing higher percentage of TAG of total lipids. On the other hand, the amount of total lipids and biomass decreased slightly. Without supplementation of convenient substrates or over-expression of the key enzymes of the route, it is difficult to obtain the dual goal of high cell density and high cellular lipid content, since the biomass and lipid production are competing for the same building blocks. Furthermore, as the TAG production pathway was not engineered as a continuum, it is possible that the potentially increased substrate flux for WS/DGAT was insufficient, due to either a naturally low expression level of WS/DGAT  or the rSupplementation of the medium with glycerol did not seem to have effect on TAG accumulation. According to analyses, glycerol had been only consumed within the first culture phase. This indicates that glycerol is consumed rather as a carbon and energy source in biomass formation than building up TAG. The lack of glycerol consumption in phase II can be related to the induction of the endogenous glycerol synthesis as a TAG backbone or just simply to the fact that cells in stationary phase do not import glycerol to the cells.In a bioenergy application point of view, few observations can be done. The results of qualitative lipid analyses show that the main constituents of the intracellular lipids are C16 and C18 fatty acids, with minor proportions of C12 and C14. The C18 and C16 fatty acids are generally the main components in vegetable oils, such as rapeseed oil, soy bean oil or palm oil, and desirable raw materials for biodiesel or renewable diesel , suggestUsing a computational analysis to screen beneficial gene deletions from a genome-wide metabolic network allows going through a number of deletions that would be impossible manually. Also, capturing effects of upstream deletions on the product yields can be difficult without modelling. However, rapid and sensitive high through-put assays are not yet available for analyzing large number of samples, although some suggestions for alternative methods have been made . In addiAcinetobacter baylyi ADP1 proved to be a potential platform for a microbial cell factory and a model system for studies involving metabolic engineering. The strain naturally produces TAG, and the lipid profile corresponds to that in vegetable oils and can potentially be used to replace or as a supplement to vegetable oil for various purposes, including biofuel applications. The TAG production of A. baylyi ADP1 was demonstrated to improve by genetic knock-outs.In this article, we demonstrated the significance of understanding the changes in natural lipid metabolism in response to simple gene knock-outs. The predictions of the constraint-based metabolic model regarding beneficial gene deletions for TAG production were experimentally verified. A. baylyi metabolism [Computational modelling and simulation was performed using a genome-wide reconstruction of tabolism . The rec(1) The main TAG producing pathway was identified, starting from glycerol-3-phosphate and acyl-CoA.R was composed of reactions consuming glycerol-3-phosphate and acyl-CoA for fatty acid, fatty aldehyde, and fatty alcohol production. Additionally, the reactions consuming TAG or TAG precursors of variable carbon chain lengths, such as 1,2-diacylglycerol, were considered.(2) The first reactions from each pathway diverging from the TAG producing pathway were selected. The set of marker reactions (3) Gene deletions were simulated by enforcing the respective enzyme-catalyzed reaction rates to zero.R were calculated using flux balance analysis [(4) The maximal achievable rates of the marker reactions in analysis .(5) If the studied gene deletions lowered the maximal achievable rate of a marker reaction considerably , the deletions were considered potentially beneficial for TAG production.In silico gene knockouts and flux balance analysis were carried out using COBRA toolbox [http://www.gnu.org/software/glpk/). The analysis considered only knock-outs of genes that have been found to be non-essential to A. baylyi [2, Na+, H+, SO42-, Fe2+, NH4+, PO4- and a carbon source. The analysis results were not dependent on the carbon source.After running the algorithm the maximal TAG production was simulated for the identified gene deletions to verify that the TAG production itself was not blocked.  toolbox  and GNU . baylyi . The modAcinetobacter baylyi ADP1 was used as the wild type strain . The single gene knock-out strains M1, M2, M3, and M4  were kindly provided by Veronique de Berardinis . In the single gene knock-out mutants, the gene in question is replaced with a gene cassette containing a kanamycin resistance gene (rkan) [et al. [The molecular work was carried out by using methods described by Sambrook et al. . For diget al. [The transformation of ADP1 was carried out by methodology described by Metzgar et al. . Brieflykan(r) was used, amplified from the plasmid pET-28  with primers ab7 and ab8 and cloned back to the plasmid in vitro using restriction enzymes XhoI and PstI and T4-DNA-ligase. The resulting plasmid was used as a PCR template for amplifying multiple cloning site (MCS) and kan(r) together with primers ab5 and ab8. Later on, cam(r)  was amplified from plasmid pAK400c with primers ab9 and ab10. Double digestions were carried out for the PCR products with restriction enzymes, and ligated in pairs. The ligation reactions were amplified by PCR with corresponding primers, digested again, and two of the pairs were ligated and amplified by PCR again. The two- and four-gene component sets were ligated and the whole gene cassette construct was amplified by PCR with primers ab57 and ab56, the final product being 2025 bp long. Purification of the PCR products was carried out in every step using PCR purification kit (Fermentas) or gel extraction kit (Fermentas). PCR products were run on 1-2% agarose  gel supplied with SYBRsafe  and visualized with SafeImager (Invitrogen).The six gene cassette components were amplified separately by PCR: flanking region Gene_I (upstream of ACIAD3381) was amplified from ADP1 by colony PCR with primers ab57 and ab58 and flanking region Gene_Z'(downstream of ACIAD3383) with primers ab55 and ab56, respectively. The promoter T5 (lac/T5) was amplified from plasmid pCSS810  with priA. baylyi strains: Na2HPO4 \u00b7 2 H2O 5.518 g/l, KH2PO4 3.402 g/l, NH4Cl 1 g/l (phase I) or 0.1 g/l (phase II), nitrilotriacetic acid 0.008 g/l, NaCl 1.0 g/l, FeCl3 0.487 mg/l, FeSO4 \u00b7 7 H2O 5.6 mg/l, MgSO4 \u00b7 7 H2O 250 mg/l, CaCl2 \u00b7 2 H2O 20 mg/l, NaCl 117 mg/l, MnSO4 \u00b7 4 H2O 0.56 mg/l, ZnSO4 \u00b7 7 H2O 0.140 mg/l, Co(NO3)2 \u00b7 6 H2O 0.150 mg/l, CuSO4 \u00b7 5 H2O 0.130 mg/l, Na2MoO4 \u00b7 2 H2O 0.120 mg/l, H3BO4 0.160 mg/l, EDTA III 22.7 mg/l. Casein amino acids were added at concentration (0.2 w-%) for the phase I cultivation. Sodium gluconate (0.11 M) and glycerol (0.05 M) were used as a carbon and energy source.The following medium composition was used for the TAG cultivation of 4Cl), in order to promote TAG accumulation [The batch cultivation was carried out in 100 ml medium/250 ml Erlenmeyer flasks. In the phase I, the strains were cultivated for 24 h in normal MA/9 medium at 37\u00b0C and 300 rpm. For phase II, the cells were collected by centrifugation  and suspended to fresh medium with reduced nitrogen concentration . The samples were analyzed for gluconate, acetate, succinate, fumarate, ethanol and lactate with LC-20AC prominence liquid chromatograph  equipped with RID-10A refractive index detector, DGU-20A5 prominence degasser, CBM-20A prominence communications bus module, and SIL-20AC prominence autosampler. Shodex SUGAR SH1011  kept at 40\u00b0C was used as a column. Sulfuric acid (0.01 N) was used as an eluent at pumping rate of 0.6 ml/min. Identification and quantification of liquid end products was based on co-chromatography of using standards. Mediums were used as controls.End-products of the cultivations were measured with high performance liquid chromatography (HPLC). Culture samples were centrifuged at 20000 g for 5 minutes. Supernatant was collected and filtered through polycarbonate filter  and dyed with iodine for visualization. Of extracted lipids, 10 \u03bcl of a sample was applied on the TLC plate. Mobile phase used was n-hexane:diethyl ether:acetic acid 90:15:1. The preparative chromatography for the isolation of the specific fraction of TAG from the total lipid extracts was carried out on Silica Gel 60 F254 10 \u00d7 20 cm glass plates with the concentrating zone 2.5 \u00d7 10 cm (Merck). The concentrated solution of the total lipid extract in chloroform  was applied as a single band to a Silica Gel plate. Tripalmityol-glycerol (Sigma) or olive oil was used as a standard. The TLC chromatogram was developed with the solvent system n-hexane:diethyl ether:acetic acid 80:20:2. Iodine was used for visualization. After evaporation of iodine TAG-zone was scraped and transferred into a Pasteur pipet containing cotton wool. TAG fraction was eluted from Silica Gel with chloroform (3 \u00d7 0.7 ml). Chloroform was purged under nitrogen and the amount of TAG was determined gravimetrically.In order to visualize the total lipid composition of the strains M1 and M2, TLC analyses were carried out using 10 \u00d7 10 cm HPTLC Silica Gel 60 F\u00ae-SiO2  into a glass pipette. The dried lipids were dissolved in 200 \u03bcl of chloroform. The silica column was washed with 3 ml of chloroform and n-hexane after which the lipid solution was transferred to the column. Three ml of n-hexane was added to the column and allowed to drain by gravity. The collected lipid fraction containing acylglycerides was purged with gentle nitrogen stream.Fractionation of ADP1 wild type lipids was conducted using a custom-made solid-phase column. The column was prepared by filling FlorisilIn order to separate and identify intracellular and membrane lipids, sonication was used for cell disruption. Four ml of PBS buffer was mixed with a cell pellet and the sonication was conducted 3 times for 5 min . After the treatment, the sample was centrifuged at 10000 g for 10 min. The supernatant (intracellular fraction) was dissolved in 5 ml of chloroform and 10 ml of methanol. The remaining pellet (membrane fraction) was dissolved in 5 ml of chloroform, 10 ml of methanol, and 4 ml of PBS buffer.3 \u00b7 MeOH (Sigma) and heated up for one hour at 100\u00b0C. One ml of distilled water and 1 ml of n-hexane were added and the reaction mixture was shaken for 2 minutes and centrifuged at 2000 g for 5 minutes. The upper n-hexane phase was collected into a new vial and evaporated under nitrogen. The n-hexane extraction and evaporation steps were repeated. The FAME sample was dissolved in dichloromethane and transferred into a GC glass vial. The sample was washed once by evaporating the dichloromethane and dissolving it again.For determination of fatty acid profiles of acylglyceride fraction and to compare the fatty acid profiles the ones of intracellular and membrane lipids, the fatty acids were transesterified to form fatty acid methyl esters (FAME). The lipids were suspended in 200 \u03bcl toluene and 100 \u03bcl BF\u00ae, Sigma) with carbon chains C10:0 - C20:0, C22:0, C24:0, C26:0 and derivatives of the chains C15 - C19 was used as a standard.The fatty acid methyl esters were analyzed with HP-GC5890 gas chromatograph equipped with flame ionization detectors (FIDs). Injection volume was 1 \u03bcl for all samples. A fused silica column HP-5MS  was used with nitrogen as a carrier gas. Hydrogen and air were used as support gases. Injector and detector temperatures were both set to constant 250\u00b0. The run was carried out using following temperature program: 50\u00b0C for 10 min, followed by 4.18\u00b0C/min for 55 min, followed by 20 min at 280\u00b0C. Bacterial Acid Methyl Ester (BAME) mix (SupelcoThe authors declare that they have no competing interests.in silico work and took part in drafting the manuscript. VS and MK supervised and coordinated the study. All authors read and approved the final manuscript.SS and VS designed the study. SS performed the molecular work, microbiological work, is responsible for the qualitative lipid analyses, and wrote the manuscript. EE carried out the quantitative lipid analyses and participated in manuscript drafting. VK, AL and TA are equally responsible for designing and performing the"}
+{"text": "Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process.Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model.TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding , GTPase activator activity , cytoskeleton , protein binding , proteinaceous extracellular matrix , ion channel/ ion transporter activity  and genes associated with developmental pathways .In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity. Tissue morphogenesis is controlled by a variety of factors including local growth factors, extracellular matrix, cell adhesion molecules and the cytoskeleton. Cadherins and tight junctions have a major role in establishing and maintaining intercellular adhesion ,2. E-cadWe have previously modelled prostate epithelial morphogenesis using 3D Matrigel culture . PrimaryThe use of human prostate tissue to grow primary cultures and patient consent procedures were approved by York Research Ethics Committee, (YREC Reference 91/7/6) and Hull and East Riding Local Research Ethics Committee (REC Reference Number 07/H1304/121). Tissue was obtained from York District Hospital, York and Castle Hill Hospital, Hull, UK. All patients who provided tissue gave their written consent. Tissues were given a unique identification number which was stored with the consent forms at participating hospitals, whilst documentation of tissue processing, experimentation and storage occurred at the YCR Cancer Research Laboratory.Primary cultures were prepared as described before . BrieflyBPH-1 cells (benign prostate cell line), primary human benign prostate epithelial cultures and primary human benign prostate stromal cultures were cultured in 3D as described previously ,10.4 cells/insert in RPMI supplemented with 10% FCS, until confluent. Epithelial cells were seeded at 5 000 cells/ml in KE2  and 4% (v/v) Matrigel. Inserts were then washed with PBS and added to epithelia plus Matrigel or blank wells, with KE2. The inserts were replaced 4, 8 and 12 days after cell seeding with fresh inserts of pre-seeded stroma. Medium was replenished at the same time through the removal of 0.5 ml spent media and the addition of 0.5 ml fresh KE2 supplemented with 4% Matrigel. Spheroids for RT-PCR were isolated from the Matrigel using BD Cell recovery solution .Briefly, Primary stromal cultures (passage 1 to 3) were seeded prior to co-culture in 0.4 \u03bcm Millicell-PCF inserts , 2 \u00d7 10Ten primary epithelial cultures (passage 1) were grown in Matrigel, with or without primary prostate stroma for 14 days, the optimum time of primary spheroid formation . Single Array pre-processing and significance analysis was performed using GeneSpring GX 10 software . Arrays were filtered on expression between the 20th and 100th percentile of the raw data. Normalization was performed by scaling and baseline transformation to the median of all samples. The experiment was analysed as a reference design. Differentially expressed genes were identified by using a paired t-test with asymptotic p-value computation and no multiple testing correction where significance level was set at p > 0.05. Genes that were > 1.1 fold up- or down-regulated between groups were selected, this was then referred to as the 'primary 1.1 fold gene list. .Three replicate cultures of BPH-1 (passage 47) were grown in 24 well plates with or without stroma  for 7 days in KE2 media. A time point of 7 days was chosen, since BPH-1 cells grow faster than primary cultures. On average, 7 day old BPH-1 acini are the same size as 14 day old primary acini. 3D acini produced by BPH-1 cells are predominantly homogeneous, therefore individual acini were not isolated, RNA was prepared from whole cultures and an Affymetrix array was performed. RNA was prepared using Illustra RNA Spin mini kit . RNA samples were analysed using Affymetrix Human Genome U133 Plus 2.0 chips . Each array contains more than 54,676 probe sets that represent more than 47,000 transcripts. The RNA hybridisation of all Affymetrix U133 Plus 2.0 arrays was performed at TF facility (University of York). The cRNA synthesis of the samples was carried out according to the manufacturer's protocol. The fluorescence intensity for each chip was captured with an (Affymetrix GeneChip Scanner 3000). Affymetrix Microarray Suite version 5.0  was used to quantitate each chip. The raw data (CEL) files, were loaded into the DNA-chip analyser software (dChip) version Feb 2009 . Normalihttp://www.affymetrix.com). Each sample and hybridization underwent a quality control evaluation mainly checking for adequate scaling factors , percentage of probe sets reliably detected , and optimal 3'/5' hybridization ratios (~1) for the housekeeping genes , poly(A) spike-in controls, and the prokaryotic controls . MAS5 normalised data were collected and analyzed using the GeneSpring GX10 Expression software . Differentially expressed genes were identified by using a two-class t test where significance level was set at p > 0.05. Genes that were > 1.1 fold up- or down-regulated between groups were selected. .Raw data was processed using the Affymetrix GCOS 1.2 software. After hybridization and scanning, probe cell intensities were calculated and summarized for the respective probe sets by means of the MAS5 algorithm. To compare the expression values of the genes from chip to chip, global scaling was performed, which resulted in the normalization of the trimmed mean of each chip to target intensity  of 500 as detailed in the statistical algorithms description document of Affymetrix . The Entrez IDs that matched were copied into a txt. file and the gene names found using RNA was prepared from BPH-1 spheroids grown in 24 well plates using Illustra RNA Spin mini kit  and grown with and without stroma . Reverse transcription was performed with RT2 PCR array First Strand Kit (SABiosciences). RT2 profiler PCR array for the human TGFB BMP signaling pathway were prepared as per manufacturers protocol. Target cDNA levels were detected using the ABI prism 7300 sequence detection system (Applied Biosciences) and normalised to HPRT, B2M, RPL13A and ACTB using the DDCt Data analysis method. The real time PCR conditions were as follows: 1 cycle at 95\u00b0C for 10 min, 40 cycles at 95\u00b0C for 15 s, and 60\u00b0C for 1 min. 49 genes appearing on the TGF beta PCR array were not differentially expressed according to the microarray data.RNA was prepared from spheroids using Illustra RNA Spin mini kit . Reverse transcription was performed with random hexamers . Quantitative real time PCR oligonucleotide primers  and hybridized their transcriptomes on an Operon microarray, which is known to be robust for low cell numbers . CompariTo verify the Operon microarray data we selected FGFBP1 (fibroblast growth factor binding protein 1), since the average expression of this genes was highly upregulated in the presence of stroma. Using matched patients samples to the microarray, we performed quantitative RT-PCR (QRT-PCR). QRT-PCR confirmed the upregulation of FGFBP1 in six primary epithelial samples in response to stromal co-culture Figure . One epiTo overcome the problems of heterogeneity we decided to analyse a prostate epithelial cell line, BPH-1, which can also grow into acinus-like spheroids in 3D culture and demonstrates increased lateral adhesions, in response to stroma . We perfThree technical replicates of BPH-1 cells were cultured in 3D with and without primary stroma , using identical culture conditions to the primary cell model. 7843 probe sets were differentially expressed between the two experimental groups (p < 0.05). Table Figure To verify the BPH-1 microarray data and in particular genes associated with TGF beta signalling pathway, we used a commercial PCR array for the 'human TGF beta/BMP signaling pathway'. The differential expression of fourteen genes was verified Figure ; BGLAP . SOX4 is an important transcription factor in development and interacts with many morphology related pathways, . SOX4 stabilises \u03b2-catenin protein and enhances \u03b2-catenin/TCF activity . Over-exX. laevis and D. melanogaster during early embryonic development and organogenesis. Therefore upregulation of MY10 found here may promote polarity and adhesion. TMOD4 is an actin filament capping protein that maintains the length of the actin filaments in skeletal muscle and in has a role in cell membrane dynamics [Actin binding and cytoskeleton genes provided the most likely set of genes to have a role in adhesion. We found up regulation of MAP2 (microtubule-associated protein 2), which is a major regulator of microtubule dynamics and is best known for its role in neuronal development . MAP2 isdynamics ,44. NoneSeveral calcium and potassium channels were up regulated on both arrays  these may provide a means of modulating cell junctions by controlling the intracellular levels of calcium and potassium . Recent Before this study, tight junctions and adherens junctions were likely candidates to be involved in increased cell to cell adhesion. They are dynamic structures linked to the acto-myosin cytoskeleton and are regulated by Rho/Ras-GTPases . MicroarWe used microarray analysis and bioinformatics to identify candidate epithelial genes which control lateral cell adhesion under stromal stimulation. We confirmed the importance of TGF beta signalling, and in particular SOX4. Analysis of genes that were common to both cell line and primary arrays found several morphology related gene clusters; actin binding, GTPase activator activity, cytoskeleton, protein binding, proteinaceous extracellular matrix, ion channel/ion transporter activity and genes associated with developmental pathways. These candidates will be investigated in future functional studies. This work highlights the complexity of any biological process and the value of combining gene array data from different models to identify important pathways and genes. Overall we have shown the complexity of stromal controlled epithelial morphology. The study of intercellular adhesion is a fast expanding field, and our identification of genes associated with actin binding, microtubules and anion signalling complements newly emerging ideas.The authors declare that they have no competing interests.KC participated in the design of the study, performed data analysis and design, prepared the primary culture samples and coordinated the Operon array study. JFP prepared samples for Affymetrix array and performed qRT-PCR assays. MG and CK coordinated and performed the Operon array. NA coordinated the Affymetrix array and was involved in data analysis. DP was involved in data analysis. SHL conceived of the study, participated in study design and co-ordination, analysed experiments and wrote the manuscript. All authors read and approved the final manuscript.Table S1: Primers and probes for QRT-PCR primer sequences.Click here for file"}
+{"text": "Adenosine monophosphate-activated protein kinase (AMPK) acts as a master mediator of metabolic homeostasis. It is considered as a significant millstone to treat metabolic syndromes including obesity, diabetes, and fatty liver. It can sense cellular energy or nutrient status by switching on the catabolic pathways. Investigation of AMPK has new findings recently. AMPK can inhibit cell growth by the way of autophagy. Thus AMPK has become a hot target for small molecular drug design of tumor inhibition. Activation of AMPK must undergo certain extent change of the structure. Through the methods of structure-based virtual screening and molecular dynamics simulation, we attempted to find out appropriate small compounds from the world's largest TCM Database@Taiwan that had the ability to activate the function of AMPK. Finally, we found that two TCM compounds, eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose, had the qualification to be AMPK agonist. One study has found that the cell \u201cstarvation\u201d signal transduction pathway reveals the process of cell \u201cstarvation\u201d signal transduction mechanisms. This finding is considered as a significant millstone to treat metabolic syndromes including obesity, diabetes, and fatty liver , 2. Aden\u03b1, \u03b2, and \u03b3 [\u03b3 subunit of AMPK [\u03b3 subunit exposes the active site, Thr172, on the catalytic \u03b1 subunit of AMPK [\u03b1 subunit is the most important. Activation of AMPK follows conformational change of \u03b1 subunit and phosphorylation at Thr172 [\u03b2 subunit is located between \u03b1 and \u03b3 subunits and is associated with the function of glycogen sensor [\u03b3 subunit and protect AMPK from dephosphorylation but cannot cause conformational change [AMPK consists of three subunits, \u03b2, and \u03b3 . Each of\u03b2, and \u03b3 , 19. The of AMPK . After b of AMPK . For allt Thr172 . The phot Thr172 . The \u03b2 sn sensor . ADP canl change .Physiological study has approved that blood glucose is partly regulated by AMPK . Activat In silico investigation of biology or computational systems biology helps us to explore the protein-protein or protein-molecule interaction [Due to progress of modern technology, the binding phenomena of protein dynamics motion and structure changing can be analyzed by computational simulation , 39. In eraction , 41. Theeraction , 43. CADeraction , 45. CADeraction , 47. Vireraction \u201350. MD ceraction . A serieeraction . Best caeraction .With progress in medical technology, many diseases can be resolved nowadays \u201356. Whenhttp://tcm.cmu.edu.tw/) to carry on AMPK agonist screening [To identify potential AMPK activators from TCM, we downloaded all small molecules from TCM Database@Taiwan . The 3D structure of rat AMPK was obtained from Protein Data Bank (PDB ID: 2Y94). The sequence of human AMPK (Q13131) and homologous crystal structure of rat AMPK (2Y94) were aligned by the modeler mode in Accelrys Discovery Studio (DS) 2.5. The identity and similarity can be calculated according to the result of sequence alignment. Homology modeling of AMPK was established by the Build Homology Models protocol in DS 2.5. The reasonable AMPK model was further validated by Ramachandran plot with Rampage protocol and Verify score with Profiles-3D protocol in DS 2.5.The ligands from TCM Database@Taiwan and the control ligand, adenosine monophosphate (AMP), were prepared for specified techniques. Ligand docking was performed by the LigandFit module in DS 2.5. The force field of Chemistry at HARvard Molecular Mechanics (CHARMm) was utilized to minimize all docking poses . AbsorptWe drew disorder disposition to exclude disordered residues by the program of PONDR-FIT in the DisProt website , 75.The package of GROMACS  was used for MD simulation. We employed SwissParam to determine topology and parameters of small compounds for GROMACS simulation. The cytoplasmic condition was set with transferable intermolecular potential 3P (TIP3P) water at 0.9% sodium chloride concentration. After docking, selected protein-ligand complexes were conducted under the following phases: minimization, heating, equilibration, and production. The minimization protocol included 500 steps of steepest descent and 500 steps of conjugated gradient. The heating time was 50\u2009ps from 50\u2009K to 310\u2009K. The equilibration time was 150\u2009ps at 310\u2009K. The production time was 5000\u2009ps with constant temperature dynamics method. The time for temperature decay was 0.4\u2009ps. We utilized the trajectory analysis to illustrate root mean square deviation (RMSD), Gyrate, solvent accessible surface (SAS), root mean square fluctuation (RMSF), total energy, database of secondary structure assignment (DSSP), matrices of smallest distance of residues for individual ligands, and protein during MD. We calculated the formation and distance of hydrogen bond (H-bond), too. Best distance of H-bond was set at 0.3\u2009nm.To analyze the ligand pathway, we employed the software of LigandPath module to illustrate the possible pathway of each ligand. A surface probe and minimum clearance were set at 6\u2009\u00c5 and 3\u2009\u00c5, respectively.According to sequence alignment between Q13131_human and template (2Y94), the overall identity was 89.4% and similarity was 89.6% . RamachaMain residues  of AMPK-modeled structure for the 2 candidates and the control were not in the disordered area, so there was no influence to the shape of the binding site .MD trajectories generated by GROMACS were illustrated. We utilized root mean square deviation (RMSD) to show the deviation degree of each ligand or protein from the beginning to the end of MD. When the ligand formed a complex with the protein (AMPK), eugenyl_beta-D-glucopyranoside, 6-O-cinnamoyl-D-glucopyranose, and the control (AMP) had first larger deviations at 1600\u2009ps, 1450\u2009ps, and 2000\u2009ps, respectively. AMP had a larger average deviation than the candidates . We util\u03b1-helix was higher than \u03b2-sheet, and \u03b2-sheet was higher than bend. The ratio of \u03b1-helix increased slightly, but the ratio of bend decreased comparatively (Root mean square fluctuation (RMSF) showed the deviation of individual residue of the protein during MD. All the compounds had similar graphs with larger fluctuations at the range between residue 320 and residue 400 . AMP hadratively . We utilratively .We showed the distance of H-bonds between eugenyl_beta-D-glucopyranoside and essential amino acids of AMPK. The O13 of eugenyl_beta-D-glucopyranoside formed H-bond with Lys39 at early stage of MD, and H34 formed H-bond with Gly22 at late stage of MD. H36 and H38 formed H-bonds with Asn138 only at initial stage of MD. H36 formed H-bond with Asn151 at early stage of MD .We showed distance of H-bonds between 6-O-cinnamoyl-D-glucopyranose and essential amino acids of AMPK. The H30 of 6-O-cinnamoyl-D-glucopyranose formed H-bond with Glu137 at late stage of MD. H26 formed H-bond with OD2 of Asp133 at early stage of MD and formed H-bond with OD1 of Asp133 at late stage of MD. H23 formed H-bond with OD2 of Asp151 at early and late stage of MD and formed H-bond with OD1 at middle stage of MD .We showed distance of hydrogen bonds between AMP and essential amino acids of AMPK. The O4 of AMP formed H-bond with Gly19 at early stage of MD, and O20 formed H-bond with Phe152 at all stages of MD. H30 formed H-bond with Glu94 at early stage of MD. O4 formed H-bond with Val90 at early stage of MD, and N14 formed H-bond with Val90 at late stage of MD. H36 formed H-bond with OD1 of Asp151 at early and late stage of MD and formed H-bond with OD2 of Asp151 at middle stage of MD .3D simulation of ligand pathway bound with AMPK helped us understand the process of combination. Eugenyl_beta-D-glucopyranoside, 6-O-cinnamoyl-D-glucopyranose, and AMP had different pathways bound with AMPK protein .http://tcm.cmu.edu.tw/). The TCM Database contained 453 kinds of herb plants, animals, and minerals. They consist of more than 20000 pure compounds. The TCM Database is a useful and precious treasury for exploring the mystery of traditional Chinese medicine.The TCM small compounds for this study were from the world's largest TCM Database@Taiwan  and rat templates  for homology modeling. The high percentage of identity and similarity of sequence alignment between Q13131 and 2Y94 indicated that the sequence alignment was reliable. The high percentage of residues in the favored and allowed area indicated that the AMPK-modeled structure was reasonable. The Verify scores of most residues were positive which indicated that the AMPK-modeled structure was reliable. We estimated that the negative values from residue 290 to residue 360 might be attributed to loss of reasonable structure from residue 311 to residue 341 illustrated in Because the catalytic Whether absorption level of ADMET, Dock scores, PLP1, PLP2 or PMF, all the top 7 TCM compounds were better than the control (AMP). Absorption level of ADMET from 0 to 3 means good to very low absorption. We selected the first 2 compounds, eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose, as candidates due to their good absorption level. Both eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose formed H-bond with Asp151. Both eugenyl_beta-D-glucopyranoside and AMP formed H-bond with Glu94. The main residues bound for the candidates and the control were located from Val18 to Asp151. The results mean that loss of reasonable structure from residue 311 to residue 341 did not disturb us for adopting the AMPK-modeled structure.By integrating the figures of RMSD, Gyrate, and SAS, individual structure of the ligand and the protein underwent certain extent change during MD. This finding was essential for explanation of AMPK activation. Activation of AMPK followed its conformational change. Further analysis of RMSD, eugenyl_beta-D-glucopyranoside, 6-O-cinnamoyl-D-glucopyranose, and the control (AMP) had first larger deviation at 1600\u2009ps, 1450\u2009ps, and 2000\u2009ps, which means that all the compounds could induce conformational change of AMPK. By further analysis of Gyrate and SAS, 6-O-cinnamoyl-D-glucopyranose had the largest ligand Gyrate and SAS values, but eugenyl_beta-D-glucopyranoside had the largest protein Gyrate and SAS values. We speculated that both the candidates could induce different kinds of conformational change of AMPK protein.All the compounds had larger fluctuations at the range between residue 320 and residue 400 in RMSF. The larger fluctuations were not reliable due to loss of reasonable structure from residue 311 to residue 341. Based on the similar graph in RMSF and total energy, we could speculate that eugenyl_beta-D-glucopyranoside, 6-O-cinnamoyl-D-glucopyranose, and the control (AMP) had the same ability to activate AMPK. Our hypothesis was further approved by DSSP and smallest distance matrices. The structure of AMPK had similar change when it complexed with the 2 candidates and the control. Eugenyl_beta-D-glucopyranoside, 6-O-cinnamoyl-D-glucopyranose, and the control had the ability to induce AMPK conformational change.According to occupancy of H-bond between eugenyl_beta-D-glucopyranoside, 6-O-cinnamoyl-D-glucopyranose, and AMP with AMPK protein, we could conclude that Asp151 was key residue for top 2 TCM candidates and the control bound with AMPK. By further analysis of distance of H-bonds between eugenyl_beta-D-glucopyranoside and essential amino acids of AMPK, the compound formed H-bond with Asp151 only at early stage of MD. At late stage of MD, the compound formed H-bond with Gly22 instead. By further analysis of distance of H-bonds between 6-O-cinnamoyl-D-glucopyranose and essential amino acids of AMPK, the compound formed H-bond with Asp151 at all stages of MD. By further analysis of distance of H-bonds between AMP and essential amino acids of AMPK, the compound formed H-bond with Asp151 at all stages of MD. Interestingly, H36 of eugenyl_beta-D-glucopyranoside formed H-bond with OD1 and OD2 of ASP151; H36 of AMP also formed H-bond with OD1 and OD2 of ASP151, but their figure patterns were quite different. However, H23 of 6-O-cinnamoyl-D-glucopyranose formed H-bond with OD1 and OD2 of ASP151 and the figure pattern was similar to that of AMP. We could confirm that all the candidates and the control formed stable complexes with AMPK. In addition, all the candidates and the control induced conformational change of AMPK due to changing position of H-bonds.http://tcm.cmu.edu.tw/) and had the ability to activate the function of AMPK. We selected eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose as candidates for further investigation. Through the methods of MD simulation consisting of RMSD, Gyrate, SAS, RMSF, total energy, DSSP, smallest distance matrices, occupancy and distance of H-bonds, and ligand pathway, we could conclude that the candidates and the control (AMP) formed stable complexes with AMPK. Asp151 was key residue for the candidates and the control bound with AMPK. These compounds also had the ability to induce AMPK conformational change. Thus eugenyl_beta-D-glucopyranoside and 6-O-cinnamoyl-D-glucopyranose had the qualification to be AMPK agonist.AMPK was a hot issue in the past decade because it plays an important role in sensing cellular energy or nutrient status. AMPK can regularize cell growth by the way of pseudostarvation leading to autophagy. Thus AMPK has become a great target for drug design of tumor inhibition. Activation of AMPK followed its conformational change. We tried to find potential compounds that could bind to AMPK by virtual screening of the world's largest TCM Database ("}
+{"text": "This work investigates emissions sampling methods employed for qualitative identification of compounds in e-liquids and their resultant aerosols to assess what capture methods may be sufficient to identify harmful and potentially harmful constituents present. Three popular e-liquid flavors  were analyzed using qualitative gas chromatography-mass spectrometry (GC-MS) in the un-puffed state. Each liquid was also machine-puffed under realistic-use flow rate conditions and emissions were captured using two techniques: filter pads and methanol impingers. GC-MS analysis was conducted on the emissions captured using both techniques from all three e-liquids. The e-liquid GC-MS analysis resulted in positive identification of 13 compounds from the cinnamon flavor e-liquid, 31 from mango, and 19 from vanilla, including a number of compounds observed in all e-liquid experiments. Nineteen compounds were observed in emissions which were not present in the un-puffed e-liquid. Qualitative GC-MS analysis of the emissions samples identify compounds observed in all three samples: e-liquid, impinge, and filter pads, and each subset thereof. A limited number of compounds were observed in emissions captured with impingers, but were not observed in emissions captured using filter pads; a larger number of compounds were observed on emissions collected from the filter pads, but not those captured with impingers. It is demonstrated that sampling methods have different sampling efficiencies and some compounds might be missed using only one method. It is recommended to investigate filter pads, impingers, thermal desorption tubes, and solvent extraction resins to establish robust sampling methods for emissions testing of e-cigarette emissions. This work is built upon the premise that current protocols commonly employed for testing alternative tobacco products, such as e-liquids used as the consumable product in conjunction with electronic cigarettes (e-cigs) may be insufficient to qualitatively identify all harmful and potentially harmful constituents (HPHCs) present in the aerosols generated by vaporizing such e-liquids. Emissions test standards must be developed for the identification of HPHCs. We further assert that such protocols must involve testing both the un-puffed consumable  and the aerosols generated under realistic  puffing conditions. While robust protocols have been established for testing conventional cigarettes, uniform standards for testing e-cigs and water pipes have not been adopted, though several variations have been proposed . While tSampling techniques also vary among previously reported studies; including Cambridge filter pads ,4,6, EpiThis investigation compares two methods for sampling the aerosol emissions from electronic cigarettes (e-cigs) for subsequent analysis using gas chromatography-mass spectrometry (GC-MS). A qualitative GC-MS analysis was performed on e-liquid samples before they were puffed, as well as corresponding emissions that were captured during the puffing process using two methods: Cambridge filter pads and a series of two impingers. The study seeks to enhance the understanding of the relationship between emissions capture methods and the compounds qualitatively identified in the emissions relative to the compounds identified in the un-puffed liquid.v/v) in methanol (HPLC grade) and placed into 1 mL GC injection vials. These diluted samples were injected directly into the GC-MS .Three flavored e-liquid samples were used in this study, including Cinnamon, vanilla, and mango. Each flavor of e-liquid was analyzed (in the un-puffed form) using samples directly from the manufacturer\u2019s containers. Each sample was diluted 1:40  with the iClearTM X.I tank , set at 7 watts. The iClearTM X.I tank is a \u201cbottom\u201d coil-style tank, which designed to deliver e-liquid to a heating coil by through fibrous wicks. The first generation RIT PESTM-1 programmable emissions system  was programed to control each discrete puff with a duration of 3.5 s and a puff flow rate of 33.8 mL/s, representative of e-cig puffing behaviors measured in the natural environment. While prior studies of natural environment topography monitoring have demonstrated significant variation in puff duration and flow rate during the course of one- and two-week observation periods [Each e-liquid was individually puffed through the same reusable e-cig device; composed of an itaste periods ,17,18,19TM-1 system inlet port. Ten pads for each e-liquid sample were exposed to 100 puffs total (10 puffs per filter pad to avoid overloading pads) using the above puffing regime. Filter pads were stored in separate sealed glass jars prior to GC-MS analysis. All ten pads associated with each e-liquid flavor were combined in a in a container with 50 mL methanol. Each sample container was placed on an orbital shaker (200 rpm) for 24 h to break down the filter pad material, followed by a wrist shaker on high speed for 15 min and another 24 h orbital shaking to ensure breakdown. The resultant samples were filtered through a 0.45 \u00b5m regenerated cellulose syringe filter and concentrated using solvent blow-off (nitrogen gas) in a 4 mL graduated concentrator to a final volume of 1 mL or less and placed into GC vials. These samples were then directly injected in the GC-MS. The combining of filters and the concentrating the final solution to 1 mL was done so that compounds in trace amounts or with weak signals could be observed during analysis.The \u201cpad sample collection method\u201d employed 48 mm silica Cambridge filter pads  to capture emissions from the air flow path between the exit of e-cig and the PESThe \u201cimpinger sample collection method\u201d used two impingers in series to maximize the collection of emissions. Impingers were created from 250 mL graduated cylinders and extra coarse fretted disks which connected between the e-cig exit and the input to puffing machine. Each impinger was filled with 80 mL methanol and the impinger system was cooled in a bath of acetone in dry ice. Samples collected from the loaded impingers were concentrated using solvent blow-off (nitrogen gas) in a 4 mL graduated concentrator to a final volume of 1 mL and placed into GC vials, which were then directly injected into the GC-MS. A Shimadzu GC-MS-QP2020  was used and the method was optimized for our study from that of Hutzler et al. [Chromatogram peaks were qualitatively identified using the 2014 NIST database and only peaks with signal-to-noise ratios greater than 3 were analyzed. We used the mass spectra from the cinnamon, vanilla, and mango samples  and the 2014 NIST database to identify several molecules of interest that correlate with the carrier liquid and flavoring agents in E-cigarettes, as well as other molecules associated with the smoking of the e-liquid and potential contaminants. Only chromatographic peaks that could be positively identified and associated with e-liquid or emissions from e-cigarette smoking were included in this study. Positive identification was concluded if the mass spectra matched at a level of at least 85% positive match with the mass spectral library and having an appropriate relative retention, as determined by the retention index of each molecule. Most compounds had better than a 95% library match. A number of peaks were positively identified, but not included because they were determined not to come from the e-liquid or the generated aerosol. The source of other compounds was the GC inlet and column and from the sampling lines. Blank runs were used to determine peaks associated with the GC column and inlet. In addition, the blank runs displayed some peaks from the sampling setup that were not associated with the generated aerosol. Compounds that came from the sampling lines were determined if the peak intensity was identical or greater in the second impinger in series as that in the first impinger.It is worth noting that we also tried a setup in the puffing machine where the aerosol first traveled through a pad and then into the impinger. Using this set-up, the pad samples showed similar molecules to prior pad samples, but the impinger did not show many molecules of interest. It was determined that the serial setup (pads flowing into impingers) was not a viable system.Many of the molecules are observed in the e-liquid and each sampling as can be seen in 4 and 5 are both identified as glyceryl 1-monoacetate. There are a number of isomers with very similar structure that all provided a high degree of match within in the mass spectral database and likely one or both or one of the structurally-similar isomers. In addition, a peak at 18.4 min was identified as a long chain aldehyde and not definitively identified. There were a number of possible aldehydes the provided high degree of spectral match so the exact identity was not determined, however, all of the high library matches were linear aldehydes. Standards could be run to determine the identity of the aldehyde.Two peaks were identified as the same compounds, peaks While the origin of many of the compounds was straight forward, the origin of all of the sampled compounds was not exhaustively investigated. The goal of this analysis was not to identify the source of a given compound but rather to demonstrate that different sampling techniques preferentially sample different compounds. Diethyl phthalate is a known plasticizer and is probably a contaminant from the supplier; however, we cannot rule out sample processing contamination, which we feel is remote, as it is observed in all samples that were processed by different means. Regardless, as it is observed in all samples it is not of great interest to this study as we were interested in the differences between the sampling techniques.There were more compounds observed and identified from the mango e-liquid and the generated aerosols than from the vanilla e-liquid trials. Similar to what was observed with the vanilla trials, many of the compounds were observed in the e-liquids and from both sampling techniques. In all, seven compounds were observed in all three runs. Five different compounds were observed in the mango e-liquid and in the aerosols sampled by the filter pads and one compounds were observed in the e-liquid and in the aerosols sampled by the impinger, but not the filter pads. One compound was observed in the aerosols sampled by the impinger and the filter pads, four were observed only from the aerosols sampled by the impinger, eleven compounds were only observed in aerosols sampled by the filter pads, and two compound was observed only in the e-liquid. The identification of these compounds can be found in The chemical makeup of the flavor of mango is more complex than that of vanilla or cinnamon and consists of a combination of many compounds. Of the compounds found in mango flavor many were observed here including: hexyl hexanoate, methyl hexanoate, hexyl acetate, citonellol, \u03b3-decalactone, \u03b3-undecalactone, \u03b4-decalactone, and potentially others. Of these, hexyl acetate, \u03b3-decalactone, \u03b3-undecalactone, and \u03b4-decalactone were found in all three samples, citronellol and hexyl hexanoate was observed in the e-liquid and from the filter pad samples while methyl hexanoate was only observed from the filter pad sampling. Due to the fact that all but the methyl hexanoate were observed in the e-liquid it is assumed that methyl hexanoate was not in fact one of the flavors added. It should be highlighted that the impinge samples missed some of the flavor compounds that were included in the aerosol as indicated by the filter pad samples.4 and 5 are both labeled 1,2-propanediol, 2-acetate and peaks 11 and 13 are both labeled glyceryl 1-monoacetate. Similarly to the vanilla trial, a number of isomers with similar structure all provided high degree of match in the mass spectral database, and one or more of the peaks are actually isomers of the identified compound. Peaks 19, 20, and 21  were all assigned the same retention index by the mass spectral software. The retention indices are based off using a DB-1 column whereas we used a DB-17 column in this work so the retention index values will not be exact and can vary compound to compound. The fact that all three compounds elute within 15 s of each other provides an additional support to their identity.Two sets of peaks were identified as the same compound in the mango experiments. Peaks As with the vanilla flavor e-liquid samples, a comprehensive analysis of the origin of all emissions was not done as the source of each compound was not the focus of this analysis. Again, the plasticizer diethyl phthalate was observed in all samples and, therefore, of low interest to this study. There were a total of ten compounds only observed in the filter samples including a number of large alcohols, \u03c4-cadinol, \u03b1-cadinol, and 1-naphthol, as well as other large compounds, including octadecanal and 9-octadecanamide that we cannot identify the source of. While we cannot rule out contamination as the source, the fact that they are observed reproducibly in subsequent runs and that they are not observed from the other e-liquids indicates that they are associated with this sample.Many of the compounds identified were observed in all three samples. In total, six out of thirteen identified compounds were observed from the e-liquid, as well as both aerosol sampling techniques including the main components of the e-liquid propylene glycol and glycerol, nicotine, and the main flavor compound for cinnamon, cinnamaldehyde, as well as a small peak from ethyl vanillin, a compound commonly used to give a vanilla flavor. There were two compounds observed only from the aerosols sampled by the impinge, three compounds from the aerosols sampled by the filter pads, and two compounds observed only from the e-liquid.As with the other e-liquids, there are a number of compounds that were observe that we cannot determine their source. As was observed with the other two e-liquids, the plasticizer diethyl phthalate was observed in all three samples for the cinnamon e-liquid. In addition, another plasticizer, tributyl acetylcitrate, was observed only in the impinger samples. It was observed reproducibly, but we do not know the source and cannot rule out contamination. In addition, tributyl prop-1-ene-1,2,3-tricarboxylate was observed from the impinger samples, but the source cannot be determined at this time.Thirty-three compounds were positively identified across the range of flavors and sampling methods studied. At least four compounds  were posDevices such as the one chosen for this study have been reported as prone to overheating the e-liquid on the coil, causing thermal decomposition and various small aldehydes, among other decomposition products that are not originally in the e-liquid. We do not believe the elements were overheating during the experiments conducted here for several reasons, including that each flavor e-liquid had its own heating element, each unit was cleaned thoroughly between trials, each tank/heating element was brand new when used, the heating elements were operated at low power , and each condition was run in duplicate or triplicate trials, and we reported outcomes only when the same results were observed (the same compounds in the same ratios) each time. We believe each trial was conducted well within the normal operating range of the device, and avoided operating the device at extremes of puff flow rate, duration, volume, and operating power in order to focus attention on the sample capture method. In addition, the compounds that would be observed due to the thermal decomposition of the e-liquid  include formaldehyde, acetaldehyde, acrolein, benzaldehyde, were not sampled for or detected, and would likely be found in the gas phase, not the particulate phase.To our knowledge, no other publication has presented similar data documenting the impact of capture system on the accuracy of HPHC identification of ENDS aerosols. Our results demonstrate the sampling method to be an important factor in proper identification of all HPHCs present in emissions from such products. Robust methods for emissions sampling are needed by the tobacco regulatory science (TRS) community, to positively identify HPHCs for toxicology studies, and inform regulation of tobacco product characteristics.Further investigation is needed to determine optimal aerosol emissions sampling methods to ensure a comprehensive identification of the molecules present in emissions from alternative tobacco product consumables  as a function of user behavior and device product characteristics . Each sampling method had advantages and disadvantages related to logistics, including sample preparation and total processing time. Certain collection methods are well suited for high flow rates, while others require more moderate rates; sampling methods have varying capture efficiencies which depend on the class of molecule(s). This study provides the foundation for future analysis, to determine which categories of molecules in aerosol emissions may be prone to capture by either the pad, impinge, or other method as a function of molecular weight, vapor pressure, or other factors.The qualitative results herein reporting presence, but not amounts, of various components of e-liquids or their aerosols gives little insight into the reasons for differing results arising from variation in the method of sample capture. However, demonstrating that the presence of compounds identified is dependent upon the sample capture method strongly supports our premise that the sample capture method is an important aspect of rigorous e-cig emissions experimental design.When using the filter pad capture method, each GC-MS qualitative analysis trial was conducted on ten pads, with each pad exposed to ten puffs. While this provided an opportunity for repeated trials across ten pads, we chose to combine all ten pads in order to concentrate the emissions products and obtain a better signal and qualitatively observe more species. The primary outcome of interest to the current study is whether the species identified with pad capture are the same as the species identified with impinger capture, resulting in a binary decision whether each compound is present or not (1 or 0). In this type of experiment design, repeated trials enhance the statistical validity of the binary comparison.Additional work is needed in the research community to establish robust emissions testing protocols which are accepted as valid by all interested parties. This study focuses on the methods employed to capture emissions samples, which is the first step in every emissions testing and sample analysis protocol. Additional elements of a robust emissions testing protocol require not only a robust sample capture method, but also require study of emissions across a range of flow conditions, devices settings, and operating conditions (such as power) which represent both the intended use of the device, as well as unintended, but observed misuse of the device.The results presented here conclusively demonstrate that molecules observed in qualitative GC-MS analysis of e-cig aerosol emissions are dependent upon the capture method employed, particularly for those molecules which are generated in the heating and aerosolization. Qualitative GC-MS analysis of the emissions samples identify compounds observed in all three samples: e-liquid, impinge, and filter pads and each subset thereof. A limited number of compounds were observed in emissions captured with impingers but were not observed in emissions captured using filter pads; a larger number of compounds were observed on emissions collected from the filter pads, but not those captured with impingers. Nineteen compounds were positively identified in the e-cig emissions (three using impinger samples and 16 using filter pad samples) which were not present in the un-puffed e-liquid. It is demonstrated that sampling methods have different sampling efficiencies and some compounds might be missed using only one method. It is recommended to investigate filter pads, impingers, thermal desorption tubes, and solvent extraction resins to establish robust sampling methods for emissions testing of e-cigarette emissions."}
+{"text": "The continuing growth of the ethanol industry has generated large amounts of various distillers grains co-products. These are characterized by a wide variation in chemical composition and ruminal degradability. Therefore, their precise formulation in the ruminant diet requires the systematic evaluation of their degradation profiles in the rumen.in situ trial to determine the degradation kinetics of the dry matter (DM) and crude protein (CP). Soybean meal (SBM), a feed with highly degradable protein in the rumen, was included as the fourth feed. The four feeds were incubated in duplicate at each time point in the rumen of three ruminally cannulated Hanwoo cattle for 1, 2, 4, 6, 8, 12, 24, and 48\u00a0h.Three distillers grains plus soluble co-products (DDGS) namely, corn DDGS, high-protein corn DDGS (HP-DDGS), and wheat DDGS, were subjected to an P\u2009<\u20090.001) and an undegradable C fraction . The filterable and soluble A fraction of CP was greatest with wheat DDGS, intermediate with corn DDGS, and lowest with HP-DDGS and SBM; however, the undegradable C fraction of CP was the greatest with HP-DDGS (41.2\u00a0%), intermediate with corn DDGS (2.7\u00a0%), and lowest with wheat DDGS and SMB (average 4.3\u00a0%). The degradation rate of degradable B fraction (%\u00a0h\u22121) was ranked from highest to lowest as follows for 1) DM: SBM (13.3), wheat DDGS (9.1), and corn DDGS and HP-DDGS (average 5.2); 2) CP: SBM (17.6), wheat DDGS (11.6), and corn DDGS and HP-DDGS (average 4.4). The in situ effective degradability of CP, assuming a passage rate of 0.06\u00a0h\u22121, was the highest (P\u2009<\u20090.001) for SBM (73.9\u00a0%) and wheat DDGS (71.2\u00a0%), intermediate for corn DDGS (42.5\u00a0%), and the lowest for HP-DDGS (28.6\u00a0%), which suggests that corn DDGS and HP-DDGS are a good source of undegraded intake protein for ruminants.Wheat DDGS had the highest filterable and soluble A fraction of its DM (37.2\u00a0%), but the lowest degradable B (49.5\u00a0%; This study provided a comparative estimate of ruminal DM and CP degradation characteristics for three DDGS co-products and SBM, which might be useful for their inclusion in the diet according to the ruminally undegraded to degraded intake protein ratio. Dried distillers grains with solubles (DDGS), generated after the fermentation and distillation in a grain-based ethanol production , are an in situ ruminal fractions, ruminal disappearance rate, and effective degradability (ED) of DM and CP in corn, high-protein corn, and wheat DDGS with those of SBM.The rapidly expanding ethanol industry will contribute significantly to an increased supply of DDGS at a competitive cost , 6. HoweIn an attempt to collect representative samples, three different batches of each sample, on three different days throughout the months of July and August 2014, were obtained through Egreen Co. . Samples of HP-DDGS and corn DDGS were originated from ethanol plants in Valero\u2019s ethanol plant in Jefferson, Wisconsin (USA) and wheat DDGS was originated from bioethanol plants located in western Canada. However, the detailed information of the DDGS co-products, including the processing conditions at the ethanol production plant, were not available for this experiment owing to the difficulty in tracing detailed international information in a practical manner.Subsamples of feeds were mixed thoroughly and hammer-milled  to pass through a 1-mm sieve, prior to being analyzed  for contin situ degradability were described earlier   . The undIn situ data for each feed were a mean of 12 observations, which were obtained over the course of two consecutive runs at two different days. The experimental design was 2 consecutive incubations\u2009\u00d7\u20094 feeds\u2009\u00d7\u20093 animal replicates\u2009\u00d7\u20092 sample replicates, giving a total of 48 observations. The data were analyzed using the PROC MIXED of SAS , where feed sources were considered fixed effects and incubation run in the rumen was assumed to be random effect. The animal data were averaged prior to statistical analysis. The model used for the analysis was: Yij\u2009=\u2009\u03bc\u2009+\u2009Fi\u2009+\u2009Rj\u2009+\u2009eij, where, Yij\u2009=\u2009the observation of the dependent variable ij; \u03bc\u2009=\u2009the overall mean of Y; Fi\u2009=\u2009the effect of feed (i\u2009=\u20094), R\u2009=\u2009the effect of incubation run as replications (j\u2009=\u20092), and eij\u2009=\u2009the random error associated with the observation ij. Mean separation was performed using the Tukey\u2019s multiple range test at 5\u00a0% significance level.The chemical composition of the DDGS co-products and SBM is presented in Table\u00a0P\u2009<\u20090.001), whereas the mean of the degradable B fraction of DM ranged from 49.5 to 69.0\u00a0% and was the lowest with corn and wheat DDGS, intermediate with HP-DDGS and the highest with SBM (P\u2009<\u20090.001). The mean values of the C fraction, or ruminally undegradable DM, was highest for HP-DDGS, followed by corn DDGS, and then wheat DDGS and SBM (P\u2009<\u20090.001).The mean DM degradation variables across the feeds are presented in Table\u00a0P\u2009<\u20090.001). The highest and lowest degradable B fraction of CP was recorded for SBM (87.2\u00a0%) and HP-DDGS (49.8\u00a0%), respectively. The range of the C fraction for CP was from 4.7\u00a0% for wheat DDGS to 41.2\u00a0% for HP-DDGS.Mean values of ruminal CP degradation variables are presented in Table\u00a0\u22121  and 3.9 to 17.6\u00a0%\u00a0h\u22121 , respectively, with the lowest rate for corn and HP-DDGS being recorded and the highest rate for SBM , and then SBM and wheat DDGS (72.6\u00a0%) (Table\u00a0The ED of DM was higher for wheat DDGS and SBM than for corn DDG and HP-DDGS Table\u00a0. The ED %) Table\u00a0.Table 3P\u2009<\u20090.001) and higher rates of disappearance resulting from HP-DDGS and SBM, or wheat DDGS, respectively.As a function of residence time in the rumen, the amount of DM and CP disappearance for the test feeds is presented in Fig.\u00a0vs. 31.4\u00a0%, and similar NDF, 34.7 and 37.5\u00a0%, respectively.The large difference in CP content across the 4 feeds was comparable to those mentioned in published reports \u201320. The Among the different types of DDGS co-products, HP-DDGS is generally recognized for its reduced concentration of EE, ADF, and NDF, because during its production process much of the fiber is removed in de-hulling . HoweverKd of the B fraction was much lower for wheat DDGS (2.7\u00a0%\u00a0h\u22121) than for corn DDGS (7.2\u00a0%\u00a0h\u22121). The range of fraction A in the DDGS co-products  is in contrast to the values reported by Kleinschmit et al. , and the higher degradable B fraction of DDGS co-products .The higher ED of DM for wheat DDGS and SBM than for corn DDG and HP-DDGS might be partially explained by its high soluble fraction [28.4\u00a0% and 37.2\u00a0% of DM for wheat DDGS and SBM \u22121, ranged from 26.1\u00a0% for SMB to 71.4\u00a0% for HP-DDGS, which indicates that HP-DDGS is a good source of UIP for ruminants. This compares with previous studies reporting that UIP constituted approximately 55.2\u00a0% of CP in HP-DDGS [KpB\u2009=\u20090.06, h\u22121, was estimated to be 57.5\u00a0%, which is comparable to the value (54.9\u00a0%) reported in the National Research Council\u2019s (NRC) Nutrient Requirements of Beef Cattle [The estimated UIP, assuming the passage rate of 0.06\u00a0h HP-DDGS  and 55\u00a0% HP-DDGS . The avef Cattle .KpB\u2009=\u20090.06\u00a0h\u22121) differed from the results reported by Mjoun et al. [KpB\u2009=\u20090.06\u00a0h\u22121): 26.1 vs. 32.3\u00a0% of CP for SBM, 57.5 vs. 52.3\u00a0% of CP for corn DDGS, and 71.4 vs. 54.5\u00a0% of CP for HP-DDGS, in the present study vs. Mjoun et al. [KpB\u2009=\u20090.06\u00a0h\u22121, is considerably higher for wheat DDGS as compared to this experiment (a 22-percentage-unit difference), however the value reported for corn DDGS was comparable to the current observation.The amount of UIP in this study , which could be due to much of their protein being heat-denatured yeast that was heated during the distillation and concentration process . This reEndosperm is mainly composed of zein protein, which is known to be resistant to ruminal degradation , 35. DurTo meet the requirements for metabolizable protein, and minimize N excretion, the dietary protein must be divided into DIP and UIP fractions, which requires a precise estimation of protein degradation in the rumen for the feed ingredients included in the diet . TherefoThe variations observed in the results of this study compared with those reported in the literature might be explained in part by differences in feed particle size or laboratory-to-laboratory variations in analytical procedures , 36. MorThe ruminal CP degradation of the DDGS co-products varied considerably in the present study, which could have been caused by variations in both the quality of the DDGS co-products and the production technology of the individual processing plants. This study showed that the observed UIP content (% of CP) was as follows: HP-DDGS (71.4\u00a0%)\u2009>\u2009corn DDGS (57.5\u00a0%)\u2009>\u2009wheat DDGS (28.8\u00a0%)\u2009>\u2009soybean meal (26.1\u00a0%), showing that HP-DDGS and corn DDGS are a good source of UIP. This information can be used as basic data for a more accurate formulation of rations for beef and dairy cattle.ADF, acid-detergent fiber; CP, crude protein; DDGS, dried distillers grains plus solubles; DIP, degraded intake protein; DM, dry matter; ED, effective ruminal degradability; EE, ether extract; HP-DDGS, high-protein distillers grains plus solubles; NDF, neutral-detergent fiber; NPN, non-protein nitrogen; SBM, soybean meal; UIP, undegraded intake protein"}
+{"text": "Salvia miltiorrhiza Bunge (Danshen). In the present study, a systematic method was developed to simultaneously isolate and purify those compounds using macroporous adsorption resins and semi-preparative HPLC with a dynamic axial compress (DAC) system. The Danshen extract  was divided into three fractions using different concentrations of alcohol  on D101 column. The content of total tanshinones of 90% alcohol eluent (TTS) was over 97%. Furthermore, the anti-inflammatory effects of those samples were investigated on LPS-stimulated RAW264.7 cells and three animal models. The results showed that the anti-inflammatory effect of TTS in vitro was superior to the one of any other sample including 0% and 45% eluent, and total tanshinones capsules. In addition, TTS exhibited a stronger anti-inflammatory effect than that of dihydrotanshinone, tanshinone IIA, cryptotanshinone, and tanshinone I, respectively. For animal models, TTS could significantly suppress xylene-induced ear oedema and rescue LPS-induced septic death and acute kidney injury in mice. In summary, the separation process developed in the study was high-efficiency, economic, and low-contamination, which was fit to industrial producing. TTS is a potential agent for the treatment of inflammatory diseases.Dihydrotanshinone, tanshinone I, cryptotanshinone, and tanshinone IIA are major lipid-soluble constituents isolated from  However, durable inflammation has more often than not lead to the inflammatory diseases, such as sepsis, endotoxemia, asthma and inflammatory bowel disease (IBD), etc.3. Specifically, sepsis is largely induced by the hyper-inflammatory responses, which involves the initiation and amplification of the innate immune system and cytokines release like TNF-\u03b1, IL-1\u03b2, IL-6 etc.4. As it stands now, there is no an effective drug with fewer side effects in the clinic to rescue septic death. Therefore, it is imperative to explore a more effective and less side effect drug for the treatment of inflammatory diseases.The inflammation is always triggered by damage to organisms, which plays a defensive role in injury or infection6. Furthermore, Danshen, as a dietary supplement, is the first traditional Chinese medicine that is documented in USP 37-NF32. Tanshinones are mainly lipophilic active constituents isolated from the root of Danshen. As it stands now, more than 40 tanshinones have been isolated and identified7. Of these tanshinones, dihydrotanshinone (DTAN), tanshinone I (TANI), cryptotanshinone (CTAN), and tanshinone IIA (TANA) are major diterpenes in Danshen9. Following the traditional application, the cardiovascular protective effect of tanshinones has been widely investigated10. The anti-cancer effect of tanshinones has drawn attentions of many researchers in recent years8. Specifically, the four compounds were always chosen as target representing Danshen to investigate its anti-inflammatory activity12. In China, total tanshinones capsules (TTC) that were just prepared by 95% alcohol extract were employed as an anti-inflammatory medicine in the market (Z13020110). However, the anti-inflammatory effect of TTC was not satisfactory due to its crude preparation. Therefore, the innovative separation and effective methods to prepare Danshen samples or obtain pure compounds from raw Danshen extract are indispensable for the research and development of Danshen products.Danshen, one of the most popular traditional Chinese medicines in Asian countries, has been used extensively for the treatment of cardiovascular diseases, cerebrovascular diseases, and various inflammatory diseases13. In addition, the advantages of macroporous adsorption resins (MARs), such as the ideal pore structure, unique adsorption properties, less solvent consumption, affable environmental management and easy regeneration make them priority for large-scale production industrially16. Therefore, it is the vital part to select optimal MARs for the enrichment of target constituents.The conventional ways to separate tanshinones were performed on extraction, multi-step open column chromatography with silicon gels, and semi-preparative HPLC. However, these assays did not fit the bill of the large-scale industrial production due to the onerous work and great consumption of solvents. Recently, macroporous adsorption resins were widely used to separate and enrich diverse compounds in industrial production for their high efficiency and low-cost qualityetc. were separated and purified using DAC columns with reversed-phase ODS19. However, as it stands now, there are no related studies to explore for purifying tanshinones using DAC columns with reversed-phase ODS. In this study, an effective preparative method was developed to simultaneously isolate and purify DTAN, TANI, CTAN, and TANA  has been developed fast in industrial separation due to its stable performance, high efficiency, and good repeatability for the large-scale production. A deluge of compounds such as pulchinenoside B4 and B5, saikosaponins A, B, and C, zopiclone, 13. For the four tanshinones, non-polar resins are more applicable to adsorption of them. In this study, seven MARs , and desorbed by gradient elution with 15%, 30%, 45%, 60%, 75%, 90%, and 100% ethanol (6 BV) at a flow rate of 4 BV/h. As shown in Fig.\u00a0The breakthrough point refers that the adsorbate concentration of eluent reaches 5% of inlet concentration. The results of leakage curve on D101 were obtained Fig.\u00a0 in termsThe adsorption kinetics curve on D101 was shown in Fig.\u00a0Equilibrium adsorption isotherms on D101 were investigated at room temperature 25\u2009\u00b0C using different concentrations of crude extract. During the dynamic adsorption, the feeding concentration and feeding volume were critical for the analytes loss. As shown in Fig.\u00a0On the basis of optimal conditions aforementioned, like the crude extract 1.8\u2009g/mL, the resin D101, the feeding sample volume 1.4 BV, the adsorption time 150\u2009min, the large-scale enrichment of the target compounds was performed on enlarged glass column. The gradient elute solution was set as 0%, 45%, and 90%, respectively. The bed volume, the volume of elute solution, and the total feeding amount of raw Danshen extract Fig.\u00a0 were enlIn the process of preparative purification, the different elution procedures were applied to obtain DTAN, TANI, CTAN, and TANA Fig.\u00a0. Finally20. LPS-induced NO production in RAW264.7 macrophages has been considered as the convenient and credible method for anti-inflammation screening21. The Griess assay for the determination of nitrite resulted from the reaction of NO with O2 was used as an effective and efficient method to quantitate NO production23. Therefore, these compounds were primarily screened with an LPS-stimulated RAW264.7 macrophage cell model for their anti-inflammatory activities. As shown in the results, TTS exhibited significant inhibitory effect on nitrite production and NO level, , NO becomes an inflammometer to modulate important cellular signaling involved in immunity and inflammationel, Fig.\u00a0. The inhANI Fig.\u00a0, which sANI Fig.\u00a0. Combinaity Figs\u00a0 and S1.F24. In terms of the advantages of xylene-induced ear oedema in mice, the anti-inflammatory model is always employed to evaluate the antiphlogistic effect for drug screenings of TTS24. In our study, TTS significantly inhibited xylene-induced ear oedema  is an abrupt or rapid loss of kidney functionvel Fig.\u00a0. In the vel Fig.\u00a0. In LPS-vel Fig.\u00a0. TTS prevel Fig.\u00a0. DEX wasvel Fig.\u00a0.Figure 7Salvia miltiorrhiza Bunge was established. The results of static adsorption/desorption and dynamic separating experiments indicated that D101 resin was superior to other six resins investigated for separating tanshinones. Further static and dynamic desorption/desorption experiments on D101 were performed to obtained optimal parameters. The further process was developed by preparative reversed-phase HPLC with a DAC column to obtain pure dihyrotanshinone, tanshinone I, cryptanshinone, and tanshinone IIA. In terms of these results, the established method was highly efficient, relatively economic, and environmentally protective, which exhibited good potential for large-scale production of these compounds for functional food and pharmaceutical application. Furthermore, TTS exhibited a significant anti-inflammatory activity in vitro and in vivo, which was superior to DTAN, CTAN, TANA, TANI, and TTC, respectively. Specifically, for three animal models, TTS significantly demonstrated to suppress xylene-induced mice ear oedema, rescue LPS-induced septic death, and reverse LPS-induced AKI. Therefore, TTS prepared from 95% alcohol extract of raw Danshen displays giant value for further research as an anti-inflammatory agent to substitute TTC in the market.In summary, a method for simultaneous purification of DTAN, TANI, CTAN, and TANA from Escherichia coli, serotype 0111:B4), Griess reagent, Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies/Gibco Laboratories . ELISA kits for IL-6, IL-1\u03b2, and TNF-\u03b1 were purchased from Neobioscience . Propitious amounts of standards were dissolved in methanol to be used as stock solutions at the concentrations of 2.34\u2009mg/mL for DTAN, 2.43\u2009mg/mL for TANI, 3.37\u2009mg/mL for CTAN and 3.67\u2009mg/mL for TANA, respectively. Ethanol  and methanol (HPLC grade) were purchased from Shanghai Chemical Reagents Ltd. .DTAN (>98%), TANI (>98%), CTAN (>98%), and TANA (>98%) purchased from Shun Bo Biological Engineering Technology Co., Ltd.  were determined by HPLC. Tanshinones capsules (TTC) were purchased from Hebei Xinglong Xili pharmaceutical co. Ltd. . LPS . AB-8 was obtained from Tianjin Nankai Hecheng S&T Co. Ltd . The specifications of these MARs were summarized in Table\u00a0The roots of Danshen were collected from Bozhou, Anhui Province, China. Its botanical origin was authenticated by Prof. Xiaoran Li in Soochow University , where the voucher specimen was deposited. The roots (1\u2009kg) were powdered, and then refluxed with 95% ethanol at the ratio 1:10  for 2\u2009h, repeating two times. The extracted liquids were pooled, filtered, and then concentrated by rotary evaporator under vacuum to remove completely the ethanol solvent. The crude extract was then diluted with deionized water at the ratio of raw material and extract to be 1.0\u2009g/mL.HPLC analysis was performed on Shimadzu Prominence LC-20A liquid chromatographic system  equipped with binary pumps, a PDA detector, and LC solution software. The Waters RP-C18 column  was employed at a column temperature of 30\u2009\u00b0C. The mobile phase consisted of methanol (A) and water  with a flow of 1\u2009mL/min using the subsequent gradient elution: 0\u201345\u2009min, 55\u201390% A. The injection volume was 20\u2009\u03bcL and the absorbance wavelength was selected at 254\u2009nm.34. The subsequent equations were used to quantify the capacities of adsorption and desorption as well as the ratios of desorption.All MARs were screened through static adsorption/desorption ratio experiments, which were performed as follows: five aliquots of 25\u2009mL sample solutions prepared as the aforementioned (one-fold dilution of stock solution) were put into 100\u2009mL flasks comprised the same amounts of various hydrated resins . The tightly sealed flasks were shaken (160\u2009rpm) for 6\u2009h at 25\u2009\u00b0C to reach adsorption equilibrium. The solutions were filtrated through 0.45\u2009\u03bcm minipore filter and detected by HPLC. Subsequently, the resins were washed with deionized water for 3 times and desorbed with 30\u2009mL 95% ethanol. Then the flasks were shaken (160\u2009rpm) for 6\u2009h at 25\u2009\u00b0C. The desorbed solutions were analyzed by HPLC. The experiments were repeated in three times. The candidate resins were screened by their properties of absorption/desorption ratiose indicates the adsorption capacity at adsorption equilibrium (mg/g dry resin); C0 and Ce are the initial and equilibrium concentrations of solutes in the solutions, respectively (mg/mL); Vi is the volume of the initial sample solution (mL); W is the dry weight of the tested resins (g).Adsorption evaluation:d is the desorption capacity after adsorption equilibrium (mg/g dry resin); Cd is the concentration of solutes in the desorption solution (mg/mL); Vd is the volume of the desorption solution (mL); D is the desorption ratio (%); C0, Ce, Vi, and W are the same as formula (1).Desorption evaluation:Dynamic adsorption and desorption experiments were performed on the open glass columns (30\u2009\u00d7\u20092.0\u2009cm i.d.) loaded with pretreated hydrated selected resins (D101 and HPD100). The 5\u2009mL stock solutions (the concentration was 1\u2009g/mL) prepared in section 2.3 were loaded on the various columns. Subsequently, the static adsorption in the glass columns was overnight to reach the adsorption equilibrium. For the elution process, the deionized water, 15%, 30%, 45%, 60%, 70%, 80%, 90%, and 100% were employed sequentially to load into the columns for 6 bed volumes (BV) with eluent rate of 2.0\u2009mL/min. The eluents were condensed by rotary evaporator and detected by HPLC. To investigate dynamic leakage curves experiments, the prepared samples were through the column loaded D101 at the flow rate of 2.0\u2009mL/min. After finishing loading sample, turn off the column till complete adsorption equilibrium. The concentration of the four compounds was detected by HPLC.35. The tightly sealed flask was shaken (160\u2009rpm) for 12\u2009h at 25\u2009\u00b0C. Then the concentrations of DTAN, TANI, CTAN, and TANA in the adsorption solution was analyzed by HPLC at certain time intervals .The adsorption kinetics curves of DTAN, TANI, CTAN, and TANA on the selected D101 resin were further researched as the following process: adding 30\u2009mL sample solution prepared as 2.3 (one-fold dilution of stock solution) into a 100\u2009mL flask comprised the same amount of selected hydrated resin 36. Briefly, adding various concentrations of solutions to a 100\u2009mL flask with the same amounts of hydrated resins . The tightly sealed flasks were shaken (160\u2009rpm) for 12\u2009h at 25\u2009\u00b0C. The adsorption solutions were analyzed by HPLC. The concentration of the loaded sample was from 0.2\u2009g/ml to 2.6\u2009g/mL.The adsorption isotherms of DTAN, TANI, CTAN, and TANA on the selected resin were investigated. The assay used was previously reportedScale-up separation was conducted by around 200 fold as that of lab conditions. D101 resin  was packed in a glass column with a Bed Volume (BV) of 20.0\u2009L (150\u2009\u00d7\u200910\u2009cm i.d.). Then 5.0\u2009L of aqueous sample solution was subjected to the column. After sample loading and adsorption equilibrium, desorption was performed smoothly with 7 BV of water, 6 BV of 45% ethanol, and 6 BV of 90% ethanol at a flow rate of 6.5\u2009L/h. The 90% ethanol effluent was dried with rotary evaporation machine. The last samples were also analyzed by HPLC to determine the contents and the recoveries of the four target compounds.The preparative HPLC was performed on a DAC column system  filled with Duke ODS gel . The column was extensively flushed with pure methanol before use. The process of preparative separation was conducted as follows: The DAC column was pre-equilibrated with 70% methanol. The 90% ethanol sample was dissolved in methanol (150\u2009mL), then filtered with 0.45\u2009\u03bcm micro membrane filter, and then loaded to the column at a flow rate of 100\u2009mL/min. The column was washed with 80% methanol (60\u2009min) to get DTAN, TANI, CTAN, and TANA in sequence. The eluting experiments were conducted at a flow rate of 300\u2009mL/min and monitored at a UV wavelength of 254\u2009nm at room temperature. The indicated peaks were collected and analyzed by HPLC to determine the contents and the recoveries of target compounds.2 in an incubator.RAW264.7 macrophages purchased from Cell Bank of the Chinese Academy of Sciences  were cultured in DMEM with 10% FBS. Cells were maintained at 37\u2009\u00b0C under a humidified atmosphere of 5% CO5 cells/well. Subsequently, 0% and 45% alcohol eluent, TTS, and TTC  were employed for co-culture with cells for 24\u2009h, and the cytotoxicity was determined using MTT assay as previously reported6.RAW264.7 cells were planted into 96-well plate at a density of 105/well in 24-well plates overnight. After pretreatment with indicated compounds  for 1\u2009h, the cells were co-cultured with LPS (1\u2009\u03bcg/mL) for 24 or 6\u2009h. The nitrite in culture media treatment for 24\u2009h was investigated by Griess reagent. The nitric oxide (NO) of cells treated with 6\u2009h were stained by DAF-FM diacetate and determined by the flow cytometry  using the FITC channel.RAW264.7 cells were cultured at a density of 5\u2009\u00d7\u2009105/well in 24-well plates overnight. Cells were pretreated with TTS for 1\u2009h and then stimulated with/without LPS (1\u2009\u03bcg/mL) for 16\u2009h. According to manufacturer\u2019s instruction, the ELISA assay was employed to detect levels of TNF-\u03b1 and IL-6 in the culture medium.RAW264.7 cells were cultured at a density of 5\u2009\u00d7\u200910BALB/c mice  were obtained from the Experimental Animal Center of Soochow University. All mice were reared in plastic cages with food and water under standard conditions (SPF) and air filtration . The study was in accordance with the Local Guide for the Care and Use of Laboratory Animals of Soochow University and was approved by the university\u2019s Ethics Committee of Experimental Animal Center of Soochow University (No. IACUC2016-21).37, BALB/C mice were administered with TTS . 12\u2009h later, 30\u2009\u03bcL xylene was applied to the posterior and anterior surfaces of the right ear of mice. The left ear was viewed as a control group. One hour later, mice were euthanized and two ear punches  were collected and weighted. The indicator of the edema was presented by the increase in the weight of right ear punch comparing with the left ear.For xylene-induced mice ear oedema model38, BALB/C mice were obtained from Experimental Animal Center of Soochow University. Sepsis was performed in mice by i.p. injection of LPS (20\u2009mg/kg), TTC  and TTS  was pretreated before LPS injection. The survival rate of mice was investigated in 7 days. The study was performed in accordance with the Local Guide for the Care and Use of Laboratory Animals of the Soochow University.For septic shock model29, Mice were randomly divided into six groups: control group , TTS , TTC , LPS group  and positive control dexamethasone (i.v.). 2\u2009h before LPS injection, TTS  and TTC  were administered mice. After 12\u2009h, blood samples were collected via retro-orbital route under anesthesia and cytokines  were examined by mouse ELISA kits. The levels of blood urea nitrogen and creatinine in serum were determined by Roche Modular P800 . Their kidneys were collected for further research.For the AKI modelThe ear or kidney tissues harvested were fixed in 10% formaldehyde. Then, the tissues were dehydrated in a series of alcohol, embedded in paraffin, and sliced. The sections were stained with hematoxylin and eosin (H&E) stain. The pathological changes of ear or kidney tissues were observed under a light microscope.*p\u2009<\u20090.05.All results were presented as means\u2009\u00b1\u2009SD. For statistical analysis, the significance of the intergroup differences was analyzed with one-way ANOVA using GraphPad Prism 6.0 software. Statistically significant difference was defined as All the detailed data and materials are available from the corresponding authors Yulin Feng or Hongzhen Tang.Supplementary Information"}
+{"text": "There is concern about recent increase and severity of sports-related injuries in children. Despite the benefits of sports participation, injuries may carry long-term health consequences. We aimed to evaluate the prevalence, characteristics and types of hospitalized sports-related injuries in children.Population-based study of all acute sports-related injuries requiring hospitalization in children 5 to 15\u00a0years of age in New South Wales (NSW), Australia, 2005\u20132013. Health information was obtained from the NSW Admitted Patient Data Collection, a census of all hospital admissions from public and private hospitals. Children with a recorded ICD10-AM injury code (S00-T79) and sport-related activity code (U50-U70) were included. Prevalence and trend in injuries by age group, sporting code, body region affected and type of injury were assessed.There was a total of 20,034 hospitalizations for sports-related injuries , involving 21,346 recorded injuries in 19,576 children. The overall population hospitalization period prevalence was 227 per 100,000 children aged 5\u201315\u00a0years in 2005\u20132013, remaining stable over time . Football codes such as rugby league/union and soccer combined represented nearly two thirds of the total (60%). The most common body regions affected were the forearm (31%) head (15%) and hand injuries (13%). Fractures accounted for 65% of injuries followed by dislocations (10%) and traumatic brain injury (10%). Compared to other age groups, children aged 5\u20138\u00a0years had double the proportion of shoulder (15% vs. 7%) while 13\u201315\u00a0year olds had higher proportion of lower-leg (14% vs. 8%) and knee (6% vs.2%) injuries. One in seven injuries sustained while playing rugby league/union, baseball and hockey were traumatic brain injuries. A total of 444 (2.2%) of children had more than one hospitalization for sports-related injuries.On average, six children were hospitalized every day for sports-related injuries in the last decade with trends remaining stable. The most common sports involved were football codes, one in three injuries involved the forearm and two thirds were fractures. These findings can be used to inform health policy and sporting governing bodies to target preventive interventions and promote safe sports participation in children.The online version of this article (10.1186/s40621-018-0175-6) contains supplementary material, which is available to authorized users. Sports participation has multiple benefits on children\u2019s psychological, physical and social capabilities , Australia, between 2005 and 2013 inclusive. Health information was obtained from the NSW Admitted Patient Data Collection (APDC). The APDC is a census of all in-patient hospital admissions from public and private hospitals that includes patient\u2019s demographic information and all diagnosis and procedures for each admission. In the APDC, diagnoses are coded according to the 10th revision of the International Classification of Diseases, Australian Modification (ICD-10-AM) and procedures coded using the Australian Classification of Health Interventions (ACHI).Injuries sustained while playing sports or during active recreation were identified using the ICD-10-AM external causes of morbidity and mortality or activity codes (U50-U71) that ascertain the relevant causal sport for injury-related hospital admissions. Some sports with similar features such as rugby league/rugby union and martial arts/wrestling were combined. We excluded injuries sustained while involved in wheeled recreation activities , using playground equipment , motorized land, water or aero sports, firearm shooting sports and those caused by anaphylactic shock or by animal encounters (e.g. dog bites or sharks) because they were unlikely to occur in an organized sporting activity.Injuries were defined by body region  and type of injury , ocular, internal organ, foreign body and drowning) following the Australian Sports Injury Data Dictionary  and patient residence . Patient residence was categorized according to the Australian Bureau of Statistics (ABS) Statistical Geography Standard using patient residential postcodes  with 95% CI) to evaluate changes in population prevalence overtime, including the log of the population as offset and stratified by age groups. We also calculated proportions of hospitalizations by location of occurrence and day of the week out of total sports-related hospitalizations. The proportion of hospitalizations with severe injuries by sporting code was also calculated. As some hospitalizations included multiple injuries we used total injuries to calculate the proportion of injuries by sporting code, age groups, body region affected, type of injury and subsequent injuries. Injuries occurring in different body regions were counted separately. Rates of subsequent hospitalized sports related-injuries were evaluated by the sporting code of the initial admission, body region and type of injury. All analyses were conducted using SAS, 9.4 .There was a total of 20,034 hospitalizations for sports-related injuries in NSW in 2005\u20132013, representing 2.7% of all hospitalizations in children aged 5\u201315\u00a0years. Hospitalizations included 21,346 injuries in 19,576 children. Multiple injuries were recorded in 1210 (6%) hospitalizations, with a median (range) of 2 (2\u20136). The child characteristics of all hospitalizations are presented in Table\u00a0n\u2009=\u2009109) to 2.0% for netball (n\u2009=\u2009852)  Fig.\u00a0. There wWhen evaluating total injuries overall, one in three injuries affected the forearm 31%), followed by the head (15%), hand (13%) and lower-leg 12%)  of children had a subsequent hospitalization for sports-related injuries, distinct to the initial admission. After evaluating subsequent sports-related hospitalizations by the initial sporting code, body region or type of injury, only a small proportion had subsequent hospitalizations with little difference across groups , importantly, severe injuries requiring lengthy hospital stay (three or more days) were not infrequent and as high as one\u00a0in seven in some sports.We found a stable prevalence of hospitalizations for sports-related injuries in the last decade; however, hospitalizations may represent the more severe spectrum of injuries and timeline assessment of total sports injuries may provide different results depending on where they were treated and the local healthcare system. In Australia, a recent study from the state of Victoria using statewide hospitalization data found that sports-related injuries increased by almost 30% in the past decade  Additional file 2:Table S1. Proportion of subsequent hospitalized sports-related injuries by sporting code, type and body region in children aged 5\u201315\u00a0years in NSW, Australia, 2005\u20132013. (DOCX 17 kb)"}
+{"text": "Interventions to reduce the morbidities and adverse health outcomes in these neonates and improve parent-infant interaction are highly important. This study was conducted to determine the effect of the Creating Opportunities for Parent Empowerment (COPE) program on the perceived maternal parenting self-efficacy of premature parents. Premature neonates are at great risk for\u00a0cerebral palsy, development. All the measurements were performed pre- and post-completion with the valid equipment and by blind assessors. This was a randomized controlled trial with equal randomization (1:1:1 for 3 groups) and parallel group design. Forty-five preterm neonates were randomly allocated to treatment (n=15), supervision (n=15) and control (n=15) groups. COPE program was provided in the form of a 4-phase educational-behavioral intervention to the treatment and supervision groups. The primary outcome was parental self-efficacy, which was assessed by the Perceived Maternal Parenting Self-Efficacy inventoryCOPE mothers reported significantly stronger beliefs regarding their parental role and have more confidence to their ability in caring of neonates compared with control mothers .An educational-behavioral intervention would strengthen mothers\u2019 belief in themselves and knowledge about their neonates and would enhance premature mothers\u2019 ability to care for their neonates as well as parent-infant interaction.  The birth of a healthy and normal neonate is a celebrated event with happiness for parents, but this is different when a neonate is born prematurely and parents face an unexpected event that involves many challenges , 2. PareParents\u2019 challenges revolve around issues such as learning to care for the newborn, obtaining information about the neonate, getting to know the baby and dealing with one\u2019s own expectations as a parent . A revieThere is considerable scientific evidence supporting the significance and necessity of increasing self-efficacy in parents , becausePremature parents need to feel efficacious in their parenting role, and parents' practical training and strategies for increasing parents and families\u2019 involvement in neonatal care (work book) have important consequences for parent and infant development .Creating Opportunities for Parent Empowerment (COPE) is an educational-behavioral intervention designed based on the presumption that providing parental supportive interventions benefits parents, neonates and families in general -14. A reMost studies on the efficacy of COPE conducted in different regions of the United States are mainly focused on the mental  and physical health of parents and their infants (weight gain) . Also, tHowever, there is a gap in the literature concerning how parents\u2019 self-efficacy is associated with caring for a preterm infant at home, especially during the first weeks and months after hospital discharge. The purpose of this study was to evaluate the effect of the COPE program on the perceived maternal parenting self-efficacy (PMP-SE) of premature parents.This was a double-blind, randomized, controlled trial conducted in the two university hospitals of Akbar-abadi and Mahdieh in Tehran, Iran, during March 2015 to September 2015. We included 45 neonates with a gestational age (GA) of under 37 weeks who were admitted to the NICUs of the two hospitals. These preterm neonates met the following inclusion criteria: (1) birthweight between 1000 and 2500 gr, (2) GA under 37 weeks, (3) 5-minute Apgar score of 7 and more, (4) no major abnormalities on brain ultrasound (grade III or IV intraventricular hemorrhage [IVH]), (5) absence of congenital anomalies or neuromuscular disorders and (6) hospitalization in NICU for at least 7 to 30 days. The exclusion criteria comprised of incurable disease, neonatal death during the study and parents\u2019 unwillingness to participate in the study for any reason.Sample size was calculated with an \u03b1-value of 5% and power of 80%. The analysis accounted for a 20% attrition rate. Fifteen neonates per group were needed to detect clinically worthwhile effects.After obtaining free and informed consent, neonate randomization was performed using\u00a0a randomized block\u00a0design, and the neonates were randomly assigned to the supervision (n=15), treatment (n=15) and control (n=15) groups. After gathering the clinical data, mothers were asked to rate their perceptions about their ability to effectively and successfully assume their caring role as a mother. PMP-SE was measured using the 20-item Efficacy subscale of the Parenting Sense of Competence scale . Items wNeonates in the control group received no additional treatment from the research therapist, but they received the routine interventions and services, and the COPE program was provided for the treatment and supervision groups. This educational-behavioral intervention program consisted of a series of CDs along with written information and reinforcing activities for parents (workbook). COPE program was delivered in four phases as follows: phase I: 2-4 days after the infant was admitted to the NICU, phase II: 2-4 days after the first phase, phase III: 1-4 days prior to the infant\u2019s NICU discharge, and phase IV: about one week after discharge. Daily strict implementation of the program was followed by the therapist in the supervision group; the therapist also provided daily comments and reviews. Follow-up assessments were completed one month after discharge by the same person, who was unaware of the group allocations . The assThe data were analyzed using SPSS. All the values were tabulated as averages (mean) with standard deviation (SD). For all the analyses, the significance level was set at 0.05.P = 0.000). The mean gestational age of the premature infants was 31.93 weeks in the control group , 33.26 weeks in the treatment group  and 33.26 weeks in the supervision group . As shown in Evidence shows that insufficient information and knowledge about neonatal care is a concerning factor for mothers in the postpartum period , 20. AccFindings of the present study showed that training programs for mothers had a positive impact on mothers\u2019 perception of their neonates\u2019 behaviors and they also improved mothers\u2019 monitoring strategies versus the control group. The present findings are consistent with the results obtained in a meta-analysis by Letarte et al. (2010). They reported that parents training had a positive effect on parents\u2019 self-efficacy and improved parent-child relationship . Parent A literature review indicated that traditional office-based family therapy services were not always useful and effective in high-risk families\u00a0. Also, tThe limitation of this study included limited sample size; thus, we recommend more comprehensive clinical trials on the effect of parent training on developmental outcomes in preterm infants.Parent training programs have been found to be very effective as most parents need careful training and support to improve their parenting skills. Thus, the present study emphasizes the need to consider learning opportunities and delivering parent training packages in hospitals and after discharge."}
+{"text": "Prompted by the 20th anniversary of Roll Back Malaria, the author recalls hypotheses concerning a major new initiative to control malaria in Africa put forward by WHO AFRO and the World Bank in 1996. These hypotheses, and the reactions to them of a panel of 18 experts, are reviewed and contrasted to the rapid progress and high ambition that characterize the field of malaria today. On November 19, 2018, the 20th Anniversary of the Roll Back Malaria Partnership to End Malaria (RBM) will be celebrated in Maputo, Mozambique. During the period leading up to the launch of RBM in New York in 1998, I was the Director for Health, Nutrition and Population at the World Bank and, in that capacity, was involved in the preparation and launch of RBM. The 20th Anniversary prompted me to look back in my files and remind myself about antecedents to the launch of this new partnership.In June 1996, Dr. Ebrahim Samba, WHO Regional Director for Africa, and I wrote to a panel of international experts as follows.DearDespite many decades of control efforts, malaria remains a leading cause of illness, death, suffering, and poverty in Africa. For reasons that have been much discussed and written about, the traditional armory of preventive approaches is not being fully or consistently used in the most affected areas. New weapons are becoming available and there is much current interest in the effectiveness of impregnated bed nets. In the medium term, an effective malaria vaccine is anticipated and its wide-spread utilization will undoubtedly assist in the control of this disease.The WHO Regional Office for Africa and the World Bank are interested in exploring a hypothesis (which we attach) with experts in health and development in Africa. WHO and the World Bank are prepared to be advised by Experts on how to intensify malaria control activities in Africa and to seek views on the most appropriate policy and approaches for reduction of malaria burden under the current economic and social environment.The purpose of this letter is to invite you, a known authority and expert in this field, to express your opinions about the attached hypothesis and related matters. What we seek is five pages of your frank personal thoughts on how it would be best to move forward internationally and nationally on malaria control in Africa. In particular, we would like your review and commentary on the hypothesis. If you agree with it, please tell us why. If you disagree with it, please tell us why and please also propose an alternative hypothesis .We hope you will be willing to contribute your wisdom and experience to this international brainstorming process. We attach a list of the others who have been invited to assist us in the same manner. We will appreciate receiving your thoughts on the subject by June 30, 1996. We will then assemble all the opinions received and come back to you with a proposed next step.not have a firm position: we do not know where this process will lead us: and we seek the best advice and opinion before making up our minds on these matters.As you will appreciate we are in a very exploratory mode. We do We send you our personal thanks for taking the time to study this letter and hope that you will be willing to assist us in the manner requested.With best regards,Yours sincerely,Dr. Ebrahim M. Samba Dr. Richard G. A. FeachemAttached to this letter were six hypotheses that Dr. Samba and I put forward for comment and reaction, reproduced below.Notwithstanding the potential of new tools  a \u201cbusiness as usual\u201d approach to malaria control in Africa will probably mean that by the year 2050 this disease continues to be a major cause of ill health, death and suffering.There is a potential for a large, long-term, focused initiative to accelerate the pace of malaria reduction.This initiative might operate on a focused geographical basis, selecting initially a small number of areas (perhaps three or four) where rapid progress in malaria control is technically feasible. The initiative would start by establishing effective malaria control in these areas and then move systematically outwards from them to eventually embrace the whole continent.An important purpose of this initiative would be: (i) to strengthen and sustain ongoing high level political and social commitment, both in Africa and among the OECD nations, to the task of malaria control and (ii) To achieve concrete results in reduction of malaria burden by using effectively the tools available  at health services and community levels.The existence of an African malaria initiative would be an incentive to well-focused malaria research investments leading to new products and tools which could be rapidly tested and applied in major ongoing control programmes.Any such initiative would need to take a 30-year time horizon and set a modest goal for the year 2010, a more ambitious goal for the year 2020, and achieve malaria control across Africa by the year 2030.The six hypotheses concerning malaria in Africa.The panel who received this letter and the hypotheses comprised 18 experts from Australia, Benin, Ethiopia, France, Ghana, Mali, Mozambique, Nigeria, Senegal, South Africa, Spain, Switzerland, UK, USA, Zambia, and Zimbabwe.There was a surprising level of agreement among the panelists and a high level enthusiasm concerning the hypotheses. To summarize the essence of the views of the panel, technical feasibility does not seem to be a stumbling block as long as ongoing evaluation and methodological issues are addressed. Ensuring high-level political commitment by African countries, and ensuring that that this is an African based and designed initiative, were deemed vital to success. The participation of OECD countries was widely endorsed as a way to ensure long-term policy and financial support, and as a means to tie the initiative closely with a research agenda.\u201cThe third hypothesis, \u2018having the initiative emanate from focused geographical areas\u2019, was much discussed. The panel supported the hypothesis but suggested conditions that would be prerequisites for the success of the initiative. Many experts mentioned that this initiative must be integrated into the existing primary health care system and that its emphasis should be placed at the district level. With this in mind, it was mentioned that significant training at all levels would need to be started as early as possible\u201d.The response from the panelists was enthusiastic. On October 1, 1996, I wrote to Dr. Samba:Dr. Samba continued to push vigorously for an ambitious new African Malaria Initiative, with support from the World Bank, the American, British and French Governments and other partners. These discussions came together in January 1997 in Dakar, Senegal, at the first meeting of the Multilateral Initiative on Malaria (MIM). The outcome of this first MIM meeting was summarized in a subsequent letter in Nature [It is interesting to reflect on how we saw the world 20\u00a0years ago. I recall that, when drafting the letter and the hypotheses to send to the expert panel, we had a strong sense of being provocative and challenging. Our suggestions came at a time of appalling levels of morbidity and mortality from malaria in Africa and a good deal of fatalism about this situation. Indeed, a number of members of the expert panel took issue with the implication in Hypothesis 2 that malaria in Africa was currently declining.Lancet Commission on malaria eradication [By today\u2019s standards, with all the remarkable progress that has been made over the past 2 decades, the hypotheses seem tame. In Hypothesis 6, our biggest ambition was to \u201cachieve malaria control across Africa by the year 2030\u201d. There was no mention of elimination. And our proposals sound very cautious in relation to the current deliberations of the Shrinking the Malaria Map [The suggestion in Hypothesis 3 concerning the establishment of bridgeheads of malaria control in 3 or 4 areas, which would then be expanded outwards, has clear resonance with the language of Roll Back Malaria and also presaged the concept of The expert panel had interesting and divergent views on the research agenda and new technology. One panelist felt \u201cpretty confident that a vaccine will be developed within 10\u00a0years and so the situation in 2050 may not be so grim\u201d. Another said that we are \u201cunlikely to have a suitable malaria vaccine in the medium term, and will face problems of cost and duration of protection\u201d. There was strong endorsement of the tailoring of solutions to local circumstances and that one size does not fit all. There was also consensus on the need for strong Africa-wide and local political commitment and ownership, and that this could not be an effort driven from Geneva or Washington.the approach is not to expand and develop work currently in hand, but to think radically about a major new effort. That effort should remain strongly in African hands. The effort would, of course, take full advantage of work already underway through WHO and national governments, but given the initiatives many integrated aspects, it should endeavour to gather broad based support and commitment  for the launch and to ensure a future for such an initiative. The tone would emphasize that the scale and ambition of this initiative were without precedent.\u201d\u201cFinally, there was wide agreement about the scale and ambition required. In further correspondence dated October 21, 1996, this was summarized as follows:In other words, a broad and bold partnership to end malaria in Africa."}
+{"text": "Over the past decades, survival rates of children born with congenital heart disease (CHD) have increased dramatically. Progress in prenatal diagnosis, less-invasive catheter techniques and perioperative intensive care as well as surgical techniques have led to an increased focus on extracardiac comorbidities, including potential neurodevelopmental sequelae associated with CHD. A growing body of literature reports impairments in early and school-age developmental outcome; however, there is a substantial variability in the spectrum of examined CHD types, assessment ages and applied test batteries. Furthermore, little information is available on executive function impairments in this population. Therefore, the aim of this systematic review is to determine the impact of CHD on intellectual outcome and executive functioning at school age and to determine risk factors for impaired outcomes by means of a systematic search.A systematic review of literature that reports neurodevelopmental outcome in children with CHD undergoing cardiopulmonary bypass surgery. Intelligence quotient or executive function scores will be considered primary outcomes. Databases such as Cochrane, EMBASE, MEDLINE and PsycINFO will be searched.The results of this systematic review will summarize the current evidence on intellectual and executive function outcome after cardiopulmonary bypass surgery in school-age children with CHD. This review will thus be the basis for better patient and parental counselling and the establishment of tailored follow-up programmes and interventional trials.CRD42019118736.In accordance with the guidelines, our systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) on January 9, 2019 (CRD42018086568). PROSPERO  Congenital heart disease (CHD) summarizes a broad array of mild to severe structural and functional congenital anomalies of the heart and great arteries. CHD occurs in about 6 infants per 1000 live births, with 1 in 1000 having a cyanotic CHD, and 2 to 3 in 1000 requiring open-heart surgery [Over the past decades, advances in cardiopulmonary bypass surgery and perioperative intensive care have lowered the mortality rates of children with CHD noticeably . This leNeurodevelopmental impairment in infancy and early childhood is often mild and characterized by delayed acquisition of motor milestones and mild cognitive impairment  but persWhereas the neurobehavioral profile of children with CHD in early infancy has been well characterized, studies reporting neurocognitive outcomes of children and adolescents with CHD remain scarce and comprise reports of predominantly small cohorts with heterogeneous CHD study populations. However, these studies show that during school age, impairments become more apparent with the increasing cognitive demands , 15.School age is also a time when executive functions are key to social development and academic achievement . ExecutiA number of demographic, foetal and perinatal as well as perioperative clinical risk factors for impaired neurodevelopment have been identified. Innate patient factors such as gender, birth weight, type of CHD, maternal education and genetic/extracardiac anomalies have been identified to be associated with neurodevelopmental and executive function outcome \u20137. FurthFurther research is warranted to better understand the impact of complex CHD on cognitive outcome and executive functioning at school age and the role of modifiable risk factors and will aid the clinicians to counsel and support patients and families accordingly. A systematic review of the current literature in this field will summarize the existing evidence for impaired cognitive and executive functioning, will address potential risk factors and by that provide the basis to develop future research questions.We aim to systematically review the literature for long-term intellectual and executive function outcomes of school-aged children with CHD, who underwent cardiopulmonary bypass surgery in comparison to control subjects or normative reference data. We aim to further examine risk factors for poor outcome, in particular modifiable risk factors, such as operative and perioperative variables. The results of this review will be the basis for better patient and parental counselling and the establishment of tailored follow-up programmes and interventional trials.The protocol for this systematic review was developed in accordance with the Preferred Reporting Items of Systematic Reviews and Meta-Analysis for Protocols 2015 (PRISMA-P)   who underwent surgical repair or palliation for CHD during infancy or childhood with cardiopulmonary bypass are considered for inclusion. Studies reporting exclusively outcome of CHD children with neurologic comorbidities  or after heart transplantation will be excluded.Studies in which both genetic syndromal and non-syndromal patient data are reported will be included and findings of the two groups extracted and analysed separately.Sociodemographic factors affecting academic performance and neurodevelopmental outcome will be considered. These include parental educational level and profession, income and environmental factors. Special educational support therapies such as early intervention as well as classroom support will be considered as exposure or confounder as appropriate. Outcomes of interventional trials will be included and extracted by intervention type. Results of the intervention group might be collated afterwards or interventional group might be accounted for in the statistical analysis.The type of cardiac defect will be considered an exposure. Other confounders including maternal factors, neonatal factors  or hypoxia (cyanosis)), perioperative characteristics and postoperative complications as well as pregnancy characteristics will be treated as confounders.Cognitive outcome in children with congenital heart disease assessed by standardized age-appropriate neuropsychological assessments and reported as intelligence quotient will be considered as primary outcome. Executive function obtained through direct assessment or questionnaire will be additionally considered as primary outcome. Studies which do not report either one of our primary outcomes will be excluded.Measures of academic achievement will be considered as secondary outcome.CochraneEmbaseMEDLINEPsycInfoThe following electronic databases will be searched:The search for relevant publications will be done using subject headings  and free text words related to congenital heart disease and intellectual as well as executive function outcome in children and adolescents. The search strategy will be adapted for each database. The search will not be restricted to study design, date of publication or language.If the full text or abstract of a reference cannot be found, authors of eligible studies will be contacted for full text or exclusion might be based on available data. If no response is received within 1\u00a0month, the study will be excluded. Reference lists of reviews, editorials and commentaries will be screened for relevant publications as stated under the section \u201cThe search strategy was developed in collaboration with a health information specialist experienced in literature search for systematic reviews and the authors. Key papers were used to derive and validate the search strategy. The database search will be carried out by the information specialist. The MEDLINE search protocol is included as Additional file 2 as an example.As we do not include grey literature, we will address reporting bias by assessing funnel plot asymmetry.www.covidence.org. Two independent reviewers  will screen titles and abstracts of a random subsample of 200 eligible studies yielded by our search strategy. Agreement on inclusion for full-text screening of >\u200980 % between the two independent reviewers will be considered as good, and the remaining number of references will be divided among the reviewing authors for further screening. Full texts of potentially relevant citations will be retrieved and examined for final inclusion. Again if >\u200980 % agreement within a subset of the eligible studies is achieved, the remainder will not be screened in duplicate. Rationale for in- or exclusion of studies will be documented, and discussion with a third author (BL) will be carried out to find consensus. The authors will not be blinded to journal titles, authors or author affiliations during the study selection process.References will be stored and managed in Endnote X8 . Duplicate publications will be automatically removed using Endnote X8. Remaining duplicates will be manually removed during the screening process, when identified. Management of citations throughout the fulltext screening and selection process will be supported by the web-based systematic review management programme on We aim to identify multiple reports of the same study by juxtaposing authors and comparing the reported study populations in terms of number, year and place of recruitment as well as outcomes. Data of multiple reports of the same study will be handled as stated in the section \u201cInformation will be extracted from the included studies using a digital data extraction form. The form will include demographic and clinical information on study subjects, details on applied assessment tools and reported outcome results. A draft of the data extraction form will be predefined and piloted and adapted after extracting data from the first ten included studies. For further detail, see the draft of the data extraction sheet . Data extraction will be carried out by two independent authors for individual subsets of the studies or in duplicate (discrepancies solved by third party) depending on the amount of included studies and the corresponding workload. The final approach will be reported accordingly in the systematic review report. In the absence of complete outcome reports or crucial information on the study population, corresponding authors will be contacted to obtain missing information. In case of multiple reports of the same study, reporting the same follow-up time point and outcome, only the most extensive report in terms of sample size will be considered for data extraction. Conversely, two separate data extraction forms will be completed if the follow-up time points or reported outcomes differ. Information from these data extraction forms might be collated afterwards or multiple reporting will be addressed through statistical analysis. Corresponding authors might be contacted to clarify questions on overlapping reports of the same study.We predefine a minimum set of information that must be extractable from the publication: age of subjects at assessment, type of CHD, cardiopulmonary bypass performed and one of the two primary outcomes reported. If the minimum dataset is not provided, corresponding authors will be contacted to retrieve missing data in order to appropriately describe the study results. If information on missing data cannot be obtained or no response of the corresponding authors is received within 1\u00a0month, the available data in each study will be used for meta-analysis and multiple imputation will be used to account for missing values if appropriate.https://www.sign.ac.uk/checklists-and-notes.html). The overall methodological quality of the studies will be ranked as \u201chigh quality\u201d, \u201cacceptable\u201d and \u201clow quality\u201d according to the criteria of the SIGN checklist. If a final rating is not possible due to missing information, corresponding authors will be contacted for clarification or quality will otherwise be rated as \u201cunclear\u201d. We do not plan to weigh study results based on the quality assessment, or to exclude studies due to low methodological quality, but will report frequencies of quality ratings and take the overall quality into consideration when discussing the results of the systematic review and meta-analysis. Risk of bias assessment will be carried out by two independent raters , and discrepancies will be solved by consulting a third party (UH).The quality of the included studies will be evaluated regarding their risk of different biases  by means of one of the SIGN checklists appropriate for study design Age at surgery Age at assessment categories Children receiving educational support versus no supportStratification by socioeconomic statusIf possible, we will carry out subgroup analyses for the following groups:A narrative synthesis of the results of the systematic review will be provided, if statistical synthesis of the quantitative data is not appropriate because of a low number of studies or heterogeneous outcomes that cannot be pooled. In this case, a predeveloped narrative synthesis method  will bePublication bias will be assessed by the graphical method of funnel plot, and the presence of bias will be visually inspected and statistically tested using the Egger test .To judge the confidence in the resulting body of evidence, the strength of the evidence will be assessed using the Grading of Recommendations Assessment (GRADE) .In the event of protocol amendments, the date of each amendment will be provided with a description of the change and the rationale. This information will be added in tabular form to the final report and manuscript of the systematic review.The results of this systematic review will contribute to a better understanding of the impact of CHD on cognitive and executive function outcomes at school and adolescent age. In particular, modifiable surgical and perioperative risk factors may be identified through this systematic review and may help to improve clinical management. The summary of the current literature will moreover aid clinicians to better understand the impact of different factors on outcome and thus help to counsel patients and their families. This information will be the basis for tailored follow-up programmes. Moreover, it will help to stratify risk groups to develop and provide early pharmacological and non-pharmacological intervention therapies. Finally, it will aid researchers to detect knowledge gaps and will help to pose future research questions.With the here outlined methodological approach to our systematic review, which is in compliance with the PRISMA-P reporting guidelines, we aim to provide methodological transparency and clarity that is crucial for future reproducibility and enhance the value and strength of the evidence we will obtain from the results of our systematic review and meta-analysis.Additional File 1: PRISMA-P\u2009+\u2009checklist_10012018.docx. Prisma-P checklist (DOCX 32 kb)Additional File 2: _20190819_revised.docx. Medline search strategy (DOCX 21 kb)Additional File 3: _20190819_revised.docx. Draft of data extraction sheet (DOCX 15 kb)"}
+{"text": "In the present version, LncBook houses a large number of 270 044 lncRNAs and includes 1867 featured lncRNAs with 3762 lncRNA\u2013function associations. It also integrates an abundance of multi-omics data from expression, methylation, genome variation and lncRNA\u2013miRNA interaction. Also, LncBook incorporates 3772 experimentally validated lncRNA-disease associations and further identifies a total of 97 998 lncRNAs that are putatively disease-associated. Collectively, LncBook is dedicated to the integration and curation of human lncRNAs as well as their associated data and thus bears great promise to serve as a valuable knowledgebase for worldwide research communities.Long non-coding RNAs (lncRNAs) have significant functions in a wide range of important biological processes. Although the number of known human lncRNAs has dramatically increased, they are poorly annotated, posing great challenges for better understanding their functional significance and elucidating their complex functioning molecular mechanisms. Here, we present LncBook ( Long non-coding RNAs (lncRNA) have a variety of functions in many important biological processes  and are To harness collective intelligence for gathering and annotating human lncRNAs, we constructed LncRNAWiki  in 2015,http://bigd.big.ac.cn/lncbook), as a complement to community-curation-based LncRNAWiki. LncBook features a comprehensive collection of human lncRNAs and systematic curation of lncRNAs by multi-omics data integration, functional annotation and disease association , CPAT , CPAT  and PLEK4), CPAT , were ushttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic  . FastaQCmmomatic  were usemmomatic  was usedmmomatic  was usedmmomatic . As a remmomatic . After r\u03c4-value \u2265\u00a00.95). To annotate methylation information of lncRNAs, bisulfite-seq data from TCGA (The Cancer Genome Atlas)  and ENCODE (The ENCyclopedia of DNA Elements) (https://www.encodeproject.org) were downloaded, covering nine cancers with both normal and cancer samples. We defined regions from \u22121500 bp relative to the transcription start sites as promoters, and calculated the methylation levels of both promoter and body regions of lncRNAs. In addition, we mapped the SNP sites in dbSNP   and GTExin dbSNP  to the lin dbSNP , pathogein dbSNP  and COSMin dbSNP  using ANin dbSNP . TargetSin dbSNP  and miRain dbSNP  were usein dbSNP .To provide high-quality annotations for experimentally validated lncRNAs, we systematically curated 1867 lncRNAs (that were sourced from LncRNAWiki ) with fuThe associations between lncRNA and disease were derived from LncRNADisease  and LncRhttp://www.mysql.org) as database engine. Web interfaces were developed by JSP (Java Server Pages) and AJAX (Asynchronous JavaScript and XML). Bootstrap (https://getbootstrap.com) was adopted as a front-end framework, which provides a series of templates for designing web pages with consistent interface components. Also, data visualization was powered by Highcharts (a charting library written in pure JavaScript), offering an easy way of adding interactive charts to any web site or application.We developed LncBook using String Boot as back-end web framework and MySQL , including 247 246 lncRNAs obtained from existing databases, 1867 from LncRNAWiki and 21 815 novel lncRNAs identified based on RNA-seq data analysis, which together belong to 140 362 gene loci. LncBook manages human lncRNAs based on transcripts, where a unique accession number prefixed with HSALNT is assigned to each lncRNA transcript entity. Likewise, the lncRNA gene has an accession number prefixed with HSALNG. In LncBook, each transcript corresponds to a specific web page containing basic information , multi-omics data , function annotations and disease associations . For any given lncRNA, LncBook profiles its expression levels across all collected tissues and visualizes its expression profile in a bar chart, greatly facilitating users to explore functional significance. Based on these expression profiles across different tissues, LncBook further identifies a total of 819 HK lncRNAs, which are consistently expressed in almost all tissues. Similarly, it also obtains 49 115 TS lncRNAs, which are expressed specifically in one or few tissues. All HK and TS lncRNAs are publicly available at om dbSNP  residingom dbSNP  overlappom dbSNP  , ceRNA (182 associations), splicing regulation (19 associations), translational control (17 associations), protein localization (4 associations)\u00a0and RNAi (3 associations). For biological process, LncBook adopts two terms, namely, pathogenic process and developmental process; function annotation of featured lncRNAs shows that most of them are involved in cancer and other diseases (3598 associations), compared to developmental process (53 associations).HOTAIR, MALAT1, H19, MEG3, CDKN2B-AS1, PVT1, NEAT1\u00a0and GAS5 are extensively studied and each of them is associated with at least 30 different diseases.Considering that most of the functionally studied lncRNAs are closely associated with human diseases, LncBook integrates 3772 lncRNA-disease associations, derived not only from LncRNADisease  and LncRhttp://bigd.big.ac.cn/lncbook/disease.Additionally, based on an abundance of methylation, genome variation and lncRNA\u2013miRNA interaction, LncBook predicts a total of 97 998 lncRNAs that are potentially associated with diseases . Briefly speaking, one lncRNA is putatively believed to be disease-associated only if any evidence for that can be obtained from methylation, genome variation and/or lncRNA\u2013miRNA interaction. For a specific lncRNA under investigation, supporting evidence can be that, for example, its methylation change relates to disease, it overlaps pathogenic variations, or it frequently interacts with disease-associated miRNAs. As a consequence, LncBook contains a collection of 97 998 disease-associated lncRNAs, where 607 are supported by three sources of evidence, namely, methylation, genome variation and lncRNA\u2013miRNA interaction, 13 257 are supported by two of them, and 84 134 are supported by only one of them. All these disease-associated lncRNAs can be found at LncBook is dedicated to the integration and curation of human lncRNAs as well as their associated data. In harmony with LncRNAWiki that is a community-curated resource, LncBook serves as an expert-curated knowledgebase that integrates a comprehensive collection of human lncRNAs and contains multi-omics data, function annotations and disease associations. The current implementation of LncBook houses a large number of 270 044 lncRNAs and includes 1867 featured lncRNAs with 3762 lncRNA\u2013function associations. It also integrates an abundance of multi-omics data from expression, methylation, genome variation and lncRNA\u2013miRNA interaction. Also, LncBook includes 3772 experimentally validated lncRNA-disease associations and identifies 97 998 lncRNAs that are putatively disease-associated. However, of note, this does not mean that these disease-associated lncRNAs play causative roles in diseases . Taken t"}
+{"text": "Children with a migrant background were diagnosed 13 months earlier than those without , and had more severe delays in language, more severe autism, no Asperger\u2019s syndrome, lower parental educational level and more frequent referrals by paediatricians. For the total sample, expressive language delay, severity of restricted and repetitive behaviours, higher nonverbal development, and paediatric referrals explained earlier diagnoses. There was a stronger effect of parental education and weaker effect of language impairment on age at ASD diagnosis in children with a migrant background. In conclusion, no delay in diagnosing ASD in children with a migrant background in a country with universal health care and an established system of paediatric developmental surveillance was found. Awareness of ASD, including Asperger\u2019s syndrome, should be raised among families and healthcare professionals.This study explored (i) differences in age at Autism Spectrum Disorder (ASD) diagnosis between children with and without a migrant background in the main diagnostic centre for ASD in Upper Austria (ii) factors related to the age at diagnosis and (iii) whether specific factors differed between the two groups. A retrospective chart analysis included all children who received their first diagnosis before the age of 10 years ( IASD and migration. A growing number of European studies, particularly from Nordic countries, suggests an increased frequency of autism in children of immigrant parents [ parents ,8,9,10.  parents ,12. Earl parents ,14, wher parents . In Euro parents . Within  parents . The pro parents .(2) Age at ASD diagnosis and predictors: The increase in ASD prevalence has been accompanied by a decrease in the age at diagnosis in many countries ,19, alth(3) Migration and age at diagnosis: Lastly, migration status may also be a cause of delayed diagnosis ,39,40,41(4) Health Care Services for Children with ASD in Upper Austria: The Austrian federal state of Upper Austria (1.48 million inhabitants) has a high proportion of immigration. In Upper Austria, 31% of all primary school children have a family language other than German . The AusThe out-patient clinic of the ISSN, situated within a public general hospital in the city of Linz, has been the major clinical focus for developmental disorders for the local community in Upper Austria in the last decade. It is the main centre where the complex multidisciplinary diagnostic evaluation needed for ASD diagnosis is regularly conducted . In the This study aimed to explore (i) whether there are differences in terms of age at ASD diagnosis between children with or without a migrant background at the principal diagnostic centre for ASD in Upper Austria, (ii) what factors might be correlated with the age at diagnosis of these children and (iii) whether predictors of age at diagnosis differ between children with and without a migrant background.A cross sectional study was used. All children of parents with and without a migrant background (see definition below) who attended the diagnostic centre of the ISSN in the study period (01-01-2013 to 31-12-2018) and received an ICD-10- diagnosis  of ASD Migration background cannot be sufficiently defined by citizenship, since there are many families with parents born outside Austria (and even more second-generation families) who have Austrian citizenship. The ISSN medical records did not contain systematic information on the parents\u2019 and children\u2019s places of birth, but detailed information about the language(s) spoken in the family. Therefore, a family was considered to have a migrant background if both parents (or one parent in a single-parent family) used a language other than German as their primary language in the family. There were no members of Austria\u2019s autochthonous ethnic minorities in the study sample.Age of diagnosis was limited to a maximum of 10 years for both samples, since there were only few individual children in our clinical population who received an ASD diagnosis after that age. All ASD cases were evaluated at the ISSN by a developmental paediatrician, a clinical psychologist and a clinical linguist using various standardised tests depending on each child\u2019s developmental level and a clinical (non-standardised) interview. The ICD-10  was usedDependent variable: chronological age of the child at first ASD diagnosis. Independent variables: grouped by (a) sociodemographic, (b) clinical characteristic and (c) referral to diagnosis.(a) Sociodemographic characteristics: Migration background status was based on language(s) predominantly spoken by the parents in the family as reported by the parents themselves. A family was considered to have a migrant background if both mother and father (or a single parent) used a language other than German as their primary language at home. The languages reported as being used at home by the study population were then categorised into five geographical regions: South-Eastern and Eastern Europe, Western and Northern Europe, Middle and East Asia, West Asia, and others see . No pare(b) Clinical characteristics: Gender was extracted from the medical record of the child. The non-verbal IQ score is the standardised non-verbal quotient of the child at the time of ASD diagnosis, either extracted directly from the evaluation report or calculated using the well-known quotient formula with the non-verbal developmental age of the child divided by the chronological age indicated in the report. The most common standardised cognitive tests reported were Mullen Scales of Early Learning (MSEL) , the Bay(c) Referral to diagnosis was either directly stated by the parents in the registration form or reported to the clinician at the evaluation (and therefore included in the diagnostic report).The clinical charts of all cases were revised and abstracted by researchers with considerable knowledge and experience in the field of neurodevelopmental disorders. To ensure accuracy, reliability and consistency in data abstraction, we developed, tested and revised a Record Review Protocol (RCR) following best practice ,66. An iEthics: This study was approved by the ethics committee (Ethikkommission der Medizinischen Fakult\u00e4t der Johannes Kepler Universit\u00e4t), Nr. 1140/2020 Version 3, following the rules of the Declaration of Helsinki of 1975 revised in 2013 .2-test was carried out. Correlations were also calculated for different age groups in order to identify possible age-dependent predictors of the age at diagnosis. For all bivariate analyses we calculated effect size measures in a correlation metric, more specifically, Pearson\u2019s r for the association between continuous variables, point biserial correlation for the association between binary variable and continuous variables and phi or Cramer\u2019s V for the association between categorical variables. Finally, we used linear regression models to evaluate the effects of all predictors simultaneously. We also tested whether the association between predictors and age at diagnosis differed by migrant status. This was achieved by including interaction terms  in the regression models [plus 8.2. [First, we separately calculated descriptive statistics  of all study variables for children with and without a migration background. Second, bivariate correlations were calculated between the age at diagnosis and the sociodemographic and clinical variables. This was done for the total sample and also separately according to migration status. In order to test for differences in correlations between the migrant and non-migrant groups, a Wald \u03c7n models . We repon models ,70. Seven models ). For exn models . Firstlyn models . Specifin models ,73, whicn models . Mplus 8lus 8.2.  was usedlus 8.2. .In p < 0.001).Of the 211 children in the sample, 58% received a diagnosis of autistic disorder, 10% of Asperger\u2019s disorder, and 32% of Pervasive Developmental Disorder\u2013Not Otherwise Specified (PDD-NOS). Children with autistic disorder were diagnosed at an average age of 39.4 months, followed by children with PDD-NOS (50.8 months) and children with Asperger\u2019s disorder (75.9 months). Notably, children with Asperger\u2019s disorder were almost exclusively from the non-migrant group; only one case in the children with migrant background subsample had received an Asperger\u2019s disorder diagnosis. The difference in the ICD-10 ASD type between the non-migrant and migrant groups is highly significant . The distributions of the age at diagnosis for the total sample and the migration-status subsamples are shown in The mean age at diagnosis was 46.7 months (SD = 22.8) for the total sample. Values ranged from 12 to 119 months. The non-migrant group was diagnosed at a mean age of 54 months as compared to 41.2 months for the migrant group. Thus, children with a migration background received their autism diagnosis significantly earlier (difference of about 13 months) than those without  and receptive  language scores were diagnosed significantly earlier. There is also a moderate correlation between RB-CSS and age at diagnosis . Children with higher RB-CSS received their diagnosis significantly earlier than those with lower RB-CSS. Similarly, analysis revealed a weak correlation between SA-CSS and age at diagnosis ; more specifically, diagnosis occurred earlier in the case of higher severity scores for social affect. Finally, children who were referred by a paediatrician received their diagnoses earlier  than the rest of the sample (M 51.16 months). In the total sample, there are moderate correlations between age at diagnosis and language scores. Children with lower expressive . Thus, children of parents with migrant status who had at least graduated high school were diagnosed at an earlier age than children of parents with migration background who had no high school diploma. In contrast, this association is of comparable strength but of opposite direction and not statistically significant in the non-migrant subsample . Second, the correlations between both ELQ and RLQ and age at diagnosis are of moderate strength and statistically significant for the non-migrant group , but weaker and non-significant for the migrant group . Finally, it is interesting to note that the correlation between SA-CSS and age at diagnosis is significant for the migrant subsample , but not for non-migrant subsample . However, these two correlations do not differ significantly.There are some notable differences between the strengths of the correlations with age of diagnosis between children with and without a migrant background. First, for migrant-background children there is a negative association between age at diagnosis and parental education . Nonetheless, there are several interesting results. First, there is no significant correlation between age at diagnosis and ELQ and RLQ, respectively, in the younger subsample, neither for children with nor for those without a migrant background. These correlations are only significant for non-migrant-background children in the older group . Thus, non-migrant-background children aged \u2265 48 months at diagnosis received their diagnoses earlier, the lower their language scores were. A somewhat similar pattern\u2014although individual correlations are not significant\u2014was found for the correlations between RLQ and age at diagnosis in the migrant group. RLQ and age at diagnosis correlate moderately  for migrant-background children aged \u2265 48 months at diagnosis, but not for those who received their diagnosis before 48 months . Second, the RB-CSS is only correlated with age at diagnosis for children who received their diagnosis at the age of 48 months or later. In detail, there are moderate to strong correlations between RB-CSS and age at diagnosis for children both with  and without  a migrant background. In the subsample of children who were younger at diagnosis, these correlations are much smaller and not significant (correlation differences between age subsamples are significant). Finally, there is a negative and significant small correlation between age at diagnosis and distance between home and hospital in the older non-migrant subsample . Thus, children from this subsample received their diagnoses later, the closer they lived to the hospital. For children with a migrant background in this age group, the correlation is not significant and positive . The correlations differ marginally (p < 0.10).p < 0.001), followed by RB-CSS  and referral by paediatrician . Interestingly, the association between IQ and age at diagnosis, which was not significant in the bivariate analysis, is negative and statistically significant , when we control for other predictors. In fact, the association becomes significant as soon as we control for LQ; thus, given a constant LQ, the higher the IQ, the younger the child at diagnosis.The results of the regression models are shown in p < 0.10) once other predictors have been controlled for. In detail, the difference in age at diagnosis, which amounted to about 13 months in the bivariate case (see p < 0.05). Thus, about half of the difference in the age at diagnosis between the migrant and non-migrant groups is due to lower language scores of the migrant subsample.The regression model shows that migrant status is only marginally significantly associated with age at diagnosis . In order to interpret this interaction, we plotted the simple slopes, that is, the associations of IQ and age at diagnosis for both high (=M + SD) and low (=M \u2013 SD) levels of LQ. Finally, some further analyses were performed to better understand the negative IQ effect in the multivariate regression model. In detail, we included an interaction term between IQ and LQ, which turned out to be statistically significant  than without (43%) a migrant background being diagnosed with ASD in our clinic. This contrasts with a reverse ratio of about 30:70 of primary non-German to German family language in Austria. On the one hand, this might be explained by the high percentage of immigrant families (56%) in the city of Linz and a closer distance to the diagnostic centre facilitating service use. On the other hand, the high percentage of children with migrant background might also be due to a higher prevalence of ASD in migrant populations, as described by some of the European studies ,10. AddiOur main finding is that for the total study sample, children with a migrant background received the ASD diagnosis significantly earlier than the rest of the sample. However, in the younger subsample, diagnosed before 48 months of age, the mean age at diagnosis was almost identical for the migrant and non-migrant groups . For the total sample, the significantly younger mean age at diagnosis in the subsample with a migrant background is due to a lack of children with less severe autism symptomatology (Asperger disorder), who are usually presented to the clinic at an age older than 4 years. In any case, contrary to findings in other health systems , there an = 1) in contrast to the non-migrant subsample (n = 21). This accords with the results of Lehti et al. in 2015 [In accordance with the literature ,79,80, w in 2015 , who fou in 2015 . HoweverBivariate and multivariate analyses that identified factors correlated with delayed age at diagnosis for the total sample demonstrated a strong impact of the severity of language problems (expressive and/or receptive) on earlier ASD diagnosis. Severe delays in expressive language are easily noticed both by parents and by professionals in health and education systems and have been described as the most frequently observed symptoms of ASD that prompt medical consultation . They haA comparison of factors related to age at identification of ASD in the migrant and non-migrant samples mostly showed significantly stronger effects of parental education in the group with a migration background. However, the correlation was small. In migrant-background families, educational disparities are often linked to pronounced deficits in (spoken and written) language that might have a particularly strong effect on access to health information.n = 23), results must be interpreted with caution. The minimal differences in factors explaining age at autism diagnosis between children with and without a migrant background are most likely a consequence of equitable access to medical care and a model of early identification with strong involvement of primary paediatric care that is well accepted.The severity of (primarily expressive) language delay had significantly stronger effects on delayed ASD diagnosis in the non-migrant sample. This effect is almost exclusively observed in the older subsample (>4 years), where\u2014due to the much higher number of children with Asperger\u2019s disorder\u2014variance in language development was significantly higher in the non-migrant sample. In school children with a migrant background, parents and professionals in health and education might ascribe language difficulties to bilingual language acquisition rather than to a developmental disorder. However, given the small size of the sample of older children with a migrant background  are usually offered at the ages of 2 and 3 years. Earlier referrals are highly unlikely. Furthermore, kindergarten, where autistic symptoms might be noticed, typically starts at the age of three. Long waiting periods for diagnostic services due to insufficient resources can cause inequalities and might be another reason that complicates the finding of significant predictors. The retrospective medical chart analysis presented has some limitations. Firstly, due to sample recruitment from a single clinic, effects of specific clinic-related factors on the age at diagnosis cannot be excluded. Secondly, another limitation of this study is its reliance on self-reported data about the parental level of occupation, since other information about Socioeconomic Status (SES), such as income or highest parental education, was not included in the medical charts. Even for information on parental occupation, data extracted from medical reports were very incomplete. Missing data, including those for other variables, were reported, and multiple imputations was used for missing values to avoid bias. Analyses of correlates of age at diagnosis by splitting the sample into two age subsamples and by migration status were interpreted with caution due to the small sizes of some of the subsamples.This is one of the rare studies investigating age at diagnosis in children with a migrant background in Europe. Our findings highlight that for them the diagnosis of ASD is not delayed in Upper Austria, where universal health care includes a system of preventive medical check-ups provided mainly by paediatricians during the first years of life. Specific effects of lower parental education on age at diagnosis in families with a migrant background point to the importance of utilising existing preventive systems for systematic ASD screening and to the need to improve parent education on child development and health care services. The almost complete absence of diagnoses of milder forms of ASD (Asperger\u2019s disorder) in children with a migrant background demonstrates the need for improving awareness of the whole autism spectrum by training professionals in health care and education and extending education to parents. Restricted and repetitive behaviours, language delay, and the combination of severe language delay with a relatively higher nonverbal development are possible symptoms of autism that can be used to identify ASD by observation by parents or professionals and by their inclusion in systematic screening tools.For children both with and without a migrant background, referrals by primary care paediatricians significantly decreased age at diagnosis. A universal implementation of a streamlined model from systematic screening to timely diagnosis is expected to further reduce age at ASD diagnosis and to facilitate earlier access to specialised intervention."}
+{"text": "A practical enantioselective total synthesis of the unnatural (+)-quinine and (\u2013)-9-epi-quinine enantiomers, which are important organocatalysts, is reported. epi-quinine enantiomers, which are important organocatalysts, is reported. The key transformation is a successive organocatalytic formal aza [3 + 3] cycloaddition/Strecker-type cyanation reaction to form an optically active tetrasubstituted piperidine derivative. This organocatalytic reaction proceeded in high yield and gave excellent enantiomeric excess with only 0.5 mol% catalyst loading. In addition, an imidate group, derived from a cyano group, was incorporated in the strategy for site-selective modification of the C4-alkyl chiral piperidine ring of quinine. Furthermore, an efficient coupling between the quinuclidine precursor and dihydroquinoline unit was achieved on a gram scale. The 15-step (LLS) synthetic protocol provided both (+)-quinine and (\u2013)-9-epi-quinine, each with 16% overall yield.A practical enantioselective total synthesis of the unnatural (+)-quinine and (\u2013)-9- The synthesis of cinchona alkaloids has advanced since the first  total synthesis of quinine (1) was achieved by Woodward and Doering.N2 cyclization (2) on a reasonable scale, we envisioned a synthetic design that could allow a practical synthesis of 2 through direct coupling between a quinoline unit and quinuclidine precursor that would expand the diversity of the aromatic portion and facilitate the development of novel organocatalysts (epi-quinine (3), which is a known precursor of urea, thiourea, and primary amine organocatalysts.9For more than a century, (\u2013)-quinine , which ilization .5 Subseqatalysts . In addi6 to be obtained, which can be transformed into quinuclidine precursor 7, a key unit of both 2 and 3. Initially, we considered coupling the quinoline fragment with quinuclidine 2-carbaldehyde; however, difficulties associated with controlling the stereochemistry of the aldehyde moiety on the quinuclidine ring were anticipated. Thus, we expected to introduce the quinoline fragment by using the thermodynamically controlled aldehyde attached on the chiral piperidine. To complete the total synthesis, direct coupling of the quinoline unit through nucleophilic addition of the metal complex of quinoline derivative to the C9 position of 7 followed by quinuclidine formation was designed.The synthetic plan for our approach is shown in 2 and (\u2013)-3 using only 0.5 mol% chiral source over 15 steps in 16% overall yield.Herein we report a practical and enantioselective total synthesis of (+)-11 by using our reported asymmetric formal aza [3 + 3] cycloaddition reaction employing diphenylprolinol diphenylmethylsilyl ether catalyst 10 (8 (1.1 equiv.), which was prepared in two steps,9 (1.0 equiv.), which was prepared in a one-pot operation from commercially available 1,3-dimethoxybenzylamine , and MeOH , provided the corresponding chiral 4-alkyl piperidine-2-ol. Cyanation of the hemiaminal moiety of the crude diastereomeric product mixture formed from the formal aza [3 + 3] cycloaddition reaction was carried out directly in a Strecker-type cyanation reaction. Thus, 6.0 equiv. of TMSCN in the presence of 1.2 equiv. of BF3\u00b7Et2O was added to the reaction mixture, and the desired cyano \u03b4-thiolactam 11 was obtained in 90% yield over two steps as a mixture of three diastereomers. Elaboration of 11 toward the tetrasubstituted piperidine intermediate 16 required site-selective reduction among the ester, nitrile, and thiolactam, followed by one-carbon elongation to form the terminal olefin. We initially tried several derivatizations with the nitrile group in place. However, most of the reaction conditions ultimately led to difficulties resulting from removal or reduction of the cyano group; therefore, we explored the derivatization of the cyano group first. After several experiments, we discovered the electron-deficient cyano group near the thiolactam was easily transformed into methyl imidate, which resists reduction conditions (see below). Thus, the diastereomer mixture of 11 was treated with DBU in the presence of MeOH to provide methyl imidate 12. At this stage, the two major diastereomers were isolated . The enantiomeric excess of each diastereomer was 94% ee. The stereochemistry of both diastereomers was determined by analysis of the coupling constants in 1H NMR spectra  and NaBH4 13 as a mixture of two diastereomers. The subsequent ester-selective reduction with diisobutylaluminum hydride  succeeded and the desired aldehyde was obtained without reduction of the imidate moiety. Subsequently, the methyl imidate moiety was hydrolyzed under mild acidic conditions . At the same time, the aldehyde was also isomerized to the thermodynamically more stable trans form, and the desired methyl ester 14 was obtained as a single diastereomer. As a result, by using the protocol reported herein, the cyano group was easily transformed into the ester via the imidate, while not having effected by the reduction.Our synthesis commenced with the enantioselective construction of the fully substituted piperidine alyst 10   because of enol formation and an unexpected degradation via retro-Mannich reaction. On the other hand, the one-carbon elongation of 14 was achieved upon treatment with Lewis acidic Tebbe reagent  to provide 15 in high yield . The C6 ester was then reduced with DIBAL-H  to provide tetrasubstituted piperidine derivative 16 in excellent yield (83%). As expected, after purification by silica gel column chromatography, the aldehyde in 16 spontaneously adopted the equatorial position in the six-membered ring system, which possesses the desired configuration for 2 and 3. Chiral piperidine 16 constitutes a potential key intermediate to prepare novel organocatalysts, and it was obtained in gram-scale quantities in eight steps in 32% overall yield from thiomalonamate 9 using only 0.5 mol% chiral source. In our established protocol, the C4 chiral center that was constructed by organocatalytic asymmetric reaction was used to control the configuration at the other two stereocenters (C3 and C6) through thermodynamic isomerization reactions. As a result, we could employ any of the diastereomers to prepare 16.The introduction of the vinyl group by using a Wittig reagent with strong basicity was not suitable for 16 by using a quinoline metal complex , all attempts failed to provide the coupling product with acceptable yield because of the unavoidable generation of side-products from the homo-coupling of quinoline.16. We then considered the use of 17 as an alternative nucleophile, given that deconjugation at the 1- and 2-positions on the quinoline ring of this compound was expected to both prevent the side reaction and increase the reactivity  provided the corresponding 4-lithiated dihydroquinoline. The addition of the latter to 16  gave the desired coupling product 18 in good yield  as a mixture of two diastereomers at the C-9 position (\u03b1/\u03b2 = 1\u2009:\u20091).Although we initially planned to introduce the quinoline derivative directly to activity . By usinachieved . Thus, t18 in hand, the stereoisomers could be separated by silica gel column chromatography, and a full optimization of the synthesis of (+)-quinine using the C-9 \u03b1-hydroxy isomer was accomplished  in the presence of Et3N (5.0 equiv.) afforded the precursor of the quinuclidine ring formation reaction. The tertiary amine was then rapidly obtained by quinuclidine formation in a thermal and neutral intramolecular SN2 reaction in toluene at 120 \u00b0C by spontaneous removal of the DMB group of the resulting ammonium salt in the presence of anisole. In this spontaneous removal of the benzyl moiety, the dimethoxy group on the benzene ring was essential; monomethoxy derivatives such as the PMB group did not have sufficient electron density to enable spontaneous removal. In addition, the avoidance of redox processes such as CAN oxidation or hydrogenation to remove the benzyl moiety was crucial to complete the total synthesis. Finally, removal of the acetyl and phenyl sulfone groups of 20 using potassium t-butoxide , provided 2 and 3, respectively, in excellent yield . The dihydroquinolines were aromatized via spontaneous aerobic oxidation or elimination of sulfinic acid under the reaction condition.At this stage, with over 2 g of 2) into (\u2013)-9-epi-quinine (3) under Mitsunobu conditions was already established,3 was a key intermediate in the recent development of a wide variety of organocatalysts such as primary amine, amide, urea, and thiourea catalysts (epi-quinine (3) was then converted into (+)-quinine (2). Thus, 3 was treated with p-nitrobenzoic acid , DEAD (1.1 equiv.), and PPh3  to provide the corresponding ester, which was then hydrolyzed with LiOH in one pot to provide 2 in 78% yield. Synthesized (+)-2 was then recrystallized after treatment with aqueous H2SO4 solution to provide enantiopure unnatural quinine sulfate hydrate (see details in the ESIThe efficient conversion of (+)-quinine . The practical enantioselective total synthesis of (+)-2 and (\u2013)-3 required only 0.5 mol% of chiral source  and was achieved in 15 steps, including Mitsunobu conversion, both in 16% overall yield from thiomalonamate 9. Our synthesis not only provides the unnatural enantiomer of quinine, which is required in many areas of current chemical endeavour, but also enables several aromatic groups to be introduced to key intermediate 16. Thus, a wide range of new catalysts that were previously difficult to derive from naturally occurring cinchona alkaloids are now available by using our method. Indeed, further development of new classes of cinchona alkaloid-mimic catalysis is under way.In conclusion, the synthesis of the title compound was achieved by using an enantioselective formal aza [3 + 3] cycloaddition/Strecker reaction sequence, followed by sequential chemoselective reduction/transformation of ester, thiolactam, and cyano groups There are no conflicts to declare.Supplementary informationClick here for additional data file."}
+{"text": "SLCO1B1), its common genetic variants OATP1B1*1b, OATP1B1*5, OATP1B1*15, as well as OATP1B3 (SLCO1B3), OATP2B1 (SLCO2B1) and organic cation transporter (OCT)-1 (SLC22A1). Previously established transporter-overexpressing cells were used to measure (i) cellular remdesivir uptake and (ii) cellular uptake of transporter probe substrates in the presence of remdesivir. There was a high remdesivir uptake into vector-transfected control cells. Moderate, but statistically significant higher uptake was detected only for OATP1B1-, OATP1B1*1b and OATP1B1*15-expressing cells when compared with control cells at 5 \u00b5M. Remdesivir inhibited all investigated transporters at 10 \u00b5M and above. In conclusion, the low uptake rates suggest that OATP1B1 and its genetic variants, OATP1B3, OATP2B1 and OCT1 are not relevant for hepatocellular uptake of remdesivir in humans. Due to the rapid clearance of remdesivir, no clinically relevant transporter-mediated drug-drug interactions are expected.Remdesivir has been approved for treatment of COVID-19 and shortens the time to recovery in hospitalized patients. Drug transporters removing remdesivir from the circulation may reduce efficacy of treatment by lowering its plasma levels. Information on the interaction of remdesivir with drug transporters is limited. We therefore assessed remdesivir as substrate and inhibitor of the clinically relevant hepatic drug uptake transporters organic anion transporting poly-peptide (OATP)-1B1 ( Remdesivir is a small molecule nucleoside analog inhibitor developed for treating diseases caused by RNA viruses such as Ebola virus . As remdSLCO1B1), OATP1B3 (SLCO1B3) and OATP2B1 (SLCO2B1), mediate the uptake of a broad range of anionic endogenous compounds and drugs from blood Sulfobromophthalein (14 Ci/mmol) (BSP) and [14C]metformin (107 mCi/mmol) were purchased from Hartmann Analytic GmbH . -17\u03b2-glucuronide (45.7 Ci/mmol) and estrone sulfate (51.8 Ci/mmol) were from PerkinElmer .Remdesivir (purity > 99%) was from Selleck Chemicals . 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline  and all other chemicals were from Merck KGaA . [2. Functionality of the cells was confirmed by measuring uptake of prototypic substrates  cells stably expressing OATP1B1 refseq (=OATP1B1*1a), OATP1B1*1b, OATP1B1*5 and OATP1B1*15 , OATP1B3ates see .2PO4, 1.5 mM CaCl2, 1.2 mM MgSO4, 5 mM glucose, pH 7.3; for OCT1: 145 mM NaCl, 5 mM hydroxyethylpiperazine ethanesulfonic acid, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, pH 7.4. Uptake was initiated by replacing this solution with uptake buffer containing different concentrations of remdesivir as indicated. Uptake was carried out at 37 \u00b0C and stopped after 10 min by removal of the uptake solution and then washing the cells twice with ice-cold uptake buffer and twice with ice-cold phosphate-buffered saline. Plates were frozen at \u221220 \u00b0C until lysis. For cell lysis, plates were thawed on ice and cells were harvested by scraping them off in 250 \u00b5L acetonitrile/H2O 1:1 (v/v)  containing 0.1% formic acid and transferring them into Eppendorf tubes. The lysis buffer contained QX as internal standard at a final concentration of 0.5 \u00b5M. Cells were disrupted by three cycles of shock freezing/thawing  and further by ultra-sonification, three times 3 sec, at 4 \u00b0C. Disrupted cell solutions were centrifuged 5 min  at 4 \u00b0C and supernatants were transferred into Eppendorf tubes and stored at \u221220 \u00b0C for analytic determination of remdesivir using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS).Uptake assays were conducted at 37 \u00b0C according to previously described methods ,18,19,20Prototypic substrate uptake was performed as described previously. For this, uptake solutions contained the respective substrate with tracer amounts of radiolabeled substrate. In case of inhibition studies, uptake solutions additionally contained different concentrations of remdesivir. Cells were incubated at 37 \u00b0C with the uptake solutions and uptake was stopped by removal of the uptake solutions and washing the cells three times with ice-cold uptake buffer. Cells were then lysed with 0.2% sodium dodecyl sulfate and intracellular radioactivity was measured by liquid scintillation counting . The following conditions were used: final concentration of 0.05 \u00b5M BSP and 10 min uptake for OATP1B1 , 5 \u00b5M esProtein contents of the lysed cells were determined with the bicinchoninic acid (BCA) assay according to Smith et al.  and as dCell viability was not determined because it is not expected that the short term incubations with remdesivir of less than 10 min will affect viability.Remdesivir was determined by UHPLC-MS-MS analysis on an Agilent 1290 infinity UHPLC system coupled to an Agilent 6495B triple quadrupole mass spectrometer  similar to a recently published method . QX was v/v) formic acid in water and (B) 0.1% (v/v) formic acid in acetonitrile as mobile phases. The gradient started at 10% B for 0.30 min, increased to 20% B to 0.35 min, then to 70% B to 1.5 min, and to 90% B to 1.8 min, remained at 90% B until 3.8 min, followed by re-equilibration to 10 % B for 1.7 min. The flow rate was 0.4 mL/min, and the injection volume was 0.5 \u00b5L. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode, using the transitions, m/z 603.2 > 228.9 for remdesivir and m/z 313.2 > 247.1 for QX. Dwell time was 100 ms. Fragmentor voltage was set at 380, and the collision energy at 32 and 40 V for remdesivir and QX, respectively.The analytes were separated on an Acquity HSS T3 column  using (A) 0.1% (v/v) containing 0.1% formic acid and 0.5 \u00b5M internal standard, in a concentration range of 5 nM to 10 \u00b5M. Calibration samples were analyzed together with the unknown samples. Calibration curves based on internal standard calibration were obtained by weighted (1/x) linear regression for the peak area ratio of the analyte to the internal standard against the amount of the analyte. The concentration in unknown samples was obtained from the regression line. Assay accuracy and precision were determined by quality controls that were prepared like the calibration samples. Data on method validation are summarized in the Remdesivir calibration samples were prepared in water: acetonitrile 1:1  and are means \u00b1 SEM. The Brown-Forsythe/Welch ANOVA test followed by a Dunnett\u2019s T3 posthoc test as multiple comparisons test were performed to determine statistical significance in all experiments. Statistical tests were two-tailed and max value was 3.7 \u00b5M after intravenous administration of clinically-used doses of 100 mg remdesivir to healthy adults [At first, accumulation of 5 \u00b5M remdesivir into cells expressing OATP1B1, OATP1B3, OATP2B1 or OCT1 was investigated. This is a relevant clinical concentration because the Cy adults . A high As statistically significant uptake was only detected for OATP1B1 and OATP1B1 has been reported as remdesivir transporter  and geneWhile the low uptake rates suggested that OATP1B1, OATP1B3, OATP2B1 and OCT1 are not relevant for hepatocellular remdesivir uptake, remdesivir might still interact with these transporters as inhibitor. We therefore investigated whether remdesivir inhibits the uptake of prototypic transporter substrates. Remdesivir had a significant concentration-dependent inhibitory effect on all investigated transporters .Remdesivir is the first FDA-approved antiviral agent for treatment of hospitalized COVID-19 patients . It expeOur data of high remdesivir uptake already into control cells and low uptake ratios at a clinically relevant drug concentration suggest that neither OATP1B1, OATP1B3, OATP2B1 nor OCT1 are important for hepatocellular remdesivir uptake in humans. Al-though the information given in the package insert of Veklury identifies OATP1B1 as transporter of remdesivir  and our We also analyzed the effect of the different OATP1B1 genetic variants OATP1B1*1b, OATP1B1*5 and OATP1B1*15 on remdesivir uptake because these had been shown to impact on the cellular uptake of clinically-used drugs, such as statins, with consequences on pharmacokinetics and drug effects ,12,13. D50 values of 2.8 \u00b5M and 2.1 \u00b5M, respectively, however without stating the prototypic substrates used for the experiments. Recently, inhibition of OATP2B1-mediated pyranine transport with an IC50 value of 3.8 \u00b5M was reported [1/2 = 1 h) [As OATP1B1, OATP1B3, OATP2B1 and OCT1 are known to mediate drug-drug interactions ,9,11,12,reported . Our dat2 = 1 h) , its pot2 = 1 h) .In conclusion, this is the first report on interaction of remdesivir with the major hepatic drug uptake transporters OATP1B1, OATP1B3, OATP2B1 and OCT1. Our data indicate that these transporters do not play a crucial role in hepatocellular uptake of remdesivir or in drug-drug interactions mediated by these transporters."}
+{"text": "Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote ( In 1965, Monod, Wyman and Changeux postulated the model of allosteric modulation in proteins . AccordiChangeux subsequently devoted much of his scientific career to the study of allosteric proteins, with specific attention paid to the nicotinic acetylcholine receptor (nAChR). This protein is a ligand-gated ion channel (LGIC) and thus in effect has no substrate, but the principle of allosteric modulation is similar in that binding of acetylcholine (ACh) shifts the thermodynamic equilibrium from a closed channel state to an open channel state through binding to a site\u00a0~50 \u00c5 away from the channel gate. The nAChR is a member of a superfamily of pentameric LGICs (pLGICs), which play major roles in fast synaptic transmission in the central and peripheral nervous systems, and are the site of action of many therapeutic drugs.3 serotonin receptor  as allosteric modulators. We discovered functionally active nanobodies, which act either as a PAM or NAM on ELIC, and determined co-crystal structures to elucidate the nanobody interactions with ELIC. One of the structures reveals an allosteric binding site located near the vestibule of the extracellular ligand-binding domain. Comparison of conservation and divergence in this site in different prokaryotic and eukaryotic receptors suggests a mechanism for achieving subtype-selective allosteric modulation across the receptor superfamily. Using cysteine-scanning mutagenesis and electrophysiological recordings, we show the vestibule site can also be targeted for modulation of the human 5-HTXenopus oocytes and employed automated electrophysiological recordings to characterize a panel of more than 20 different ELIC nanobodies. While none of the nanobodies had any functional effect on ELIC when applied alone, we found that co-application with the agonist GABA evoked a response that broadly falls into three categories. One type of nanobody enhanced the agonist-evoked response, while a second type of nanobody diminished the agonist-evoked response, and the\u00a0third type had little to no effect. From these classes, we selected several enhancers (PAMs) and inhibitors (NAMs) for a detailed electrophysiological characterization as potential allosteric modulators. In parallel, we conducted X-ray diffraction screening of ELIC plus nanobody co-crystals for structural elucidation. From this selection, we obtained a PAM-active nanobody (PAM-Nb) as well as another NAM-active nanobody (NAM-Nb) and determined their structures bound to ELIC by X-ray crystallography.In this study, we took advantage of nanobodies, which are high-affinity single chain antibodies derived from llamas; they have been widely employed to facilitate structural studies  and also50-value of 5.37\u00a0\u00b1\u00a00.03 (EC50: 4.2 \u03bcM) and Imax\u00a0=\u00a0257 \u00b1 14% . In contrast, co-application of a range of NAM-Nb concentrations demonstrates that NAM-Nb decreases the agonist response  and Imax\u00a0=\u00a034 \u00b1 2% (n\u00a0=\u00a06). Unlike competitive antagonists, which fully inhibit the agonist response at saturating concentrations, the inhibition of NAM-Nb levels off at 70% of the response with GABA alone, consistent with the mode of action of certain NAMs. We further investigated the effects of PAM-Nb and NAM-Nb on the GABA concentration-activation curve and obtained results that support their effects as positive and negative allosteric modulators  or negative (NAM-Nb) allosteric modulators, we solved X-ray crystal structures of ELIC in complex with the PAM-Nb or NAM-Nb, respectively . The strA more detailed analysis of the interaction interface between both nanobodies and ELIC reveals remarkable features . The PAMThe mode of interaction of the NAM-Nb with ELIC is distinct from the PAM-Nb . The intThe pores of both Nb complexes resemble previous structures of ELIC, with narrow constriction points at the 9\u02b9, 16\u02b9, and 20\u02b9 positions , suggestTo further investigate the possible conservation of the vestibule binding site in different prokaryote and eukaryote pLGICs, we performed a systematic analysis of the vestibule site architecture in all currently available pLGIC structures. The results from this analysis show that the outer rim of the vestibule site, which corresponds to residues N60-F95 in ELIC, can adopt one of three possible conformations .3AR  and F125 (bottom), respectively, as well as residues deeper into the vestibule site, N147, K149 and L151 on the \u03b26-strand and Y86 on the \u03b22-strand  of the concentration-activation curve after MTSEA-biotin modification as well as a shift of the EC50-value to lower concentrations  versus pEC50: 4.96\u00a0\u00b1\u00a00.04, n\u00a0=\u00a05 (EC50: 11 \u03bcM)), indicating a strong PAM-effect at this position . The L151C mutation alone causes a\u00a0~50 fold increase of the EC50-value compared to wild type (EC5098 \u03bcM versus 1.8 \u03bcM), suggesting that this mutation impacts function, possibly because L151 contributes to a hydrophobic patch in the center of the subunit (it is\u00a0<4 \u00c5 from I100 and L131), and this, or the mutation itself, could affect the adjacent Cys-loop and loop B. Modification of this residue with MTSEA-biotin appears to change the EC50 (11 \u03bcM) back toward wild-type value (1.8 \u03bcM). Mutants T112C and K149C also showed a lesser but significant decrease of the EC50-value after MTSEA-biotin, namely 6.7 \u03bcM  versus 2.9 \u03bcM  for T112C  versus 2.1 \u03bcM  for K149C , \u03b17 nAChR modulation using a fragment-based screening approach (2+-mediated inhibition of the \u03b11 GlyR (Representative current traces of channel responses to increasing concentrations of serotonin (5-HT) are shown in trations   and incubated in an orbital shaker overnight at 28\u00b0C. Cells were harvested by centrifugation, resuspended in TES buffer  and incubated for 1 hr. To this fraction four times diluted TES buffer was added and incubated for 1 hr. This fraction was cleared by centrifugation at 10,000 g. The supernatant was incubated with Ni Sepharose 6 Fast Flow resin  and incubated for 1 hr at room temperature. The beads were washed with buffer containing 20 mM TRIS pH 8.0, 300 mM NaCl and 40 mM imidazole. Protein was eluted with the same buffer supplemented with 300 mM imidazole. The eluted protein was concentrated to less than 1 ml on a 3 kDa cut-off Vivaspin concentrating column (Sartorius) and further purified on a Superdex 75 10/300 GL column  equilibrated with 10 mM Na-phosphate (pH 8.0) and 150 mM NaCl. Peak fractions corresponding to nanobody were pooled and spin-concentrated to\u00a0~50 mg/ml.A series of nanobodies were individually expressed in the periplasm of Xenopus oocytes, we used the pGEM-HE expression plasmid . 2 ng of mRNA per oocyte was injected into the cytosol of stage V and VI oocytes using the Roboinject automated injection system (Multi Channel Systems). Oocyte preparations and injections were done using standard procedures . Cells were superfused with standard OR2 solution containing 82.5 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mg/L BSA and 5 mM HEPES buffered at pH 7.4. Cells were held at a fixed potential of \u221280 mV throughout the experiment. Agonist-evoked current responses were obtained by perfusing oocytes with a range of GABA concentrations in OR2 solution. Different nanobodies diluted into OR2 solution were tested at a range of concentrations by pre-incubation with nanobody alone and followed by a co-application of nanobody with 5 mM GABA. Data acquired with the HiClamp were analyzed using the manufacturer\u2019s software (Multi Channel Systems). Concentration-activation curves were fitted with the empirical Hill equation as previously described  and incubated in an orbital shaker at 20\u00b0C overnight. After cell lysis, membranes were collected by ultracentrifugation at 125,000 x g and solubilized with 2% (w/v) anagrade n-undecyl-\u03b2-D-maltoside  at 4\u00b0C overnight. The cleared supernatant containing the solubilized MBP-ELIC fusion protein was purified by affinity chromatography on amylose resin (New England Biolabs). Column-bound ELIC was cleaved off by 3CV protease in the presence of 1 mM EDTA + 1 mM DTT at 4\u00b0C overnight. A final purification step was carried out on a Superdex 200 Increase 10/300 GL column  equilibrated with buffer containing 10 mM Na-phosphate pH 8.0, 150 mM NaCl, and 0.15% n-undecyl-\u03b2-D-maltoside . Peak fractions containing pentameric ELIC were pooled, concentrated to\u00a0~10 mg/mL and relipidated with 0.5 mg/mL E. coli lipids (Avanti Polar Lipids). Nanobodies were added at a 20% molar excess calculated for monomers and incubated at room temperature 2 hr prior to setting up crystallization screens with a Mosquito liquid handling robot (TTP Labtech). Crystals for the ELIC complex with PAM-Nb grew at room temperature in the presence of 0.1 M GABA, 0.2 M Ca(OAc)2, 0.1 M MES buffer pH 6.5% and 10% PEG8000. Crystals for the ELIC complex with NAM-Nb grew at room temperature in the presence of 0.1 M Na2SO4, 0.1 M bis-trispropane pH 8.5% and 10% PEG3350. Crystals were harvested after adding cryo-protectant containing mother liquor gradually supplemented with up to 25% glycerol in 5% increments. Crystals were then plunged into liquid nitrogen and stored in a dewar for transport to the synchrotron.Purified ELIC protein was produced as previously described, but with minor modifications . In brieDiffraction data for the ELIC+PAM-Nb structure were collected at the PROXIMA 1 beamline of the SOLEIL synchrotron . Diffraction data for the ELIC+NAM-Nb structure were collected at the X06A beamline of the Swiss Light Source . Both structures were solved by molecular replacement with Phaser in the CCP4 suite  using thXenopus oocytes were purchased from Ecocyte (Germany) and stored in ND-96  containing 2.5 mM sodium pyruvate, 50 mM gentamicin and 0.7 mM theophylline. cDNA encoding human 5-HT3AR was cloned into the pGEM-HE expression plasmid (3AR current recordings were obtained using a Roboocyte voltage-clamp system (Multi Channel systems) at a constant voltage clamp of \u221260 mV. Oocytes were perfused with ND-96 with no added calcium, and 5-HT  was diluted in this media. Oocytes were tested at 10 \u03bcM 5-HT before obtaining concentration-response curves. MTSEA-biotin (Biotium) was diluted immediately prior to application into calcium-free ND-96 solution at a concentration of 2 mM from a stock solution of 500 mM in DMSO. Analysis and curve fitting was performed using Prism v4.03 (GraphPad Software). Concentration-response data for each oocyte were normalized to the maximum current for that oocyte. Data are presented as the mean\u00a0\u00b1\u00a0standard error of the mean (SEM) with the raw data points overlaid as a dot plot in the relevant figures.Stage V-VI  plasmid . MutantsXenopus electrophysiology experiments were repeated three\u00a0to\u00a0eight times. The number of \u2018n\u2019 is mentioned in the relevant sections of the main text. We define each separate oocyte recording as a biological repeat. No data were excluded, unless the oocyte gave no detectable current. All electrophysiology experiments were conducted on automated devices, either the HiClamp or the Roboocyte, so essentially there was no human bias in recording of these data. Data are presented as the mean\u00a0\u00b1\u00a0standard error of the mean (SEM) with the raw data points overlaid as a dot plot. Statistical comparison between groups of data was performed using an unpaired two-tailed t test or an ANOVA followed by a Dunnetts multiple comparisons test as appropriate; the significance value p is mentioned in the relevant sections of the manuscript.All The X-ray diffraction data sets were collected from single crystals and typically the data set with the highest resolution was used for structural elucidation. Equivalent reflection data were recorded multiple times in agreement with the rotational symmetry of the crystal packing. The relevant data multiplicity value for each data set is mentioned in the crystallographic table . All asp In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Using a combination of X-ray crystallography and targeted mutagenesis combined with functional assays, this manuscript identifies two nanobodies that allosterically modulate the bacterial pentameric ligand-gated ion channel (pLGIC), ELIC, and examines the structural mechanisms underlying their functional effects. One nanobody acts as a positive allosteric modulator (PAM) and the other a negative allosteric modulator (NAM). X-ray crystal structures reveal that the NAM-nanobody binds to a novel site in the upper extracellular domain vestibule of ELIC. The PAM-nanobody binding site overlaps with a previously-identified allosteric drug site in the related nicotinic acetylcholine receptor. Guided by the structures, the authors examine the vestibule binding site in the related serotonin type 3 receptor and show that perturbing this region  alters channel activation suggesting the site might represent a new region to target for drug development. The data provide new information about mechanisms underlying allosteric modulation of pLGICs and will be of interest to many in the field.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kenton Swartz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Alexander Sobolevsky (Reviewer #1); Alexandru Radu Aricescu (Reviewer #3).Thank you for submitting your article \"A functionally conserved mechanism of modulation via a vestibule site in pentameric ligand-gated ion channels\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The major results and conclusions are:1) Identified two nanobodies that bind to ELIC: one that acts as a positive allosteric modulator (PAM) and the other as a negative allosteric modulator (NAM).2) Solved high-resolution structures of ELIC bound to PAM-nanobody and to NAM-nanobody.3) The NAM-nanobody binds to a novel site in the upper ECD vestibule of ELIC.4) Covalently linking a bulky reagent MTSE-biotin into a pocket in the vestibule region in the 5HT3R modulates serotonin-evoked currents suggesting the upper ECD vestibule might represent a new target for developing drugs.The authors' conclusions are supported by the experimental data. The manuscript is well written and offers new insights into a novel intra-vestibule binding site on pentameric ligand-gated ion channels (pLGICs).Addressing the following essential revisions will strengthen the manuscript.Essential Revisions:1) Revise title so it is less general and does not overstate manuscript's main conclusion. Abstract should be revised and describe specific experiments done and results of these experiments. Do not generalize results to all pLGICs.2) Previous work from Hassaine et al., 2014 (5HT3R) and Miller et al., 2018 (bioRxiv) have reported that nanobodies modulate pLGIC function \u2013 please mention this point and add references.3) A modulatory drug vestibule site may not be a general feature of all pLGICs. For GABA-A receptors, vestibule is occupied by glycans and thus is not accessible. Authors need to be careful not to generalize the conclusions drawn from their work to all pLGIC members.4) Authors need a more thorough analysis/discussion of residues involved in positive versus negative modulation. Figure 2 needs to be revised and/or an additional supplementary figure should be included to highlight the residue side chains that interact with the nanobodies versus small ligands. Compare and contrast nanobody interaction residues with CU2017 and flurazepam contact residues. Providing dose-response curves in the presence of the nanobodies would provide additional mechanistic insight into their mode of action.5) Please include/discuss whether there any examples of allosteric modulators that bind in the vestibule of other members of the pLGIC superfamily e.g. nAChRs, GABARs, GlyRs, 5HT3Rs. Could pLGICs with omega-in and omega-out conformations also be modulated via the vestibule?6) Include data showing effects of MTSEA-biotin on wild-type ELIC (control). Provide rationale for using MTSEA-biotin as the modifying reagent. Summarize SCAM results for the tested cysteine mutant receptors in a table.7) NAM nanobody blocks vestibule but only 35% of the GABA evoked current can be blocked. Additional discussion is needed to support idea that current enters via fenestrations at subunit interfaces. Show on ELIC structure where this could take place along with assessments of ion sizes in relation to fenestration/tunnel dimensions. Alternative mechanisms are possible and should be discussed. Could NAM-NB binding restrict flexibility in the ECD, thereby reducing the efficacy of the orthosteric agonist? Is it possible that the NAM-Nb-bound vestibule is occluded in the apo/desensitized states but not in an open state, due to an agonist-induced conformational change?8) The authors' need to elaborate more about omega loop and how they envision this region of the protein regulating channel activation and drug modulation. Are conformations of the omega loop different in GluCl apo, open, desensitized conformations, GLIC apo, open, desensitized structures, and in different GABA-A receptor conformations. Do they imagine this loop intrinsically flexible? Do you think only omega-open channels are drug targets? Previous studies on nACHR  and GABA-A receptors  have mutated this region and should be discussed.9) Flurazepam is a 'classic' benzodiazepine targeting an extracellular binding site between \u03b1- and \u03b3-subunits of GABA-A receptors, quite far from the (occluded) \u03a9-out vestibular site. It will be useful for the general reader, if you mention that flurazepam has more than one binding site in the pLGIC family. It should be clearly stated that flurazepam site in ELIC is not the site that corresponds to its action on the eukaryotic GABAR.10) L151C modification by MTSEA-biotin has large functional effects. Can the authors comment on why? The cysteine substitution of L151 alone has large effects on function. Please discuss.11) Figures 1 and 4 \u2013 please include the number of experiments performed for each of the dose response curves. Data are mean +/- SEM?12) NAM nanobody site in ELIC does not overlap with previously identified flurazepam site. NAM nanobody site sits above the cavity that binds flurazepam. Please comment on and discuss.13) Previous studies have shown that targeted modification of introduced cysteine residues by sulfhydryl reactive reagents throughout the ECD and TMD of many different pLGICs alter channel activation and/or drug modulation. It is unlikely that all of these sites are targets for drug development. The SCAM experiments are interesting and demonstrate that structural perturbations at these residues effect receptor activation but authors need to be careful about interpreting the data as evidence for a conserved allosteric binding site in the vestibule. Essential Revisions:1) Revise title so it is less general and does not overstate manuscript's main conclusion. Abstract should be revised and describe specific experiments done and results of these experiments. Do not generalize results to all pLGICs.We have revised the title and Abstract as requested.2) Previous work from Hassaine et al., 2014 (5HT3R) and Miller et al., 2018 (bioRxiv) have reported that nanobodies modulate pLGIC function \u2013 please mention this point and add references.These references are now cited and discussed in the subsection \u201cConclusion\u201d.3) A modulatory drug vestibule site may not be a general feature of all pLGICs. For GABA-A receptors, vestibule is occupied by glycans and thus is not accessible. Authors need to be careful not to generalize the conclusions drawn from their work to all pLGIC members.A receptors .We agree that modulation via the vestibule site may not be a general feature of pLGICs and have been more cautious in this revised version, where we have also mentioned N-linked glycan access restriction of the vestibule in GABA4) Authors need a more thorough analysis/discussion of residues involved in positive versus negative modulation. Figure 2 needs to be revised and/or an additional supplementary figure should be included to highlight the residue side chains that interact with the nanobodies versus small ligands. Compare and contrast nanobody interaction residues with CU2017 and flurazepam contact residues. Providing dose-response curves in the presence of the nanobodies would provide additional mechanistic insight into their mode of action.These are good suggestions. We have now analyzed residues involved in PAM-Nb and NAM-Nb interactions in more detail and compared them to CU2017 (NAM) and flurazepam (PAM) interactions, respectively. This is discussed in the third paragraph of the subsection \u201cCrystal structures of ELIC in complex with a PAM- or NAM-type nanobody\u201d, and new Figure 2\u2014figure supplement 1-2.We have also determined GABA concentration-activation curves in the presence of nanobodies as suggested, and indeed this has revealed further mechanistic insight consistent with their mode of action as allosteric modulators. This is discussed in the last paragraph of the subsection \u201cIdentification of nanobodies active as allosteric modulators on ELIC\u201d, and new Figure 1\u2014figure supplement 1.5) Please include/discuss whether there any examples of allosteric modulators that bind in the vestibule of other members of the pLGIC superfamily e.g. nAChRs, GABARs, GlyRs, 5HT3Rs. Could pLGICs with omega-in and omega-out conformations also be modulated via the vestibule?We now describe several known examples of vestibule site modulators in different members of the pLGIC superfamily in the last paragraph of the subsection \u201cCysteine-scanning mutagenesis in the vestibule site of the human 5-HT3A receptor\u201d.Regarding the modulation of \u03a9-in and \u03a9-out conformations, our structural analysis shows that vestibule access for these is restricted. It is perhaps possible (as we discuss in the revised manuscript) that ligand-induced conformational changes of the \u03a9-loop could occur, permitting vestibule site access, but in the absence of any evidence this is too speculative to consider in any detail.6) Include data showing effects of MTSEA-biotin on wild-type ELIC (control). Provide rationale for using MTSEA-biotin as the modifying reagent. Summarize SCAM results for the tested cysteine mutant receptors in a table.3 receptor, not to ELIC. The data are now summarized in a new Supplementary file 3 along with data from wild type 5-HT3R . We also provide a rationale for using MTSEA-biotin in the first paragraph of the subsection \u201cCysteine-scanning mutagenesis in the vestibule site of the human 5-HT3A receptor\u201d.We assume this remark relates to the 5-HT7) NAM nanobody blocks vestibule but only 35% of the GABA evoked current can be blocked. Additional discussion is needed to support idea that current enters via fenestrations at subunit interfaces. Show on ELIC structure where this could take place along with assessments of ion sizes in relation to fenestration/tunnel dimensions. Alternative mechanisms are possible and should be discussed. Could NAM-NB binding restrict flexibility in the ECD, thereby reducing the efficacy of the orthosteric agonist? Is it possible that the NAM-Nb-bound vestibule is occluded in the apo/desensitized states but not in an open state, due to an agonist-induced conformational change?A receptor structure (PDB 4COF), although slightly smaller in radius. This provides additional support for an alternate ion pathway in the NAM-Nb bound ELIC state. This analysis was conducted using the program CAVER, which also revealed a partially-blocked ion conduction pathway along the extracellular vestibule entrance. This result was also confirmed using the program HOLE and could provide an additional explanation for the partial reduction of the GABA-evoked current in the presence of NAM-Nb. Finally, we also agree with the reviewer that the NAM-Nb could restrict flexibility in the ECD, thereby limiting a gating transition to an open/active conformation. This is now discussed in the last paragraph of the subsection \u201cCrystal structures of ELIC in complex with a PAM- or NAM-type nanobody\u201d.We analyzed ELIC structures in apo as well as nanobody-bound structures and found lateral fenestrations at the same location as in the \u03b23 GABAWe have updated the pore radius profiles throughout the study with the results from HOLE and CAVER, as the program CHAP appears to produce deviating results for the vestibule entrance see .8) The authors' need to elaborate more about omega loop and how they envision this region of the protein regulating channel activation and drug modulation. Are conformations of the omega loop different in GluCl apo, open, desensitized conformations, GLIC apo, open, desensitized structures, and in different GABA-A receptor conformations. Do they imagine this loop intrinsically flexible? Do you think only omega-open channels are drug targets? Previous studies on nACHR  and GABA-A receptors  have mutated this region and should be discussed.We have elaborated on this in the revised manuscript. A comprehensive analysis of all pLGIC structures available to date, including >100 GLIC structures, revealed no clear correlation between the \u03a9-loop conformation and apo, open, desensitized structures in GluCl or GLIC, or any other pLGICs. Analysis of the average B-factor per residue, which is used as an indicator of vibrational movement, did reveal that in certain structures the \u03a9-loop has enhanced B-factors relative to other parts of the structure. Some of the most striking examples are the \u03a9-in loop of the a3 subunit in the a3b4 nAChR  , the \u03a9-out loop in POPC-bound GluCl   and the \u03a9-open loop in GLIC T25\u2019A  . These examples also represent possible intermediate or end states of the gating cycle, suggesting that the \u03a9-loop could show enhanced movement during the gating reaction. This is consistent with the very relevant studies mentioned by the reviewers, which are now cited and discussed (subsection \u201cSubtype-dependent vestibule site access in different prokaryote and eukaryote receptors\u201d).9) Flurazepam is a 'classic' benzodiazepine targeting an extracellular binding site between \u03b1- and \u03b3-subunits of GABA-A receptors, quite far from the (occluded) \u03a9-out vestibular site. It will be useful for the general reader, if you mention that flurazepam has more than one binding site in the pLGIC family. It should be clearly stated that flurazepam site in ELIC is not the site that corresponds to its action on the eukaryotic GABAR.ARs. See the third paragraph of the subsection \u201cCrystal structures of ELIC in complex with a PAM- or NAM-type nanobody\u201d.We now state explicitly that flurazepam has multiple binding sites and that the vestibule site in ELIC does not correspond to the high affinity binding benzodiazepine binding site in human GABA10) L151C modification by MTSEA-biotin has large functional effects. Can the authors comment on why? The cysteine substitution of L151 alone has large effects on function. Please discuss.50-value compared to wild type (EC50 98 \u00b5M versus 1.8 \u00b5M) suggesting that this mutation impacts function. In the revised manuscript we suggest it might be because L151 contributes to a hydrophobic patch in the center of the subunit (it is < 4 \u00c5 from I100 and L131) and this \u2013 or the mutation itself \u2013 could affect the adjacent Cys-loop and loop B. This is discussed in the last paragraph of the subsection \u201cCysteine-scanning mutagenesis in the vestibule site of the human 5-HT3A receptor\u201d.This is indeed interesting as the L151C mutation alone significantly increases the EC11) Figures 1 and 4 \u2013 please include the number of experiments performed for each of the dose response curves. Data are mean +/- SEM?The number of experiments is now mentioned in the text as well as the summarizing SCAM table (new Supplementary file 3). Data are presented as the mean \u00b1 standard error of the mean (SEM) with the raw data points overlaid as a dot plot in the relevant figures. We now mention this specifically in the last paragraphs of the subsections \u201cIdentification of nanobodies active as allosteric modulators on ELIC\u201d , \u201cCysteine-scanning mutagenesis in the vestibule site of the human 5-HT3A receptor\u201d , the Materials and methods section, and in the \u201cTransparent Reporting Form\u201d.12) NAM nanobody site in ELIC does not overlap with previously identified flurazepam site. NAM nanobody site sits above the cavity that binds flurazepam. Please comment on and discuss.The NAM-Nb interaction site is the a\u20191-helix in ELIC (N60-N69), which forms the top of the vestibule binding site. The flurazepam site and NAM-Nb site are adjacent to each other, see new Figure 2\u2014figure supplement 2. This is discussed in the third paragraph of the subsection \u201cCrystal structures of ELIC in complex with a PAM- or NAM-type nanobody\u201d.13) Previous studies have shown that targeted modification of introduced cysteine residues by sulfhydryl reactive reagents throughout the ECD and TMD of many different pLGICs alter channel activation and/or drug modulation. It is unlikely that all of these sites are targets for drug development. The SCAM experiments are interesting and demonstrate that structural perturbations at these residues effect receptor activation but authors need to be careful about interpreting the data as evidence for a conserved allosteric binding site in the vestibule.3 receptor has an accessible vestibule site that very closely resembles ELIC. At the functional level, the mutagenesis and SCAM experiments also closely mimic the potentiation of ELIC by flurazepam. Combined, these data suggest that receptors with an accessible vestibule such as the 5-HT3 receptor could be targets for drug development.The reviewer is correct that SCAM experiments should be interpreted with caution, but at the structural level the 5-HT"}
+{"text": "Genetic factors contribute to the development of autism spectrum disorder (ASD), and although non-protein-coding regions of the genome are being increasingly implicated in ASD, the functional consequences of these variants remain largely uncharacterized. Induced pluripotent stem cells (iPSCs) enable the production of personalized neurons that are genetically matched to people with ASD and can therefore be used to directly test the effects of genomic variation on neuronal gene expression, synapse function, and connectivity. The combined use of human pluripotent stem cells with genome editing to introduce or correct specific variants has proved to be a powerful approach for exploring the functional consequences of ASD-associated variants in protein-coding genes and, more recently, long non-coding RNAs (lncRNAs). Here, we review recent studies that implicate lncRNAs, other non-coding mutations, and regulatory variants in ASD susceptibility. We also discuss experimental design considerations for using iPSCs and genome editing to study the role of the non-protein-coding genome in ASD. Autism spectrum disorder (ASD) is a neurodevelopmental disorder with complex genetic underpinnings, and our current understanding of specific genetic risk for ASD comes from the studies of rare mutations affecting DNA that encodes protein-coding exons and genes . HoweveAlthough genetically modified rodents can be invaluable model systems to explore functions of ASD-associated protein-coding genes , human rInduced pluripotent stem cells (iPSCs) can produce inexhaustible supplies of personalized neurons that are genetically matched to individuals with ASD or unaffected individuals . CRISPR writers, erasers, and readers of H3K4 methylation in ASD [ASD is now increasingly considered a disorder of synaptic connectivity, and the growing list of ASD-relevant genes has largely converged on two biological processes: synaptic transmission and regulation of gene expression , 48, 49.n in ASD  has led n in ASD . Neuronan in ASD .CHD8, DIPK2A/C3orf58, and NRXN1) or neuronal function . It was initially unknown whether non-coding variants would play a substantial role in ASD, but subsequent WGS studies suggest that approximately 5% of ASD cases may be accounted for by non-coding variants [cis-regulatory elements like promoters, enhancers, and RNA regulatory sequences in ASD, and many of the genes that are regulated by these elements have been functionally or genetically linked to ASD , which identified 15 intergenic loci that were sites of recurrent genomic rearrangement found in ASD subjects . Most ofvariants . Also, tvariants , 6. Furtvariants , althougvariants , 55\u201357. variants , 58. IntDLG2 and NR3C2, which have both been implicated in brain function or neurodevelopment [WGS studies have revealed extensive evidence for ASD-associated non-coding variants in transcriptional regulatory elements like promoters and enhancers, which are functionally annotated based on transcriptomics data and chromatin state analyses \u201362. WGS elopment .LEO1 gene, which was previously implicated in ASD by exome sequencing [LEO1 and MAPK6) in carriers of the deletion who had ASD. The deletions encompass a predicted regulatory element that interacted with the promoters of both LEO1 and MAPK6 [Several ASD-associated variants of transcriptional regulatory elements have been shown to directly affect gene expression. For example, one ASD-associated single nucleotide variant mapped to a predicted transcription factor binding site and drove aberrant expression of a reporter gene in the developing mouse forebrain, where the reference sequence was not active . Reportequencing . Interesnd MAPK6 . This rend MAPK6 . TogetheEIF4G1 and EIF4G3 is impaired in ASD, and deletion of the Eif4g1 microexon in mice led to prolonged neuronal activation, altered synaptic plasticity, and impaired social interactions [Misregulation of RNA splicing has also been implicated in ASD (reviewed in ), althouractions . These mGRIN1 [PTEN [SMEK1 [MBD5 [WGS studies have begun to uncover ASD-associated non-coding variants in splice signals and untranslated regions. For example, intronic single nucleotide variants that were predicted to alter splicing of transcripts from synaptic genes like GRIN1 , the ASDN1 [PTEN , and theN [SMEK1 . WGS stuN [SMEK1 , 5, partN [SMEK1 . The stuN [SMEK1 . MoreoveK1 [MBD5 \u201316  , and miRfeatures . Mice wifeatures , 72. Thefeatures , the majfeatures . Dozens features , 76. ForNK3 mRNA . Also, cNK3 mRNA . DespiteNRXN2 and CNTNAP2 [Several WGS or whole-exome sequencing studies have reported genetic variants predicted to affect miRNAs or miRNA target sequences. One study specifically tested the hypothesis that ASD-associated synonymous variants in coding sequences may affect miRNA binding sites, although no significant enrichment for miRNA binding sites was detected . The miR CNTNAP2 . TogetheMost of ~ 16,000 lncRNAs encoded by the human genome , 81 havePTCHD1-AS [PTCHD1-AS in ASD and also implicated the uncharacterized lncRNA AK127244 [MSNP1-AS [LINC00689, and LINC00693 [lnc-NR2F1) was shown to regulate autism risk genes and promote maturation of mouse stem cell-derived neurons [PTCHD1-AS (described below) provide direct evidence that ASD-associated non-coding RNAs directly regulate neurodevelopmental processes relevant to ASD.Data from genetic \u201322, 24 aTCHD1-AS , which iTCHD1-AS . A recenAK127244 , 19. GenINC00693 , althougThe aforementioned studies suggest that non-coding variants play important roles in the development of ASD, although the functions of these variants and the regulatory elements they disrupt remain largely unknown. Next, we discuss recent advances in cellular reprogramming and CRISPR technologies that are poised to greatly advance our understanding of ASD-associated non-coding variants.22q13.3+/\u2212, SHANK3\u2212/\u2212), impairments in neurotransmitter release , or hypofunction of excitatory NMDA receptors (PTCHD1-AS)  or hyperfunction of NMDA receptors (EHMT1+/\u2212) (Table Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human iPSCs, have the capacity to differentiate into unlimited supplies of brain cells and therefore have tremendous potential for modeling ASD . To dateThe protein-coding variants modeled to date are known or predicted to be of high penetrance, whereas the penetrance is unknown for most non-coding variants. Therefore, iPSC experiments modeling of non-coding variants must be carefully designed to minimize heterogeneity and experimental noise. Here, we discuss experimental design considerations for using iPSCs to model ASD, with a specific focus on challenges associated with modeling the consequences of non-coding variants.Published hPSC models of ASD have used two primary approaches: personalized iPSCs from donors with ASD or genome editing to introduce specific ASD-associated variants in reference lines. Modeling ASD with iPSCs typically uses a case-control model, where iPSC-derived neurons from people with ASD are compared to neurons from people who do not have ASD. These studies are often stratified by gene or shared neurodevelopmental phenotypes Table , and conCHD8 mutations revealed similar misregulated genes in monolayer neurons and organoids [CHD8+/\u2212 neurons included the lncRNA DLX6-AS1, which was also misregulated in organoids from people with idiopathic ASD and macrocephaly [NRXN1, STXBP1, SHANK3, and NLGN4  in PSCs or neural progenitor cells results in homogeneous populations of excitatory cortical neurons that mature in only 3\u20134 weeks [Direct conversion inherently overcomes heterogeneity by using specific transcription factors to swiftly generate pure populations of post-mitotic neurons , 96. Ect\u20134 weeks , 102. Ne\u20134 weeks . Direct \u20134 weeks  and astr\u20134 weeks . Due to \u20134 weeks .As a first step in deciding which approach to use for modeling a potential regulatory variant, it is important to determine when and where the regulatory element is active. Publicly accessible transcriptome data from the developing and adult human brain and from differentiating iPSC-derived neurons \u2013112 can We anticipate that non-coding variants will affect neuronal gene expression, but the design of expression analyses with iPSC-derived neurons can influence the interpretation of results. The heterogeneity that results from directed differentiation is undesirable for transcriptomics , 41 and PTCHD1-AS [cis-acting effects of non-coding variants on the expression of target genes, it may also be necessary to perform allele-specific gene expression analyses, which also benefits from improved sequencing coverage [Another consideration when modeling transcriptional consequences of non-coding variants in ASD is the need for substantial sequencing depth in transcriptomic analyses. Detection of alternatively spliced exons and low abundance lncRNAs requires thorough read coverage. Indeed, we recently found that a read depth of 60 million paired-end reads per sample was necessary to detect the ASD-associated lncRNA TCHD1-AS . Furthercoverage .iPSC models have confirmed the hypothesis that synaptic dysfunction underlies ASD Table , but theh) channels were observed in human and mouse neurons with SHANK3 mutations [EHMT1 also share decreased network burst frequency and increased NMDA receptor activity [STXBP1 [NRXN1 [SHANK2 [Some of the phenotypes observed in iPSC-derived neurons recapitulate those observed in mouse models, although contrasting phenotypes have also been reported from these two modeling approaches. Similar functional impairments in excitatory synapses and in hyperpolarization-activated cation , neurons can be plated on a grid of microelectrodes to simultaneously record extracellular voltage changes within a synaptic network . Multi-wCNTN5 and EHMT2 exhibited hyperactive networks [MEAs have recently been used to explore ASD-associated action potential firing and connectivity phenotypes Table , which hnetworks . iPSC-denetworks .MEA phenotyping is attractive for modeling ASD because the simple non-invasive recordings facilitate higher throughput applications than imaging or patch-clamp electrophysiology. However, a careful experimental design will be necessary to overcome extensive technical and biological variability in baseline MEA metrics . To overcome this variability, we recommend using isogenic controls when possible, performing ASD/control analyses on the same plate to account for batch effects, and establishing a schedule to ensure consistent latencies between media renewal and MEA recordings. Previous work in hPSC models of ASD has shown that some synaptic phenotypes can be rescued Table . The medTUNA [lnc-NR2F1 [LINC00473 is a primate-specific lncRNA that is robustly induced by synaptic excitation of human iPSC-derived neurons [NEAT1 is a highly abundant lncRNA that is downregulated in response to neuronal depolarization and interacts with epilepsy-associated potassium channels to regulate the excitability of human iPSC-derived neurons [Several recent studies have reported potential roles for miRNAs and lncRNAs in ASD-associated processes like neurodevelopment. Global expression analyses revealed that several miRNAs change in expression during differentiation of iPSC-derived neurons , 126. ExTUNA  and lnc-nc-NR2F1  regulate neurons  and may  neurons . NEAT1 i neurons . TogethePTHCD1-AS [PTCHD1-AS, along with iPSCs from three unaffected individuals. These iPSCs were differentiated into forebrain neurons, and phenotypic analyses revealed pronounced deficits in excitatory synaptic function, including decreased frequency of mEPSCs and diminished amplitude of NMDA-evoked currents. We also used genome editing to replace a critical exon of PTCHD1-AS with a premature polyadenylation sequence, which recapitulated the mEPSC frequency impairment and confirmed the importance of PTCHD1-AS in excitatory synaptic function. Our work with PTCHD1-AS therefore provides proof of principle that ASD-associated non-coding variants can have pronounced phenotypic consequences in human iPSC-derived neurons.We recently reported a human iPSC approach for modeling ASD-associated non-coding variants focused on the lncRNA THCD1-AS . We genecis functions at the endogenous site of lncRNA synthesis [Future analysis of non-coding variants in ASD will benefit from the concurrent application of CRISPR-based tools  for artiynthesis .ASD-associated enhancers and splice sites can also be evaluated using CRISPR-based approaches. Enzymatically inactivated Cas9 can be fused to catalytic domains that add or remove histone modifications to directly manipulate enhancer function. For example, Cas9-mediated recruitment of catalytic domains of p300 and HDAC8 has been used to artificially activate or block dynamically regulated enhancers in mouse neurons . CRISPR Although lncRNAs are defined in part by their limited protein-coding potential, recent results have challenged the notion that all lncRNAs are devoid of translated open reading frames. Several approaches have been developed for characterizing the translational landscapes of human cells, resulting in the surprising discovery that some lncRNAs are associated with ribosomes and may therefore undergo translation . HistoriContinued WGS will invariably lead to increasing numbers of ASD-associated non-coding variants being discovered. iPSCs and genome editing provide exciting opportunities to model the consequences of these variants in human neurons and for correlating gene expression changes with functional differences in synaptic connectivity. Careful experimental design and use of well-selected experimental controls (including isogenic controls when possible) will reduce experimental noise and heterogeneity, leading to more sensitive analyses. Determination of the phenotypic consequences of non-coding variants will provide insights into both the neuronal dysfunction that underlies ASD and the mechanisms governing the regulation of human genetic information."}
+{"text": "We infer that this pattern can be understood as a difference in the spectrum of subsistence activities employed in the Loess Plateau and the Yangtze-Huai regions, which can be partly explained by differences in environmental conditions. We argue that regional differentiation in dietary tradition are not driven by differences in the conventional \u201cstages\u201d of shifting modes of subsistence , but rather by myriad subsistence choices that combined and discarded modes in a number of innovative ways over thousands of years. The introduction of wheat and barley from southwestern Asia after 2000 cal BC resulted in the development of an additional east to west gradient in the degree of incorporation of the different staple products into human diets. Wheat and barley were rapidly adopted as staple foods in the Continental Interior contra the very gradual pace of adoption of these western crops in the Loess Plateau. While environmental and social factors likely contributed to their slow adoption, we explored local cooking practice as a third explanation; wheat and barley may have been more readily folded into grinding-and-baking cooking traditions than into steaming-and-boiling traditions. Changes in these culinary practices may have begun in the female sector of society.We conducted a meta-analysis of published carbon and nitrogen isotope data from archaeological human skeletal remains (n = 2448) from 128 sites cross China in order to investigate broad spatial and temporal patterns in the formation of staple cuisines. Between 6000\u20135000 cal BC we found evidence for an already distinct north versus south divide in the use of main crop staples (namely millet vs. a broad spectrum of C Staple foods pass through a long transformative process as they are acquired, prepared, and distributed by human societies, and the performances of staple food preparation and presentation are intimately connected with social relationships . Recent Cultivation of staple cereals has played a vital role in the development of many aspects of Chinese culture from prehistory to today. Globally, the process employs millions of people and presently feeds 20% of the world\u2019s population . Cerealsc. 18,000 years ago, associated with hunter-gathers  and [N] of the potential dietary resources, which we calculated from data available in the United States Department of Agriculture (USDA) Nutrient Database following Koch and Phillips [4 plants to human diet varies by just 4% among the models. We conducted Markov Chain Monte Carlo (MCMC) sampling within MixSIAR using the \u201cvery long\u201d chain length, which includes running three replicate chains, each with 1,000,000 draws, a burn-in of 500,000, and a thinning rate of 500. We used Gelman-Rubin diagnostics to confirm model convergence [To estimate the proportional contributions of potential plant and animal food resources to past human diets at Xinglonggou\u2014one of the oldest sites at which humans were using millet as a staple food\u2014we used the Bayesian stable isotope mixing model MixSIAR , 28 follPhillips . To accoPhillips , 31. We Phillips , but fouvergence . Althoug\u03b413C and \u03b415N values reflect both dietary protein and dietary non-protein disproportionately , and within the geographic framework of the three regions described above: the broad Loess Plateau, the Yangtze-Huai Region, and the Continental Interior .3 diet (mean \u03b413C < -17\u2030). In the Loess Plateau, human values from Xiaojingshan, Baijia and Beiliu are consistent with a mixed C3-C4 diet , while at Xinglonggou and Xinglongwa, people have carbon isotope values indicative of a C4-plant dominated diet (mean \u03b413C > -12\u2030). The two regions have significantly different \u03b413C and \u03b415N values .Carbon and nitrogen isotope values measured in human bones are reported from five sites dating to the period between 6000\u20135000 cal BC . With th\u03b413C values measured in human bone collagen are consistent with a C4 diet with relatively high \u03b415N values  [3 diet and relatively low nitrogen isotope values . Carbon isotope ratios in humans seemingly suggest that humans directly consumed millet as a staple food, perhaps on a daily basis. Nitrogen isotope ratios on the other hand, suggest that the animal protein consumption at Xinglonggou was also significant, with a human-animal collagen offset of about 5 \u2030 in \u03b415N values.Xinglonggou I  provides a unique case study, as there are additionally data available from a range of both plant and animal dietary sources. At Xinglonggou,  n = 32) . The maj4 plants to human diets at this site. The results suggest that the proportional contribution of C4 plants (likely millet) to human diet at Xinglonggou was between 52\u201362% . Twenty-seven out of thirty populations from the Loess Plateau show significant consumption of C4 plants , suggesting that there is probably northward expansion of rice cultivation at this time  suggests that their energy comes from a mixture of C3 and C4 resources. Humans from Jiangzhai and Shijia, on the other hand, plot more closely to the C4 protein line and their position along the y-axis, with apatite \u03b413C values > -5\u2030, suggests their dietary energy is derived primarily from C4 energy sources. Both of these sites are located on the Loess Plateau and these results help to clarify that some humans from this time period and region were likely consuming fully C4 diets. The individual from Banpo, another Loess Plateau site, tells a slightly different story because they fall between the C3 and C4 protein lines, suggesting a mixed protein diet; their apatite \u03b413C value is similarly suggestive of mixed C3 and C4 energy sources. Although this method is not quantitative, it nonetheless allows for energy and protein resources to be evaluated separately, allowing for a deeper understanding of past human diet than bulk collagen isotope values provide. Indeed, these data suggest that later in the period of 5000\u20132000 cal BC, some humans on the Loess Plateau were consuming millet directly as well as animals provisioned with millet (Jiangzhai and Shijia).Some interesting patterns emerged in the collagen versus apatite \u03b4 methods . At Jiah3 plants) [4 or mixed C3-C4 consumption. Conversely, humans from the Continental Interior exhibit a broader spectrum of dietary habits including predominantly-C3, mixed C3-C4, and predominantly-C4 diets. The two regions show statistically significant differences in \u03b413C and \u03b415N values . Human data from 39 out of 43 sites from the Loess Plateau suggest that millet consumption was very significant (\u03b413C > -12\u2030), while human data from 19 out of 28 sites from the Continental Interior are consistent with C3 or C3-C4 mixed diets (\u03b413C < -12\u2030). The significantly different \u03b415N values between the two regions could be the result of a combination of several factors, including variable animal protein input, differences in crop \u03b415N values caused by variable soil 15N enrichment, or aridity in the Continental Interior. The earlier north to south divide in staple crop use is accompanied by a new divide between the east and the west  \u201349. The west see .\u03b413C and \u03b415N values  could indicate that females had greater access to newly introduced C3 crops than males (n = 293). When gendered differences are considered at the provincial level, differences  are evident in several provinces where males display higher access to C4 resources and protein products  [4 foods. Broomcorn and/or foxtail millet were documented at all four northern sites  in high quantities [4 cereal has been identified in the plant macrofossil assemblages from this time period. There is microbotanical evidence for job\u2019s tears (a C4 plant) at Xinglonggou [Coix, job\u2019s tears were unlikely to be cultivated on a large enough scale to become a staple cereal 7500 years ago. The tradition of consumption of C4 crops as staple foods emerged in this period and was particularly pronounced among the Xinglongwa cultural communities. At Xinglonggou, we estimated the proportional contribution of C4 plants to human diet to be greater than 50%, nearly two-times more significant than herbivores, the second most important dietary resource.It is no exaggeration to say the millennium between 6000 and 5000 cal BC is crucial to understanding the origins of farming activities in East Asia , 16. Thes, nuts) . In the antities , 51. No glonggou , howeverAbove all, the distinct subsistence modes between north and south in Neolithic China are driven by regional environmental differences. The lower catchment of the Yangtze and Huai Rivers was an intricate deltaic wetland crisscrossed by hundreds of distributaries, merging and diverging with seasonal flooding. People in this region relied overwhelmingly on wetland resources, including rice\u2013an aquatic plant\u2014for their subsistence. In contrast, landscapes in the north form a single relatively uniform semi-arid zone across the Loess Plateau. From early on, millet cultivation became the key component of agrarian based subsistence in the Loess Plateau. Within this perspective, the broad spectrum of subsistence activities in the Yangtze-Huai within an environmental mosaic consisting of swamps, marshes, fens and wetlands can be seen as the mirror image of unified agrarian practices based on millet grain in the northern Loess Plateau. That is, both of these highly sustainable systems in the north and south took advantage of the subsistence options their landscape setting provided. And this arrangement seems to have persisted for another 3000 years . The regional difference in dietary tradition between north and south, along with the variation within each region, challenges the conventional \u201cstages\u201d of shifting modes of subsistence\u2013hunting, foraging, pastoralism, and farming\u2013in an evolutionary framework. Both historical and archaeological evidence shows that peoples moved fairly readily between distinctive modes of subsistence and the same people might have practiced more than one subsistence mode in a single lifetime , 54. In 4-dominated diet to a mixed C3-C4 diet at this time was concurrent with \u201cHolocene Event 3\u201d at 4200 BP  [The rapid adoption of wheat and barley as staple foods in the Continental Interior by 2000 cal BC contrasts the very gradual pace of the adoption of these western crops in the Loess Plateau. In a recent review focused on northern China, the authors noted that the shift from a C,250 BC) . A globa,250 BC) . But thiAn alternative interpretation lies in the deep-seated East and West culinary distinction. As established, boiling and steaming of grains and other foods appear to have been and remained the predominant East Asian methods for preparing foods. By contrast, cereals in southwestern and Central Asia such as wheat and barley were processed for a flour-focused food system. Such an East-West culinary distinction can be traced back to the pre-agricultural Palaeolithic . These cModern Chinese cuisine formed over thousands of years through the development of diverse regional subsistence systems and cuisines, which were further influenced by food traditions from other parts of the world. Our results help to illustrate the ways in which both environment and culture contributed to shaping the Chinese staple food system over the past 8000 years. A distinct north versus south divide in Chinese ancient staple cuisines was already evident isotopically between 6000\u20135000 cal BC and became more pronounced between 5000\u20132000 cal BC. We infer that this pattern is better understood as a difference in the spectrum of subsistence activities, which was partly driven by environmental differences between the Loess Plateau and the Yangtze-Huai region. The introduction of wheat and barley from southwestern Asia after 2000 cal BC resulted in the development of an additional east to west gradient in the degree of incorporation of the different staple products into human diets. We argue the regional differences in dietary tradition between and within the three broad regions throughout the Neolithic and the Bronze Age could not be understood in the conventional \u201cstages\u201d of shifting modes of subsistence: hunting-foraging-pastoralism-farming. Instead the same people might have practiced more than one subsistence mode and combined them in a number of innovative hybrids that co-existed over thousands of years. The rapid adoption of wheat and barley as staple foods in the Continental Interior by 2000 cal BC contrasts the very gradual pace of the adoption of these western crops in the Loess Plateau. Apart from the possible environmental and social drivers, we explored a third explanation that these novel grains may have at first been ignored as a staple grain because of their incompatibility with local culinary practice; people of the Loess Plateau belonged to a boiling-and-steaming culture while those in the Continental Interior belonged to a grinding-and-baking culture into which wheat and barley were more readily folded. Finally, in some cases in the Loess Plateau, newly introduced staple cereals from the West were consumed by females to a greater extent than males, suggesting that the female sector of society may have pioneered the innovations in culinary practice.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 TableData are from archaeological human skeletal remains (n = 2448) from 128 sites across China.(XLSX)Click here for additional data file.S2 TableSee  for deta(XLSX)Click here for additional data file.S3 TableA-D. ANOVA results by province. Results of comparisons of human \u03b413C (Table A in S3 Table) values and \u03b415N (Table B in S3 Table) by province in time period II , and results of comparisons of human \u03b413C (Table C in S3 Table) values and \u03b415N (Table D in S3 Table) by province in time period III .(XLSX)Click here for additional data file.S4 TableA-H. ANOVA results by sex. Results of comparisons of male and female \u03b413C (Table A in S4 Table) and \u03b415N (Table B in S4 Table) values by region in time period II ; ANOVA comparisons of human \u03b413C (Table C in S4 Table) and \u03b415N (Table D in S4 Table) values by sex and province in time period II ; ANOVA comparisons of male and female \u03b413C (Table E in S4 Table) and \u03b415N (Table F in S4 Table) values by region in time period III ; and ANOVA comparisons of human \u03b413C (Table G in S4 Table) and \u03b415N (Table H in S4 Table) values by sex and province in time period III .(XLSX)Click here for additional data file.S5 TableResults from the model run highlighted in green are reported in the text.(XLSX)Click here for additional data file."}
+{"text": "Translational failures in anti-adhesion molecule therapies after stroke reveal the necessity of developing new strategies that not only interrupt leukocyte recruitment but also consider the inhibition of endothelial cell inflammation, verification of therapeutic time window, and normal function maintenance of circulating leukocytes. Our study focused on the potential therapeutic value of CD151 downregulation in improving current anti-adhesion molecule therapies.Lentivirus intracerebroventricular administration was conducted to inhibit the CD151 expression and observe its functional influence on neurological injuries and outcomes. Then, immunohistochemistry and myeloperoxidase activity assessment were performed to explore the effects of CD151 expression on neutrophil and monocyte recruitment after rat cerebral ischemia. Primary rat brain microvascular endothelial cells were subjected to oxygen glucose deprivation and reoxygenation to elucidate the underlying working mechanisms between CD151 and VCAM-1.The CD151 downregulation remarkably reduced neurological injuries and improved neurological outcomes, which were accompanied with reduced neutrophil and monocyte infiltration after the CD151 downregulation. The VCAM-1 expression was remarkably decreased among the adhesion molecules on the endothelial cell responsible for neutrophil and monocyte infiltration. The activation of p38 MAPK and NF-\u03baB pathways was restricted after the CD151 downregulation. p38 MAPK and NF-\u03baB inhibitors decreased the VCAM-1 expression, and p38 acted as an upstream regulator of NF-\u03baB. However, CD151 downregulation did not directly influence the neutrophil and monocyte activation.Overall, CD151 regulated the expression of adhesion molecules. It also played a critical role in suppressing VCAM-1-mediated neutrophil and monocyte infiltration via the p38/NF-\u03baB pathway. This study possibly provided a new basis for improving current anti-adhesion molecule therapies.The online version contains supplementary material available at 10.1186/s12974-021-02171-6. Leukocyte infiltration after cerebral ischemia is linked to secondary injury after ischemic stroke . It is aNew evidence suggests the emerging role of tetraspanin in the interaction of immune and endothelial cells. As a member of the tetraspanin family, CD151 is known as a modulator of the activities of different transmembrane protein families; it also functions as a partner of adhesion molecules during chronic inflammation . CD151, This work aimed to investigate the therapeutic value of CD151 downregulation and its function in leukocyte infiltration after cerebral ischemia. Herein, we specifically focused on the changes in the expression of TEM components and other adhesion molecules in endothelial cells in neutrophil and monocyte recruitment after experimental stroke.A total of 239 specific pathogen-free male Sprague-Dawley rats were included, and a mortality of around 6% was observed. The rats weighing 250\u2013260\u2009g were first purchased from the Beijing Vital River Laboratory Animal Technology Co. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Tsinghua University. The animals were placed in a house with a 12-h light/dark cycle condition, a temperature of 23\u2009\u00b0C \u00b1 3\u2009\u00b0C, a relative humidity of 50\u201360%, and ad libitum access to water and chow. All the rats were randomly assigned to different groups. All the experiments followed a double-blind method.A rat experimental ischemic stroke model was established as previously described . After lThe lentivirus for expressing the CD151 shRNA (LV CD151 shRNA) and the scramble lentivirus with an inert random shRNA sequence (LV Vehicle) were purchased from GenePharma Corporation . Both the two lentiviruses expressed GFP tag. The lentivirus shRNA information is presented in detail in Table SThe rats were divided into three groups, namely, lentivirus vehicle injection and sham surgery (LV Vehicle + Sham), lentivirus vehicle injection and MCAO (LV Vehicle + MCAO), and lentivirus CD151 shRNA injection and MCAO (LV CD151 shRNA + MCAO).9 TU/ml) or 8\u2009\u03bcl of LV vehicle (1 \u00d7 109 TU/ml) at a rate of 0.5\u2009\u03bcl/min. After 5\u2009min, the needle was slowly withdrawn to prevent reflux.The rats were transfected with the lentivirus through ICV injection 7\u2009days before the MCAO surgery. Briefly, they were anesthetized with 2% sodium pentobarbital (60\u201380\u2009mg/kg) and placed in a stereotaxic apparatus . The ICV injection was performed using a 10-\u03bcl syringe, and coordinates were 2\u2009mm lateral to the bregma, 1.5\u2009mm posterior to the bregma, and 4\u20135\u2009mm deep from the dura. The rats received either 8\u2009\u03bcl of LV CD151 shRNA  in the cryosections of the brain tissues 7\u2009days after lentivirus injection Fig.\u00a0c.Fig. 1The rats were sacrificed 24\u2009h after reperfusion. As previously described , the braThe ischemic and contralateral hemispheres were collected 24\u2009h after reperfusion . The wetg for 10\u2009min. The supernatant (25\u2009\u03bcl) was mixed with ethanol (100\u2009\u03bcl), and the absorbance at 632\u2009nm was measured using a spectrophotometer . The content of Evans blue was quantified using a standard curve and expressed in nanograms of Evans blue per gram of brain tissue.The rats were injected with Evans blue  injection through the tail vein 1\u2009h before they were sacrificed. Before the brain tissue was harvested, the rats were perfused with phosphate-buffered saline (PBS) to remove the circulating dye. Then, the brain tissue was homogenized in 50% trichloroacetic acid (2\u2009ml), and the homogenate was centrifuged at 13,000Neurobehavioral tests, including Longa\u2019s score, Bederson score, forelimb placing test, and beam walking test, were conducted 24 and 72\u2009h after reperfusion. All the assessments were performed by an investigator blinded to the group treatment.Longa\u2019s scoring system  was adopted to evaluate neurological deficits .The Bederson score  was used to evaluate the overall neurological function .A forelimb placing test was carried out to assess the response to sense tactile stimulation from the vibrissae and the eyes. The test was composed of four parts. First, the rats were held gently by the tail, and their heads were slowly lowered to the table surface. Normally, the rats stretched out their forelimbs to the surface with symmetrical movements in the air. Second, the rats\u2019 forelimbs were placed at the edge of the table and gently pushed against the edge. Normally, the rats successfully resisted pushing. Third, the rats were placed parallel to the table edge, and their forelimbs were forced to the edge. Normally, the rats withdrew their paws immediately. Fourth, the rats\u2019 hindlimbs were placed on the table edge, and each paw was taken down alternately. Normally, the rats repositioned their legs quickly. Scores were recorded as follows: normal performance, 2 points; delayed (>\u20092\u2009s) and/or incomplete performance, 1 point; and no performance, 0 points .The beam walk test was used to evaluate motor coordination. The rats were first trained for 3\u2009days before MCAO surgery and pre-tested to pass through the beam  voluntarily without a slip. Motor performance was scored as follows: 0, no attempt to stay on the beam; 1, attempted to stay on beam but no movement; 2, attempted to cross the beam but failed; 3, crossed the beam but the contralateral hindlimb slipped by >\u200950%; 5, crossed the beam but the contralateral hindlimb slipped by <\u200950%; and 6, crossed the beam without a slip .Three 15-\u03bcm-thick brain tissue cryosections  from each brain were obtained 24\u2009h after reperfusion. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and NeuN labeling  were processed for neuronal apoptosis analysis in accordance with the manufacturer\u2019s instructions . Immunofluorescence images were captured using Axio Scan.Z1 . For each section, the average number of TUNEL-positive neurons was calculated from six randomly chosen fields from the penumbra. The average number of the three sections was recorded as the neuronal apoptosis value for each brain.Three 8-\u03bcm-thick brain tissue cryosections  from each brain were collected 24 or 72\u2009h after reperfusion. Myeloperoxidase (MPO) and CD115 were used as biomarkers to identify infiltrating neutrophils and monocytes, respectively. Endogenous peroxidase was blocked with a peroxidase blocking solution  for 30\u2009min after 4% PFA fixation. Then, all the sections were washed with PBS containing 0.1% Tween-20  and incubated with the primary antibody of MPO  or CD115  for 60\u2009min at 21\u2009\u00b0C and with the secondary antibody for 60\u2009min. An immunoenzyme polymer  was used for MPO or CD115 and developed in diaminobenzidine to visualize immunoreactivity. Immunohistochemistry images were collected using Pannoramic SCAN . For each section, the average number of MPO-positive cells or CD115-positive cells was calculated from six randomly chosen fields from the penumbra. The average of the three sections was recorded as infiltrating neutrophils for each brain.A commercially available MPO activity kit  was used to reveal neutrophil activation. The ischemic hemisphere was harvested at 24 or 72\u2009h after reperfusion, and MPO assessment was performed in accordance with the manufacturer\u2019s instructions. Briefly, the total protein was extracted using the reagents in the kit, and the absorbance at 645\u2009nm was measured using spectrophotometer . The results were presented as units per gram of the brain.2, and 5% CO2, glucose-free Dulbecco\u2019s modified Eagle medium, and reoxygenation for 24\u2009h in a complete medium were performed.The primary rats\u2019 BMVECs were commercially purchased . The LV CD151 shRNA and the LV Vehicle used for in vitro transfection were the same as those used in the in vivo experiments  at a multiplicity of infection of 50. Transfection efficiency was tested using the expression of a green fluorescent protein through immunofluorescence by Olympus IX81 . The CD151 expression was determined using Western blot  was used for total protein extraction from the brain tissue and cultured primary BMVECs. The NE-PER nuclear and cytoplasmic extraction reagents  were used for fresh cultured primary BMVECs, and the Minute\u2122 Cytosolic and Nuclear Extraction Kit for Frozen/Fresh Tissues  was utilized for frozen brain tissues. The protease and the phosphatase inhibitor cocktails  were added to the protein extraction steps. All the procedures were conducted in accordance with the manufacturer\u2019s instructions.Protein samples (20\u2009\u03bcg) were separated through 8\u201312% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked with the NcmBlot blocking buffer  for 10\u201320\u2009min at room temperature, stripped with the NCM Western blot stripping buffer , and incubated overnight at 4\u2009\u00b0C with the following primary antibodies: anti-CD151, VCAM-1 , ICAM-1 , E-selectin , CD9 , p-p38 , p38 mitogen-activated protein kinase , p-JNK , Jun N-terminal kinase , p-ERK , extracellular-related protein kinases , nuclear factor kappa-B (NF-\u03baB) inhibitor \u03b1 , p65 , YY1 , VLA4 , LFA1 , CD44 , PSGL1 , \u03b2-actin , and glyceraldehyde-3-phosphate dehydrogenase . The membranes were incubated with secondary antibodies  and visualized using an enhanced chemiluminescent substrate . The optical densities of the bands were scanned and quantified using image analysis systems . \u03b2-actin, GAPDH, and YY1 were set as internal controls.ROS in HL-60 and THP-1 cultured with BMVEC supernatant for 72\u2009h were measured with a ROS assay kit . All the steps were performed in accordance with the manufacturer\u2019s instructions. Briefly, the culture medium was first removed, and the cells were washed with PBS. Dihydroethidium (DHE), diluted with RPMI-1640 to a final concentration of 10\u2009\u03bcM, was applied to resuspend the cells that were then incubated at 37\u2009\u00b0C for 40\u2009min. Fluorescence was read with a spectrophotometry  at 518\u2009nm for excitation and 605\u2009nm for emission.A human neutrophil extracellular traps (NETs) ELISA kit  was used to test the NET concentration in HL-60 supernatant after 72\u2009h of culture with BMVEC supernatant. A human sCD14 ELISA kit and a human sCD163 ELISA kit  were used to detect THP-1 activation after 72\u2009h of culture with BMVEC supernatant. All the steps were conducted in accordance with the manufacturer\u2019s instructions.The RT-PCR procedure is presented in detail in the Additional file.P < 0.05 was considered significant.All data were presented as mean \u00b1 SD and analyzed using GraphPad Prism 8 . One-way ANOVA or unpaired t test was applied to determine the significance of differences among different groups. Tukey\u2019s test was used for multiple comparison. Neurological outcomes were examined at 24 and 72\u2009h after reperfusion to investigate the effect of the CD151 downregulation on experimental stroke outcomes in rats. The overall design of the in vivo experiments is shown in Fig.\u00a0The infarct volume of the LV CD151 shRNA + MCAO group was significantly smaller than that of the LV Vehicle + MCAO group  and NF-\u03baB pathways are required for VCAM-1 regulation . Thus, tIn our study, the activation of MAPK kinases  and NF-\u03baB  was first tested in the infarcted hemisphere in vivo. In vivo data . Table S1. Detailed information of lentivirus CD151 shRNA sequences. Figure S1. Different lentivirus CD151 shRNA transfection effectiveness in vitro. Figure S2. Lentivirus CD151 shRNA transfection effectiveness assessment in vivo. Figure S3. Lentivirus transfection effectiveness in vitro. Figure S4. CD151 expression assessment at observation time points in vivo and in vitro.Figure S5. p38 and NF-\u03baB activation were restrained in vivo after CD151 knockdown. The MAPK kinase  activation was evaluated  using infarcted hemisphere (n = 6 per group) or enriched endothelial cells  from infarcted hemisphere (n = 3 per group), * and **vs. LV Vehicle + MCAO indicate p < 0.05 and 0.01, respectively. The NF-\u03baB pathway activation evaluated using the (d) I\u03baB \u03b1 degeneration and the p65 translocation from the (e) cytoplasm to the (f) nucleus in infarcted hemisphere (n = 6 per group), *, ** and *** vs. LV Vehicle + MCAO indicate p < 0.05, 0.01 and 0.001, respectively. Figure S6. Anisomycin increased the phosphorylation of both p38 and JNK in BMVECs. Cultured primary BMVECs were treated with 1\u03bcM anisomycin for 3 h (n = 3 per group). An increase in the phosphorylation of p38 (a) and JNK (b) were observed, ** vs. control group indicate p < 0.01."}
+{"text": "Measuring hospital efficiency is one of the way how to use resources.The optimal hospital performance is the goals of healthcare policymakers. This study aimed to the current study was conducted to evaluate the efficiency the current study was conducted to evaluate the efficiency and assess the association between hospital size and hospital area population with technical efficiency in public hospitals.In this descriptive-analytical study, the statistical population consisted of 15 public hospitals in the west of Iran. First, the data envelopment analysis (DEA) method was used to evaluate technical efficiency. inputs included staff and beds, and outputs consisted of the number of surgeries, the number of patients, and the average length of stay. Then, according to the public ownership of all hospitals, their educational and therapeutic activities, as well as their size and population were considered as the environmental factor affecting efficiency. Thus, regression was applied to measure their effects on efficiency.The average technical efficiency of the studied hospitals, the average management efficiency, and the average efficiency of the scale were 0.935, 0.961, and 0.987, respectively. Out of the total evaluated hospitals, six and nine hospitals had an efficiency of less than one and one, respectively. Moreover, the size of the hospital and the population as the environment variable were significant in the Tobit model. Our regression demonstrated that although the size of the hospital is positively associated with its technical efficiency, the hospital population negatively affects hospital efficiency.According to the size and area population of the hospitals, they decrease their inputs to maximize their efficacy by optimizing their surplus amounts. Tobit regression analysis concludes that hospital size and population covered by the hospital significant effect on hospitals' efficiency. Health has a significant impact on the infrastructure of different parts of society based on sustainable social, economic, political, and cultural development . The proConsidering that the health change plan was implemented in May 2014 with particular effects on hospitals, the portion of health expenses has increased out of gross domestic incomes, and that of the hospitals has risen through this statistic more than ever . In IranThere is a pressing need concerning the optimal use of scarce sources and improvement of efficiency for providing health treatment cares. Accordingly, some measures are taken into account to prevent or decrease the waste of resources allocated to the health treatment system, helping in providing services as better as possible, developing availability, and improving hospital service quality . TherefoIn the economic literature, efficiency is the minimum use of inputs for a certain level of output. In other words, it is the increasing of outputs with a certain level of inputs. Inputs are the same as manufacturing factors such as energy, initial materials, capital, and labour force in the manufacturing process of commodities and services for generating outputs . Efficiency is an appropriate criterion for measuring the acquirement of the best output by limited inputs. Further, it is a new approach to peoples\u2019 work and life . The ratData envelopment analysis determines whether the considered decision-making units consider the efficiency line. It is non-parameter linear planning that estimates the frontier production function. The difference between this study and previous studies is Tobit regression, which can measure the effect of environmental factors on the efficiency level. The environment in this context is the factor that can affect firm efficiency although it is not part of the applied inputs. Furthermore, the assumption is that it is not under management control. Environment variables are ownership, the number of customers, and the firm position and size. Thus, if a dependent variable of a critical limit, Tobit regression or censored regression, will apply to review linear relationships . ConsideThe population of this descriptive study includes all public hospitals (N\u2009=\u200915) in the west of Iran. Due to ethical considerations, the name of the hospital is specified in alphabetical order in this study. Data collection tools for the theoretical framework of the research are scientific and library documentation. Given that data are collected from hospitals by standard tables of the Ministry of Health and Medical Education, there is no need to determine validity and reliability. Some reports were used to collect data consisting of data in 2018 such as general particularities of the hospital, the number of fixed and active beds, all patients (outpatients and inpatients), the number of staffs , the number of surgeries , and the length of stay.m, s, and n are the number of inputs, the number of outputs, and the number of units, respectively. The existence of a free variable with a w signal is the difference of this relation with the constant returns to scale. Therefore, the variable w signal determines returns to scale for every unit. The type of returns to scale represents a decrease, the scale to scale is fixed, and the type of returns to scale increases if w\u2009<\u20090, w\u2009=\u20090, and w\u2009>\u20090, respectively [Data envelopment analysis was used to analyze data and assess the efficiency level by Deap 2.1, assuming variables return to scale, which was of input-based type. The mathematical relation of data envelopment analysis is as follows:ectively . Scale eectively . Tobit rectively . FurtherAccording to technical efficiency, the results concerning the efficiency and rankings of the hospitals are presented in Table The calculated values by data envelopment analysis are presented as numbers 0\u20131. Further, Six hospitals with a lack of maximum technical efficiency. The minimum technical efficiency was 0.544, which was related to hospital No.15. Moreover, the scale efficiency of 60 and 40% of nine and six hospitals3 was 1 and less than 1, respectively. Furthermore, 73.3 and 26.7% of 11 and 4 hospitals had management efficiency of 1 and less than 1, respectively. In total, six and four hospitals were inefficient in technical efficiency and management efficiency, respectively. The excess of the related hospitals is specified by the number of active beds and staff based on management efficiency. Excess inputs are shown in Fig.\u00a0According to the vertical axis representing the excess input, the most and least excess inputs of the number of staff belonged to hospitals No.13 and No.15, respectively. Further, the most and least excess inputs of the number of active beds were related to hospitals No.13 and No.15, respectively.Hospital ownership, type of activity, the population covered by the hospital, and hospital size are not under management control. However, the public ownership of all hospitals and their educational and therapeutic activities were specified in this research. Thus, the size of the hospital and its population were considered as the environmental factors affecting efficiency. Moreover, Tobit regression was employed to assess their effect on efficiency(y*).But we only observe y\u2009=\u2009max. The Tobit model uses MLE to estimate both \u03b2 and \u03c3 for this model.Furthermore, the T-test was applied to demonstrate the significance of coefficients. The results are provided in Table According to the very significance of hospitals in providing health treatment services, as well as the health management system of every country, data envelopment analysis can be a huge step by providing the possibility for comparing, ranking, and patterning capability. Thus, it will pave the way for improving the functions of hospitals, particularly in the health treatment section. Based on the limitations of data envelopment analysis, the total considered organizations should be more than or equal to 3 times the inputs and outputs. Therefore, due to technical limitations, there is no possibility to choose more than five variables owing to the number of hospitals . In the Thus, hospitals must decrease their excess inputs to achieve maximum efficiency. Furthermore, the Tobit test results concerning environment variables in the studied method suggested that the size of the hospital and the population have a significant effect on efficiency. Therefore, the size of the hospital and the population of the location of the hospital will affect efficiency, which is out of management control.In general, six hospitals were inefficient in technical efficiency. Inefficiency was also observed in five and four hospitals in terms of scale efficiency and management efficiency, respectively. The size and population of the hospital as environmental factors have significant effects on hospital efficiency. Hospitals with the efficiency of less than one had different initial and optimal values, and there was also excess input. Therefore, the mentioned hospitals should decrease the initial values of their inputs to achieve maximum efficiency. According to the limitation of data envelopment analysis in this research, there was no possibility of choosing more than five variables due to the limited number of public hospitals in the west of Iran."}
+{"text": "Although research on the association between subjective views of aging (VOA) and survival is scarce, more negative VOA have been found to be associated with increased all-cause mortality, even after controlling for possible confounders. Longitudinal studies on the predictive association of VOA with survival in individuals aged 80 years or older are, however, very limited. Thus, the aim of this study was to link adults\u2019 awareness of age-related change (AARC), a multidimensional measure of adults\u2019 subjective VOA, to survival time across a 3.5-year observation interval in advanced old age. To put the AARC construct in context, the study also considered related psychosocial concepts  essential for coping with late-life challenges as potential behavioral predictors of longevity. Data came from a representative panel study that included persons living in community and institutional settings. A total of 1,863 interviews were conducted at wave 1. This study used meta-data from wave 2 fieldwork 2 years after the initial assessment and death records obtained during panel maintenance after 3.5 years to estimate determinants of survival. Results showed that loss-related VOA indicated increased risk to survival, whereas gain-related VOA were predictive of longer survival. Both perceived age-related losses and perceived age-related gains exerted a significant independent effect on late-life mortality over and above socio-demographic background characteristics, perceived control, engagement with life, as well as health status. These findings suggest that the multidimensional examination of very old adults\u2019 VOA may help to better understand successful longevity in the Fourth Age. Advanced old age is frequently seen as the most vulnerable period of the human lifespan because many adults experience multimorbidity, functional disability, motor and sensory impairment, significant cognitive decline, and frailty. Psychological conceptualizations of what has also been named the \u201cFourth Age\u201d to a large extent echo this biomedical loss perspective of very late life, resulting in what This article addresses whether psychological resources also account for significant variance in predicting survival in advanced old age. An important background for addressing this question is that the predictive strength of social factors, such as income, education, and marital status, for survival is much weaker in those 80+ than in younger age groups . In addiAgainst this background, the primary aim of this study was to link awareness of age-related change (AARC), an established concept to assess older adults\u2019 subjective views of aging (VOA) in a multidimensional way , to survConsidering in our study the connection between subjective VOA and late-life survival is based on the observation that individuals reflect on their own development and try to understand their own aging as they move across the adult lifespan . Thus, aA vast body of research has documented that more negative VOA are associated with a range of unfavorable developmental outcomes, such as poorer physical and mental health, and poorer cognitive functioning, including cognitive pathology  and negative (AARC-Losses) perceptions and interpretations of events, behaviors and sensations across various life domains . Previous research in younger age groups has shown that perceived age-related gains and losses co-exist even within behavioral domains and have different antecedents and different associations with developmental outcomes, including depression, psychological well-being, and self-rated health . Thus, AImportant for assessing AARC in very old individuals, In addition to AARC as a predictor of survival, we focused on two areas of psychosocial resources that have shown an association with survival in younger age groups and, therefore, may also be relevant for predicting survival in advanced old age. First, we addressed how very old individuals manage to maintain a self-view of a purposeful, valuable life, as seen from an individual and societal point of view. In a meta-analysis of ten prospective studies with more than 136,000 participants, Second, another major psychological challenge is to what extent very old adults are able to exert control over their lives and keep track of current societal developments. This point addresses the core challenge whether and to what extent individuals in the Fourth Age can maintain feelings of agency and avoid feelings of being dependent on others. With respect to societal development, feeling distant and disconnected from major trends, such as globalization or communication technology, may result in possible alienation and perceived obsolescence. Adverse effects of alienation have been described in the context of suicide ideation in subpopulations with mood disorder . HoweverThe aims of this study were to examine the contribution of very old adults\u2019 subjective VOA as predictors of survival time across a 3.5-year observation interval. Individuals\u2019 subjective VOA, as operationalized in terms of AARC-Gains and AARC-Losses, were incorporated into a set of established socio-behavioral predictors of survival. We expected subjective VOA to show substantial associations with survival time. Moreover, we expected positive (AARC-Gains) and negative facets (AARC-Losses) of individuals\u2019 subjective VOA to contribute independently to the prediction of survival in those in advanced old age, because these predictors have been shown to be differentially related to developmental outcomes in younger age groups. We expected a remaining increment of predictive power due to AARC even after controlling for other major survival-relevant psychological resources. Therefore, we assumed that attribution of perceived change to aging itself should evolve from a process of integrating knowledge of conditions more prevalent with age  and experiences in handling such change .Data came from a representative panel study on quality of life (QoL) and well-being of very old adults conducted in Germany\u2019s most populous state, North-Rhine Westphalia . For theSD = 4.5 years; range: 80.1 to 102.9 years). A total of 211 interviews (11.3%) were conducted in nursing homes. The sample included 176 interviews with proxy informants  where target persons were willing to be included in the study but were not able to conduct the 90 min interview themselves due to severe mental or physical health constraints.A total of 1,863 computer-assisted personal interviews were conducted at participants\u2019 homes to assess a wide array of individual QoL resources  and subjective QoL outcomes . The study protocol also included objective testing such as a screening for mild cognitive impairment (MCI). Informed consent was given by all participants after written and verbal explanation of the study aims and procedures. Mean age of the realized sample at the time of the interview at wave 1 was 87.0 years  of respondents had died. The study was approved by the ethical board of the medical faculty at the University of Cologne (Protocol #: 17-169).not at all) to 5 (very much). A sample gain item (INT+ domain) is, \u201c\u2026I appreciate relationships and people much more.\u201d A sample loss item (LIFE- domain) is, \u201c\u2026I have to limit my activities.\u201d The 10-item short form of the Awareness of Age-Related Change scale AARC-SF;  was usedAdditional psychosocial resources predictive of survival in very old age were assessed in terms of appraisal of life  and control .(no), 1 (neither/nor), 2 (yes) is suggested for use in very old respondents  to 4 (very much). Scale consistency was moderate for this perceived obsolescence composite in the current sample  and comparable to Cronbach\u2019s alpha of 0.72 reported for the 5-item obsolescence subscale by  to 4 (very much).The Valuation of Life Scale VOL;  was usedpondents , 2013. GPerceived control was assessed using the Internal and External Control Beliefs scale IE-4, , with tw(not possible without help), 1 (some help needed), 2 (no help needed). Reliability of the ADL and IADL scales in the current sample was high .Adults\u2019 self-reported performance on Basic Activities of Daily Living ADL;  and InstFurther, the number of self-reported currently treated health conditions was used as an indicator of multimorbidity. Hence, this measure refers to a subset of medical conditions with high salience for the individual in everyday life. This index was modified from the Self-Administered Comorbidity Questionnaire SCQ;  to inclu(no cognitive impairment) to 7 (most severe). In terms of cognitive status, the DemTect was developed as a brief screening tool for MCI and early stages of dementia . The tesCharacteristics of the very old population estimated based on (1) wave 1 participants, (2) those willing to be contacted again for future waves, and (3) respondents still alive approximately 3.5 years after wave 1 are shown in Compared to the full wave 1 sample, the subsample of respondents willing to be contacted again for a second interview allowed for unbiased population estimates with respect to gender, multimorbidity, appraisal of life, perceived control, as well as AARC-Gains and AARC-Losses, as judged from almost completely overlapping 95% confidence intervals of parameter estimates in the initial and panel subsample. In addition, estimates for age composition , ADL/IADL independence, cognitive function and living arrangement  indicated no significant selectivity of those willing to be contacted again.2 = 20.7, df = 18, p = 0.29).Information on survival status and survival time was collected across the study interval from different sources. Fieldwork metadata from contacting via mail and subsequent home visits or hotline responses from relatives included documentation of dropout due to death of respondents at wave 2, sometimes accompanied by more specific information . Additional postal registry data on survival status and date-of-death was obtained in March 2021 during panel maintenance. Of all 237 participants who had died since wave 1 according to information collected during wave 2 face-to-face fieldwork (i.e. home visits), valid death dates could be obtained for 141 individuals. Selectivity analyses found only very limited evidence of selective availability of death dates in this subsample of wave 2 non-respondents and potential bias in the subgroup with observed survival time . More spn = 96 and n = 3, respectively). Interval-censoring occurred for 8 cases where individuals were known to be alive at wave 2  and deceased by March 5, 2021 , but no death date could be obtained.Inability to retrieve the exact date-of-death for some respondents known to have died before wave 2 or by March 5, 2021, respectively, and the fact that most participants were still alive 3.5 years after initial assessment led to a complex scheme of censoring with respect to survival time in this study . A totalIn a first step, we describe levels of socio-behavioral factors of longevity in a subsample of wave 1 respondents known to have survived at least 3.5 years from the initial interview.In a second step, we used accelerated survival time analysis  gives the event-time-ratio (ETR) that represents the factor by which time-to-death itself is accelerated  or slowed down  in a \u201ctreatment\u201d group compared to a reference group. Hence, an ETR of 1.5 describes a protective factor that increases survival time by 50 % compared to that in the reference group, whereas, an ETR of 0.75 describes a risk factor that reduces survival time to 75 % of that in the reference group. Inspection of the cumulative distribution function of time-to-death for different distributional assumptions in an intercept-only model indicated that both the Weibull and log-normal function may be used to fit the data, but that the log-normal function was better suited to model shorter survival time  model, the parametric AFT model offers more efficient estimation  when distributional assumptions are met. Whereas, semi- or non-parametric models offer a limited range of possible censoring schemes, AFT can handle interval- and left-censored time-to-event data in addition to the more common right-censored scenario . Hence, time see . The invtime see .All survival analyses controlled for socio-demographic background characteristics, such as age, gender, or living in an institution, and used participants\u2019 subjective VOA, life appraisals, control beliefs as well as cognitive and health resources as predictors. Predictors were introduced block-wise in a predetermined order to estimate the unique relevance of VOA among the behavioral indicators of survival and the robustness of parameter estimates.All analyses also include available proxy information. As to be expected, our study was faced with a classic trade-off: On the one hand, it may be seen critical to involve external individuals also when it comes to self-referential information such as the AARC-SF. However, we always involved proxy persons that were very familiar with the target individual. On the other hand, doing so can be seen as a needed strategy to counteract a likely under-representation of a seemingly most vulnerable part of the very old population. Besides such a meta-methodological argument, we were able to establish measurement equivalence of the AARC-Gains and AARC-Losses scales across self- and proxy-report using multi-group structural equation modeling with equality constraints see .All analyses used calibration weights to correct for the disproportional sampling design and survey nonresponse at wave 1 to allow for unbiased population estimates . All anaM = 3.2, 95% CI [3.2\u20133.3]) than AARC-Losses . High average initial levels were found for the VOL subscales, indicating considerable engagement with life and optimism in very old age . Nevertheless, the average level of feelings of obsolescence and anomia observed in this sample was close to scale midpoint , indicating a fair amount of perceived discrepancy of values and lifestyle in today\u2019s very old adults compared to current society. In a related vein, most respondents showed only moderate levels of feeling needed by society . With respect to health and functioning, the survivor subgroup reported, on average, 3.4 treated health conditions (95% CI [3.2\u20133.6]) as an indication of multimorbidity. Need for assistance with basic ADL was lower  than need for assistance with more complex IADL . Almost three out of four respondents in the survivor group showed age-adequate cognitive function in the screening, whereas, only one out of 10 respondents in this group were screened for probable beginning AD at the initial interview. Hence, the survivor subsample had better cognitive functioning than the average population 80+ with respect to the estimated prevalence of cognitive impairment . Because fieldwork for initial interviews span 196 days, survival time for those 1,215 participants still alive on March 5, 2021 was 1,201.5 days on average, with a range from 1,105 to 1,301 days.Estimated event time ratios (ETR) from the AFT model are given in Results showed a rather consistent pattern of associations of socio-demographic background variables with 3.5-year survival in very old age across all models. Specifically, an estimated ETR of 0.72 to 0.75 for men indicated that survival time was 25 to 28 percent shorter in very old men compared to women. Younger age was also consistently and positively associated with survival time. Expected survival time was 65% longer in the 85\u201389 age group than that for the oldest-old (90+), and 132% longer in the youngest participants (80\u201384 years). Acceleration of time-to-death was observed for respondents in institutional settings  compared to community-residing participants in all models except model 5 that also included physical and cognitive health status as predictors.Awareness of age-related gains (AARC-Gains) and AARC-Losses were independent and significant predictors of survival time over and above socio-demographic background characteristics (Model 2). Reporting more AARC-Gains at wave 1 was associated with longer survival time, whereas, reporting more AARC-Losses was associated with a reduction in survival time of approximately comparable magnitude. Adding psychosocial resources in the following models, effects of VOA on estimated survival time stayed virtually the same when different aspects of appraisal of own life (Model 3) and different facets of perceived control over life (Model 4) were added to the list of predictors. Notably, AARC-Losses remained a significant independent predictor of survival in the oldest-old even when multimorbidity, ADL/IADL independence and cognitive status were controlled for (Model 5). However, AARC-Gains no longer was a significant independent predictor in the full set of socio-behavioral factors of survival. Of note, parameter estimates were virtually unaltered in subsequent sensitivity analyses that excluded proxy reports see .Results for the set of predictors tapping appraisals of one\u2019s own life were both less consistent across models with different competing predictors and showed smaller associations with survival time. The ERT for the \u201cengagement with life\u201d subscale of the VOL scale was consistently estimated below 1.0 , indicating shorter survival time after initial interview in those reporting feelings of self-efficacy in overcoming problems and difficult situations. However, a significant effect could only be observed after controlling for perceived control (Model 4), suggesting some degree of overlap between engagement with and perceived responsibility for one\u2019s life. Results also suggested a potential marginal association between feeling needed by society and reduced survival time with ERTs of 0.93, 0.90 and 0.88, respectively. These effects, however, failed to reach statistical significance in all models.Results with respect to different facets of perceived control in very old age were mixed. Neither internal nor external control beliefs contributed significantly to the prediction of late-life survival unless effects of health resources were controlled for in Model 5. Specifically, once health resources were taken into account, respondents who reported higher dependency of personal life outcomes on chance or luck (rather than on own action or those of powerful others) showed significantly longer survival times (ERT: 1.18) after the initial interview.Not possible without help to 1 = Some assistance needed) was associated with a 75% increase in survival time (ERT: 1.75). Whereas, estimates for IADL and levels of cognitive functioning also pointed in the expected direction, benefits of age-adequate cognitive functioning for survival time did not meet the.05 level of statistical significance. It is noteworthy that considering health status differences at the initial interview in Model 5 significantly attenuated some of the effects reported for less comprehensive models. Not surprisingly, the effect of living in an institution on survival time diminished when taking health and functional status into account. Similarly, the protective effect of positive VOA and the adverse effect of engagement with life on survival time became non-significant when health differences were controlled for.Of the proposed health and functioning predictors of mortality, only basic ADL independence was found to be significantly associated with survival time in this sample. A 1-point increase in ADL performance  can be found below: GESIS Datenarchiv, K\u00f6ln. ZA7558 Datenfile Version 1.0.0, The studies involving human participants were reviewed and approved by the ethical board of the medical faculty at the University of Cologne (Protocol #: 17-169). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.RK initiated the study, collected the data, conducted the analyses, and wrote the manuscript. H-WW and MD wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. Here, we conducted biased chemoreceptor screening to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. We identified three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, as well as the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, we confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature. Dhakal et al. use chemoreceptor screening on three ionotropic receptors in Drosophila, IR25a, IR76b, IR51b to evaluate their impact on avoidance behaviors against nitrogenous waste products. The results of electrophysiology assays show that ammonia compounds are aversive in taste by directly activating bitter-sensing gustatory receptor neurons. These organic compounds can be recycled as a source of nitrogen groups such as amines, amides, and anilines, which are central for developmental and physiological processes in both plants and animals5. In nature, ammonia concentration is in the range of 10\u201320\u2009mM in the cassava plant leaf and the cattle manure7.Nitrogen is an essential building block for the synthesis of DNA and protein and is the most abundant element in the Earth\u2019s atmosphere. Therefore, the nitrogen cycle is instrumental in maintaining healthy ecosystem dynamics. Animals and many plants produce nitrogenous wastes throughout their life histories. Ammonia and urea are the decomposition byproducts of protein11. Moreover, although the anatomy and molecular basis of taste perception in vertebrates and invertebrates are evolutionarily distinct, they share a few similar fundamentals14. Fruit flies (Drosophila melanogaster) possess specialized taste neurons on their labella, legs, pharynx, wing margins, and ovipositor17. Flies can sense sweetness, bitterness, sourness, saltiness, and water22. Major gustatory organs, such as the labellum and legs, have evolved to recognize chemicals via several channels and receptors, such as gustatory receptors (GRs), odorant receptors (ORs), ionotropic receptors (IRs), transient receptor potential (TRP) channels, and pickpocket ion channels (PPKs)28. Most taste sensilla harbor four distinct GRNs, of which two are attractive GRNs (sweet-sensing and water-sensing GRNs) and two are aversive GRNs 31. Moreover, these neuronal circuits have been found to be distinct, as attractive or aversive GRNs can be independently activated and behaviorally controlled by artificially expressing temperature-activated TRPA1, capsaicin-activated rat TRPV1, or light-activated channelrhodopsin-233.Animals, including insects, rely on chemoreception for feeding, mating, and escaping from predators35. Recent studies on the mechanisms of taste perception indicate that saltiness, sourness, amino acids, and other chemical cues are directly sensed by taste IRs37. In nature, amine-containing compounds not only elicit aversive responses but have also been identified as important kairomones in host-seeking insects. For instance, insects and some disease vectors are attracted by the odor of ammonia and amines, which are excreted through sweat41. Ammonia and putrescine are olfactory cues for many anthropophilic insects, including Anopheles mosquitoes41. However, high concentrations of ammonia and urea have been found to dramatically decrease the fecundity and egg viability of Drosophila suzukii42. The production of urea in insects has been attributed to the catalysis of arginine hydrolysis43. In contrast, blood-feeding insects such as Aedes aegypti can efficiently detoxify ammonia-containing compounds. Specific mechanisms have evolved to tightly regulate the synthesis and excretion of nitrogenous waste and avoid the toxic effects that may result from abnormally high ammonia concentrations in tissues44.Among these chemoreceptors, IRs are broadly expressed in the peripheral sensory systems involved in chemosensation, thermosensation, and hygrosensation38; however, the cellular and molecular mechanisms that enable the taste perception of these compounds remain largely uncharacterized. Here, we elucidated the gustatory mechanisms by which Drosophila (fruit flies) sense ammonia and its derivatives in plant- and animal-derived decay products. Using a combined behavioral and electrophysiology approach, we discovered that flies perceive and avoid ammonia and its derivatives as bitter tastants via bitter-sensing GRNs. We also elucidated molecular sensors that are required for the gustatory perception of naturally occurring nitrogenous waste products.The above-described studies highlight the critical role of nitrogen-containing compound perception in insects, in addition to the more widely characterized perception of sweetness, sourness, saltiness, bitterness, and water. Previous studies have investigated the mechanisms of ammonia and amino group olfactory perception in Drosophila antennae and other insects46. However, only a few studies have assessed the gustatory mechanisms of ammonia perception47. The labellum is the major taste organ in D.melanogaster and possesses 31 taste sensilla in each hemisphere16. Each sensillum is named according to its length and position . All sensilla have one sweet gustatory receptor neuron (GRN), but only S-type and I-type sensilla have bitter-sensing GRNs16. We found that nitrogenous wastes such as ammonium sulfate [(NH4)2SO4], ammonium chloride (NH4Cl), urea, and putrescine can activate S6 sensilla, but not L4, in a dose-dependent manner  gene to inhibit each category. All four chemicals induced action potentials in the S6 sensilla of the controls (w1118 and UAS-Kir2.1/+) , whereas the other neurons exhibited similar responses to those of the controls49. The results of these electrophysiological tip recordings were further confirmed by our behavioral assays , ammonium chloride (pH 5.9), and putrescine (pH 5.5) are slightly acidic in solution, whereas urea (pH 7.6) is slightly basic. To address whether the pH affected oviposition behavior and binary food choice assay, we tested ammonium sulfate which were adjusted to neutral pH by adding ammonium hydroxide  Fig.\u00a0, b. Thesays Fig.\u00a0. Flies aays Fig.\u00a0. Female ays Fig.\u00a0. Howeverion Fig.\u00a0, we testion Fig.\u00a0. We foun29  using labellum, leg, and antenna samples  or bitter-sensing (Gr33a-GAL4) receptors to detect whether Ir51b participates in the GRN-mediated perception of bitter substances using tubulin as an internal control  were involved in the gustatory detection of nitrogenous waste products via heteromultimeric channel formation; however, ammonia sensing could not be recapitulated in sweet-sensing GRNs.To investigate the genetic recapitulation of ammonia-taste receptors, we assessed whether these three genes were sufficient to elicit taste-induced avoidance of nitrogenous compounds in flies. I-type sensilla in the fly\u2019s labellum possess only two GRNs, whereas L-type and S-type sensilla harbor four GRNs Fig.\u00a0. We thenlla Fig.\u00a0. HoweverGr5a-GAL4. This indicates that additional channel subunits required for the responses to the nitrogenous waste compounds must be present in S-type and I-type GRNs. Therefore, this study has a limitation to claiming that the IRs are receptor ion channels for nitrogenous waste compounds, because we cannot rule out the function of IRs in transduction pathways. Future works should focus on finding additional channel subunits to prove that nitrogenous waste compounds can directly activate these IRs by heterologous expression.Overall, we identified three IRs to sense nitrogenous waste compounds in S-type. Furthermore, three IRs were enough to induce ammonium sulfate response in I-type sensilla. However, the combination of three IRs was insufficient to elicit physiological responses to ammonium sulfate in L-type sensilla when driven with 51. However, recent studies have indicated that IRs are likely the most ancient chemoreceptors and thus predate ORs and GRs, as their existence can be traced back prior to the deuterostome-protostome split27.Chemical sensation is an essential modulator of physiology and behavior. In invertebrates, the vast majority of chemical stimuli in the environment are recognized by members of two evolutionarily related chemosensory receptors: the ORs and the GRs52. Females of some uricotelic muscid flies are reportedly attracted to ammonia when searching for suitable oviposition sites53. In this case, ammonia acts as a chemical attractant that enables some parasitic organisms to detect their hosts. However, ammonia can also be used to kill or repel bed bugs, ants, rats, fleas, and snakes. Insect survival may also vary depending on ecological niche or host characteristics; however, the proliferation of insect populations is generally thought to be highly host-dependent. Here, we demonstrated that fruit flies avoided nitrogenous waste products both when laying eggs and when selecting their food, as these compounds are potentially toxic. Despite the differences in the mechanisms of chemical sensation between arthropods and humans, the identification of ammonia-associated taste sensors in insects provides important insights into how animals perceive and react to specific chemicals.Ammonia can act as a kairomone, and therefore some species, such as flour mites, are attracted to the microbial degradation products of certain amino acidsw1118.Unless otherwise indicated, all flies were maintained at 25\u2009\u00b0C under a 12\u2009h light/12\u2009h dark cycle. Both male and female flies were used randomly in our experiments. The control strain used in this study was Ir7a1, Ir47a1, Ir52a1, Ir56a1, Ir60b3, Ir94a1, Ir94c1, and Ir94h1 strains20. The Ir7g1 (BL42420), Ir10a1 (BL23842), Ir21a1(BL17171), Ir48a1 (BL26453), Ir48b1 (BL23473), Ir51b1 (BL10046), Ir52b1 (BL25212), Ir56b1 (BL27818), Ir62a1 (BL32713), Ir67a1 (BL56583), Ir75d1(BL24205), Ir92a1(BL58205), Ir94b1(BL23424), Ir94d1 (BL33132), Ir94g1 (BL25551), and Ir100a1 (BL31853). Gr2a1 (BL18415), Gr10a1 (BL29947), Gr22f1 (BL43859), Gr23a1 (BL19287), Gr28bMi (BL24190), Gr36b1 (BL24608), Gr36c1 (BL26496), Gr58b1 (BL29065), Gr59a1 (BL26125), Gr77a1 (BL26374), Gr93d1 (BL27800), Gr94a1 (BL17550), Gr97a1 (BL18949), Ir8a1 (BL41744), Ir85a1 (BL24590), UAS-hid (BL65403), and UAS-Kir2.1 (BL6596) strains were obtained from the Bloomington Drosophila Stock Center. Additionally, we obtained the following mutants from the Korea Drosophila Resource Center: Gr28a1, Gr36a1, Gr39b1, Gr59c1, and Gr89a1. We previously described the Gr8a154, Gr33a148, Gr47a155, Gr66aex8356, Gr33aGAL4 48, Gr98b157, Ir76b129, Ir76b-GAL429, and UAS-Ir76b29. K. Scott provided ppk23-GAL458 and ppk28-GAL418. H. Amrein gave \u2206Gr32a, Gr66a-GAL4, and Gr5a-GAL460. L. Vosshall provided Ir25a223. We obtained Gr22e1 (140936) from Kyoto Drosophila Stock Center.We previously described the Sucrose , sulforhodamine B , ammonium sulfate (CAS No 7783-20-2), ammonium chloride (CAS No 12125-02-9), putrescine (CAS No 333-93-7), and urea (CAS No 57-13-6) were purchased from Sigma-Aldrich Co. Brilliant blue FCF  was purchased from Wako Pure Chemical Industry Ltd.Ir51b2 knock-in-GAL4 mutant line was created via ends-out homologous recombination as previously described61. Concretely, we amplified 2.94\u2009kb upstream and 3.05\u2009kb downstream of genomic fragments through PCR and subcloned the DNA into the pw35-GAL4 vector48. Right arm extension included 3050\u2009bp and left arm extension included 2944\u2009bp along with the ATG start codon. GAL4 was inserted by replacing 1017\u2009bp of genomic regions to preserve the reading frame of the ATG start codon. The construct was injected into w1118 embryos by the Korea Drosophila Resource Center (KDRC).The Gr5a-GAL4/UAS-hid, and Gr33a-GAL4/UAS-hid adult flies. Total RNA was extracted using TRIzol (Invitrogen) and cDNA was synthesized using AMV reverse transcriptase (Promega). To perform the RT-PCR, we used the following Ir51b primers: 5\u2032-GGC GCT AAC AAA CGC TGC TTAC -3\u2032 and 5\u2032-CAG AGC TGA CAG TAT CCA ACC AA-3\u2032. The tubulin primers were 5\u2032-TCC TTG TCG CGT GTG AAA CA-3\u2032 and 5\u2032-CCG AAC GAG TGG AAG ATG AG-3\u2032. RT-PCR products were obtained after 35 cycles. Each samples were repeated at least three times. Intensity measurement was done by using ImageJ (Fiji) application and then Ir51b RNA level in each sample was normalized by the internal control, tubulin.Labellum, leg, and antenna samples were dissected from approximately 30 control, 29. First, 5\u20137-day-old mixed gender  flies were starved in a vial containing water-soaked Kimwipe paper for 16\u201318\u2009h in a dark and humid chamber. Each experiment was conducted using 50\u201370 flies. We then prepared two food options, both containing 1% agarose: one contained only sucrose and the other contained sucrose mixed with nitrogen-containing chemicals. These food sources were colored with either blue  or red food-grade dye . These two food preparations were dispensed into a 72-well microtiter dish  in an alternative position. We briefly anesthetized the starved flies and introduced them into the food dish, after which we immediately transferred them to an incubator for 90\u2009min. The flies were euthanized in a \u221220\u2009\u00b0C freezer for at least 2\u2009h. Then, the abdomen color was classified as \u201cblue,\u201d \u201cred,\u201d or \u201cpurple\u201d using a stereomicroscope. The preference index (PI) was calculated using following equations:To perform binary food choice assays, we followed a previously described protocol29. First, 4\u20136-day-old flies were anesthetized on ice. A reference glass electrode filled with Ringer\u2019s solution  was inserted into the thorax of the flies. The glass electrode was gently pushed towards their proboscis without causing any severe damage to the GRNs on the proboscis. Approximately 4\u20136 flies were used for each experiment. Using an electrophysiology system, we activated the S-type, I-type, and L-type taste sensilla on the labella of flies for 5\u2009s using a mixture of tastants with 30\u2009mM tricholine citrate (TCC). The recording electrode (10\u201320\u2009\u03bcm tip diameter) was connected to a preamplifier , and the signals were collected and amplified by 10x using a signal connection interface box (Syntech) in conjunction with a 100\u20133000\u2009Hz band-pass filter. Recordings of action potentials were acquired using a 12\u2009kHz sampling rate and analyzed using the Autospike 3.1 software (Syntech). We then counted the action potentials for 50\u2013550\u2009ms and presented doubled values of the period per second in the figures. Each consecutive recording was performed with an approximately 1\u2009min gap between each stimulation. The sample numbers (n) in each experiment indicate the number of animals. The same procedure was repeated on different days and using different setups.Tip recording assays were conducted as described in a previous study62. The flies were first starved for 20\u201324\u2009h in a vial with water-soaked Kimwipe paper. The flies were then briefly anesthetized on ice and fixed on a glass slide using glue. A fine Kimwipe paper wick was then used to deliver the initial 100\u2009mM sucrose stimulus to the flies. Only flies that showed a positive PER to sucrose were considered for the next test. Taste stimuli were delivered to the labellum at least three times to avoid false-positive responses. At this point, only the flies that exhibited a positive PER to the experimental solutions  were deemed PER positive. A total of 10\u201315 flies were evaluated per experiment, after which PER percentages were calculated. At least six replicates were performed for each strain.The PER assay was performed as previously described63. A total of 15 female and 15 male newly hatched flies were transferred into a new food vial supplemented with dry yeast and kept in a normal light/dark cycle for two days. Prior to the assays, the experimental animals were acclimatized in 1% agarose containing a test food choice for 5\u20136\u2009h. Two food options were then provided, one containing only sucrose and another containing a mixture of sucrose and a nitrogen-containing chemical, both of which were dispensed on a Petri dish  divided into two equal halves. The agarose food was allowed to solidify and then transferred to an egg-laying chamber ; the acclimated flies were transferred into the chamber thereafter. The flies were then allowed to lay eggs overnight inside of the incubator. The next day, the number of eggs deposited on each side of the chamber  was counted, and the ovipositional preference index was calculated as follows: Oviposition preference assays were conducted as described in a previous studyD.melanogaster as a model animal. Both sexes of experimental animals were considered randomly for the experiments we performed. All the experiments were conducted at laboratory conditions. Based on the previous studies, we determined at least six replicates per genotype were enough to verify behavioral data, where as at least 10 animals per genotype were enough in electrophysiological recordings. We performed three replicates to analyze Ir51b expression for RNAi analysis. We met enough sample size to make our data more reliable in each figure. Each experiment was conducted for at least two different days. No data was excluded from the analysis. Each data points represents a real value. Average of all the replicates for that specific genotypes were presented. All error bars represent the standard error of the mean (SEM). Multiple comparisons were then evaluated using single-factor ANOVA coupled with Scheffe\u2019s post hoc test. Asterisks indicate statistical significance . Statistical analyses were performed using Origin Pro 8 for Windows .We selected Further information on research design is available in the\u00a0Supplementary information.Description of Additional Supplementary Files.Supplementary Data 1.Reporting summary."}
+{"text": "There were no significant differences in steatosis or liver enzymes or in outcomes between control and intervention cohorts. A high level of patient acceptability was reported. Integrating telephone-delivered coaching provided non-inferior care and high levels of patient satisfaction. Telephone coaching aligned with the principles of an obesity service should be trialled to improve patient access to obesity interventions.Australia has one of the highest prevalences of obesity in the developed world with recognised gaps in patient access to obesity services. This non-randomised before and after study investigated the health benefits and patient acceptability of integrating the Get Healthy Service, a state-funded telephone-delivered coaching service in Australia, as an adjunct to multidisciplinary care for adults attending a public obesity service. Forty-one participants received multidisciplinary care alone while 39 participants were subsequently allocated to receive adjunctive treatment with the Get Healthy Service. Weight, body mass index, glycosylated haemoglobin, measurement of hepatic steatosis and liver enzymes were collected at baseline and 6 months. Participant evaluation was obtained post intervention. Statistically significant reductions from baseline were achieved for both control and intervention with respect to weight (\u22126.7 \u00b1 2.2 kg,  Australing rate .2 or BMI \u2265 35 kg/m2 with comorbidities) and often complex health care needs [While national guidelines recognise the key role that primary care plays in identifying and implementing lifestyle interventions to support overweight and obesity management ,6, primare needs ,8,9.Specialist obesity services (SOS), often found in public hospital systems, generally comprise of a multidisciplinary team (MDT) and are recommended for managing patients with severe obesity ,5,6. SpeTelephone-delivered health coaching is a well-established care model to support dietary and lifestyle changes . Not onlThe Get Healthy Information and Coaching Service (GHS) is a government-funded telephone-delivered information and coaching service available to adults within New South Wales (NSW), Australia. The GHS offers 10 telephone calls delivered over a 6-month period targeting healthy eating, physical activity and achieving and sustaining a healthy weight. Participants receive counselling from a personal health coach with university qualifications in dietetics, exercise psychology and/or psychology for the duration of the coaching period. Enhancement programs are also available for specific health conditions and populations and have been successfully integrated into routine care across several health settings. The Type 2 Diabetes Prevention Program has shown clinical improvements in anthropometric and lifestyle risk factors for adults at risk of T2D , The GetDespite the effectiveness of the GHS in supporting individuals\u2019 efforts to achieve and sustain moderate positive behaviour changes and reductions in chronic disease risk factors , no rese\u00ae, and liver enzymes alanine aminotransferase (ALT) and gamma-glutamine transpeptidase (GGT), which are commonly elevated in MAFLD. It is hypothesised that participants who complete the GHS coaching program as an adjunct to MDT care would achieve greater weight loss with associated greater reductions in HbA1c, liver stiffness, ALT and GGT enzymes when compared to participants who receive standard MDT care alone.This is the first study to investigate the health outcomes and patient acceptability of integrating telephone-delivered coaching using the GHS as an adjunct to MDT care for adults attending a SOS. Due to the association between obesity, T2D and MAFLD, secondary outcomes of this study included improvements in glycosylated haemoglobin (HbA1c), liver stiffness as assessed by FibroScan2) with two obesity-related complications  or Class II obesity (BMI \u2265 35 kg/m2) with co-existing T2D and no major mental health, drug or alcohol abuse/addiction. Study participants were consecutively recruited from May 2019 to October 2019. Participants who required individualised nutrition counselling were excluded from this study .Participants were recruited from the Blacktown Metabolic and Weight Loss Program, Blacktown-Mt Druitt Hospital, New South Wales, Australia. This SOS accepts patient referrals from any physician or specialist provided patients are over 18 years of age with Class III obesity  prior to dietetic intervention.Patients attended an initial dietitian-led group education session to commence a very low-calorie diet  consisting of a total meal replacement program or a low-calorie diet  consisting of a partial meal replacement program for 6 months. The patients were provided the option between a VLCD or LCD to allow for individual goals and circumstances. The participants also had access to weekly dietitian-led patient support groups to allow for regular monitoring and counselling to improve adherence to dietary interventions, though data on compliance was not collected due to the expected bias from patient recall. Following this, study patients were transitioned into dietitian-led group programs focusing on a staged food reintroduction, dietetic counselling and weight maintenance support. Individuals in the study were also referred to psychologists for assessment and cognitive behavioural therapy group programs ; this comprised 6 1 h sessions in principles of emotional regulation, mindfulness, managing depression, anxiety and eating behaviours. Physiotherapy interventions were also included in the standard of care for high-risk patients  and referred for assessment and supervised exercise programs (8 \u00d7 1 h onsite gym session) and 6 educational 1 h group sessions. The interventions described occurred over the course of the patients one-year program enrolment, after which time the patient is discharged from the SOS back to primary care or a select number of patients are eligible to receive publicly funded bariatric surgery. The latter was decided based on the patient\u2019s age, comorbidities, level of engagement and perceived benefit within a multidisciplinary case format discussion. Adherence to psychology and physiotherapy interventions was strongly advised, though data on adherence was not specifically collected.The intervention group consisted of the MDT care described above with adjunct telephone-delivered coaching using the GHS. At the initial dietitian-led group education session, participants were provided with an overview of the GHS with the option to participate. Interested participants were referred to the GHS via a customised handover form. The standard GHS consists of 10 health coaching calls with enhancement programs offering up to 13 coaching calls delivered over a 6-month period. To provide the intensive contact required for this high-risk population, the enhanced call cycle protocol with a tapered schedule was utilised for this study. Participants were provided with the option to opt-out of the GHS at the screening call, after which participants who agreed to participate in the program were contacted by the GHS once a week for the first 6 weeks, with the following 7 phone calls completed on a fortnightly basis. The completion of the GHS coaching was defined as completing all 13 coaching calls\u2014or if the participant reported they had reached their health goals, the option of early graduation was offered.To ensure consistency of clinical practice guidelines, training was provided to the GHS coaches by the SOS dietitian prior to the intervention period. The GHS coaching therefore targeted the intensive dietary interventions set by the SOS in line with best practice for obesity management ,6,7. The\u00ae, which non-invasively measures liver stiffness as a marker of liver fibrosis and hepatic steatosis was performed by research staff to measure median stiffness and controlled attenuation parameter (CAP) median changes. Data collection for the outcomes described were collected at consent (baseline) and repeated at 6 months. Participant acceptability of the GHS was measured using semi-quantitative methods administered by a blinded evaluator at the end of the GHS intervention period. Participants scored their response on a Likert scale from 1 (strongly disagree) to 5 (strongly agree), in addition to open-ended questions included for free comment.The primary outcome measure of this study was weight loss with weight and BMI changes obtained by dietitians using digital scales and a wall mounted stadiometer as part of patient support groups. To ensure consistency, patient support groups were scheduled at the same time each week and patients were advised to wear the same clothing and footwear. Secondary outcome measures included improvements in glycaemic control for those with T2D or prediabetes, liver stiffness, ALT and GGT enzymes and participant acceptability. For participants with prediabetes or T2D, HbA1c was measured by routine blood collections in addition to ALT and GGT enzymes and FibroScan\u00ae changes as well as ALT and GGT enzymes between control and intervention groups were calculated using a paired t-test. To determine differences in the same outcomes between the control and intervention arms, a mixed analysis of variance model (ANOVA) with Tukey\u2019s tests were used to correct for multiple comparisons. A p < 0.05 was taken to be statistically significant. Data were computed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA). Responses to the questionnaires were calculated using mean +/\u2212 SD using Excel (Microsoft\u00ae 2021).Differences between parameters achieved in the control and intervention arms from baseline to 6 months in total body weight, BMI, HbA1c, FibroScanA total of 80 participants were eligible to participate in the study from 1 May 2019 to 31 October 2019 . Among tp = 0.01) whereas those in the intervention arm lost \u221212.6 \u00b1 3.2 kg  at the end of the 6-month study period. The differences between both arms did not reach statistical significance  and intervention , respectively, with no statistically significant changes between cohorts .Compared to baseline, patients within the control group lost \u22126.7 \u00b1 2.2 kg  in the intervention group. Similar  reductions were seen in the control group following the study intervention . Between-group differences did not reach statistical significance .With respect to glycaemic control, differences in HbA1c were seen post-intervention (6.5 \u00b1 0.4% vs. 5.8 \u00b1 0.2) with a mean absolute difference of \u22120.7 \u00b1 0.2%  and intervention  at the end of the study period, but the differences between groups failed to reach statistical significance (p = 0.49). However, due to body habitus and large abdominal girths, FibroScan\u00ae scores could only be reliably obtained for 21 participants across the sample. The same observation was seen for CAP (p = 0.9) and intervention groups  although it did not reach statistical significance. GGT levels also decreased but failed to reach statistical significance in the both control  and intervention groups .There were trends for reduction in FibroScan for CAP . ALT simOf the 14 patients who completed the GHS coaching, a 100% response rate was achieved and showed enthusiasm for the GHS coaching with strong participant satisfaction reported . ParticiThe integration of telephone-delivered coaching into an intensive-lifestyle MDT program did not yield significant differences in health risk factors when compared to intensive lifestyle MDT care alone. Clinical within-group improvements were demonstrated for weight loss, improvements in glycaemic control as well as reductions in liver stiffness. A high level of patient acceptability, however, was reported on the addition of telephone-delivered coaching to MDT care. This is an important finding and consistent with the literature which also found the support and rapport built by the GHS coaches increased participant adherence and motivation . As disc2) or class II and below obesity categories (BMI < 40 kg/m2) [Despite being a public health priority, there is limited research on the role of telephone-delivered interventions for populations with severe obesity, with the majority of existing literature targeting populations in overweight (BMI 25\u201329.9 kg/m0 kg/m2) . While c0 kg/m2) , while tWhile weight loss was observed across both groups, participants who completed the telephone-delivered coaching in addition to MDT care achieved 8.88% total weight loss while participants who received MDT care alone reported an average of just 4.44% total weight loss over the 20-week intervention period. This is an important finding as a weight loss of 5% or more of initial body weight is considered a successful and clinically meaningful weight reduction leading to a decreased risk for development or improvement of obesity-related risk factors for many patients . While wDiRECT trial demonstrated that a structured and intensive weight management intervention can lead to sufficient weight loss and T2DM remission, with results maintained over 2 years [DiRECT study are comparable to the present study, consisting of a flexible VLCD, stepped food reintroduction and structured support for long-term weight loss maintenance. While HbA1c reductions were observed across both groups in the present study, participants who completed the telephone-delivered coaching in addition to MDT care achieved significant reductions in HbA1c as opposed to participants who received MDT care alone, strengthening the beneficial role of the GHS in populations at risk of and with established T2D.Previously considered a permanent and progressive disease requiring lifelong treatment, the  2 years ,45. The \u00ae may require longer to be fully appreciated. These results substantiate prior studies on the effect of telephone-delivered interventions on increased self-efficacy in adherence to diet, physical activity, and healthy behaviours [In parallel to the obesity epidemic, MAFLD has become a global health hazard, leading to inflammation and fibrosis, cirrhosis, liver failure, hepatocellular carcinoma and early mortality . Lifestyhaviours , as wellhaviours .There are several limitations to this study. Firstly, this was a non-randomised before and after study with the recruitment of the two cohorts occurring at different time points. Seasonal differences, holidays and the global COVID-19 pandemic may have impacted compliance to interventions and access to MDT care. Participant compliance with intensive dietary interventions also relied upon self-reported data obtained in weekly patient support groups. In addition, while participants who completed telephone-delivered coaching achieved higher reductions across all primary outcomes, the differences were not significant due to the small sample size. Only participants who graduated from GHS coaching and remained part of the SOS at 6 months were included in the follow-up data collection and analysis. As the sample sizes were small, this limits the generalizability of the findings and made it impossible to perform intention-to-treat analyses. This may reflect a biased, highly motivated group. While the study duration was appropriate to investigate early weight loss, long-term follow-up is required to investigate the effect of adjunct telephone-delivered coaching on long-term obesity management; however, the trends across the primary outcomes in this small pilot study provide reassurance of the utility of this program. More clinically significant results may be obtained with a trial of a longer duration. Beyond gender and age, no further patient demographics were collected as part of this study with recommendations for future research to investigate the effect of demographics such as ethnicity, education levels and socioeconomic status on outcome measures. Furthermore, controlling for baseline body weight, the time of year of intervention, ethnicity and physical activity was not possible due to COVID-19 restrictions at the time of the study.No significant differences in health risk factors were found with the integration of telephone-delivered coaching as an adjunct to MDT care for adults attending a public SOS. The high level of patient acceptability, however, indicates that telephone coaching aligned with the principles of an obesity service should be trialled to improve patient access to obesity interventions and is especially relevant in the Australian setting with many patients living in remote or in rural areas, especially in the current time of a global pandemic, which imposes challenges on many health services."}
+{"text": "The two predominant pathophysiological defects resulting in glucose intolerance are beta-cell dysfunction and insulin insensitivity. This study aimed to re-examine beta-cell function and insulin sensitivity across a continuum from normal glucose tolerance (NGT) to early type 2 diabetes (T2DM) employing highly specific insulin, C-peptide and intact proinsulin assays.A total of 104 persons with NGT, 85 with impaired glucose tolerance (IGT) and 554 with newly diagnosed T2DM were investigated. Following an overnight fast, all underwent a 4-h standardised mixed meal tolerance test (MTT), and on a second day, a sub-group underwent a frequently sampled insulin-modified intravenous glucose tolerance test (FSIVGTT) over a 3-h period. The participants were stratified according to fasting glucose and BMI for analysis.The MTT revealed that increasing FPG was accompanied by progressively elevated and delayed postprandial glucose peaks. In parallel, following an initial compensatory increase in fasting and postprandial insulin responses there followed a progressive demise in overall beta-cell secretory capacity. FSIVGTT demonstrated a major reduction in the early insulin response to IV glucose in persons with IGT accompanied by a dramatic fall in insulin sensitivity. Beyond pre-diabetes, ever-increasing fasting and postprandial hyperglycaemia resulted predominantly from a progressively decreasing beta-cell secretory function.This study utilising improved assay technology re-affirms that beta-cell dysfunction is evident throughout the spectrum of glucose intolerance, whereas the predominant fall in insulin sensitivity occurs early in its evolution.The online version contains supplementary material available at 10.1007/s00592-021-01785-9. Glucose homeostasis is regulated by a feedback loop, involving beta-cell secretion and insulin sensitivity to maintain glucose concentrations within a tight range , 2. GlucHistorically, it has been shown that in the early stages in the development of glucose intolerance, fasting hyperglycaemia (impaired fasting glucose) is predominantly due to insulin resistance , 7. HoweMany of the pathophysiological studies that highlighted the roles of beta-cell dysfunction and insulin resistance in the development of glucose intolerance were conducted 20 or more years ago , 8\u201316. TMore recent studies , 17, 18 Most recently, there has been a shift in focus towards recognising sub-groups of individuals with glucose intolerance ranging from pre-diabetes to overt type 2 diabetes with their respective risk of developing complications, based on their beta-cell function and insulin sensitivity \u201321.Therefore, there is a need to revisit this area utilising the recently available more specific immunoassays and standardised challenge tests to re-affirm or modify the historical findings. The data presented here describe the glycaemic and beta-cell secretory responses to two different challenge tests: a standardised mixed meal tolerance test (MTT) and a frequently sampled insulin-modified intravenous glucose tolerance test (FSIVGTT). These were performed within a few days of each other, in people with normal glucose tolerance (NGT), IGT and a large cohort of newly diagnosed, treatment na\u00efve participants with T2DM, not exposed to lifestyle change or pharmacological intervention. Glucose, insulin, C-peptide and intact proinsulin profiles were derived, and indices of beta-cell function, including the \u2018gold-standard\u2019 disposition index, and insulin sensitivity are described. The participants were also stratified according to their FPG and BMI to view the findings in the context of increasing glycaemia and obesity.Data collection was carried out between 1981 and 2007. A total of 743 participants were recruited within 2\u00a0weeks of diagnosis: 104 with NGT, 85 with IGT and 554 newly diagnosed with T2DM, all confirmed by a 2-h 75-g oral glucose tolerance test. Those with T2DM were referred directly from primary care with minimal advice and no pharmacological intervention given. All were confirmed GAD autoantibody negative.Ethical approval was received from South Glamorgan/Bro Taf Research Ethics Committee, and all participants provided informed consent. Procedures were conducted in accordance with the principles of the Declaration of Helsinki (1996) and Good Clinical Practice.Following a 10-h overnight fast, all subjects underwent a standardised MTT. A FSIVGTT was also performed in a sub-set of 286 subjects . All subjects had an intravenous cannula inserted for blood sampling, kept patent with a slow-running saline infusion. Baseline blood samples were taken (\u221230 and \u221215\u00a0min) before the meal was given (0\u00a0min) which was consumed within 10\u00a0min. Further samples were taken at regular intervals over the 4-h postprandial period (Supplementary Table 2) for determination of plasma glucose, insulin, C-peptide and intact proinsulin.Baseline fasting samples were taken , before 300\u00a0mg/kg body mass of 50% dextrose was administered intravenously over 2\u00a0min. At the 20-min time point, an intravenous infusion of insulin was given  . Blood sBlood was collected into tubes containing fluoride oxalate for determination of glucose and into lithium heparin for insulin, C-peptide and intact proinsulin, separated in a refrigerated centrifuge and the plasma decanted into labelled tubes and stored at \u221220\u00a0\u00b0C until assay.Glucose was determined using a glucose oxidase method . The insulin, C-peptide and intact proinsulin samples were measured using highly specific immunoassays. The earliest samples were measured as described by Sobey et al. [max) was the peak postprandial concentration and time to maximum (Tmax), the time (in minutes) to reach peak. Areas under the response curves (AUC) were calculated using the trapezoidal rule. Insulin/glucose ratio (representing the insulin response relative to the ambient glucose level) was calculated by dividing insulin by the corresponding glucose value (pmol/mmol). Acute insulin response (AIR) was the insulin Cmax during the first 10\u00a0min post-glucose bolus during the FSIVGTT.Maximum concentration (C1), an indicator of beta-cell function, was derived by modelling the glucose and C-peptide levels before and during the MTT as described by Hovorka et al. [Postprandial beta-cell responsiveness  was estimated using the minimal model method \u201328, utilNormality was assessed using the Shapiro\u2013Wilk test and QQ plots. Normally distributed data are displayed as the mean (standard deviation) and compared using t tests. Non-normally distributed data presented as the median (interquartile range) and compared using Kruskal\u2013Wallis with Bonferroni adjusted post hoc analysis.1) Glucose ToleranceNGTi) ii) IGTiii) T2DM-GT1 (FPG <8.5 mmol/L)iv) T2DM-GT2 (FPG 8.5\u201311.6 mmol/L) andv) T2DM-GT3 (FPG >11.6 mmol/L)2) BMIi) IGT-OB1 and T2DM-OB1 (BMI <27.7kg/m2),ii) IGT-OB2 and T2DM-OB2 (BMI \u226527.7 to 31.9kg/m2), andiii) IGT-OB3 and T2DM-OB3 (BMI >31.9kg/m2)Participants were divided into sub-groups according to glycaemic status. The IGT group contained both those with isolated IGT and IFG/IGT; as subsequently no differences in beta-cell function or insulin sensitivity were observed between these groups, the groups were combined for analysis. Further sub-division into tertiles of FPG within the T2DM group or BMI within the IGT and T2DM sub-groups was performed to ensure similar numbers could be included for analysis in each comparator group.Demographic data for the respective glycaemic sub-groups are displayed in Table The plasma glucose, insulin, C-peptide and proinsulin responses to the MTT are displayed in Fig.\u00a0Responses to the FSIVGTT are displayed in Fig.\u00a02, the fasting and peak glucose was lower than for BMI\u2009<\u200927.7\u00a0kg/m2. In T2DM, there were a progressive increase in fasting and peak insulin with increasing BMI and a similar increase in the insulin/glucose ratio (Supplementary Table 3a). In both IGT and T2DM, fasting and peak C-peptide and intact proinsulin levels were increased in those with the highest BMI .The plasma glucose, insulin, C-peptide and proinsulin responses to the MTT are displayed in Supplementary Fig.\u00a01(A\u2013D). Increasing BMI was not associated with any change in either fasting or peak glucose in subjects with IGT; however, in those with T2DM, with BMI\u2009\u2265\u200927.7\u00a0kg/mResponses to the FSIVGTT are displayed in Supplementary Fig.\u00a01 (E\u2013H). No differences in the glucose peak were observed with increasing BMI in either the IGT or T2DM groups; however, a significant increasing trend in acute insulin response was observed with increasing BMI in both IGT and T2DM .Insulin sensitivity and beta-cell function calculated during the FSIVGTT and MTT are displayed in Fig.\u00a0Insulin sensitivity and beta-cell function calculated during the FSIVGTT and MTT are displayed in Supplementary Fig.\u00a02A and 2B, respectively. In both IGT and T2DM, SI was only reduced in those with the highest BMI. Disposition index did not change significantly with increasing BMI in IGT; however, a significant reduction was observed in T2DM with the greatest BMI (Supplementary Table 3b).The two predominant defects in the pathogenesis of glucose intolerance are beta-cell dysfunction and reduced insulin sensitivity. The reported influence of these defects, however, can depend on multiple factors including the stage of development of glucose intolerance, assay methodology and challenge tests employed and the clinical circumstances of the individual under investigation. Consequently, making direct comparisons between studies is limited.We present here an assessment of the glycaemic and hormonal responses to two different carbohydrate challenge tests, with derived indices to represent both beta-cell function and insulin sensitivity, applied across a spectrum of participants with NGT, IGT and newly diagnosed, treatment na\u00efve T2DM, collected over a period of over 20\u00a0years. The assay antibodies employed for determination of the hormonal parameters were highly sensitive and specific, without the high cross-reactivities observed in earlier assays used in many previous studies. By stratifying the participants according to glycaemic status, the changes in beta-cell function and insulin sensitivity responses to both carbohydrate challenge tests could be viewed in the face of deteriorating glucose control.In response to the meal, with worsening glycaemia, the postprandial glucose levels were seen to reach progressively increased and delayed peaks. In parallel, insulin concentrations followed the well-recognised \u2018Starling\u2019s curve of the pancreas\u2019 pattern , where aUnlike insulin, the fasting and postprandial proinsulin remained significantly elevated compared to NGT in all of the glucose-intolerant sub-groups. This difference, added to the high cross-reactivity with proinsulin in early and less specific insulin immunoassays, may partly explain discrepancies in the literature with respect to changes in \u2018insulin\u2019 response with worsening glycaemia. In addition, the continued elevation of proinsulin in all of the glucose-intolerant sub-groups may offer the potential for its use as a biomarker of glucose intolerance.Following administration of the glucose bolus during the FSIVGTT, all of the glucose-intolerant participants displayed a severely blunted acute insulin response that continued to decrease to almost non-existent levels as FPG continued to increase in T2DM.A modelled index of beta-cell function  was estimated for all subjects. By utilising peripheral C-peptide rather than insulin concentrations during the MTT in the model, the pre-hepatic beta-cell response was determined. A hyperbolic decrease in beta-cell function was observed between NGT and IGT and subsequently between IGT and T2DM, continuing to fall significantly in each increasing T2DM FPG sub-group. Insulin sensitivity, derived from the FSIVGTT, also fell in a hyperbolic manner, falling rapidly at lower FPG levels, reaching a nadir in T2DM, all the significant changes occurring during the transition from NGT to IGT and subsequently to T2DM. In contrast to beta-cell function, no further significant changes were observed with increasing FPG in the participants with T2DM.Our findings that the decrease in beta-cell function is relentless with increasing FPG, whereas the decrease in insulin sensitivity is predominantly in the early stages of glucose intolerance (IGT), reaching a nadir on the advent of T2DM corroborate those observations from previous studies , 31 thatBeta-cell function and insulin sensitivity displayed a hyperbolic relationship. In NGT, a decreased beta-cell function was associated with an increase in insulin sensitivity and vice versa. In those with IGT, a similar relationship was observed; however, the relationship was shifted downwards towards the origin, with a further shift in those with T2DM. This is in agreement with previous oral glucose and euglycaemic clamp studies which noted the relationship , 33.The disposition index is considered the \u2018gold-standard\u2019 method of assessing beta-cell function as it \u2018corrects\u2019 for the prevailing insulin sensitivity. While displaying a similar overall trend to the more independent measure of beta-cell function, the magnitude of fall of disposition index, however, was far greater at lower FPG, falling by approximately 80% in IGT, in agreement with previous findings of an 80\u201385% loss of beta-cell function in the upper tertile of IGT . This hiThe main limitation of this study is its cross-sectional nature and long duration. Further limitations include the minimal number of individuals with isolated IFG in the analysis and also the difference in observed BMI between the NGT and glucose-intolerant groups, thus identifying the impact of increasing obesity on both insulin sensitivity and insulin secretion across the entire glycaemic continuum is difficult. The study does, however, have strengths including the large cohort of study participants, with a broad spectrum of glucose tolerance. Those, who were IGT and T2DM, were all newly diagnosed, had received minimal lifestyle advice and were all treatment na\u00efve at the time of investigation. Participants undergoing the meal tolerance test all consumed the same standardised mixed meal, while those who had the intravenous glucose tolerance test all underwent the insulin-modified protocol, allowing more successful modelling of the insulin sensitivity. Those that underwent both carbohydrate challenge tests did so within a few days, ensuring that the derived indices of insulin sensitivity and beta-cell function were performed in the same participants under similar conditions, and also allowed calculation of the disposition index.In summary, in this large cohort of untreated participants spanning the spectrum of glucose tolerance, increased glycaemia was associated with decreased insulin sensitivity and beta-cell function. The changes in insulin sensitivity were predominant in the early stages, i.e. at the lower levels of FPG observed between NGT and IGT, whereas beta-cell function continued to decrease with increasing glycaemia, and would therefore determine the progression of the disease. We feel these findings present a definitive view of the contribution of both decreased insulin sensitivity and beta-cell dysfunction to the development of glucose intolerance, established as they are in participants who were newly diagnosed, not influenced by lifestyle or pharmacological intervention and determined using well-characterised specific methodologies. The findings also continue to endorse the overall traditionally held views of the pathophysiology of glucose intolerance.Supplementary file1 (PPTX 340 KB)Supplementary file2 (PPTX 88 KB)Supplementary file3 (DOCX 28 KB)Below is the link to the electronic supplementary material."}
+{"text": "In vitro assays were used to evaluate the ability of these polysaccharide coated biocompatible, water-soluble, magnetic nanoparticles to deliver drug therapy across a model of the BBB. As a drug model, dopamine hydrochloride loading and release profiles in physiological solution were determined using UV-Vis spectroscopy. Cell viability tests in Human Lung Microvascular Endothelial (HLMVE) cell cultures showed no significant cell death, morphological changes or alterations in mitochondrial function after 24 and 48 h of exposure to the nanoparticles. Evidence of nanoparticle interactions and nanoparticle uptake by the cell membrane was obtained by electron microscopy (SEM and TEM) analyses. Permeability through a BBB model (the transwell assay) was evaluated to assess the ability of Fe3O4@CMC nanoparticles to be transported across a densely packed HLMVE cell barrier. The results suggest that these nanoparticles can be useful drug transport and release systems for the design of novel pharmaceutical agents for brain therapy.Sustained and safe delivery of therapeutic agents across the blood\u2013brain barrier (BBB) is one of the major challenges for the treatment of neurological disorders as this barrier limits the ability of most drug molecules to reach the brain. Targeted delivery of the drugs used to treat these disorders could potentially offer a considerable reduction of the common side effects of their treatment. The preparation and characterization of carboxymethyl cellulose (CMC) coated magnetic nanoparticles  cell barrier BBB model. Consequently, there is a pressing need to develop more efficient non-evasive and brain-directed therapies for neurological disorders.Neurodegenerative disorders such as Parkinson's and Alzheimer's diseases are the leading cause of disability and the second cause of mortality worldwide; these disorders now affect more than 250 million people globally. This number is expected to rise substantially as current population growth and the increase in life expectancy mean that more people will reach the age ranges where these disorders are prevalent.3 Development of drugs for the CNS is extremely costly. Only 3\u20135% of the pharmaceuticals intended for brain delivery reach the market, as most are unable to cross the BBB in vivo.4,5 Although it is well known that larger molecules (\u223c100%) have restricted BBB permeability, most small molecules (>98%) are also unable to cross the BBB. An estimated 95% of the small molecules in drug discovery libraries have very limited ability to permeate the BBB, tempering their pharmacological usefulness for the treatment of neurological disorders.4While brain-targeted drug delivery has been gaining increasing attention, these strategies pose a significant challenge for drug developers. Due to the very high cost of their development and the potential for undesired side effects and long-term health risks, new pharmaceutical formulations for the treatment of neurological disorders have the lowest approval rate in the drug development pipeline.6 Therefore, research in the field of nanotechnology has started to focus on the generation of nanostructured drug delivery carriers capable of crossing the BBB and delivering drugs to specific sites in the brain. Nanoscale drug delivery devices can absorb and carry drugs and then due to their ultra-tiny volume they can pass through the smallest capillary vessels to penetrate cells and tissue gaps. Their size allows them to avoid rapid RES clearance, so their duration in the bloodstream is greatly extended. The increased safety, efficacy, and bioavailability of nanoparticles (NPs) make them attractive options in the investigation of pharmacological therapies for neurological disorders.7Successful development of CNS drugs requires an understanding of both the pharmacological target and achieving sufficient permeability across the BBB to attain effective therapeutic concentrations in the brain. The functional complexity of this barrier demands the development of different strategies to effectively overcome it. One such strategy involves the design of more efficient drug delivery systems that can transport promising therapeutic and/or imaging agents over the BBB.8 Although brain targeting delivery systems can enhance the distribution of therapeutic drugs in the brain, an important factor to consider in the design of these systems is nanoparticle accumulation. As the treatment of chronic neurological disorders often requires long-term and frequent drug administration, nanoparticles could potentially build up in the body, causing undesirable side effects. To provide biological safety, the materials used for these systems should be biodegradable, compatible with the metabolic system, able to be eliminated from the brain and have a high potential for biological and biomimetic effects. Recently, research has focused on the use of natural materials for the fabrication of nanocarriers, as they inherently possess many of these qualities.Targeted delivery of these systems significantly reduces the required dosage, which may decrease the undesired effects of the treatment of these disorders such as adverse reactions and toxicity.3O4) SPIONs have received widespread acceptance within the scientific community as their magnetic properties make them highly attractive for biomedical applications, especially as agents for MRI and targeted drug delivery.9,10 The biocompatibility of these particles is frequently enhanced with organic or inorganic coatings that allow their suspension in aqueous or organic media.11 After drug molecules are attached to these delivery systems, the biodegradability, pH, ion and/or temperature sensibility of the materials can be used to activate drug delivery at a controlled and sustained rate to the target areas of the brain.12 An advantage of biopolymer-coated magnetic nanoparticles is that they have shown lower toxicity levels compared to those with bare cores. This phenomenon is attributed primarily to the spontaneous aggregation of the bare cores induced by the presence of plasmatic proteins and salts in biological media. The addition of polymeric layers confers an \u201canti-aggregation\u201d barrier to the magnetic cores, as well as an appropriate surface for functionalization.13Magnetite  (PEG) was often attached to the surface of these NPs as a \u2018\u2018stealth layer\u2019\u2019 to decrease protein adsorption and to increase their concentration in the in vivo circulation.16\u201318,22 However, there were a number of drawbacks to these particles, particularly concerning their biocompatibility and biodegradability, which may result in adverse side effects, whereas, natural polymers are proving to be a very attractive option for coating NPs as their chemical similarity to biomolecules already present in extracellular matrices affords the resulting nanomaterial high bioactivity and biocompatibility.The first nanocarriers were coated with artificial23\u201326 while comparatively little work has been done with other polysaccharides. Polysaccharides have been reported to reduce unspecific protein adsorption and increase plasmatic life.27 Furthermore, the biodegradability of these materials not only facilitates the eventual clearance of the nanocarrier but can be exploited to trigger drug release and activation by using certain enzymes to produce controlled degradation of the coating.28\u201331 These properties, together with their ability to interact with certain protein/cell surfaces and reduce particle aggregation make polysaccharides very interesting materials for the construction of brain-targeted NPs.The contemporary nanoparticle drug delivery field is studying several of these natural polymers as alternatives to PEG. Most of this work has focused on chitosan-based materials,32 Although this biopolymer is well known and highly versatile, successful uses of CMC for biomedical applications of nanoparticle technologies are quite limited. CMC can be used for the modification of magnetite nanoparticles as the high density of carboxylate ions per chain allow it to be physically adsorbed or conjugated on the surface of the nanoparticles, yielding a highly stable, water-soluble colloidal solution, and it is easy to modify with a reporter or targeting species.33\u201338 Studies exploring brain delivery systems indicate that nanoparticle surface charge has an important role in determining cellular uptake and the interactions between particles and cells.39 The negative charge of the CMC coating not only has a favorable impact on the nanoparticles' colloidal stability,39,40 it favors less plasma protein adsorption and therefore increases the plasma circulation time of the particles and confers other properties that make CMC a suitable coating material for in vitro and in vivo applications in the investigation of brain-targeted magnetic nanoparticles.41Carboxymethyl cellulose (CMC) is produced from the reaction of cellulose (from wood pulp or cotton fibers) under basic conditions with chloroacetic acid.42\u201345 Although these advances have become very popular, there are still a few important drawbacks to their use, such as drug-release failure resulting from the high stability of the generated bonds or reduced circulation times of the NPs due to alteration of the physicochemical properties when the ligands are attached.46 These drawbacks offset the potential benefits of active targeting as they affect the bioavailability and specific molecular recognition ability of the NPs.47In the past decade, enhanced properties such as adhesion of ligands  on the NP surface, ligand density, and NP shape have been shown to improve the transport of NP formulations through the BBB as well as improving molecular recognition and controlled release in specific targets.48\u201353 Most recently our research has focused on exploring alternative biocompatible polymeric coatings for these nanoparticles.54,55 Here, the preparation and characterization of CMC coated magnetite nanoparticles (Fe3O4@CMC) and the evaluation of their performance as drug delivery systems for dopamine are presented. Fluorescence and electron microscopy (SEM and TEM) studies were used to analyze the cell viability of HLMVE cell cultures exposed to the magnetic nanoparticles and their interaction with cell components. The ability of these CMC coated SPIONs to move through a BBB model was determined using the transwell assay. The results of our in vitro assays suggest that these systems may be useful for drug delivery to the brain. Furthermore, the simplicity of preparation, plentiful supply and the safety and biocompatibility of the components of these CMC coated magnetite NPs could offer a considerable reduction in the cost of the developmental phase of brain-targeted pharmaceuticals using these delivery systems.Our group has been exploring the biomedical applications of magnetic nanoparticles as drug carriers, MRI contrast agents and for magnetic hyperthermia.3\u00b76H2O, NH4OH, Na2SO3, 3-aminopropyl-trimethoxysilane (APTMS), carboxymethyl cellulose (CMC) and fluorescein were purchased from Sigma-Aldrich; fluorescent dyes 4,6-diamidino-2-phenylindole (DAPI)  and rhodamine phalloidin  were used as received without further purification. Water was doubly deionized, rendering conductivity in the range of 16\u201318 M\u03a9. Stock 2 M aqueous solutions of FeCl3 (dissolved in HCl 2 M) and Na2SO3 were freshly prepared. Glassware was cleaned with concentrated HCl, rinsed with deionized water and dried before use.Analytical grade FeCl3O4) nanoparticles were prepared based on a previously reported method.48,56 In summary, 1.08 g of FeCl3 was dispersed in 10 mL of DI water and stirred at 500 rpm until complete dissolution. Separately, 0.3975 g of FeCl2 was dissolved in 10 mL of DI water and stirred at 500 rpm until complete dissolution. Then, the Fe(ii) solution was added rapidly to the Fe(iii) solution while stirring strongly, and immediately afterward 2.5 mL of NH4OH (30%) was added. A black suspension formed and the recovered black solid was washed several times with deionized water. Finally, the nanoparticles produced with this method were separated by centrifugation, vacuum-dried at room temperature and stored. The nanoparticle size, as determined by TEM, was in the range of 11\u201317 nm. A strong tendency to agglomerate was observed in these nanoparticles. DLS analysis of the uncoated magnetic nanoparticles in DI water determined the average hydrodynamic diameter to be 30 nm.Magnetite (Fe57100 mg of the previously prepared magnetite was ground in an agate mortar with anhydrous toluene (3 mL) until a fine powder was obtained. Then, the powder was transferred to a 250 mL round bottom flask and dispersed in 57 mL of anhydrous toluene. A volume of 20 \u03bcL of 3-aminopropyltrimethoxysilane (APTMS) was added to this black suspension and stirred at 60 \u00b0C for 4 hours. Finally, the magnetic precipitate was magnetically decanted, washed twice with absolute ethanol, and then vacuum-dried at 50 \u00b0C for 30 minutes. Labeling with fluorescein was achieved following the well-known EDC/NHS coupling protocol of adding fluorescein (acid form) during silanization of the magnetite nanoparticles in a mixture of water/DMF.3O4 and Si\u2013Fe3O4 were coated with CMC following the same procedure. 100 mg of dried, finely ground magnetic nanoparticles were dispersed in 10 mL of DI and sonicated for 10 min. An aqueous 0.5% solution of sodium carboxymethyl cellulose (NaCMC) was prepared by dissolving 10 mg of NaCMC in 10 mL of DI, mechanically stirring until complete dissolution and then adding the NaCMC solution dropwise to the magnetic nanoparticle suspension and stirring at 500 rpm for 10 h. After this time, the material was magnetically decanted and washed once with DI. It was then dried in a vacuum oven at 50 \u00b0C for 20 min which yielded a dark brown powder. DLS analysis of CMC coated magnetite in aqueous suspensions shows hydrodynamic radii in the range from 40 to 120 nm, depending on the coating time, with zeta potential values (\u03b6) of \u221250 to 70 mV.Both pure Fe\u22121. Dynamic light scattering (DLS) and zeta potential (\u03b6) measurements were performed using a Nanotrac Wave II (Microtrac) instrument, working at 28 \u00b0C in DI water as the dispersing medium, with a red laser of 780 nm, 3 mW. The crystalline phase of the iron oxide nanoparticles was identified by powder X-ray diffraction (XRD). The patterns were collected between 20 and 70\u00b0 (2\u03b8) using a Bruker-AXS D5000 diffractometer on ground powders in a quartz sample holder using the Cu K\u03b1 line source (\u03bb = 1.5418 \u00c5); step scan = 0.02; step time = 0.6 s. Scanning electron microscopy (SEM) images were obtained using a Tescan VEGA-II microscope, with an accelerating voltage of 20 kV. SEM specimens were dispersed in ethanol by ultrasonication, and a few drops were deposited on a graphite film adhered to an Al pin. The size and morphology of the NP were determined by transmission electron microscopy (TEM) using a JEOL JEM-120EXII electron microscope. TEM samples were prepared by placing one drop of a dilute suspension of magnetic nanoparticles in water on a carbon-coated copper grid and allowing the solvent to evaporate at room temperature. The average particle size was evaluated by measuring the largest internal dimension of \u223c200 particles. High-resolution transmission electron microscopy (HRTEM) analyses were performed using a JEOL Model JEM2010 electron microscope operated at 200 kV accelerating voltage.Fourier Transform Infrared (FT-IR) spectra of the nanostructured materials were recorded using a Varian Scimitar FTIR spectrophotometer equipped with an ATR detector and recorded in the region of 3000\u2013600 cm\u22121 penicillin, 50 mg mL\u22121 streptomycin, and supplemented with 5% fetal bovine serum (FBS), at a final concentration of 10%. All the media, serum, and antibiotics were provided by Life Technologies . Cell cultures were performed in a 5% CO2 atmosphere at 37 \u00b0C and maintained in an incubator. For the experiments, the cells were seeded into 24-well plates at an initial density of 1 \u00d7 104 cells per well. Treatments were initiated three days after plating (approximately 70% confluence). For certain experiments , the cells were seeded on 13 mm square glass coverslips placed into the wells. To analyze the internalization of nanoparticles, HLMVE cells were grown on coverslips and incubated for 24 h with different concentrations of nanoparticles .HLMVE cells, obtained from the Centre for Cell Engineering at the University of Glasgow, were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) with 50 units mLFor SEM analysis, after incubation, the containing medium was removed; the cells were washed three times with PBS and fixed with 1 mL of 1.5% glutaraldehyde in cacodylate buffer and 2% sucrose at 4 \u00b0C for 10 min. The cells were osmicated first with 1% osmium tetroxide, and then with 2% uranyl acetate for 5 min. After this, each slide was transferred into a Petri dish containing hexamethyldisiloxane (HDMS), dried in a desiccator and sputter-coated with a thin layer of gold in preparation for scanning electron microscopy (SEM) analysis. For TEM analysis, 13 mm Thermanox coverslips for seeding cells were used in 24-well plates; cell fixation was carried out following the previously described procedure. The samples were then dehydrated and embedded in EPON-812 resin, then frozen in liquid nitrogen and sectioned using an ultramicrotome. Slices were mounted on copper grids and analyzed by transmission electron microscopy.2. Next, the wells were washed with Ham's F-10 medium and 1 mL of staining solution  was added to each well and incubated for 1 h at 37 \u00b0C. For each sample, the assay was performed in duplicate. The cytoskeleton and cell nuclei were stained with rhodamine phalloidin (200 \u03bcL) and DAPI (50 \u03bcL), following standard procedures.58,59 Briefly, the cells were fixed in glutaraldehyde and, permeabilized ; 0.476 g HEPES and pH adjusted to 7.2, followed by the addition of 0.5 mL Triton X). Non-specific binding sites were blocked by incubation with PBS/1% BSA for 5 minutes at 37 \u00b0C prior to incubation with rhodamine phalloidin for 1 hour at 37 \u00b0C . Following washing, the cells were further incubated with DAPI mounting medium (Vector Laboratories). All images were viewed using an Axiophot fluorescence microscope.The cells were seeded on 13 mm square glass coverslips, placed into 24-well plates and incubated for 24 h. Then, the medium was replaced with fresh medium containing nanoparticles and incubated for 24 h, at 37 \u00b0C and 5% CO\u22121 in PBS) for 1.5 h at 37 \u00b0C and 5% CO2. Then 100 \u03bcL of DMSO was added to each dish to dissolve the formazan crystals that formed in the cells. The absorbance was measured with a microplate reader (Tecan Spectra Fluor spectrophotometer) at 570 nm for each well. Cell survival was determined by the percentage of absorption of treated cells in comparison with that of control cells (incubated without nanoparticles) and was calculated using the following equation:The viability of the HLMVE cells was determined using a standard methylthiazol tetrazolium bromide (MTT) assay. Briefly, this involved incubation of the cells with unloaded or dopamine-loaded nanoparticles in 24-well plates for 24 and 48 h, after which MTT was added to each well . The total amount of dopamine loaded onto the nanoparticles used for cell exposure never exceeds 0.13 mg per mg of magnetite.The results are the mean value and standard deviation (SD) obtained from three repetitions of the experiment (5 HLVMEC cells per well were overlaid with 125 \u03bcg mL\u22121 growth factor-reduced Matrigel (diluted in HamF10) and placed in a 24-well plate, at 37 \u00b0C and 5% CO2, and incubated for 24 h. For each sample, the assay was performed in duplicate. After incubation, the medium was replaced with fresh medium containing magnetic nanoparticles labeled with fluorescein (fSi\u2013Fe3O4), at a concentration of 1 mg mL\u22121. To evaluate the integrity of the monolayer as a BBB model the transendothelial electrical resistance (TEER) was measured using an EVOM2 epithelial voltmeter with an STX2 electrode (World Precision Instruments) at 12.5 Hz, as reported previously.60 These measurements were taken during three stages of the test: before addition of the magnetic nanoparticles, and after 24 and 48 h of incubation with them. After exposure to the magnetic nanoparticles, the culture medium in the bottom of the well was recovered and transferred to a tube and pelleted by centrifugation at 1200 rpm for 3 min. The supernatant was eliminated, and 30 \u03bcL of the pellet was transferred to a glass slide for analysis by fluorescence microscopy.Cell culture inserts (transwells) with a density of 1 \u00d7 10\u22121. A UV-visible spectrophotometer was used to determine the absorbance of the above concentrations at 280 nm using DI water as a blank. These concentrations were used to plot a calibration curve. The tests were carried out at room temperature (25 \u00b0C) and pH 7.0. For loading the nanocarrier, 10 mg of dried CMC coated magnetite nanoparticles (Fe3O4@CMC), prepared as previously indicated, were dispersed in 50 mL of an aqueous solution containing dopamine (30 \u03bcg mL\u22121) at pH 7.0, and mechanically stirred at 200 rpm for 10 h. After this time, the nanoparticles were magnetically decanted and washed with ice-cold water, and then dried for 2 h in a vacuum oven at 50 \u00b0C. The amount of dopamine loaded onto the nanoparticles was determined spectrophotometrically (at \u03bb = 280 nm) by measuring 1 mL of aliquots at spaced intervals  during the incubation time. The amount of dopamine entrapped within the nanoparticles was calculated from the difference between the total amount of dopamine (M1) used to prepare the nanoparticles and the amount of dopamine present in the aqueous phase (M2). The following formula was used:To determine the performance of the NPs as drug carrier and release systems, dopamine hydrochloride (Aldrich) was dissolved in DI water and then diluted to obtain concentrations within the range of 1 to 10 mg mLTo determine dopamine release kinetics, 10 mg of the dried drug-loaded nanoparticles were dispersed in 50 mL of DI water at room temperature and pH 7. The concentration of free dopamine in the aqueous solution was determined using a UV spectrophotometer under constant mechanical stirring (200 rpm), for 4 h. 1 mL aliquots were taken at different time intervals ; the amount of released dopamine was determined using the previously established calibration curve.3O4, and silanized magnetite, Si\u2013Fe3O4, were synthesized as superparamagnetic carrier cores through the chemical coprecipitation method.56,61 These magnetic nanoparticles were coated with the polysaccharide carboxymethyl cellulose (CMC) by an electrostatic adsorption method.62 Silanization of the magnetite nanoparticles was tested to ascertain if it improved the stability of the NPs as well as the immobilization of CMC on the nanoparticle surface. No significative differences were found between the non-silanized and silanized CMC-coated nanoparticles. The hydrodynamic radii were similar in both cases (around 110.0 \u00b1 10.0 nm), and the aqueous suspensions were stable for several days at room temperature, which agrees with their highly negative zeta potential values. The morphology and size distribution of the nanomaterials were characterized by SEM and TEM. 3O4, Fe3O4@CMC, and Si\u2013Fe3O4@CMC nanoparticles); the particles are mostly spherical and show a narrow size distribution, with average sizes of 19.90 nm \u00b1 3.06 nm (Fe3O4), 14.05 \u00b1 1.70 nm (Fe3O4@CMC) and 14.96 \u00b1 4.16 nm (Si\u2013Fe3O4@CMC). Smaller nanoparticle average sizes were obtained when the nanoparticles were coated with CMC, which may be the result of reduced agglomeration of these NPs in solution. The average sizes of these magnetic nanoparticles are in the range required for superparamagnetism, as well as for biomedical applications.63Nanoparticles of magnetite, Fe\u22121. New vibrational bands appear at frequencies between 800 and 1100 cm\u22121 due to the presence of aminopropylsilane on the surface (\u22121 associated with C\u2013O and C\u2013C bonds due to the presence of CMC on the nanoparticle surface (\u03b6) measurements.The nanoparticle's crystalline phase was identified by X-ray diffraction (XRD) . The XRD surface  and betw surface . The pol\u22121 of magnetic nanoparticles in the cell culture medium for 24 h. Analysis by confocal fluorescence microscopy of cells stained with calcein AM and ethidium homodimer-1 showed isolated live cells (green), negligible aggregation of the nanoparticles and no evidence of dead cells (red) after 24 h of incubation  were loaded with dopamine  containing Ham's F-10 medium at 37 \u00b0C and 5% CO2 (1 \u00d7 105 cells per well). The cells were incubated for 24 h, after this time the medium was replaced with fresh medium containing 0.1 mg mL\u22121 of fluorescein-labeled Fe3O4@CMC or Si\u2013Fe3O4@CMC nanoparticles.An 2. 24 h after the addition of magnetic nanoparticles the TEER values of Fe3O4@CMC and Si\u2013Fe3O4@CMC were on average 212 \u00b1 5 \u03a9 cm2 and 195 \u00b1 5 \u03a9 cm2, respectively, while after 48 h they were on average 197 \u00b1 13 \u03a9 cm2 and 185 \u00b1 12 \u03a9 cm2. The resulting TEER value of \u223c200 \u03a9 cm2 is considered consistent with the formation of an intact BBB. Aliquots were taken from the lower chamber after 24 and 48 h and analyzed with a fluorescence microscope. Fluorescein labeling of the magnetic nanoparticles was used for monitoring their transmigration from the upper to lower chamber in the transwell inset through the BBB model. In 3O4@CMC and Si\u2013Fe3O4@CMC), even after just 24 h. This result indicates that the fluorescein labeled, CMC coated magnetic nanoparticles can cross through the densely packed barrier of HLMV endothelial cells used as a BBB model. Silanized magnetite nanoparticles coated with CMC had a higher transmigration rate than those that were not silanized, and they had a lower propensity for aggregation in the medium. The values of TEER were near the standard 200 \u03a9 cm2 at the beginning of the experimental period and remained close to this value after 24 and 48 h, suggesting that exposure to the fluorescein labeled, CMC coated magnetic nanoparticles did not compromise the integrity of the BBB model. The results obtained from this BBB model are in agreement with those previously obtained by Thomsen and co-workers, who studied the uptake and transport of SPIONs through an in vitro BBB model made of human brain capillary endothelial cells (HBCEC).64The integrity of the grown BBB model was evaluated using TEER measurements at the onset and during the experiments, and these were compared to the control (non-cultured wells). Before the addition of fluorescein-labeled magnetic nanoparticles, the TEER value for the non-cultured wells (control) was on average 219 \u00b1 7 \u03a9 cm3O4@CMC system was selected for the evaluation of dopamine loading and release. For this evaluation, magnetic nanoparticles were loaded with dopamine. Up to 0.13 mg of dopamine was loaded in the nanocarrier per mg of magnetite. This amount was calculated by considering the total amount of dopamine available in the solution and the amount of magnetite dispersed in it. 3O4@CMC over a period of 10 h and the dopamine release profile over 4 h at room temperature and pH 7.0. A drug LE% of 84.6% was estimated ; this was likely to have been the dopamine adsorbed on the surface of the carrier. The remaining dopamine released at a slower rate during the next few hours, which can be associated with the release of the dopamine in the polymeric coating, reaching almost an invariant concentration in solution after 4 hours. The sustained-release pattern suggests that this system may be a good candidate for controlled drug delivery and release, although the release profile in physiological solution still needs to be determined.The Festimated , which iQt = Q0 + K0t; first order, ln\u2009Qt = ln\u2009Q0 + Ket and Higuchi, Qt = Q0 + Kht1/2). Considering the R2 values, the calculated zero-order (R2 = 0.926) and first-order (R2 = 0.718) models were not appropriate to describe the drug release kinetics. The release kinetics appeared to be square root time-dependent, as in the Higuchi model (R2 = 0.992), suggesting that diffusion plays an important role in the release of the drug.Dopamine release kinetics were analyzed using various mathematical models  or high (\u221215 to \u221245 mV) negative zeta (\u03b6) potentials and even moderate (up to 15 mV) or higher positive \u03b6-potentials73\u201376 have been able to cross the BBB and deliver drugs to the brain.77,78 This may be due to the greater cellular uptake profile of positively charged NPs in brain microvessel endothelial cells compared with negatively charged IONPs of similar size.79,80 Experiments using magnetic field assisted permeability show increased uptake for nanoparticles with a greater negative charge, suggesting that negatively charged NPs are likely to follow a paracellular route, which makes them more suitable for magnetic assisted drug targeting.81 An in vitro study of carboxymethyl dextran-coated NPs concludes that the uptake of most negatively charged particles seems to occur via non-specific interactions.39The surface charge of CMC coated magnetite NPs is dependent on the pH and the amount of CMC and may be tailored to improve their BBB permeability. Although studies have shown that NPs with high positive charge are immediately toxic to the BBB,\u03b6-potentials of CMC/Fe3O4 NPs make these particles apt for another interesting application for brain-targeted magnetic nanoparticles. As recently reported by Dante et al., the NP surface charge is key for the modulation of neuronal electrical activity.82 Their findings regarding selective NP\u2013neuron interactions open up the possibility for novel applications of NPs in neuroscience, specifically for the design of NPs capable of neuronal subtype-specific targeting. This research suggests that negatively charged NPs could be used in long-term imaging as markers of active neurons, which would enable visualization of the aberrant increased neuronal activity of neurological disorders. In addition, the increase in neuronal activity produced by these NPs could be used to increase the activity of inhibitory neurons with reduced excitability, which is a hallmark of the severe forms of epilepsy.83 These NPs may eventually be utilized to modulate the balance between excitation and inhibition in the brain, which is a significant factor in most neurological diseases.The characteristics of these versatile NPs make them apt for multi-modal functions. In addition to their potential for drug delivery to the brain, they can serve simultaneously as magnetic resonance imaging contrast agents. Furthermore, radiocontrast agents may be attached to these delivery systems, which would facilitate better imaging and diagnosis of neurodegenerative disorders. The negative Superparamagnetic nanoparticles coated with a layer of the biocompatible polysaccharide CMC were prepared and fully characterized. Cell viability assays, as well as a complete analysis of their interaction with the cell membrane, cell internalization and ability to pass through a model of the blood-brain barrier (BBB), were performed in cell cultures. No indication of toxicity was found, and our results indicated that the nanoparticles accumulated in endosomes. Clear evidence of crossing through a barrier of densely packed endothelial cells was observed; these results indicate that CMC coated magnetic nanoparticles may show great potential as drug delivery systems for neurological treatments. Further testing is required for the continued development of this drug delivery system.3O4 NPs can be tailored for use in different applications and are very attractive for brain-targeted magnetic nanoparticle research. After the attachment of drug molecules to these delivery systems, the neutral to negative surface charge of the CMC coating may facilitate BBB crossing.82,84 The biodegradability of the coating of the nanoparticles allows the drug to be delivered at a controlled and sustained rate to the target site in the brain. In addition to the already proven advantages of paramagnetic NPs for drug delivery, the ferrite cores allow magnetic field targeting that could favor both permeability of the BBB and specificity of the drug release site.64,85 The CMC coating creates a hydrophilic surface that avoids agglomeration, increases nanoparticle dispersion in physiological solution and extends bioavailability; it also provides an appropriate surface for functionalization with targeting moieties to trigger drug release or an enzymatic stimulus, which would allow for even more precise targeting of specific cells. Targeted delivery of these systems would significantly reduce the required dosage of the therapeutic agent, which may decrease the undesired effects of the treatment of neurological disorders such as adverse reactions and toxicity. Furthermore, the simplicity of preparation, plentiful supply and the safety and biocompatibility of the components of these CMC coated magnetite NPs could offer a considerable reduction in the cost of the developmental phase of brain-targeted pharmaceuticals using these delivery systems.In summary, the physicochemical properties of CMC/FeThe manuscript was written with the contribution of all authors. All authors approved the final version of the manuscript.All authors declare no competing interest."}
+{"text": "Participants received either the POD Adventures intervention delivered over 4 weeks or usual care comprising information about local mental health services and national helplines. Outcomes were assessed at two timepoints: baseline and 6 weeks post-randomisation.n = 5), and only four control arm participants completed outcomes. No qualitative interviews or participant satisfaction measures were completed because participants could not be reached by the study team.Seventy-nine classroom sensitisation sessions reaching a total of 1575 students were conducted. Ninety-two self-initiated study referrals (5.8%) were received, but only 11 participants enrolled in the study. No intervention arm participants completed the intervention. Outcomes at 6 weeks were not available for intervention arm participants (Despite modifications to address barriers arising from COVID-19 restrictions, online delivery was not feasible in the study context. Low recruitment and missing feasibility and acceptability data make it difficult to draw conclusions about intervention engagement and indicative clinical outcomes. Prior findings showing high uptake, adherence and engagement with POD Adventures when delivered in a school-based context suggest that an online study and delivery posed the biggest barriers to study participation and engagement. There have been considerable psychosocial impacts linked to suspended routines and recreation, and rising concerns for family income and health.1 The pandemic has also been linked with rising incidence of some mental disorders among adolescents and exacerbations in pre-existing mental health problems.12Public health measures, such as lockdowns and school closures, have severely affected many adolescents during the COVID-19 pandemic, despite relatively little mortality and morbidity arising directly from infection.14 Reviews of digital mental health interventions have consistently raised concerns about the accessibility and reach of digital technologies, especially among disadvantaged groups,15 and difficulties in keeping participants engaged irrespective of social background.16 Promising engagement approaches have recently emerged ; however, evidence is scarce on effectiveness and uptake, especially in LMICs. A recent review of 18 systematic reviews and meta-analyses of digital mental health interventions for adolescents found no studies from low-resource settings.18 Another comprehensive review of 83 studies of digital mental health interventions for children and young people found only one report from an LMIC.19COVID-19 disruptions have accelerated the transition to online delivery of mental healthcare.www.podadventures.in) is part of the PRIDE research programme (2016\u20132022), which was designed to address the scarcity of evidence-based interventions for common adolescent mental health problems in India, and low-resource settings more broadly. PRIDE involved the development and evaluation of a suite of transdiagnostic psychological interventions to be delivered by non-specialist (\u2018lay\u2019) counsellors in under-resourced school settings.22The current study describes a pilot feasibility and acceptability trial of \u2018POD Adventures\u2019, a gamified problem-solving intervention delivered via a smartphone app and supported by non-specialist counsellors. The intervention was aimed at a target population of secondary school students in India during the COVID-19 pandemic. POD Adventures . Findings showed that the intervention was highly acceptable, engaging and feasible to deliver in school settings. Indicative clinical outcomes showed significant reduction in problem severity and mental health symptoms after 4 and 12 weeks.26 The timing of the COVID-19 outbreak meant that a planned randomised controlled trial designed to evaluate this offline, in-school mode of delivery of POD Adventures had to be modified to fit around extended school closures, which were instigated in India from March 2020 onward (and remained in place for nearly 2 years). The specific objectives of this modified trial were to assess whether the feasibility and acceptability of POD Adventures would be replicated when delivered online and with remote telephone-based support.POD Adventures is grounded in stress-coping theory,We conducted a parallel, two-arm, individually randomised controlled pilot trial with outcomes assessed at two timepoints: baseline and 6 weeks post-randomisation. Originally designed as a full-scale trial intended to take place in person from June 2020, we modified the original protocol into a remotely delivered online pilot trial before trial registration or any participants enrolling in the original trial.27 Trial findings have been reported according to CONSERVE (CONSORT and SPIRIT Extension for RCTs Revised in Extenuating Circumstance) guidelines for trials, modified for COVID-19.28The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human patients were approved by the Institutional Review Boards of Sangath (the implementing organisation in India) , Harvard Medical School (the sponsor), London School of Hygiene and Tropical Medicine (collaborator) and the University of Sussex (collaborator). This trial was registered at ClinicalTrials.gov (identifier NCT04672486). Additional permissions were obtained from all participating schools. The pilot protocol has been previously published.26 The schools comprised adolescents from both centrally located urban and remote rural areas of the state.The trial was conducted during partial  and complete COVID-19 school closures between December 2020 and May 2021 in 11 co-educational, government-aided, English-medium secondary schools in Goa, India, with an overall sampling frame of approximately 2500 students. Schools had an average of 230 students within grades 9\u201312. Goa is one of India's most urbanised states, and offered a suitable context in which to evaluate an online intervention intended for low-resource settings. Goa was also the setting of an earlier uncontrolled evaluation of the offline version of POD Adventures.We recruited participants who (a) were enrolled in grades 9\u201312 (ages 13\u201319 years) in collaborating schools; (b) had access to an internet-enabled Android smartphone with a valid telephone number for the duration of the pilot trial; (c) were able to read and understand English as a primary language and (d) were willing to provide written assent/consent (including from a parent/guardian (\u2018caregiver\u2019) for participants aged <18 years). We excluded students who were unable to comprehend the intervention materials (e.g. owing to a reading or hearing disability or inability to comprehend English) or were identified as having an elevated risk of self-harm or suicide and requiring external referral, based on a brief screening questionnaire and follow-up structured interview during study enrolment.Recruitment involved a brief 20- to 30-min sensitisation session delivered to individual classes either online  or, where social distancing policies allowed, in school with a slideshow and brief video containing information about the study; and, where feasible, distribution of a downloadable or printed information flyer via school-moderated email/WhatsApp groups to enrolled students, explaining the study and how to participate.Interested students were directed to visit the study website from their homes and using their own or borrowed devices. The website could be accessed in a language of their choice . Students first completed a short eligibility assessment consisting of questions related to their age, class and language. Eligible students were then prompted to watch an animated video and/or read information about what study participation entailed. Ineligible students were provided with a downloadable information flyer containing details about local and national services and helplines.Potential participants were guided through a brief online registration process and asked to provide basic demographic details and a telephone number, and to create a password for their use of the study website. This information was sought to be able to contact potential participants to obtain assent or caregiver/parental consent. Following registration, assent/consent was obtained through a web-based consent form via the study website, which was e-signed and dated by participants and the caregiver/parent. Consent was obtained from participants aged \u226518 years and assent from those aged <18 years. For participants aged <18 years, web-based caregiver/parental consent was followed by a confirmatory telephone call from the study team within 2 working days. A toll-free telephone helpline was also made available for prospective participants to ask questions or seek technical support for registration.The research team implemented the study in line with local and national public health guidance and made every effort to minimise in-person visits to schools unless specifically requested by the school authorities. Fieldwork safety training was provided to all study team members.The POD Adventures intervention comprised an app and brief counsellor guidance via telephone. Participants also received information about local mental health service providers and government provided/affiliated helplines .21 The app was offered in English text with English, Konkani or Hindi voiceover options. Formative work conducted in Goa as part of the intervention design process indicated an overall student preference for English as the primary language of the POD Adventures app. Voiceovers in Hindi and Konkani were added to further assist with comprehension for students who spoke one of these local languages at home.21 Guidance was offered in a language of the participants\u2019 choice.The content of the POD Adventures app comprises two sections: \u2018Adventures\u2019 teaches problem-solving concepts and methods through contextually appropriate stories and games; and \u2018My POD\u2019 guides a individual through the application of step-by-step problem-solving procedures for their own prioritised problem(s). The description of the app has been published elsewhere.The intervention was initiated by watching a 1 min pre-recorded orientation video via the study website and a brief 10\u201315\u00a0min on-boarding telephone call with a counsellor, in which the counsellor offered an overview of the intervention and worked with the participant to identify and prioritise a target problem(s). The counsellor also provided the participant with app download instructions, and participants were expected to download the app from the study website. Participants were then encouraged to work at their own pace through the Adventures content, and apply the steps of problem-solving to their own prioritised problem(s) in the My POD section of the app. During the fourth week of the intervention or after completing both sections of the app, whichever was first, a brief review call was scheduled between the counsellor and participant via text message or a telephone call. The purpose was to discuss the participant's progress, overall learning and their plan for managing future problems.For the duration of the study, each participant received a weekly reminder via text message containing encouragements to use the app. They also received a notification reminder to use the app if they did not log in for 5 consecutive days. In addition, counsellors proactively made telephone calls to participants who did not use the app despite reminders. On-demand telephone support from a counsellor was offered for addressing technical problems and clarifying app content throughout the study. A troubleshooting guide about installing the app, resetting passwords and online connectivity problems was made available for participants on the study website.29 and 1 year of experience in facilitating use of the POD Adventures app in school-based group sessions.26 Counsellors received a 4-day training built around a printed intervention manual. Training included an orientation to the intervention and its contents, and detailed instructions about how to conduct a classroom sensitisation session, procedures involved in session-by-session guidance, participant safeguarding and arrangements for providing technical app-related support to participants. The counsellors\u2019 supervision consisted of weekly peer group supervision meetings (lasting approximately 1 h), moderated by a psychologist. In each meeting, counsellors discussed progress of individual participants, reviewed fidelity checklists from the telephone sessions and identified areas where troubleshooting or support might be required by participants.Guidance was provided by multilingual non-specialist counsellors. Counsellors had 2 years of experience in delivering a face-to-face  problem-solving intervention,Control arm participants received enhanced usual care, comprising a digital flyer with information about and contact details for local mental health service providers and government-provided and -affiliated mental health helplines.We collected descriptive sociodemographic data about the selected school populations and adolescents registering for the study. Enrolled participants were also asked to respond to four questions about their mobile phone and internet ownership and use.Feasibility of research procedures was assessed with routinely logged frequencies and proportions of eligible/ineligible self-referrals (with reasons for ineligibility), assenting/consenting participants (with reasons for not assenting/consenting), randomised participants (with reasons for not randomising) and completed outcome assessments (with reasons for non-completion).Feasibility of intervention delivery was assessed with routinely logged frequencies and proportions of participants who logged into the app at least once, completed individual sections of the app and completed the intervention overall . Granular data on participants\u2019 use of the app was also recorded via integrated analytics software. Exploratory variables of interest included knowledge of problem-solving assessed through multiple-choice quizzes, and self-reported use of problem-solving in real-world situations (extracted from the My-POD section of the app).29 with four additional forced-choice items that asked specifically about the experience of using the POD Adventures app. Qualitative interviews were also planned to investigate participants\u2019 experiences of online research procedures in both trial arms, with additional questions planned about the acceptability of the POD Adventures app and counsellors\u2019 input for intervention arm participants. Interviews were to be conducted via telephone within 2 weeks of completing the follow-up assessment with a subsample of participants, purposively selected from both trial arms.Participant satisfaction data was intended to be collected from participants in the intervention arm at 6 weeks, using an adapted eight-item participant satisfaction measure that had been used in previous PRIDE studies,30 and self-reported depression and anxiety ).31 Assessments were carried out at two timepoints: baseline (pre-randomisation) and post-intervention follow-up (6 weeks after randomisation).Indicative clinical outcomes were assessed with two validated self-report questionnaires that measure psychosocial problem severity (Youth Top Problems)32 which recommended a sample size of 70 participants (35 per arm) to estimate the s.d. for a continuous outcome with adequate precision for a pilot RCT.We used a confidence interval approach for the calculation of sample sizes for external pilot randomised controlled trials,Each participant was allocated a unique, anonymised identification number after registering on the study website. Upon completion of consent, a notification was sent to the study data manager via a secure web portal designed for the study data collection.The randomisation algorithm was computer-generated and stratified by school grade, using randomly sized blocks of 4, 6 and 8. Randomisation was performed by the data manager on this platform, and the outcome of allocation was communicated to the participants through a telephone call from a researcher and an SMS text message alert, both of which informed the participant to log in to the study website for information about their allocation. The study website consisted of a personalised dashboard that directed the participant to their next step.Participants and counsellors were not blinded to allocation status. However, other members of the research team  remained blind to participation allocation status.The first participant was enrolled on 28 January 2021, and their 6-week assessment was completed on 4 April 2021. Baseline data was collected via the study website. Participants received an automated SMS alert to initiate the baseline assessment after completing the above-mentioned assent/consent procedures. A researcher additionally contacted participants by telephone to remind them to complete the baseline assessment if it had not been completed within 2 days.The follow-up assessment was initiated by SMS text message invitation exactly 6 weeks after randomisation. This message was followed up by a telephone call from a researcher following a standardised script that asked participants to complete the assessment. Automated SMS text message reminders were sent to participants every 3 days over the next 2 weeks or until the follow-up assessment was completed on the study website. Researchers made a minimum of four telephone attempts following the due date, with a maximum allowance of 2 weeks.The research study team received training on conducting remote and in-person recruitment activities, such as sensitisation sessions; participant consent procedures via the study website and telephone; providing telephone reminders for assessments and technical troubleshooting via the toll-free study helpline. Supervision of the research team consisted of weekly meetings (lasting approximately 1 h), moderated by the study coordinator. In each meeting, the team reviewed participant enrolment progress, data collection procedures and any technical difficulties encountered that required troubleshooting.The study was hosted on the servers of Sangath, the implementing organisation based in Goa, India. These servers were encrypted, with data back-ups occurring daily. The study web portal and its associated data were accessible only to authorised and approved personnel.The statistical analysis was mainly descriptive in nature, aiming to provide estimates of key feasibility and acceptability parameters and indicative clinical outcomes. The outcome measures were summarised at baseline and at 6-week follow-up, by trial arm. These were summarised by mean (s.d.), median (interquartile range), or number (percentage) values overall, and stratified by age, gender and baseline outcome score.n\u00a0=\u00a069, 75%) originated from in-person sensitisation sessions, followed by referral forms via drop boxes placed in schools . Only five referrals (5.4%) were made through the toll-free helpline following online sensitisation.Overall, 79 sensitisation sessions  were conducted, reaching a total of 1575 students. From the sensitised sample, 92 referrals (5.8%) were received, all self-initiated by students. Most referrals (n\u00a0=\u00a03). No students were excluded because of risk. Of the 34 eligible referrals, 16 (45%) completed consent procedures and the remaining 18 referrals did not enrol. Reasons for non-participation included students who were uncontactable (n\u00a0=\u00a09), unable to access a telephone/internet (n\u00a0=\u00a04), examinations (n\u00a0=\u00a02), problem resolved (n\u00a0=\u00a02) and parent consent denied (n\u00a0=\u00a01). The mean time taken from referral to randomisation was 6.8 days (s.d.\u00a0=\u00a08.8).From the referred sample, 38 students (41.3%) completed the online eligibility self-screener. One student was excluded because of literacy difficulties, and three were excluded because of lack of access to a smartphone   . All par.\u00a0=\u00a07.8) . BaselinProcess indicators for the intervention group are summarised in Data about participants\u2019 use of the app captured via an integrated analytics software was excluded from this analysis because of very low completion of the app and non-completion of sections such as multiple-choice quizzes, or self-reported use of problem-solving in real-world situations.No outcome data were available for intervention arm participants, none of whom could be reached to complete assessments.This study aimed to evaluate the feasibility and acceptability of POD Adventures, an app-based adolescent mental health intervention, when delivered online accompanied by telephone support from counsellors during the COVID-19 pandemic in India. Despite a range of modifications to address barriers arising from COVID-19 restrictions, online remote delivery was not found to be acceptable or feasible for the target population in the study context.N\u00a0=\u00a0248) of POD Adventures, which was delivered offline on school premises before the COVID-19 pandemic. The latter study had comparatively higher rates of self-referral (18.2 v. 5.6% in the current study) and intervention completion (93% v. no completers in this study). Qualitative interviews with participants further showed that the app was easy to use, engaging and helpful in solving their problems when used offline, and the brief guidance provided by counsellors was experienced as adequate and helpful whether provided individually or in small groups.26The current results contrast remarkably with feasibility and acceptability findings from a large school-based cohort study  and/or the timing of the study. Regarding study timing, it is notable that the research took place during the especially severe second COVID-19 wave in India, which included widespread lockdowns and school closures in the context of overwhelmed health systems.33 Greenhalgh et al, in three reviews of technology diffusion and implementation,35 have shown that the implementation of a new technology as part of changes to healthcare services is inherently very challenging. These reviews highlight that those innovations requiring changes in organisations or the wider care system have a poor track record of adoption because of the dual challenge of non-adoption by individuals and difficulties with spread or scale-up. They further emphasise that it is not only individual factors that make or break a technology implementation effort but the dynamic interplay between these factors. The more complex an innovation or the setting in which it is introduced, the less likely it is to be successfully adopted or scaled up. They also emphasise that methodologically robust randomised controlled trials alone will not elucidate these complex interactions, and emphasise the need for more studies that are interdisciplinary, non-deterministic, locally situated and designed to examine the recursive relationship between human action and the wider system context.36 Further, findings from the current study are consistent with those from recent reviews that show that digital interventions may achieve greater uptake and sustained engagement when delivered in structured and guided settings, such as schools or clinics.19 Furthermore, these reviews have concluded that interventions involving educational programmes completed in the participant's own time (i.e. at home without supervision) are not effective. Li et al found that prior experiences of accessing mental health counselling may make students more open to online services,37 a finding that was not relevant to the present sample because they had limited experiences of in-person mental health services. Given the prior findings that showed high uptake, adherence and engagement with POD Adventures when delivered in schools,26 it seems likely that an online study format and remote delivery, rather than the app content, were the biggest barriers to study participation and engagement. Further, a systematic review by Garrido et al found that study drop-out rates for digital interventions for depression and anxiety with young people tended to relate more to recruitment methods, especially in the case studies completed by participants at home/in their own time, than to non-engagement of the interventions themselves.16The findings of this study are broadly aligned with research related to the non-adoption or scale-up of many promising technological innovations in healthcare.38 In the current study, self-referring students who were unable to enrol in the study reported lack of access to a smartphone or internet as the most common reason for non-participation. Even among those participants who did enrol in the study, it is possible that they faced recurring or intermittent difficulties with accessing a smartphone and/or internet connectivity. For intervention arm participants in particular, telephone guidance and text messages may not have been readily accessible because of sporadic telephone access.Findings from the implementation of remote learning approaches in schools in LMICs around the world have revealed extremely low rates of smartphone and internet access, ranging from 2 to 6% of young people with access at home. This has impeded learning and school participation throughout the COVID-19 pandemic.Another key barrier may have been limited sensitisation in a sample with little to no prior experiences of formal mental health interventions. It is possible that the brief classroom sensitisation session provided as part of recruitment was insufficient in building an understanding of the intervention features or potential benefits when delivered online.40Recent research shows that enhancing mental health literacy and addressing concerns about the implications of making use of online help-seeking  may help build demand from young people.41 emphasising the ongoing need for in-person interventions as well as the development and evaluation of technologies that are context-specific; for example, use of web browser-based rather than app-based interventions, as they do not require downloading or regular updates or work on any smartphone device.41This study suggests there may be a gap between the potential that online digital interventions offer and the reach and uptake of these technologies, especially for adolescents from LMICs,41 Future evaluations of POD Adventures  should systematically record and report use of different languages where these can be selected by users. Interventions which explicitly aim to reduce demographic disparities related to intervention retention  should be evaluated.43 Finally, judicious use of technology for trial procedures and the use of hybrid approaches, which include in-person interaction at school, may help to conduct evaluations.44There is also a pressing need to distinguish digital mental health interventions by their content and delivery characteristics. Findings from this pilot trial suggest that online delivery of POD Adventures was not feasible, despite earlier evidence that the content of the app was useful, appropriate and potentially effective in the same population. Furthermore, distinct adaptations that account for population-specific needs of adolescents  are needed.In conclusion, findings from this pilot trial are inconclusive about whether the key barriers to adolescent participation were a result of difficulties accessing online research procedures, intervention delivery or a combination of both, which may have been exacerbated by COVID-19 pandemic conditions. More studies with this age group and in similar settings are needed to establish the generalisability of these findings."}
+{"text": "The ability of pathogens to develop drug resistance is a global health challenge. Severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102) presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody (mAb) therapies and vaccine\u2010induced sera. Decoy nanoparticles\u2014cell\u2010mimicking particles that bind and inhibit virions\u2014are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, quantitative understanding as to how design features impact performance of these therapeutics is lacking. To address this gap, this study presents a systematic, comparative evaluation of various biologically\u00a0derived nanoscale vesicles, which may be particularly well suited to sustained or repeated administration in the clinic due to low toxicity, and investigates their potential to inhibit multiple classes of model SARS\u2010CoV\u20102\u00a0virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus\u2010decoy binding affinities. In addition, these cell\u2010mimicking vesicles effectively inhibit model SARS\u2010CoV\u20102\u00a0variants that evade mAbs and recombinant protein\u2010based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS\u2010CoV\u20102\u00a0and other viral infections. This study elucidates design rules for building \u201cdecoy\u201d nanovesicles that bind and inhibit infection by severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102)\u00a0particles. Key findings are that biologically\u00a0derived decoy vesicles are potent inhibitors regardless of vesicle subtype or virus\u2010vesicle binding affinity, and these decoys are effective even against viral mutants that are resistant to soluble protein or monoclonal antibody therapeutics. The development and approval of monoclonal antibody (mAb) therapies that neutralize virions emerged as a viable treatment early in the pandemic and continues to play an important role in treating severe forms of the disease. Unfortunately, circulating viral strains contain mutations in key glycoprotein residues that have rendered many mAb treatments in development, and six out of eight of the United States Food and Drug Administration (FDA) authorized mAbs, ineffective. The use of mAb cocktails and the development of broadly neutralizing mAbs that target conserved viral epitopes hold promise in overcoming such challenges, but these remain susceptible to escape or have not yet been evaluated clinically. Therapies capable of combating circulating and evolving viral strains are urgently needed to supplement the use of vaccines and current antiviral treatments.The Coronavirus\u201019 (COVID\u201019) pandemic has killed over 5.8\u00a0million people globally and dramatically underscored the need for therapeutics for treating infectious disease.1 The  In the case of SARS\u2010CoV\u20102, viral entry into a cell is mediated by the binding of the viral Spike glycoprotein (Spike) to the human protein angiotensin\u2010converting enzyme 2 (ACE2) on the cell surface. As the first step in the viral replication cycle, the Spike\u2010ACE2\u00a0binding interaction represents an attractive therapeutic target. In the context of SARS\u2010CoV\u20102, decoy nanoparticles may exploit this interaction by presenting ACE2\u00a0on the decoy surface to bind Spike and inhibit cellular infection by SARS\u2010CoV\u20102. This type of strategy has shown promise in other disease contexts, such as in human immunodeficiency virus (HIV) treatment. The decoy strategy is particularly attractive because it might be robust to evolutionary escape by pathogens\u2014mutations that reduce decoy\u2010pathogen binding affinity  will concomitantly attenuate pathogen\u2010cell binding. Such mutations are thus likely to decrease viral fitness such that these viral variants are unable to outcompete decoy\u2010susceptible variants. Another potential advantage is that if more infectious variants evolve through increasing the affinity with which the pathogen binds a host receptor, such a variant would likely be equally or more susceptible to inhibition by decoys.A promising complement to mAb therapies is cell\u2010mimicking \u201cdecoy\u201d systems\u2014nanoparticles that display host cell receptors on their surface to mimic a cell and bind pathogens.14 In  The most prominent biological nanoparticles are extracellular vesicles (EVs)\u2014nanometer\u2010scale particles released by all cells which mediate intercellular transfer of biomolecules. EVs have recently been investigated as infectious disease decoys in part because, in contrast to most synthetic vehicles, EVs uniquely exhibit low toxicity and low immunogenicity, and these properties are likely to be of central importance for particles to be administered via sustained infusions or repeat injections, as is envisioned for decoy applications. Although decoy nanoparticles have yet to be evaluated clinically, it seems likely that this approach would be most beneficial for patients experiencing severe or prolonged infections that are not controlled by either their immune system or available antiviral agents.Although any particle that displays a pathogen's cognate receptor may serve as a decoy particle, biologically\u00a0derived nanoparticles that closely resemble the membrane environment  of a natural host cell are of particular interest.12 The was supported in early studies that demonstrated ACE2 EVs bound the SARS\u2010CoV\u20102\u00a0Spike protein and were capable of inhibiting SARS\u2010CoV\u20102\u00a0pseudotyped lentivirus transduction in vitro. ACE2 EV viral inhibition was later interrogated as a function of dose, providing the community the first quantitative benchmark of decoy potency. Subsequent work demonstrated efficacy against replication\u2010competent SARS\u2010CoV\u20102\u00a0in vitro and suggested that ACE2 EVs were safe and effective against pseudotyped virus when delivered intranasally in a rodent model. Recent work showed that intravenously administered ACE2 EVs lowered the viral load of authentic SARS\u2010CoV\u20102\u00a0in a mouse model, reduced the levels of pro\u2010inflammatory cytokines in lung tissue, and mitigated lung tissue injury. These studies validated the fundamental concept that decoy EVs could address this disease and raised a number of interesting questions. However, the diversity of experimental systems and designs employed across these studies makes it difficult to synthesize the results of these efforts to evaluate the relationship between specific EV design choices and efficacy. Moreover, we lack understanding as to how decoy EV performance varies across various emerging viral strains, which is an open question of recognized importance. Resolving these knowledge gaps could help improve development and facilitate deployment of decoy EV treatments for SARS\u2010CoV\u20102, novel variants thereof, and perhaps novel viral infections.The COVID\u201019\u00a0pandemic sparked a flurry of decoy EV research that has dramatically advanced the decoy nanoparticle field. Initial speculation that ACE2\u2010containing EVs (ACE2 EVs) might inhibit viral infection26 wasFigure\u00a0In this study, we systematically evaluate the relationships between design features of decoy EVs and performance characteristics vis\u2010\u00e0\u2010vis inhibition of a model SARS\u2010CoV\u20102\u00a0lentivirus Figure\u00a01. We co22.1 Cell lines were analyzed for ACE2\u00a0expression, surface display, and EV loading. HEK293FTs did not endogenously express ACE2\u00a0at an appreciable level, while both engineered lines expressed high amounts of ACE2\u00a0relative to Calu\u20103s, a model ACE2\u2010expressing lung cell line  cells to stably express a codon\u2010optimized version of the wild\u2010type ACE2\u00a0protein (WT\u2010ACE2) via lentiviral\u2010mediated gene delivery. In parallel, we generated a stable cell line expressing a mutant version of the ACE2\u00a0gene (Mut\u2010ACE2) that binds to the SARS\u2010CoV\u20102\u00a0Spike protein with higher affinity than does WT\u2010ACE2 and an ultracentrifugation EV fraction (UC\u2010EVs)   contained between 500\u00a0and 2000\u00a0ACE2\u00a0molecules  Figure\u00a0, and bot) Figure\u00a0. The sigs Figure\u00a0. We notes Figure . The abo2.3Figure\u00a0 We utilized a second generation lentivirus system to generate lentiviral particles pseudotyped with the SARS\u2010CoV\u20102\u00a0Spike protein (Spike\u2010lenti), wherein an \u201cinfection\u201d event causes genomic integration and expression of an enhanced yellow fluorescent protein (EYFP) reporter . We chose to use a FLAG\u2010tagged SARS\u2010CoV\u20102\u00a0Spike construct that contained a D614G mutation and lacked a 19\u00a0amino\u2010acid C\u2010terminal sequence because both of these choices have been reported to improve pseudotyping efficiency. We also generated model recipient cells by engineering HEK293FTs to stably express ACE2. In our hands, this combination produced a detectable but low titer of functional lentivirus: \u2248101\u00a0transducing units mL\u22121 (TU\u00a0mL\u22121) evaluated on ACE2\u2010expressing HEK293FTs compared to a typical yield of \u2248105\u00a0TU\u00a0mL\u22121\u00a0for a vesicular stomatitis virus G (VSV\u2010G) pseudotyped virus applied to HEK293FT recipient cells  values, which were on the order of 1\u00a0\u00d7\u00a0107\u00a0particles for all cases , but ID50\u00a0is not an absolute measurement of potency . Moreover, we propose that ID50\u00a0is a more appropriate metric than is the commonly used half\u2010maximal inhibitory concentration (IC50) metric, since these experiments involve relatively small numbers of discrete particles . Nonetheless, we analyze both metrics for comparison . Thus, we hypothesize that in the aforementioned report, ACE2\u00a0loading per EV was subsaturating. Since the absolute amount of protein loaded per vesicle may scale with vesicle size, this effect must be considered when comparing vesicle populations that differ in size. However, the EVs investigated in this study are comparable in size  and extruded through 100\u00a0nm filters to generate ACE2\u00a0NVs of comparable diameter to EVs and facilitate comparison across vesicle types. Particles exhibited a similar mean size to EVs, although the NV size distribution was narrower  compared to the parental Spike protein. The second mutant contains a point mutation, F486S, that abrogates inhibition by sACE2, likely by altering the Spike RBD and affecting ACE2\u00a0receptor engagement (F486S). Although this mutation has not been reported in circulating SARS\u2010CoV\u20102\u00a0strains, sACE2\u00a0treatments currently under development would likely be ineffective against a strain that has or develops this mutation. We cloned the aforementioned mutants into the same backbone as our D614G  Spike protein and generated Spike\u2010lenti for each variant.Given the diversifying SARS\u2010CoV\u20102\u00a0strains in circulation, we next investigated how decoy EV potency varies across Spike mutant variants. Many such mutants have been identified,54 and50\u00a0value for the strain considered by the ID50\u00a0value of a reference, parental strain ; we term this metric \u201crelative resistance.\u201d A relative resistance value of one indicates that the Spike\u2010lenti variant is equally susceptible to the decoy EV (compared to the reference Spike\u2010lenti), a value less than one indicates that this variant is less resistant to the decoy EV, and a value greater than one indicates that this variant is more resistant to the decoy EV. Relative resistance is defined to facilitate quantitative comparison across strains and treatments by minimizing dependency on virus\u2010to\u2010virus variability  which can impact ID50. Relative resistance is also useful because it enables comparison across treatments which might be defined in distinct natural units of concentration . To evaluate whether any differences in decoy potency were due to different viral titers across viral strains, we also calculated ID50s normalized to the viral quantity added (ID50\u00a0TU\u22121) and a relative resistance calculated from ID50\u00a0TU\u22121\u00a0metrics  in Tables  or decrease (F486S) the Spike\u2010ACE2\u00a0binding affinity. These data demonstrate that decoy vesicles, in a SARS\u2010CoV\u20102\u00a0context, are capable of inhibiting viral mutants that are resistant to clinically authorized mAb treatments. Furthermore, these data provide evidence that vesicles displaying wild\u2010type host cell receptors are capable of potently inhibiting viral strains that prove refractory to soluble, protein\u2010based therapeutics. Importantly, this approach circumvents the need for separately engineering a Spike\u2010binding protein in a manner that could be immunogenic and problematic, particularly in the context of sustained or repeated administration.We then evaluated the ability of WT\u2010ACE2\u00a0UC\u2010EVs, Mut\u2010ACE2\u00a0UC\u2010EVs, and sACE2\u00a0to inhibit this panel of Spike\u2010lenti variants ; this combination of drug resistance properties integrates features of both the F486S and Beta strain previously investigated. The emerging Lambda variant is less well\u2010studied, but preprints suggest high transmissibility and moderate immune evasion. Spike\u2010lenti containing the RBD mutations for these variants was generated as follows: Delta , Delta\u2010plus: , and Lambda . Viral inhibition experiments were performed with WT\u2010ACE2\u00a0UC\u2010EVs using the parental D614G strain as the reference strain  has been reported across several studies. Moreover, 75% of neutralizing mAbs treatments authorized by the FDA fail to inhibit Omicron in vitro. Although untested here, we hypothesize that variants such as Omicron would be similarly inhibited by decoy vesicles because this variant requires ACE2\u00a0for entry, the Omicron Spike\u2010ACE2\u00a0affinity is similar in strength to that of the Beta strain evaluated here, and ACE2\u2010based soluble protein inhibitors confer neutralization of Omicron.Given the promising activity of WT\u2010ACE2\u00a0EVs against our test strains , we next sought to extend our investigation to naturally emerging and prominent Spike mutants. In particular, the Delta variant of SARS\u2010CoV\u20102\u00a0rapidly became the dominant strain in 2021\u00a0due to high transmissibility and resistance to mAb and vaccine\u2010induced sera neutralization.2, 32\u00a0area of exposed outer membrane, on average. This density of ACE2\u00a0display could theoretically facilitate the binding of a decoy vesicle to a virion at several attachment points . We hypothesize that the lipid bilayer structure of vesicles enables decoy ACE2\u00a0receptors to diffuse across the vesicle surface and improve their likelihood of encountering a Spike protein in trans, particularly after an initial vesicle\u2010virion contact has occurred. It is interesting to speculate that decoy particles that contain a fluid bilayer membrane may possess advantages\u2014in terms of either avidity or inhibition mechanism\u2014over nanoparticle systems with fixed protein\u2010attachment points, but this possibility requires further investigation. By comparing ID50\u00a0values for vesicle\u2010mediated inhibition  likely account for these quantitative differences, highlighting the importance of evaluating any given design choice using apples\u2010to\u2010apples comparisons. We speculate that the high avidity of decoy vesicles for their target also explains why decoys effectively inhibit strains bearing Spike variants that bind ACE2\u00a0with reduced affinity . Thus, the effects of avidity render binding between any one EV and any one virus effectively independent of modest changes in ACE2\u2010Spike affinity. This hypothesis (and the importance of avidity) is bolstered by a recent report demonstrating that EVs with low levels of ACE2 (1\u20135\u00a0ACE2\u00a0proteins per vesicle\u2014two orders of magnitude lower than we observed) conferred neutralization in a manner that depended on the Spike\u2010ACE2\u00a0affinity. The efficacy of high\u2010avidity particles to inhibit SARS\u2010CoV\u20102\u00a0variants mirrors observations with HIV, suggesting that this phenomenon could indeed be a general advantage for this type of antiviral inhibitor. An interesting structural consideration is that the SARS\u2010CoV\u20102\u00a0Spike protein quaternary structure is a trimer with three RBDs; all of which may be bound to ACE2\u00a0at the same time. Exploring how various potential modes of avidity and molecular rearrangement with the viral and vesicle membranes contribute to the efficacy of decoy vesicles is an exciting avenue for future research.We speculate that avidity is largely responsible for the efficacy of decoy nanoparticles and confers advantages in terms of potency and robustness to drug resistance compared to soluble receptor protein decoys. Given our estimated levels of ACE2\u00a0loading ; this gene was cloned into a modified pcDNA 3.1\u00a0backbone (Clontech\u2010Takara) with a beta\u2010globin intron in the 5\u2032 untranslated region for pseudotyping lentivirus. The Tet3G transactivator (pLVX\u2010EF1a\u2010TET3G) and cognate TRE3GV promoter (pLVX\u2010TRE3G) (Takara) were cloned into modified pGIPZ and pLVX (Takara) backbones, respectively. In this context, the Spike protein (Addgene plasmid # 145032) was cloned downstream of the TRE3G promoter. Cloned plasmids used in this study were sequence\u2010verified, and maps are available in Data Escherichia coli were used for transformation of all plasmids and subsequently grown at 37\u00a0\u00b0C.Plasmids used in this study were generated using standard polymerase chain reaction techniques and/or type II and IIs restriction enzyme cloning. Restriction enzymes, Phusion DNA polymerase, T4\u00a0DNA Ligase, and Antarctic phosphatase were purchased from NEB. psPAX2\u00a0and pMD2.G plasmids were gifted by William Miller from Northwestern University and DsRed\u2010Express2\u00a0was purchased from Clontech\u2010Takara. WT\u2010ACE2\u00a0and Mut\u2010ACE2\u00a0gene fragments were codon optimized and synthesized by Thermo Fisher and cloned into a pGIPZ backbone (Open Biosystems). The Spike protein from pcDNA3.1\u2010SARS2\u2010Spike was a gift from Fang Li ;45 thi DNA purity and concentrations for relevant experiments were measured with a NanoDrop 2000 (Thermo Fisher Scientific).Plasmid DNA used to generate lentivirus, for viral inhibition assays or for cell\u2010line engineering, was prepared using a polyethylene glycol precipitation protocol.71 DNA\u22121\u00a0glucose from Sigma (G7021), 3.7\u00a0g\u00a0L\u22121\u00a0sodium bicarbonate from Fisher Scientific (S233), and 100\u00a0U\u00a0mL\u22121\u00a0penicillin and 100\u00a0\u00b5g\u00a0mL\u22121\u00a0streptomycin (15140122), 4\u00a0mm L\u2010glutamine (25030\u2010081), and 10% fetal bovine serum (FBS) (16140\u2010071) from Gibco. Lenti\u2010X cells were grown in the HEK293FT formulation supplemented with 1\u00a0mm sodium pyruvate from Gibco (11360070). Calu\u20103s were grown in minimum essential medium from Gibco (41500\u2010018) supplemented with 1.5\u00a0g\u00a0L\u22121\u00a0sodium bicarbonate and the pH was brought to between 7.0\u00a0and 7.4\u00a0with HCl. Calu\u20103\u00a0media was further supplemented with 1\u00a0mm sodium pyruvate, 10% FBS, and 100\u00a0U\u00a0mL\u22121\u00a0penicillin and 100\u00a0\u00b5g\u00a0mL\u22121\u00a0streptomycin. In some cases denoted below, HEK293FTs were briefly cultured in phenol\u2010red free DMEM from Millipore Sigma (D2902). This DMEM formulation was supplemented with 4\u00a0mg\u00a0L\u22121\u00a0pyridoxine\u2010HCl from Millipore Sigma (P6280), 16\u00a0mg\u00a0L\u22121\u00a0sodium phosphate from Millipore Sigma (S5011), 3.7\u00a0g\u00a0L\u22121\u00a0sodium bicarbonate, 3.5\u00a0g\u00a0L\u22121\u00a0glucose, 100\u00a0U\u00a0mL\u22121\u00a0penicillin, and 100\u00a0\u00b5g\u00a0mL\u22121\u00a0streptomycin, 4\u00a0mm L\u2010glutamine (25030\u2010081), and 10% FBS. Cells were maintained in a 37\u00a0\u00b0C incubator held at 5% CO2. Spike\u2010expressing cells were induced for at least 24\u00a0h prior to assays requiring Spike expression with doxycycline at 1\u00a0\u00b5g\u00a0\u00b5L\u22121. Doxycycline was purchased from Fisher Scientific (BP2653\u20105) and resuspended in sterile, nuclease\u2010free water prior to use.HEK293FT cells were purchased from Thermo Fisher/Life Technologies. HEK293T Lenti\u2010X cells were purchased from Takara Bio. Calu\u20103s were purchased from ATCC (# HTB\u201055). HEK293FTs and engineered HEK293FTs were grown in a base Dulbecco's modified eagle medium (DMEM) formulation (Gibco 31600\u2010091). Base medium was further supplemented with 3.5\u00a0g\u00a0L6\u00a0HEK293FTs were plated in 10\u00a0cm tissue culture (TC)\u2010treated plates and allowed to attach for 5\u20138\u00a0h. Cells were then transfected via calcium phosphate method. Briefly, DNA  were diluted with sterile H2O and added to CaCl2 (2\u00a0m) to achieve a final concentration of 0.3\u00a0m\u00a0CaCl2. DNA\u2010containing sample was then added dropwise to an equal\u2010volume of 2\u00d7 HEPES\u2010buffered saline  and pipetted four times to mix. After 3\u20134\u00a0min, the solution was vigorously pipetted eight times and 2\u00a0mL of transfection reagent per 10\u00a0cm dish was added dropwise to cells. The plates were gently swirled and incubated overnight at 37\u00a0\u00b0C with 5% CO2. The medium was replaced the morning after transfection and cells were incubated for an additional 28\u201330\u00a0h. Conditioned medium containing lentivirus was harvested, clarified via centrifugation at 500\u00a0\u00d7\u00a0g for 2\u00a0min at 4\u00a0\u00b0C, and purified through a 0.45\u00a0\u00b5m polyethersulfone filter from VWR (28143\u2010505). Lentivirus was further concentrated via ultracentrifugation at 100\u00a0420\u00a0\u00d7\u00a0g for 90\u00a0min at 4\u00a0\u00b0C in a Beckman Coulter Optima L\u201080\u00a0XP model and using a SW 41\u00a0Ti rotor. Lentivirus was stored on ice until use. 105\u00a0HEK293FT parental cells were plated for transduction \u224824\u00a0h in advance in a 12\u00a0well TC\u2010treated plate. At the time of transduction, media was aspirated and concentrated lentivirus was added; DMEM was used to bring final volume to 1\u00a0mL per well. 2\u00a0days later, drug selection on cells began and continued for at least 1\u00a0week. ACE2\u2010expressing cell lines were selected using 1\u00a0\u00b5g\u00a0mL\u22121\u00a0puromycin from InvivoGen (ant\u2010pr). Inducible Spike\u2010expressing cell lines were generated from HEK293FTs by inoculating cells with two lentiviruses\u2014one delivering the doxycycline\u2010inducible Tet\u2010On 3G transactivator and one delivering the Spike protein downstream of the TRE3G promoter. These concentrated viruses were added at 1:2\u00a0volume ratio, respectively. The cell line was selected using the aforementioned timeline but with 1\u00a0\u00b5g\u00a0mL\u22121\u00a0blasticidin S from Gibco (A11139\u201003) and 2\u00a0\u00b5g\u00a0mL\u22121\u00a0hygromycin B from Millipore Sigma (400053).HEK293FT cells were used to produce lentivirus for stable cell line generation. 5\u20136\u00a0\u00d7\u00a010e method.71 Bri6\u00a0cells mL\u22121. 100\u00a0\u00b5L of each cell suspension (if two different cell types were incubated) or 200\u00a0\u00b5L of the cell suspension (for control wells with only one cell type) were then added to phenol red\u2010free DMEM (300\u00a0\u00b5L) in a non\u2010TC\u2010treated 24\u2010well plate such that the final volume was 500\u00a0\u00b5L. Cells were incubated at 37\u00a0\u00b0C for 15\u00a0min and hand\u2010shaken every 5\u00a0min. At 15\u00a0min, wells were imaged on a Keyence BZ\u2010x800\u00a0microscope using BZ Series Application software v01.01.00.17\u00a0and using a PlanApo 4\u00d7 objective with a numerical aperture of 0.2.Cells were grown in 10\u00a0cm dishes and harvested with a brief trypsin incubation (<30\u00a0s) followed by quenching with phenol red\u2010free DMEM. Cell suspensions were vortexed to break up clumps, counted, and then diluted to 1\u00a0\u00d7\u00a010m EDTA, 0.05% bovine serum albumin), and then samples were centrifuged at 150\u00a0\u00d7\u00a0g for 5\u00a0min at 4\u00a0\u00b0C. After decanting the supernatant, cells were resuspended in FACS buffer (50\u00a0\u00b5L) and blocked with 10\u00a0\u00b5L of 1\u00a0mg\u00a0mL\u22121\u00a0IgG  for 5\u00a0min at 4\u00a0\u00b0C. After blocking, 2.5\u00a0\u00b5L of 0.2\u00a0\u00b5g\u00a0\u00b5L\u22121\u00a0\u03b1\u2010ACE2\u00a0antibody  was added and incubated for 30\u00a0min at 4\u00a0\u00b0C. Cells were washed three times by adding cold FACS buffer (1\u00a0mL), centrifuging cells at 150\u00a0\u00d7\u00a0g for 5\u00a0min at 4\u00a0\u00b0C, and decanting supernatant. Cells were resuspended in one drop of FACS buffer prior to analytical flow cytometry.2\u00a0days prior to assay, cells were plated into 12\u00a0well tissue culture treated plates such that they were 80\u201395% confluent at time of harvest. Medium was aspirated, cells were harvested with 1\u00a0mL cold fluorescence\u2010activated cell sorting (FACS) buffer . The next morning, the media was replaced with HEK293FT DMEM (18\u00a0mL) supplemented with 10% EV\u2010depleted FBS . After 22\u201328\u00a0h, conditioned medium was harvested as previously reported. Briefly, the supernatant was clarified by sequential centrifuge spins for 10\u00a0min at 300\u00a0\u00d7\u00a0g and 20\u00a0min at 2000\u00a0\u00d7\u00a0g. HS\u2010EVs were pelleted by a subsequent centrifugation at 30\u00a0min for 15\u00a0000\u00a0\u00d7\u00a0g in a Beckman Coulter Avanti J\u201026XP centrifuge using a J\u2010LITE JLA 16.25\u00a0rotor. The supernatant was centrifuged at 120\u00a0416\u00a0\u00d7\u00a0g\u00a0for 135\u2009min in a Beckman Coulter Optima L\u201080\u00a0XP model using a SW 41\u00a0Ti rotor to pellet UC\u2010EVs. All centrifugation was performed at 4\u00a0\u00b0C. EVs were resuspended via gentle pipetting in the conditioned cell medium remaining in their respective vessel.15\u00a0\u00d7\u00a010reported.72 Bri8\u00a0particles mL\u22121\u00a0in PBS before recording data. Samples were infused at an injection rate setting of 30, imaged with a camera level setting of 14, and analyzed at a detection threshold setting of 7. Three 30\u00a0s videos were captured for each sample; vesicle concentrations and size histograms were determined from the average values of the three videos.Vesicle concentration and size were measured using a NanoSight NS300  running software v3.4\u00a0and a 642\u00a0nm laser. Vesicles were diluted to between 2\u00a0and 10\u00a0\u00d7\u00a01010\u00a0\u00b5L of purified vesicles was placed onto a carbon\u2010coated copper grid  for 10\u00a0min before being wicked away with a piece of filter paper. The grid was dipped in PBS twice to remove excess proteins from the media and was allowed to dry for 2\u00a0min. Next, uranyl acetate (10\u00a0\u00b5L of a 2\u00a0wt% solution) was placed on the grid for 1\u00a0min, before again being wicked away with filter paper. The grid was allowed to fully dry for 3\u00a0h to overnight at room temperature. Bright\u2010field TEM imaging was performed on a JEOL 1230\u00a0TEM. TEM operated at an acceleration voltage of 100\u00a0kV. All TEM images were recorded by a Hamamatsu ORCA side\u2010mounted camera or a Gatan 831\u00a0bottom\u2010mounted CCD camera, and AMT imaging software.m NaCl, 50\u2009mm Tris\u2010HCl pH 8.0, 1% Triton X\u2010100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with protease inhibitor (Pierce/Thermo Fisher #A32953). After a 30\u00a0min incubation on ice, lysates were centrifuged at 14\u00a0000\u00a0\u00d7\u00a0g for 20\u00a0min at 4\u00a0\u00b0C. Protein concentration for each sample was evaluated using a bicinchoninic acid (BCA) assay (Pierce/Thermo Fisher #23225). Samples were kept on ice until use or frozen at \u221280\u00a0\u00b0C for long term storage.To generate cell lysates, HEK293FTs were washed with cold PBS and lysed with ice\u2010cold radioimmunoprecipitation assay buffer  at a concentration of 0.2\u20131\u00a0\u00d7\u00a0107\u00a0cells\u00a0mL\u22121\u00a0buffer; typical volumes at this stage were 5\u201315\u00a0mL. Lysis continued on ice for at least 30\u00a0min. Samples were then sonicated in an ice\u2010cold water bath  at medium power. Samples were sonicated for 10\u00a0s and allowed to recover on ice for 50\u00a0s; this process was repeated a total of six times such that all samples were sonicated for 1\u00a0min. Samples were then clarified via successive centrifugation steps at 4\u00a0\u00b0C in Beckman Coulter Avanti J\u201026XP centrifuge using either a J\u2010LITE JLA 16.25\u00a0rotor or a JA\u201014.5\u00a0rotor: 3250\u00a0\u00d7\u00a0g for 5\u00a0min and 20\u00a0000\u00a0\u00d7\u00a0g for 30\u00a0min. Subsequent ultracentrifugation of pelleted membrane fragments at 80\u00a0000\u00a0\u00d7\u00a0g for 90\u00a0min. PBS was completely aspirated and the samples were resuspended in PBS (30\u201360\u00a0\u00b5L per ultracentrifuge tube). Samples were then extruded to 100\u00a0nm by passing samples seven times through a 100\u00a0nm polycarbonate filter (Whatman #800309) installed in an Avanti Mini Extruder. Samples were then concentrated \u224810\u00d7 in Amicon Ultra\u20100.5\u00a0mL filter using a 10\u00a0kDa molecular weight cutoff (Millipore Sigma #UFC5010) per manufacturer instructions; filters were prerinsed with PBS immediately prior to use.HEK293FTs were plated in 10\u00a0cm dishes and grown for 2\u00a0days until reaching 80\u201395% confluency. The day of harvesting, the medium was aspirated and the cells were washed in PBS. Cells were briefly trypsinized (\u224830\u00a0s) before quenching with EV\u2010depleted DMEM. Cells were pelleted via centrifugation at 150\u00a0\u00d7\u00a0 tablets)50 at 8\u2013109\u00a0particles). For western blots comparing the protein content of cell lysates, equal amounts of total protein as determined by BCA were prepared and loaded into gels . For western blots comparing protein in cell lysates to protein in vesicles, a fixed number of vesicles and a fixed amount of cell lysate were loaded into each well: 4.8\u00a0\u00d7\u00a0108\u00a0particles and 3\u00a0\u00b5g protein, respectively. A detailed western blot protocol was reported and was followed with the subsequent modifications. In most cases, the following reducing Laemmli composition was used to boil samples ; in some cases, a nonreducing Laemmli composition (without DTT) was used . Primary antibody was added in 5% milk in TBST, rocking, for 1\u00a0h at room temperature and then washed three times with TBST for 5\u00a0min each. Secondary antibody in 5% milk in TBST was added at room temperature for 1\u00a0h or overnight at 4\u00a0\u00b0C. Membranes were then washed three times with TBST for 5\u00a0min each. The membrane was incubated with Clarity Western ECL substrate (Bio\u2010Rad) and imaged on an Azure c280 running Azure cSeries Acquisition software v1.9.5.0606. The Azure software and ImageJ were used to analyze the resulting TIF files and adjust brightness and contrast where necessary. Specific antibodies, antibody dilution, heating temperature, heating time, and Laemmli composition for each antibody can be found in Table For western blots comparing the protein content of vesicles, equal numbers of vesicles as determined by NTA were prepared and loaded into the gel  as a function of the amount of ACE2\u00a0added in number of molecules (assuming a 115\u00a0kDa size for sACE2), and a linear regression was performed to generate a calibration curve. The estimated number of ACE2\u00a0proteins in vesicle lanes was determined from the band intensities and calibration curve, and an inverse regression was performed to estimate uncertainty associated with the calibration curve. Estimated number of ACE2\u00a0proteins per vesicle was then calculated by dividing the estimated ACE2\u00a0proteins per lane by the number of vesicles added to that lane. Error was propagated throughout each calculation, and final error associated with the average number of ACE2\u00a0molecules per vesicle was determined by adding\u2010in\u2010quadrature the propagated error and the calculated standard error of the means.Digital membrane images were analyzed in ImageJ using the analyze gel function.73 Ban6\u00a0Lenti\u2010X cells were plated 24\u00a0h prior to transfection of viral plasmids unless otherwise stated; 5\u20136\u00a0\u00d7\u00a0106\u00a0HEK293FTs were plated in 10\u00a0cm TC\u2010treated plates and allowed to attach for 5\u20138\u00a0h. Cells were then transfected via calcium phosphate method as discussed above. Here, 3\u00a0\u00b5g Spike envelope protein, 8\u00a0\u00b5g psPAX2\u00a0packaging vector, 10\u00a0\u00b5g of transfer plasmid encoding an EYFP transgene, and 1\u00a0\u00b5g of DsRed\u2010Express2\u00a0transfection marker were used. To generate mock lentivirus, the 3\u00a0\u00b5g Spike envelope protein was replaced with an empty pcDNA 3.1\u00a0vector (Clontech\u2010Takara). The plates were incubated overnight at 37\u00a0\u00b0C with 5% CO2. The medium was replaced the morning after transfection and cells were incubated for an additional 32\u00a0h prior to harvesting unless otherwise stated. In some cases, medium was replaced, cells were incubated for an additional 24\u00a0h, and virus was harvested a second time. Conditioned medium containing lentivirus was harvested, clarified via centrifugation at 500\u00a0\u00d7\u00a0g for 2\u00a0min at 4\u00a0\u00b0C, and purified through a 0.45\u00a0\u00b5m polyethersulfone filter (VWR #28143\u2010505). Where required, Spike\u2010lenti was concentrated using Amicon Ultra\u201015\u00a0centrifugal filter units with a 100\u00a0kDa cutoff (Millipore Sigma #UFC910024). Samples were centrifuged at 4\u00a0\u00b0C in a Beckman Coulter Avanti J\u201026XP centrifuge using either a J\u2010LITE JLA 16.25\u00a0rotor or a JA\u201014.5\u00a0rotor at 5000\u00a0\u00d7\u00a0g until concentrated \u224810\u201350\u2010fold  and stored on ice at 4\u00a0\u00b0C for up to 1\u00a0week or at \u221280\u00a0\u00b0C until use. To determine functional viral titer, unless otherwise stated, virus was diluted in DMEM and pipetted into a 96\u2010well plate, centrifuged at 500\u00a0\u00d7\u00a0g for 1\u00a0min at 4\u00a0\u00b0C to remove bubbles, and immediately incubated at 37\u00a0\u00b0C for 1\u00a0h. WT\u2010ACE2+ HEK293FTs were trypsinized briefly, counted, and 4\u00a0\u00d7\u00a0103\u00a0cells were plated on top of the virus such that the final volume was 200\u00a0\u00b5L. After 16\u00a0h, media was aspirated and fresh DMEM (200\u00a0\u00b5L) was added. Cells were harvested for flow cytometry 3\u00a0days after inoculation.HEK293FT or HEK293T Lenti\u2010X cells were used to produce Spike\u2010lenti for optimizing viral production; Lenti\u2010X cells were used to generate Spike\u2010lenti for all viral inhibition experiments. 5\u20136\u00a0\u00d7\u00a0102O at a stock concentration of 0.25\u00a0mg\u00a0mL\u22121\u00a0per manufacturer instructions and then diluted further in DMEM prior to inhibition experiments. Vesicles or sACE2\u00a0were then serially diluted in DMEM. Spike\u2010pseudotyped lentivirus was added to each sample at a projected MOI between 0.02\u00a0and 0.15\u00a0and mixed with pipetting. 175\u00a0\u00b5L of the mixed sample were transferred to TC\u2010treated 96\u2010well plates, centrifuged at 500\u00a0\u00d7\u00a0g for 1\u00a0min at 4\u00a0\u00b0C to remove bubbles, and incubated at 37\u00a0\u00b0C for 1\u00a0h. WT\u2010ACE2\u00a0expressing HEK293FTs were briefly trypsinized (<1\u00a0min), quenched with DMEM, and counted. Cells were diluted in DMEM and added to plates such that 4\u00a0\u00d7\u00a0103\u00a0cells were plated per well in 25\u00a0\u00b5L media resulting in 200\u00a0\u00b5L media total per well. Approximately 16\u00a0h later, the media was replaced with fresh DMEM (200\u00a0\u00b5L) and the cells were cultured for an additional 2\u00a0days (\u224872\u00a0h post inoculation). Cells were harvested for flow cytometry via trypsin, quenched with phenol red\u2010free DMEM, and diluted with at least five volumes of FACS buffer in FACS tubes. Samples were centrifuged at 150\u00a0\u00d7\u00a0g for 5\u00a0min at 4\u00a0\u00b0C, the supernatant was decanted, and the samples were stored at 4\u00a0\u00b0C until flow cytometry analysis.Stock vesicle concentration was determined by NTA, and samples were then diluted in DMEM such that each vesicle sample had the same concentration (in units of vesicles per volume). SACE2  was resuspended in sterile, nuclease\u2010free HAnalytical flow cytometry was performed on a BD LSR Fortessa Special Order Research Product (Robert H. Lurie Cancer Center Flow Cytometry Core); EYFP expression and Alexa Fluor 488\u00a0staining were measured using the fluorescein isothiocyanate channel from a 488\u00a0nm excitation laser and captured using a 505\u00a0nm long pass filter and a 530/30\u00a0nm bandpass filter. Approximately 5000\u201310\u00a0000\u00a0single cells were analyzed for each sample on FlowJo software v10. As illustrated in Figure  Curves were then fit with a four parameter, nonlinear regression in GraphPad Prism 9.2. Convergence criterion was set to \u201cStrict\u201d with 10\u00a0000\u00a0maximum iterations, and the regression was constrained as follows: \u201cBottom\u201d\u00a0=\u00a00, \u201cTop\u201d\u00a0=\u00a0100, \u201cIC50\u201d\u00a0>\u00a00. Relative resistance metrics were calculated by dividing the ID50\u00a0of a strain of interest by the ID50\u00a0of a reference, parental strain . Where reported, viral titer was calculated by determining MOI for experimental conditions with less than 30% cells transduced , assuming a Poisson distribution. When normalizing ID50\u00a0values to viral TU as reported in the Supporting Information, the viral titer for each condition was calculated as described above using the lower limit (lowest inhibitor concentration) case from the corresponding dose response curve. Band intensities from semiquantitative western blots were evaluated using ImageJ, and error was propagated using MATLAB as discussed above. In all other cases, error was propagated using standard propagation rules in Microsoft Excel.Unless otherwise stated in the relevant figure caption, data were provided as mean\u00a0\u00b1\u00a0standard error of the mean, and derived parameters  were presented as best\u2010fit parameter estimates \u00b1\u00a095\u201399% confidence interval. The number of replicates performed was stated in the relevant figure captions. Generally, viral inhibition experiments were performed in biological triplicate, and two independent replicates of each experiment were performed. In flow cytometry experiments evaluating the dose\u2010response curves of viral inhibitors, the percent of transduced cells for a given treatment was normalized by the percent of transduced cells determined from that treatment's largest dilution as depicted in Figure 74 CurThe authors declare no conflict of interest.T.F.G., D.M.S., N.P.K., and J.N.L. conceived the initial project. T.F.G. performed the experiments. R.E.M. performed transmission electron microscopy. T.F.G., D.M.S., N.P.K., and J.N.L. planned and analyzed experiments. T.F.G., D.M.S., N.P.K., and J.N.L. wrote the manuscript. N.P.K. and J.N.L. supervised the work.Supporting InformationClick here for additional data file.Supplemental Table 1Click here for additional data file.Supplemental Table 2Click here for additional data file."}
+{"text": "Angiopoietin-like 4 (ANGPTL4) is highly expressed in a variety of neoplasms and promotes cancer progression. Nevertheless, the mechanism of ANGPTL4 in ovarian cancer (OC) metastasis remains unclear. This study aimeds to explore whether ANGPTL4 regulates OC progression and elucidate the underlying mechanism.ANGPTL4 expression in clinical patient tumor samples was determined by immunohistochemistry (IHC) and high-throughput sequencing. ANGPTL4 knockdown (KD) and the addition of exogeneous cANGPTL4 protein were used to investigate its function. An in vivo xenograft tumor experiment was performed by intraperitoneal injection of SKOV3 cells transfected with short hairpin RNAs (shRNAs) targeting ANGPTL4 in nude mice. Western blotting and qRT-PCR were used to detect the levels of ANGPTL4, CDH5, p-AKT, AKT, ETV5, MMP2 and MMP9 in SKOV3 and HO8910 cells transfected with sh-ANGPTL4 or shRNAs targeting ETV5.Increased levels of ANGPTL4 were associated with poor prognosis and metastasis in OC and induced the angiogenesis and metastasis of OC cells both in vivo and in vitro. This tumorigenic effect was dependent on CDH5, and the expression levels of ANGPTL4 and CDH5 in human OC werepositively correlated. In addition, CDH5 activated p-AKT, and upregulated the expression of MMP2 and MMP9. We also found that the expression of ETV5 was upregulated by ANGPTL4, which could bind the promoter region of CDH5, leading to increased CDH5 expression.Our data indicated that an increase in the ANGPTL4 level results in increased ETV5 expression in OC, leading to metastasis via activation of the CDH5/AKT/MMP9 signaling pathway.The online version contains supplementary material available at 10.1186/s13048-022-01060-7. Ovarian cancer (OC) is one the deadliest gynecological tumors . AlthougAngiopoietin-like 4 (ANGPTL4) is a secreted protein, that is cleaved into two active peptides; the N-terminal domain is an effective inhibitor of lipoprotein lipase (LPL) activity and regulates lipid composition and energy homeostasis . The C-tP\u2009<\u20090.05) when the gene copy number was over 2.0-fold and recurred more than three times. Among the differentially expressed genes, ANGPTL4, which has been reported to be highly expressed in a variety of neoplasms and can promote cancer angiogenesis and metastasis, was found to exhibit significantly increased expression in OC metastasis \u2009=\u20090.71 (0.55\u20130.93)) \u2009=\u20090.76 (0.6\u20130.97)) \u2009=\u20090.68 (0.51\u20130.89))  using IHC for CD31. Compared with that in the control group, the number of CD31-positive microvessels in the LV-shANGPTL4 group was significantly reduced Fig.\u00a0\u00a0A. We alr\u2009=\u20090.1215, P\u2009=\u20090.0005 Fig.\u00a0To investigate novel signaling pathways downstream of ANGPTL4 in OC, we subjected all significantly upregulated and downregulated genes   plasmids expressing shRNAs targeting ANGPTL4 into OC cells. ETV5 short interfering (si)RNA and negative controls were purchased from RiboBio . We obtained shANGPTL4 plasmids and negative controls from OBIO . The protocols involving all cell lines received ethical approval from the Human Research Ethics Committee of Shanghai General Hospital affiliated to Shanghai Jiao Tong University.The human OC cell lines SKOV3, H08910, Hey, A2780 and A2780/DDP (cisplatin-resistant cell line) were cultured in RPMI 1640  medium. SKOV3 cells were obtained from the FuHeng Cell Center . HO8910 cells were obtained from Procell Life Science & Technology. An immortalized ovarian epithelial cell line (Moody) and HUVECs were conserved in our laboratory and had been purchased from the American Type Culture Collection (ATCC), these cell lines were cultured in DMEM:F12 . All cell lines were cultured according to standard protocols and maintained at 37\u00a0\u00b0C under 5% COThe tissue microarray (TMA) included 97 OC tissues and 2 normal ovarian tissues and was purchased from the Shanghai Weiao Biological Company. Eighteen normal ovarian tissue samples were collected from the Department of Gynecology and Obstetrics, Shanghai General Hospital, between 2018 and 2019. Four pairs of metastatic foci and primary foci from OC samples were collected for high-throughput sequencing after surgery at Shanghai General Hospital from April 2017 to December 2018. None of the patients received any preoperative treatment. Samples were cryopreserved in liquid nitrogen. All patients signed informed consent forms. This study was approved by the Institutional Research Ethics Committee of Shanghai General Hospital.Total RNA was isolated using an RNeasy mini kit . The TruSeq\u2122 RNA Sample Preparation Kit  was used to synthesize the paired-end library according to instructions in the sample preparation guide. The library was constructed and sequenced by Sinotech Genomics Co., Ltd. . Differential mRNA expression was analyzed by R language packages. Differentially expressed RNAs with a |log2(FC)| value\u2009>\u20091 and a q value\u2009<\u20090.05 were regarded as significantly differentially expressed.Lentivirus vectors encoding human shRNAs against ANGPTL4 and an empty vector (LV-shCon) were purchased from OBIO . Cells were stably transfected with lentivirus, grown and harvested after puromycin selection for 14 days. Details of the commercial antibodies are shown in Table\u00a0\u2212\u0394\u0394Ct method. All primers sequences used are shown in Table\u00a0Using TRIzol reagent , total RNA was isolated according to the manufacturer\u2019s instructions, and qRT-PCR was performed with TB Green Premix Ex Taq  on a 7500 real-time PCR system  and was determined by the 2Cellular extracts containing the same amount of protein were separated on SDS-polyacrylamide mini-gels and transferred to PVDF membranes  for 90\u00a0min at 300 mA. The membranes were blocked with 5% skim milk at room temperature for 1\u00a0h and then incubated with specific primary antibodies at 4\u00a0\u00b0C overnight. Then, they were washed with TBST buffer3 times (10\u00a0min each) and incubated with secondary antibodies  at room temperature for 1\u00a0h. ECL chemiluminescence (Millipore) was used to detect proteins.ANGPTL4, CDH5 and MVD-related protein levels were analyzed by IHC as previously reported . The per5 cells/100 \u00b5l were seeded in the upper chambers of 24-well plates  with serum-free medium. RPMI 1640 medium containing 10% FBS was added to the lower chamber. After 24\u00a0h, the cells in the upper part of the chamber were removed, and the cells in the lower part of the chamber were fixed with formaldehyde and stained with crystal violet. In the invasion test, the upper chamber was precoated with Matrigel , and cells were seeded in the upper chamber in serum-free medium. Medium containing 10% serum was added to the lower chamber. After 48\u00a0h, invaded cells were fixed and stained with crystal violet. The cells were counted under a microscope.For Transwell migration assays, 1\u2009\u00d7\u2009105 HUVECs was added to the upper chamber, and 800\u00a0\u00b5l tumor supernatant was added to the lower chamber and incubated at 37\u00a0\u00b0C with 5% CO2 for 24\u00a0h. Cells were incubated with a subsequent tumor procedure as previously described.HUVEC migration assay was performed using Falcon\u2122 Cell Culture Inserts (BD353097) according to the manufacturer\u2019s instructions. Then, 200\u00a0\u00b5l of serum-free medium containing 1\u2009\u00d7\u2009105 cells per well in 6-well plates, a 100\u00a0\u00b5l pipette tip was used to create 3 wounds devoid of cells, and medium without FBS was added. Images were captured at 0 and 24\u00a0h, and wound widths were quantified and compared to baseline values.The wound healing experiment was performed by plating 1\u2009\u00d7\u200910The proliferative ability of HUVECs after coculture with CM from different cells was determined by an EdU proliferation assay (RiboBio). After pretreatment as described above, HUVECs were incubated in 50\u00a0M EdU for 2\u00a0h and then fixed, permeabilized, and stained following the manufacturer\u2019s instructions.4 cells/well in 96-well plates were cultured in 250 ng/ml rhANGPTL4 or tumor supernatants from each cell line for 6\u00a0h. The plates were precoated with 100\u00a0\u00b5l of Matrigel (BD Bioscience) at 37\u00a0\u00b0C for 1\u00a0h. After 6\u00a0h of incubation, images of the tubules were acquired and analyzed by Image-Pro Plus software and tubules were quantified by counting the number of tubes in 10 randomly chosen fields of view. Data were obtained from three independent experiments.HUVECs at a density of 1\u2009\u00d7\u200910http://www.kmplot.com. Progression-free survival , disease-specific survival  and overall survival  were analyzed using TCGA data.ANGPTL4 expression data in OC were retrieved from TCGA . Then, the correlation between ANGPTL4 expression and CD31 expression and the correlation between ANGPTL4 expression and VEGFA expression were assessed. Survival curves were determined by the Kaplan-Meier method with the website: For CoIP assays, SKOV3 cells were lysed on ice in lysis buffer  for 30\u00a0min with occasional vortexing, and centrifugation was performed for 12\u00a0min to remove cellular debris. After preclearing, 500\u00a0\u00b5l of protein lysate was immunoprecipitated with anti-CDH5 antibody  and protein A Sepharose beads. The immunoprecipitates were then probed with anti-ANGTPL4  and anti-CDH5  antibodies. The precipitates were separated by SDS-PAGE and analyzed by immunoblotting.ChIP assays were performed with a ChIP Kit (Millipore) following the manufacturer\u2019s protocol. Protein and DNA were crosslinked in 1% formaldehyde, with glycine used to terminate the crosslinking reaction, after which the crosslinked molecules were extracted with SDS lysis buffer, and sheared by sonication. An ETV5 antibody (Proteintech 66657-1-lg) was used for immunoprecipitation. After purification of the precipitated DNA, PCR was conducted. The primer sequences used for PCR are listed in Table\u00a06 cells/100 \u00b5l) were intraperitoneally (i.p.) injected into 5-week-old BALB/c nu/nu female mice (8 mice per group). After 4 weeks, the animals were anesthetized and killed with an excess of 2% pentobarbital sodium (0.5 ml), and death was then confirmed with cervical dislocation. The intraperitoneal tumor nodules were extracted and weighed.All animal experiments were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Department of Laboratory Animal Science, School of Medicine, Shanghai Jiao Tong University. SKOV3-LV-shCon and SKOV3-LV-shANGPTL4 cells  was used as instructed by the manufacturer quantify the secretion of ANGPTL4 in cell culture medium.Control and LV-shANGPTL4 groups of SKOV3 and HO8910 cells were seeded at a density of 1\u2009\u00d7\u2009106 in 60-mm Petri dishes and cultured in RPMI 1640 medium for 48\u00a0h. The CM was collected and centrifuged at 3000\u00a0rpm for 10\u00a0min at 4\u00a0\u00b0C.All data are presented as the means\u2009\u00b1\u2009SD. Data from two groups were compared by two-tailed Student\u2019s t-test. GraphPad Prism 6.0  was used for all statistical analyses. Differences for which the P value was <\u20090.05 were considered statistically significant.Additional file 1:\u00a0Supplementary Figure 1.\u00a0A. ELISAshowing the level of extracellular ANGPTL4 proteins in the culture media ofSKOV3 cells and HO8910 cells transfected with control or ANGPTL4 shRNA andincubated for 2 days . B. Recombinant ANGPTL4 rescued changes in HUVEC tube formation activity due to treatment with LV-SKOV3-shANGPTL4 CMas shown using the tube formation assay. Representative images are shown. Scale bars, 400\u00a0\u00b5m.C. Recombinant ANGPTL4 rescued changes in HUVEC migration activity due totreatment with LV-SKOV3-shANGPTL4 CM as shown using the transwell assay.Representative images are shown. Scale bars, 100\u00a0\u00b5m.\u00a0Supplementary Figure 2.\u00a0A.Top network identified by IPA. Gene signatures of metastatic sites and primarysites of OC. B. The binding sites of ETV5 to the CDH5 promoter as predicted bythe online Jaspar website (http://jaspar.generge.net/).\u00a0Supplementary Figure 3.\u00a0The expression level ofANGPTL4 is independent of each other with VEGFA.\u00a0A. VEGFA mRNA expression level after knock downANGPTL4 in OC cells. Data represent mean\u00b1SD of three independent experiments.B. Expression level of ANGPTL4 after adding 250ng/ml bevacizumab in OC cellculture medium. Data represent mean\u00b1SD of three independent experiments. C. Anelevated ANGPTL4 in ovarian cancer increases the expression of CDH5 byup-regulating ETV5 which could binding to CDH5 promoter region, would activateAKT followed by induction of MMP9. At the same time, high expression of ANGPTL4can promote angiogenesis of ovarian cancer.\u00a0Additional file 2.\u00a0Additional file 3.Additional file 4."}
+{"text": "The time between the CMRd and CMRf was 5 months [3\u201311]. The LV ejection fraction (LVEF) was 55 \u00b1 6 and 59 \u00b1 4%, p = 0.008, respectively, and 94.1% of the patients showed late gadolinium enhancement (LGE) and myocardial edema on the CMRd. Significantly lower FTc  and DLSc  were observed with respect to the controls. Significant increases in the FTc  and DLSc  were found between the CMRd and CMRf, which were unrelated to the LGE. The LVEF correlated well with the FTc (r = 0.840) and DLSc (r = 0.760). Both techniques had excellent reproducibility, with high intra-  and inter-observer  correlation. There was correlation between the LV DLSc/FTc and LVEF in the patients with acute myocarditis according to the CMRd and CMRf.This study sought to examine the correlation between left ventricular (LV) myocardial feature tracking (FT) and deep learning-based strain (DLS) analysis in the diagnostic (CMRd) and follow-up (CMRf) cardiac magnetic resonance imaging of patients with acute myocarditis. The retrospective study included 17 patients with acute myocarditis and 20 healthy controls. The CMRd took place within 14 days of symptom onset, while the CMRf took place at least 2 months after the event. The global-circumferential FT (FTc) and global-circumferential DLS (DLSc) were analyzed. The continuous variables were compared using paired  Myocarditis is an inflammatory disease of the heart muscle that involves multiple etiological factors, including autoimmune disorders, infections , and cardiotoxic drugs/toxins . Its dia2-STIR and T2-map) and myocardial fibrosis .The strength of CMR lies in its excellent capacity for tissue characterization and cardiac functional assessment, which permits the determination of the presence/absence of the previously well-established modified Lake Louise Criteria  through Recently, the analysis of myocardial deformation by means of feature tracking  has become possible using the basic cine sequences acquired in CMR studies, and this technique has proven useful in the assessment of subclinical cardiac pathology ,6. WhileThe aim of the present study was to evaluate the applicability of the FT and DLS methods for the CMR of patients hospitalized for acute myocarditis with clinical/analytical expression/diagnostic CMR (CMRd) and its evolution in follow-up CMR (CMRf). We compared the findings obtained using the two advanced deformation analysis techniques with the CMR available in our center.The research protocol for this retrospective observational study was approved by the ethics committee of the national reference center (n\u00ba14/22). We included patients with a diagnosis of acute myocarditis who were hospitalized between 2016 and 2021 and who had CMRf performed during follow-up at least 2 months after the event ,10. All The exclusion criteria included cardiomyopathy and a previous history of ischemic or valvular heart disease, as well as the general contraindications for CMR. The baseline characteristics of the patients were extracted from their medical records. All the patients gave prior consent for CMR and the use of their imaging data for educational/scientific purposes. No external funding was received for any aspect of this work.\u00ae, Bayer Schering Pharma AG, Berlin, Germany; 1 mmol/mL), the LGE images were acquired, with the same planning as used for the cine images, using a T1-weighted inversion recovery (IR) gradient-echo T1-weighted sequence.The imaging studies were performed on a 1.5T GE Optima MR450w  MRI unit using a 32-channel multi-element surface antenna and electrocardiographic synchronization. The cine images were obtained in expiratory apnea using the conventional SSFP sequences in 4-, 3-, and 2-chamber longitudinal slices, and in 10\u201315 contiguous short-axis slices covering both ventricles from the base to the apex. Approximately 8\u201310 min after the intravenous infusion of 0.15 mmol/kg gadobutrol (Gadovist2-weighted sequences and short tau inversion recovery (STIR) sequences, which were obtained prior to the gadolinium administration, in the short axis from the base to the apex, as well as the T2-map sequences in the end-diastole in the basal, middle, and apical cut in the short axis, and a longitudinal cut in 4 chambers, using the T2-weighted TSE (turbo spin echo) sequences with different T2 times , with the repetition time (TR = 1 \u00d7 R-R). The T1-mapping was performed with a modified Look-Locker IR sequence using a 3(3)5 scheme in the same four planes acquired for the T2-map prior to and 15 min after the contrast infusion.For the assessment of the edema, we used both the classic T2-STIR sequences was visually assessed and with agreement between the two experts, whereas the mapping analysis was performed by tracing a region of interest (ROI) in the septum on each of the acquired slices. The LGE in each segment was visually classified as subepicardial, intramyocardial, subendocardial, transmural, or non-enhancement. The endocardial and epicardial contours in the LGE short-axis sequences were manually traced, and the percentage of enhanced mass volume was calculated using the full width at half maximum (FWHM) method.All the CMR studies were analyzed by a cardiologist (*) (EACVI European CMR level 3 accreditation) and a radiologist (**) with CMR experience . The cardiac functional analysis was performed retrospectively using the dedicated advanced analysis software CVI42 . The left (LVEF) and right (RVEF) ventricular ejection fractions were measured from the cine sequences using the disc summation method. The myocardial edema on the TThe feature tracking was performed using the advanced cardiac analysis software CVI42 . The global circumferential FT (FTc) was performed on the short-axis, 4-, 3-, and 2-chamber views in expiratory apnea using the cine SSFP sequences. For this purpose, the contours were traced along the LV endocardial and epicardial border in both the end-diastole and end-systole in all the basal slices  and in a 4-, 3-, and 2-chamber reference slice. The contours subsequently propagated automatically through all the phases, and in the case of an erroneous propagation, it was manually edited in the respective slice, again propagating automatically. The FTc was obtained from the derivative of the tracking of the features within the myocardium (defined by the inputted endocardium and epicardium contours) throughout the cardiac cycle, resulting in an overall FTc value.All the DLS analyses were performed using the advanced research-use-only strain feature hosted on the Arterys platform  and developed by the AiDA Laboratory. The model was based on artificial intelligence or deep learning, and it inferred the 2D myocardial velocity fields from the short-axis cine SSFP image series. These velocity fields were then used to calculate the pixel-wise myocardial strain rate and strain, thereby providing the regional circumferential and radial strain measurements. The algorithm was trained using the coregistered 4D Flow and short-axis cine SSFP images acquired during the same examination for each patient with full left-ventricle coverage. Once the LV endocardial and epicardial border at the end-diastole and end-systole, as well as the RV inferior\u2013anterior septal junction points, had been traced, a mathematical algorithm automatically calculated the myocardial deformation values for each frame of the cardiac cycle, obtaining among them the 2D global circumferential DLS (DLSc). It is not possible to calculate the global longitudinal DLS using this technology at the present time. Finally, the FTc and DLSc results between the CRMd and CRMf were compared.t-test or the Wilcoxon test, while the categorical variables were analyzed using McNemar\u2019s test. Pearson\u2019s or Spearman\u2019s correlation was used to evaluate the correlation between the continuous variables, after checking the normality of the variables. The absolute intraclass correlation coefficient (ICC) values were calculated to determine the intra- and inter-observer reliability. Receiver operating characteristic (ROC) plots were used to determine the FTc and DLSc values that diagnosed circumferential myocardial deformation involvement with better specificity and sensitivity, and the area under the curve (AUC) and its 95% confidence interval (CI) was calculated. The data analysis was performed using SPSS, version 25.0  at a 5% significance level.The descriptive statistics are presented as the absolute (n) and relative (%) frequency for the categorical variables and as the mean \u00b1 standard deviation (SD) or median  for the continuous variables, depending on the parametric or non-parametric behavior of the variables, respectively. The continuous variables were compared using Student\u2019s paired A total of 17 patients with acute myocarditis and 20 healthy individuals were included in this study. The baseline characteristics of the patients in the study are shown in The morphofunctional data from the CMR findings are shown in 1 , T2 map , and extracellular volume (ECV) . In total, 94.1% (16/17) of patients presented with signal hyperintensity in the T2-STIR sequences in the regions with LGE compatible with myocardial edema on the CMRd. Only two patients (11.8%) presented with segmental contractile alteration, such as septal hypokinesia, on the CMRd.Regarding the quantitative map values, there was a normalization of the values between the CMRd and CMRf for the native myocardial Tp = 0.016) and DLSc  values between the CMRd and CMRf, increasing by >15% of their relative value in 29% (5/17) (FTc) and 25% (4/17) (DLSc) of the patients, respectively. At the same time, we found good correlation between the techniques in those patients in whom there was no increase in the FTc/DLSc . Our results show that those patients in whom there was an improvement in the strain values according to the DLSc technique also showed an improvement according to the FTc technique, meaning they were equally reproducible in the opposite case .In our sample, the FTc/DLSc improvement was not related to the evolution of the percentage of LGE between the CMRd and CMRf (FTc p = 0.001) and DLSc \u221238.1 \u00b1 5.2% vs. \u221241.3 \u00b1 4.5% (p = 0.015). The ROC curves determined that values of \u221217% for the FTc  and \u221238% for the DLSc  were the best cut-off points for diagnosing circumferential myocardial deformation involvement in this setting in our population  and inter-observer  variability with both techniques.The main findings of our study are the following. We found excellent reproducibility with high intra-/inter-observer correlation for the DLSc and FTc. In addition, the DLSc/FTc values were lower in the patients with acute myocarditis than in the healthy group, and there was good correlation between the DLSc and FTc findings and the LVEF. Finally, no relationship was found between the LGE grade and DLSc/FTc-based improvement in our population.Previously, to the best of our knowledge, only two groups have assessed patients after admission for acute myocarditis using CMR-FT follow-up. In the first of these, Luetkens et al.  observedIn agreement ,14,15,16Among the aspects to be highlighted, we would mention that despite representing different strain values for the DLSc than for the FTc , we obtained good correlation between the two strain values in the CMRd and CMRf, showing a relative increase of more than 15%  with botWhile we have to consider that our sample was composed of patients who met the strict criteria for a diagnosis of acute myocarditis at the time of hospital admission (94.1% with the presence of edema in the STIR and LGE sequences on the baseline CMR and elevated troponin T), we did not find correlation between having a higher percentage of LGE and presenting with worse FTc/DLSc values in the CMRf, with the caveat that we cannot assess the direct correlation between the presence/absence of LGE and the DLSc/FTc strain values. Likewise, we did not find correlation between the evolution of the degree of LGE between the CMRd and CMRf and the improvement of strain by both techniques (FTc/DLSc).Regarding the relationship of edema and LGE with FT, there are also contradictions according to previous studies. In a study sample where 77% of the patients had LGE and 75% showed myocardial edema in CMR, Luetkens et al.  found thSeveral groups have highlighted the importance of determining whether or not the technique has added value in patients with myocarditis and preserved the LVEF, with the impact on the subclinical evaluation of these patients that this would imply. Studies such as that by Gatti et al.  using anTo the best of our knowledge, this is the first study in patients with a follow-up CMR after hospitalization for acute myocarditis that performed a comparative analysis of myocardial deformation simultaneously between the FTc and DLSc techniques. Our findings reflect a good concordance of results between the two analysis techniques despite the disparity between their values, showing a decrease in both with respect to the control group and the good correlation of both techniques with the LVEF, with excellent intra- and inter-observer variability as long as the same tool was used for the follow-up.Our study has several limitations that should be considered. First of all, it is a single-center retrospective study with a limited number of patients. However, considering the strict inclusion criteria for our sample , we consider that it was adequate for the preliminary analyses, which will hopefully open the door for future multicenter studies. Nevertheless, given the relatively small number of patients, the generalizability of our cut-off points for diagnosing circumferential myocardial deformation involvement should be treated with caution. On the other hand, there is a gender difference between the normal and myocarditis patients in our sample that should be taken into consideration. Some authors, however, have shown that there are no significant differences between the circumferential strain values by gender . We consWhile we acknowledge that we used two different myocardial deformation analysis techniques in the CMR, the purpose of the study was to assess the correlation between their results, which demonstrated their feasibility and reproducibility as long as the same tool is used during their follow-up.The assessment of LV myocardial deformation by means of CMR strain DLSc-FTc in patients with acute myocarditis showed good correlation between their values and with the LVEF in the CMRd and CMRf. Lower DLSc/FTc was observed in this scenario in the CMRd compared with the healthy group, with a subsequent increase in the CMRf, which was unrelated to the degree of LGE in our population."}
+{"text": "Salmo salar) exposed to different light conditions were analyzed, including a developmental series and a circadian profile. The results showed that genes mediating nonvisual photoreception are present prior to hatching when the retina is poorly differentiated. The clock genes were expressed early, but the circadian profile showed that only two clock genes were significantly cycling before first feeding. Few genes were differentially expressed between day and night within a light condition; however, many genes were significantly different between light conditions, indicating that light environment has an impact on the transcriptome during early development. Comparing the transcriptome data from constant conditions to periodicity of white light or different colors revealed overrepresentation of genes related to photoreception, eye development, muscle contraction, degradation of metabolites and cell cycle among others, and in constant light, several clock genes were upregulated. In constant white light and periodicity of green light, genes associated with DNA replication, chromatin remodeling, cell division and DNA repair were downregulated. The study implies a direct influence of light conditions on the transcriptome profile at early developmental stages, by a complex photoreceptive system where few clock genes are cycling.Light cues vary along the axis of periodicity, intensity and spectrum and perception of light is dependent on the photoreceptive capacity encoded within the genome and the opsins expressed. A global approach was taken to analyze the photoreceptive capacity and the effect of differing light conditions on a developing teleost prior to first feeding. The transcriptomes of embryos and alevins of Atlantic salmon ( Light conditions, including, periodicity, intensity and spectrum, are environmental factors that have driven evolution and shaped the organisms photosensory system and regulation of biological processes from behavior to gene expression. In this study, we have taken advantage of early development in Atlantic salmon, an organism with life history closely linked to changing light environment, that develop to an advance stage prior to first feeding. The data represent a long-term exposure trail of 4 months, which is independent of disturbing effects of feeding, giving the opportunity to gain insight into the direct effects of different light conditions on the transcriptome profile. The transcriptomic data reveal that the photoreceptive capacity and a non-cycling clock system develop early. Overall, many genes representing various pathways were significantly different between light conditions. In our opinion, this holistic dataset and results represent a very important step to gain better overview of how different light conditions impact the transcriptome during development. Light is one of the most important environmental cues known to modulate the behavior, physiology and gene expression of organisms. In nature, the daily solar cycle provides a predictable rhythm of light and dark periods that allows for the entrainment and regulation of many circadian and circannual biological processes by non-image forming photoreception ,2. KnowlDanio rerio) In silicodatabase . Note thdatabase , these wdatabase  during tdatabase .P-values based on Kendall\u2019s rank correlation coefficient statistical analyzes [Cycling transcripts were identified by JTK_CYCLE v3.1 in R, which is designed to efficiently identify and characterize cycling variables in large collection of data, such as genome-scale data sets. JTK_CYCLE v3.1 effectively distinguishes rhythmic and non-rhythmic transcripts by determining analyzes . NormaliS1 Figmax = ~610 nm), at medium intensity and the narrow bandwidth spectrum of blue (\u03bbmax = ~450nm), green (\u03bbmax = ~535nm), and red (\u03bbmax = ~660nm) LEDs. C) Pictures of eggs in the egg incubators with medium white, blue, green, and red light.A) Schematic illustration of the experimental setup showing constant or changing light environments. Embryos and alevins were exposed to different lighting regimes from fertilization to first feeding (121 days). The experiments were divided across three parameters: (i) different photoperiods (i.e. continuous white light (LL), continuous darkness (DD), or a white light:dark (LD) cycle of 14:10); (ii) intensities of LD white light, consisting of high (LDH), medium (LDM) or low (LDL) light levels; (iii) a LD cycling condition with medium intensity of light of different wavelengths of the visible spectrum, namely (blue (LDB), green (LDG), red (LDR). The arrows indicate sampling points during development, 255 dd (40 days), 379 dd (60 days), 555 dd (90 days) and 690 dd (113 days). The circadian sampling for LDM at 690 dd is highlighted by wider boxes indicating the sampling points for the 24 h series. All light regimes were sampled at 18:00 and 02:00. B) Spectrum of the white LED, warm white 2700K (\u03bb(TIF)Click here for additional data file.S2 FigA) Venn diagram of all genes expressed at different developmental stages, only genes that had a total count greater than 10 within a developmental stage were included. Only a few genes are unique for each color-coded developmental stage  and 33,377 genes (87.5%) being expressed at all four stages. B) Bar chart of differentially expressed genes (DEGs), comparing 255 dd, 379 dd and 555 dd to 690 dd. C) Venn diagram of Gene Ontology (GO) terms comparing 255 dd, 379 dd and 555 dd to 690 dd. The diagrams show that 51.3% of upregulated and 43% downregulated terms are shared between the different comparisons.(TIF)Click here for additional data file.S3 FigThe heatmap is shown by individual normalized counts, scaled by row.(TIF)Click here for additional data file.S4 FigThe heatmap is shown by individual normalized counts, scaled by row.(TIF)Click here for additional data file.S5 FigThe heatmap was made by comparing the mean count for each developmental stage.(TIF)Click here for additional data file.S6 FigComparing A) continuous darkness (DD) B) periodicity of blue light (LDB) C) periodicity of green light (LDG) D) periodicity of red light (LDR) to periodicity of white light (LDM). The scale indicates the logfold2 change and the color code indicate upregulated (blue) and downregulated (red) genes.(TIF)Click here for additional data file.S1 TableThe normalized counts are for the LDM at 255 dd, 379 dd, 555 dd and 690 dd.(XLSX)Click here for additional data file.S2 TableThe table contains the normalized counts for the LDM circadian series and day and night samplings under DD, LL, LDH, LDL, LDB, LDG and LDR conditions.(XLSX)Click here for additional data file.S3 Tabletmtopsin3a1 is not annotated and opn9 is incorrectly annotated in the Ensembl database. These genes are identified by LOC-ID.The LOC-ID, Ensembl GeneID, chromosome location, strand and Ss4R duplication pair are included. Note that (XLSX)Click here for additional data file.S4 TableNumber of differentially expressed genes (DEGs) and pathway enrichment analyzes comparing 255 dd, 379 dd, and 555 dd to 690 dd, listing both upregulated and downregulated Gene Ontology (GO) terms together with Ensembl GeneIDs for each DEGs.(XLSX)Click here for additional data file.S5 TableThe counts were obtained from (XLSX)Click here for additional data file.S6 TableResults from JTK_CYCLE (p < 0.05) showing cycling genes. Lines indicate the (p < 0.01) and (p < 0.001) and PER show the periodicity of 20 h, 24 h or 28 h.(XLSX)Click here for additional data file.S7 TableIn the circadian series the different sampling points were compared to either 10:00, 02:00, 18:00 or an altering control and the number of DEGs are listed. The table also include the upregulated or downregulated DEGs from the analyzes within a light stimulation (02:00 vs 18:00) and comparing different light conditions to DD, LL or LDM at 690 dd shown in .(XLSX)Click here for additional data file.S8 TableThe result of clusterProfiler for differing photoperiods  and wavelength experiments .(XLSX)Click here for additional data file."}
+{"text": "This research aims to explore the role of Tanshinone IIA (Tan IIA) and microRNA (miR)-30b-5p in chemoresistance of colorectal cancer (CRC). The expression levels of miR-30b-5p and apoptosis and caspase activation inhibitor (AVEN) was detected by reverse transcription-quantitative polymerase chain reaction assay. The cell proliferation and apoptosis were examined by 3--2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays. The target relationship between miR-30b-5p and AVEN was confirmed by Dual-luciferase reporter assay. Transwell assay was performed to assess CRC cells\u2019 metastasis. Western blot was carried out to measure the apoptosis-related protein. The results showed that miR-30b-5p was lowly expressed in oxaliplatin-resistance CRC cells SW480 (SW480/R) compared to SW480 cells. Overexpression of miR-30b-5p significantly suppressed the malignant biological behaviors of SW480/R cells and significantly promoted the sensitivity of SW480/R cells to oxaliplatin by down-regulated AVEN expression. Besides, Tan IIA treatment upregulated miR-30b-5p expression in SW480/R cells. Moreover, miR-30b-5p upregulation strengthened the promoting effect of Tan IIA on the sensitivity of SW480/R cells to oxaliplatin. In conclusion, Tan IIA and miR-30b-5p could reverse oxaliplatin resistance of CRC cells and may thus be potential treatment strategies for treating patients with CRC. CRC is MicroRNAs (miRNAs) are composed of 19\u201325 nucleotides and are involved in the regulation of gene expression at the post-transcriptional process by binding to the 3\u2032-untranslated region (3\u2032-UTR) of mRNA of the target gene  and miR-Tanshinone IIA (Tan IIA), a compound isolated from the dried root and rootstock of Salvia miltiorrhiza (Danshen) . RecentlApoptosis and caspase activation inhibitor (AVEN) is essential for its anti-apoptotic function ,23. In aTherefore, this study aims at figuring out whether Tan IIA regulates chemoresistance of CRC via the miR-30b-5p/AVEN axis.22.12 at 37\u00b0C. Oxaliplatin-resistant CRC cell lines (SW480/R) were induced by oxaliplatin . Generally, the oxaliplatin-resistant cell lines were developed from SW480 cells by stepwise exposure to increasing concentrations of oxaliplatin from 0.2 to 2\u2009\u00b5M, and the drug-resistant cell lines SW480/R was established after 6 months.Human CRC cell line SW480 were purchased from the Chinese Academy of Science, Shanghai Institute of Biochemistry and Cell Biology. SW480 cells were maintained in Dulbecco\u2019s modified eagle medium, high glucose  supplemented with 1% penicillin and streptomycin  and 10% fetal bovine serum  in a humidified atmosphere of 5% CO2.22, transfected cells were harvested for subsequent use.For regulation of expression of miR-30b-5p, miR-30b-5p inhibitor and negative control oligonucleotide NC inhibitor (miR-30b-5p inhibitor: 5\u2032-AGAACAGUGAAAUUUCCAGUCC-3\u2032 and inhibitor control: 5\u2032-CAGUACUUUUGUGUAGUACAA-3\u2032), mimic of miR-30b-5p (5\u2032-UGUAAACAUCCUACACUCAGCU-3\u2032) and mimic control (5\u2032-UACUGAGAGACAUAAGUUGGUC-3\u2032) were purchased from Ribobio . For overexpression of AVEN, AVEN-plasmid were also purchased from Ribobio (Santa Cruz Biotechnology). All these vectors and reagents were transfected into cells and were grown to 70\u201380% confluence with Lipofectamine 6000 . After incubating for 48\u2009h at 37\u00b0C and 5% CO2.3\u00ae (Invitrogen), and RNA was reverse-transcribed into cDNA with the QuantiTect Reverse Transcription Kit . The expression of miRNA was detected by Hairpin-itTM miRNAs Quantitation Kit  and the RT-qPCR was carried out using SYBR green reagents . The 2\u2212\u0394\u0394Ct method was employed to calculate the relative expression levels. The following primers were used in PCR assay: U6: 5\u2032-CGCTTCGGCAGCACATATACTA-3\u2032 (F) and 5\u2032-CGCTTCACGAATTTGCGTGTCA-3\u2032 (R); \u03b2-actin: 5\u2032-CCTCGCCTTTGCCGATCC-3\u2032 (F) and 5\u2032-GGATCTTCATGAGGTAGTCAGTC-3\u2032 (R); AVEN: 5\u2032-GCGCCGGTTGAAGATGACA-3\u2032 (F) and 5\u2032-TGCAGAGCTAAGGAGGACACT-3\u2032 (R); miR-30b-5p: 5\u2032-CGCGTGTAAACATCCTACA-3\u2032 (F) and 5\u2032-CAGTGCGTGTCGTGGAGT-3\u2032 (R).According to the manufacturer\u2019s protocol, total RNA was extracted from the AR42J cells, respectively, by using TRIzol2.43 cells per well and incubation with MTT solution (10\u2009\u03bcL) for 2\u2009h at 37\u00b0C and 5% CO2 in the dark. Optical density values were detected at 570\u2009nm using ultraviolet spectrophotometer .The capability of cell proliferation was tested by MTT assay. After Tan IIA stimulation and transfection, SW480/R cells were made into single-cell suspension and seeded to 96-well plates with 5 \u00d7 102.56 Tan IIA-treated and transfected SW480/R cells were collected and treated with 500\u2009\u03bcL of buffering agent containing 5\u2009\u03bcL of Annexin V-fluorescein isothiocyanate and 5\u2009\u03bcL of propidium iodide  at room temperature in the dark for 20\u2009min. Then, cell apoptosis rate was analyzed by flow cytometry (FCM) .Following trypsinization and centrifugation, 1 \u00d7 102.6http://www.targetscan.org/vert_72/). The 3\u2032-UTR of AVEN, which contains miR-30b-5p binding site or mutated target site, was synthesized and cloned into the pGL3-basic plasmid  to construct the reporter vector AVEN-WT or AVEN-MUT, and SW480/R cells were co-transfected with the aforementioned reporter vectors and miR-30b-5p mimic or mimic control. After 48\u2009h transfection, dual-luciferase gene reporter system  was applied for detecting of luciferase activity, and the data were normalized to Renilla luciferase activity.The complementary sequences between AVEN and miR-30b-5p were predicted using the TargetScan . Equivalent amounts of protein were separated by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes . After blocking with 5% non-fat milk solution, these bands were incubated first with antibodies at 4\u00b0C overnight, membranes were washed with PBST followed by incubation with the appropriate secondary antibody for 4\u2009h at 4\u00b0C. Protein bands were imaged with ECL . The following primary antibodies were used: anti-cleaved-caspase3 , anti-AVEN , and anti-GAPDH . GAPDH was taken as an inherent reference.2.8Transwell assay was described previously . Briefly2.9t tests or one-way ANOVA analysis followed by Tukey\u2019s test were used for the statistical analysis. P < 0.05 was perceived statistically significant.All data were analyzed using SPSS 17.0 software. Each experiment was repeated at least three times, and the data were presented as mean value \u00b1 standard deviation (SD). Student\u2032s 33.150 value of SW480/R was higher compared to that of SW480 , and analyzed the sensitivity of oxaliplatin in SW480 cells. MTT assay results showed that cell viability of SW480/R was higher than that of SW480 with the treatment of different concentrations of oxaliplatin from 0 to 30\u2009\u03bcM . The IC5of SW480 . RT-qPCRof SW480 .3.2Bioinformatics assay was executed to expound the molecular mechanism of miR-30b-5p in oxaliplatin-resistant CRC cell. By using the TargetScan, we discovered that miR-30b-5p harbored the potential AVEN bind sites . To furt3.3RT-qPCR analysis certified that miR-30b-5p expression level in SW480/R cells was upregulated upon miR-30b-5p mimic transfection, compared with that in the mimic control group . RT-qPCR3.4We co-transfected miR-30b-5p mimic and AVEN-plasmid into SW480/R cells. The data depicted that cell viability was strikingly suppressed by miR-30b-5p mimic, whereas it was reversed by AVEN-plasmid . Besides50 value was lower compared to mimic control groups, the aforementioned results were rescued by AVEN overexpression .3.750 value also decreased, the phenomenon was strengthened by miR-30b-5p mimic co-transfection (To research the effect of miR-30b-5p in oxaliplatin sensitive to Tan IIA treated SW480/R cells, SW480/R cells were treated with 16\u2009\u03bcM Tan IIA and co-transfected with miR-30b-5p mimic or miR-30-5p inhibitor, then were treated with different dosages of oxaliplatin for 48\u2009h. MTT analysis certified that cell viability of Tan-IIA-induced SW480/R cells was lower than that in the control group, and ICsfection .50 value also decreased, the phenomenon was reversed by miR-30b-5p inhibitor co-transfection (Conversely, MTT analysis revealed that cell viability of Tan-IIA-induced SW480/R cells was lower than that of control group, and ICsfection .4Currently, CRC is the common malignant tumors in the world . OxaliplRecent data have revealed that Tan IIA had anti-chemoresistance activity of various cancer cells. Li and Lai reported that Tan IIA could enhance the sensitivity to doxorubicin in doxorubicin-resistant breast cancer cells . Guo et MiRNAs are a short, non-coding RNA which affect expression of target by inhibiting translation or degrading messenger RNA . There i5miR-30b-5p markedly inhibited the malignant biological behaviors of SW480/R cells by targeting AVEN, and Tan IIA inhibited the malignant biological\u00a0behaviors of SW480/R cells via the upregulation of miR-30b-5p. Tan IIA significantly enhanced the sensitivity of SW480/R cells to oxaliplatin, and this enhancement could be further promoted by miR-30b-5p upregulation. Therefore, miR-30b-5p combined with Tan IIA may be a new therapeutic strategy for the treatment of oxaliplatin-resistant CRC."}
+{"text": "The instruments used to measure presenteeism are all flawed and only incompletely measure the concept of presenteeism in employees of the general population. As a result, the concept of presenteeism is not measured, and in most of these instruments, the population for which the instrument has been developed differs from the nursing population. The present research was conducted to design and validate the instrument for evaluating presenteeism in nursing.The present study was part of an exploratory sequential mixed study. In this study, the instrument for measuring the level of presenteeism among nurses was developed and validated based on the results of the qualitative stage. To this end, the instrument\u2019s psychometric properties were investigated using face, content, and construct validity, as well as reliability through internal consistency and stability.In this study, an instrument containing 17 items and three dimensions  with favorable validation characteristics was developed. Therefore, the instrument was able to explain 56.375% of the total variance. Furthermore, Cronbach\u2019s alpha and McDonald\u2019s omega coefficients were 0.881 and 0.815, respectively. The intra-cluster correlation coefficient (ICC) was also reported as 0.972 for the entire instrument, with a 95% confidence interval of 0.941 to 0.987.Based this study, it was possible to measure the level of nurses\u2019 presenteeism through an instrument with favorable psychometric properties. This study helps health managers lay the groundwork for designing a system for measuring presenteeism among Iranian nurses using the developed instrument.The online version contains supplementary material available at 10.1186/s12912-023-01454-y. Presenteeism is the staff\u2019s physical presence at the workplace with reduced performance . In othePresenteeism among nurses will gradually lead to the destruction of the desired medical organization through its destructive consequences. The medical organization suffering from these unhealthy conditions will lead to increased human errors, decreased productivity and job satisfaction, and various other unknown consequences \u20136. It's Most instruments to measure presenteeism establish incomplete conceptual compatibility with the main concept. In other words, instead of directly measuring the concept of presenteeism, they indirectly measure its few consequences \u201315. MoreIn the search to identify instruments measuring presenteeism, we came across the Work Limitations Questionnaire (WLQ). Evidence indicates that this instrument has mainly been used to measure employees\u2019 presenteeism . This inAnother instrument was the Work Productivity and Activity Impairment (WPAI) scale, developed in 1993 to investigate employees\u2019 productivity and performance . SimilarEndicott Work Productivity Scale (EWPS) was another instrument used for this purpose. This instrument was developed in 1997 in a population of depressed patients, and its primary application was to measure productivity in patients with depression. This instrument is used in clinical trial studies in the field of psychiatry in order to investigate the effect of psychiatric interventions on depressed patients\u2019 work productivity , 19.One other instrument was the Stanford scale, developed in 2002. Melancon et al. state that this instrument is invalid for measuring presenteeism and lacks the ability to measure this concept. On the other hand, this instrument has likewise been designed with an emphasis on measuring performance and productivity in the general population with at least a high school education and does not investigate and measure the concept of presenteeism in nursing , 20.Baris et al. conducted a study entitled \"Development and psychometric validation of the Sickness Presenteeism Scale-Nurse\". However, it is important to note that the tool developed in this study may not be suitable for the context and culture of Iran. A critical review of the study indicates that cultural factors and contextual differences can influence the manifestation and perception of presenteeism among Iranian nurses. Therefore, there is a need to develop a new tool that is specifically tailored to the Iranian context. Such a tool would accurately measure presenteeism among nurses in Iran, capturing the unique aspects and nuances of presenteeism in the Iranian healthcare setting. This would ensure that the data collected from such a measurement tool are more accurate and reliable, which would be beneficial for research and policy-making purposes .In addition, these instruments have been criticized regarding methodological quality; in most cases, the criticisms are so deep that critics, including Ospina, Thompson, and Nuben, in their independent studies, believe presenteeism instruments suffer a crisis in the quality of methodology. Accordingly, issues such as failure to measure content validity, construct validity, internal consistency, and test\u2013retest reliability are evident in these instruments , 22.According to what has been stated, the instruments used to measure presenteeism are defective, and they merely incompletely measure the concept of presenteeism among employees of the general population. As a result, this concept is not measured, and in most instruments, the population for whom the instrument has been designed is not trained in nursing. On the other hand, the concept of presenteeism is affected by the context, and the administrative, cultural, and social background of Iran\u2019s medical organizations vary from many western countries where these instruments have been developed. Consequently, the present research was conducted to design and validate the presenteeism instrument in nursing.To design and validate presenteeism scale in nursing.Does the presenteeism scale in nursing have validity in terms of face, content, and construct?Does the presenteeism scale in nursing have reliability in terms of internal consistency and stability?Research questions:The present study was a quantitative part  of an exploratory sequential mixed study through which an instrument for measuring the rate of presenteeism among nurses was developed and validated from August 2021 to February 2023 based on the results of the qualitative phase. In this study, the inductive-deductive method was used to generate items  formula, in which N is the number of items, Sum h*2 is the sum of items shared, and r is the total item loadings. AIC's optimal level was considered between 0.15 and 0.5, while the acceptable omega was greater than 0.7 , 36. CroFor the test\u2013retest method, 30 nurses completed the developed instrument twice, with a two-week gap. The Intra-Cluster Correlation Coefficient (ICC) was calculated using a two-way mixed model and based on Absolute Agreement, with an ICC of 0.75 and higher being suitable . The appThe intra-cluster correlation coefficient alone provides the relative reliability of the instrument . In ordeSD pooled is calculated using the formula SD Pooled\u2009=\u2009(SD1\u2009+\u2009SD2) /2.It should be noted that in this formula, In this study, the floor and ceiling effects were calculated based on the percentage of respondents obtaining the lowest or highest possible score, respectively. Moreover, the criterion for the presence of floor and ceiling effects in this study was considered to be at least 15%.In the present study, the weighting of items was performed based on the factor analysis results. All steps are provided in Appendix In this study, in order to score the instrument, a method known as Simple Linear Transformation was used . Using tThe obtained score ranged between 0 and 100, and a higher score indicated more presenteeism.The initial version of the instrument consisting of 30 items was developed using the inductive-deductive method. After merging overlapping items and removing unrelated ones, the research team created a pool of 48 items that were later reduced to 30 items.Face validity was determined through qualitative and quantitative methods. Seven items were modified based on interviews with ten nurses to improve their clarity. All items had an item impact score above 1.5, indicating their appropriateness from the respondents' perspective.Content validity was also determined qualitatively and quantitatively. After interviewing ten faculty members, three items were modified and revised. The Content Validity Ratio (CVR) stage removed seven items due to scoring less than 0.62, leaving 23 items to be assessed for CVI. Based on the K* statistic, two more items were removed, and the instrument entered the reliability determination stage with 21 items. The entire instrument's content validity index was reported as S-CVI/Ave\u2009=\u20090.98.The initial reliability of the instrument was established using Cronbach's alpha coefficient, which was reported as 0.889. Removing any item did not significantly improve the coefficient. Additionally, the Corrected Item Total Correlation was investigated, and no item reported a correlation lower than 0.3, making no item a candidate for elimination. With 21 items remaining, the instrument entered the construct validity stage.The study used exploratory factor analysis to determine the construct validity of the presenteeism instrument for nursing, with an initial sample size of 320 nurses. After removing indifferent participants and outliers, the final sample size was 302 individuals, with a mean age of 37.36\u2009\u00b1\u20095.54\u00a0years. Of the participants, 46% were male and 54% were female, while 52% were married and 48% were single. The mean work experience was 12.03\u2009\u00b1\u20095.54\u00a0years.P\u2009<\u20090.0001). The Screen Plot showed visually that three factors could explain the factor construct of the instrument  of 0.972 for the entire instrument, with a 95% confidence interval of 0.941 to 0.987. The standard error of measurement for the entire instrument was estimated to be 1.411 Table .Table 6Floor and ceiling effects were calculated for the entire instrument and each factor, but were not evident in all cases Table . The iteP\u2009<\u20090.001). Therefore, the preferred method is weighting items based on factor analysis.The statistical analysis results based on the Friedman test indicate a significant difference in ranking between the two weighting approaches, 'fixed weights equal to one' and 'weighting items using factor analysis', with observed differences leading to rankings in both approaches  developed by Endicott et al., two items\u2014\"at work, you forget to contact other units of the factory\" and \"at work, you forget to respond to requests from the production manager\"\u2014were relatively consistent with some items in the present tool's ionnaire .Another item mentioned in the Endicott's tool aligned with the current tool was \"I don't focus on my work duties at work,\" which could be combined with items from our tool like \"I am distracted and not focused at work\" and \"I lack concentration in my work, so I may repeat specific care\"; Therefore, our tool covered the lack of concentration more comprehensively than Endicott's tool .In the Lerner et al. work limitations questionnaire, we found the item \"At work, I don't focus my mind on my work,\" which was aligned with the concentration-based items in our tool. However, none of the tools used in presenteeism research addressed delays among nurses' decision-making, poor prioritization of clinical tasks, repetition of clinical tasks, or slowness in performing clinical tasks, and none of them addressed bedside nursing care .Existing tools only focused on office spaces, workshops, and industrial factories, where workers interact with machines, attend meetings, and engage with production units. However, they did not address the unique human interactions between nurses and patients at the bedside.imperfect emotional presence, and it consists of five items that explain 19.003% of the instrument's total variance. This dimension indicates that nursing professionals may lack dynamism and vitality in their work. When nurses show up to work with a masked face, lost smile, and withered emotions, they resemble programmed, soulless, emotionless robots who merely complete tasks without considering the human aspect of the nursing profession. As a result, compassionate care cannot be provided effectively, and nurses might not understand patients' vulnerability, suffering, and grief accurately. Kim and colleagues argue that presentism translates to a loss of the spirit of nursing, where nurses do not have the chance to manifest their caring hearts, leading to excessive spiritual-emotional fatigue that eventually leads to demoralization of the care provided [The present tool's second dimension was provided .This dimension of the present tool appears unique to the nursing profession because nursing deals with the human aspect of healthcare, the concepts of feeling, art, tenderness, and spirit of care. It is this emotional presence that distinguishes the nursing profession from other industrial jobs. Industrial workers deal with raw materials, industrial machinery, clients, and other industrial units but not with human beings. Florence Nightingale emphasized that nurses must use their hands, heart, and mind to create an improved and healing environment for patient care. In other words, it is the nurse's heart that covers a special aspect of their work duties that cannot be found in any industry-related jobs . Holistiimperfect movement presence. This dimension encompasses issues such as feeling pain in specific physical positions, loss of physical strength, inability to stand for extended periods, and difficulty performing independent skills. None of the presenteeism tools available completely or ideally cover issues related to an individual's physical presence at the workplace. However, Reilly et al. created a tool titled \"Work Productivity and Activity Impairment (WPAI)\" which examines a limited aspect of an individual's physical performance in life. The tool includes an item that emphasizes physical activities outside the workplace, such as working at home, shopping, taking care of children, and participating in sports [The third dimension of the tool was n sports . Howevern sports . In thisThe limitation of the present study was the risk of contracting the coronavirus disease during data collection. In order to minimize this risk, an effort was made to use an electronic questionnaire to collect data at the construct validity stage. In other stages of data collection, wearing a mask, maintaining a distance of at least two meters from the participant, and taking into account proper ventilation in the interview location, were considered.It is recommended that the instrument developed in the present study be used in other studies on nursing. Furthermore, it is advised that an instrument be designed in future studies to determine the causes of presenteeism based on the antecedents of the concept.In the present study, an instrument including 17 items and three dimensions was developed that enjoys good validity and reliability and provides the possibility of measuring presenteeism in nurses. The present instrument can help healthcare managers obtain information about the level of presenteeism among nurses since the assessment of the current situation can be the first step in developing management plans related to presenteeism. Moreover, this instrument can be used in a wide range of research related to presenteeism; as a result, the implementation of research related to presenteeism in Iran will be facilitated, and a significant step will be taken in developing and promoting this concept in Iranian research.Additional file 1:Appendix 1. The steps of item weighting in this study.Additional file 2:Appendix 2. The final version of the presenteeism scale in nursing."}
+{"text": "A wide range of factors influence coordination and continuity of care. The aim of this study was to explore how management continuity of cardiovascular-related ambulatory care is influenced by the following network characteristics: presence of a case coordinator, network reciprocity, network composition and team climate.This cross-sectional observational study included three written surveys. The primary outcome management continuity of cardiovascular care was measured with the team/cross-boundary scale in the Nijmegen Continuity Questionnaire. The final analysis comprised a multivariate linear multilevel model with the predictors: presence of a case coordinator, network reciprocity, network composition and team climate.Eighteen general practices with 83 health workers and 340 patients participated. The linear multilevel regression analysis showed a positive influence of team climate on cross-boundary continuity of care . No statistically significant influence was measured for the other predictors.To improve integrated care, therefore, emphasis should also be placed on promoting the team climate within individual practices. Regarding network characteristics, further research is needed, especially in larger practices.This study showed that team climate had an independent, relevant and statistically significant association with cross-boundary continuity of cardiovascular ambulatory care. Cardiovascular diseases continue to have a high prevalence and are one of the leading causes of death worldwide. With the increase in multimorbidity, the care of patients with cardiovascular diseases is becoming more complex and requires the integration of various health workers to achieve optimal patient care 2. Optima468Continuity of care has been defined by Haggerty et al.  as a thr10The focus in this study is on management continuity in ambulatory cardiovascular care, which is challenging in healthcare systems that lack clinical guidance that is shared across healthcare providers. Many factors influence provider\u2019s views on clinical management, including vocational training and socialisation, the influence of peers, and other considerations. For example, a previous study with claims data regarding physicians-networks showed that the prescription of a new medication for heart failure is influenced by network structures. When physicians were connected to other physicians through common patients who were already prescribing the new medication, the likelihood of prescribing the new drug increased . HoweverIn this study, we focused on the role of ambulatory care providers\u2019 interaction networks. Based on empirical and theoretical work, we developed a conceptual model to explain the impact of professional interaction networks of health workers on care delivery and healthcare outcomes. Health workers conduct various activities, including diagnosis, counselling, treatment and prevention, and these activities must be coordinated. We hypothesized that coordination of care is influenced by the following factors: presence of a case coordinator , networOver time, network reciprocity within interactions between network members increases mutual trust and reduces risk of defecting behaviours. Research in evolutionary biology has shown that, over time, network reciprocity is crucial for the emergence of altruistic cooperation and that it may even counterbalance short-term individual benefits of non-cooperation behaviours . Under cA moderate change of network composition as opposed to no change makes it possible to select co-operators and unselect non-co-operators, while maintaining the favourable effects of repeated interactions on cooperation . This chIn addition to these structural network factors, a good climate and team culture in teams can enhance management continuity as well as experience of healthcare providers . AccordiThe aim of this study was to explore how management continuity of cardiovascular-related ambulatory care is influenced by the following network characteristics: presence of a case coordinator, network reciprocity, network composition and team climate.The study was conducted in accordance with the Declaration of Helsinki and received approval from the Ethics Committee of the Medical Faculty of Heidelberg (ID: S-726/2018) and from the respective State Medical Chambers. Due to the anonymity of the patient survey, the Ethics Committee of the Medical Faculty of Heidelberg approved a waiver for informed consent. Additionally, participants were informed about this waiver in writing and that returning the questionnaire was sufficient. We registered the study prospectively on 07/11/2019 at the German Clinical Trials Register under the ID: DRKS00019219. The STROBE reporting guideline [This cross-sectional observational study explored the influence of coordination work on management continuity of cardiovascular care in German primary care and consisted of three written surveys. The three-year 2019\u20132022) ExKoCare project aimed to recruit a sample of 40 general practices in the German states of Baden-Wuerttemberg , Rhineland-Palatinate , and Saarland  [\u20132022 ExKWe aimed to recruit 40 general practices and anticipated a low participation rate of 5%. The general practices were recruited from a clustered, stratified sample from a total of 25 counties. The counties were chosen with regard to population density to ensure that rural and urban areas were equally represented. We aimed to contact each general practice via fax or email. The owners of each general practice were identified through the publicly available online physician\u2019s databases of the three states. This led to an initial sample of 1,617 practices  . Some ban = 208) to participate in the survey. Data were collected using a written pseudonymized questionnaire. All participants gave written informed consent for the study.After the practice owners consented to the ExKoCare study, all health workers over the age of 18 of the general practices were contacted personally in writing and invited (n = 600). Based on previous research, we expected a response rate of 30%, and so we intended to invite 50 patients per practice. The research team assisted the practices via phone in identifying potential study participants to ensure that the inclusion criteria  were met. A list of potential participants was compiled from the physicians\u2019 billing systems. This system lists all patients who have been billed for services, regardless of whether they regularly visit this doctor. Then, up to 50 patients were selected by selecting every 3rd patient from a starting point specified by the researcher. If there were fewer than 50 patients on the list, all were selected. Before this selection, the physician was asked to check for the cognitive ability to complete a questionnaire and potential contra-indications. Patients were sent the questionnaire by post, and an anonymous return envelope addressed to the research department was included, so that the practices did not know which patients participated.After the health worker survey was completed, all participating practices were contacted and invited to support patient recruitment. Initially, the aim was to recruit 15 patients from each general practice  or with I do not know.The outcome of the study was management continuity of cardiovascular care, which was measured from the patient\u2019s perspective with the team/cross-boundary scale in the Nijmegen Continuity Questionnaire (NCQ) 22. The NThe English version of all 12 items in the NCQ was translated carefully and independently by CA and PH using a forward translation into German and a backwards translation into English. After the independent translations, consensus discussions were held and a common German version was produced. A test involving interviews with six patients did not identify any significant lack of clarity . For thiPresence of care coordinator was measured in the practice questionnaire using a dichotomous item (yes/no) that indicated the presence of a case manager for cardiovascular care.Reciprocity indicates the percentage of returned relationships in a directed network. The value range is between 0 and 1. The network was constructed based on the questionnaire in which the health workers were asked about the weekly exchange of information within their general practice. They were asked to mark the persons with whom they exchange information weekly [n weekly .Change of network composition was measured on a 5-point Likert scale, with 1 indicating little change in the last two years regarding the collaboration with cardiologists outside the health worker\u2019s own general practice and 5 indicating a high level of change. This predictor was also used to measure network changes within the general practice and among health workers outside the general practice.Team climate within the general practice was measured with the short version of the Team Climate Inventory scale [disagree to 5 = agree. We translated the questionnaire into German using the forward-backward method, which was completed in each part by two individuals. For each participant, the mean of the 14 items was calculated. One missing value was permitted. The mean value for each practice was then determined. Higher scores indicated a better team climate. Thus, a mean with a standard deviation for each practice was available with a possible range of 1 to 5.ry scale . This qunetwork size that indicates the number of health workers in each general practice. In addition, the number of physicians and non-physician professionals was measured. From the patient questionnaire, we included the number of chronic diseases (from a list of ten).We included SDs were calculated. Next, descriptive statistics were calculated for the predictors, and then the patient-reported outcome and team and cross-boundary continuity of care with means and SDs were calculated. The final analysis comprised univariate and multivariate linear multilevel regression analyses. Before this, the independent variables were tested for multicollinearity, which was assumed when the correlation coefficient was larger than 0.6. The two final models included the following predictors: presence of care coordinator (yes or no), reciprocity (range 0\u20131), change of network composition (range 1\u20135) and team climate (range 1\u20135), which should express the cooperation work. Additionally, the models were adjusted for network size and number of chronic diseases. In the first model, the dependent variable was the cross-boundary continuity of care between general practice and cardiologist practice; in the second model, it was team continuity of care within the general practice. Based on the nested data in GP practices, the interclass correlation coefficient was calculated to compute the proportion of variance explained by the GP practice in the total variance. All analyses were performed with the statistics software R (version 4.0.2) using RStudio (version 1.2.5033). The significance level was set at an alpha of 0.05.First, we conducted a descriptive analysis of the study population. According to the measurement level, relative and absolute frequencies and mean values with n = 24) were not complete, which led to their exclusion from this analysis. From the 18 practices, a total of 93 health workers and 596 patients were invited to participate in the study. With response rates of 89.3% and 57.0%, respectively, 83 health workers and 340 patients were included in the study. Data were collected from November 2019 to December 2021 , that was predominantly during the COVID-19 pandemic. After exclusion due to unavailable fax numbers or incorrect deliveries, we contacted 1,511 family practices, from which 42 took part in the ExKoCare project (response rate 2.8%). Eighteen general practices had collected all data for this study and were included in the present analysis. Due to the increased workload during the COVID-19 pandemic, the data from the other practices (SD 1.2). In addition to the measured network characteristics (SD 0.4). With a maximum value of 1, the average network reciprocity was 0.6 (SD 0.3). Patients (n = 231) reported cross-boundary continuity of care between family doctor and cardiologist with a mean of 3.8 (SD 0.8) and team continuity of care within the general practice (n = 270) with a mean of 4.0 (SD 0.7).The practices examined had an average network size of 5.2 . No statistically significant influence was measured for the other predictors  completed the questions about cross-boundary continuity of cardiovascular care. A further issue is that only few confounders could be included in the analysis due to the sample size. It is therefore important to conduct further research with larger samples to demonstrate effects of network mechanisms on coordination work.The findings suggest, that especially in smaller single-handed practices the network structures play a subordinate role compared to the team functioning, since all interact frequently and there is little explicit coordination. It is possible that we would have been more likely to find a statistically significant effect of network mechanisms in a sample with larger practices.A specific issue is that single-handed practices constituted almost 80% of the sample of practices. Although Germany has a high proportion (60%) of individual practices , group pAnother limitation is that the team climate questionnaire was not explicitly validated for the setting due to low response rate.This exploratory study showed that team climate had an independent, relevant and statistically significant association with cross-boundary continuity of cardiovascular ambulatory care. The hypotheses regarding network structures were not confirmed, but it can be supposed that the structures and division of tasks in practices are becoming increasingly important due to the expansion resulting from mergers of single-handed practices."}
+{"text": "Chronic kidney disease is classified as a civilization disease and is being diagnosed in an increasing number of patients. Hypertension and diabetes mellitus often coexist in hemodialyzed patients. The aim of the present study was to identify publications on the oral cavity status of multimorbid hemodialyzed adult patients additionally diagnosed with hypertension and/or diabetes mellitus, published between 2012 and 2022 to establish evidence of the impact of hypertension and diabetes mellitus on the oral status of hemodialyzed patients. Scopus and Web of Science databases were searched. Eight articles were included in the review. In total, 3 articles discussed oral hygiene in hemodialyzed patients, 4 discussed periodontal status, 3 discussed mucosa condition and saliva parameters, and 3 discussed the problem of Candidiasis infections. The conclusions were as follows: there is still a limited number of publications discussing the oral status of hemodialyzed patients diagnosed with hypertension; involved articles have proven that coexisting diseases can influence the oral cavity status of hemodialyzed patients and cause periodontal disorders, lower hygiene status, saliva parameters and make the risk of Candida infections higher. Chronicommended . Almost ommended .There are some publications available describing the poor condition of the oral cavity in hemodialyzed patients ,6,7,8. RMany researchers have also investigated the correlation between diabetes mellitus and oral cavity status. The most frequently observed oral complications were an increased frequency of caries occurrence, xerostomia, periodontal diseases in the form of gingivitis and periodontal disease, taste disorders, and burning mouth syndrome. Diabetic patients were also more prone to infections .Medications used in the treatment of hypertension may lead to gingival overgrowth, xerostomia, salivary gland swelling, lichenoid reactions, taste disorders, and parathesis . Most siChronic kidney disease is classified as a civilization disease. It affects more and more people. In 2017, 850 million people were diagnosed with chronic kidney disease. Hemodialysis is the most common out of the available kidney replacement therapy methods . Results of the cross-sectional study in 2018 showed that the median country-specific use of hemodialysis was 298.4 per million in the population [The aim of our study was to identify publications regarding the oral status of hemodialyzed patients suffering from diabetes mellitus and/or hypertension published between 2012 and 2022 (with the usage of Web of Science and Scopus databases) and establish the evidence of the impact of hypertension and diabetes mellitus on the oral status of hemodialyzed patients. We aimed to assess the comparability of the chosen studies. The authors also wanted to assess how the experience of particular members of our research team influenced the conducting of particular stages of the review process.The protocol of the research was prepared on the basics of PRISMA guidelines ,13,14 F. The revInclusion criteria:Original articles discussing oral manifestations observed in adult patients (older than 18 y.o.) receiving hemodialysis and diagnosed with diabetes mellitus and/or hypertension, articles written in English, articles published between 1 January 2012 and 17 February 2022, articles with their full text available, articles assessed as satisfactory with the Newcastle\u2013Ottawa Scale.Exclusion criteria:Case reports, reviews, or non-human studies.The MeSH  indexation was used in order to choose appropriate keywords for database searching. My own experience in the research field resulted in widening the range of keywords and including three that were not available in the MeSH . As a result, the following terms were used: chronic kidney disease or hemodialysis and oral health or oral status; chronic kidney disease or hemodialysis and periodontal status or periodontal disease; chronic kidney disease or hemodialysis and oral hygiene; chronic kidney disease or hemodialysis and caries; chronic kidney disease or hemodialysis and mucosa; chronic kidney disease or hemodialysis and saliva.Studies were screened by title and abstract due to the PICO  criteria ,16. The <0.00\u2014poor;0.00\u20130.20\u2014slight;0.21\u20130.40\u2014fair;0.41\u20130.60\u2014moderate;0.61\u20130.80\u2014substantial;0.81\u20131.00\u2014almost perfect.The search was conducted on 17 February 2022. Article selections were performed separately by two independent researchers (A.T and D.M.) who were calibrated. The agreement between them was calculated with the usage of Cohen\u2019s Kappa value, which is a commonly used comparative scale ,17. The The following information was extracted from the chosen studies: the year of the study, the country where it was conducted, the characteristics of both the examined and control groups, the observed status of periodontium, mucosa, saliva, oral hygiene, and the presence of Candida infection. Data were extracted by one researcher (A.T.). The extracted information was checked by another coauthor (M.T.) in order to eliminate the risk of bias.9\u201310 points: very good quality;7\u20138 points: good quality;5\u20136 points: satisfactory quality;0\u20134 points: unsatisfactory quality ,18.The reliability of these studies was performed with the use of the Newcastle\u2013Ottawa Quality Assessment Scale for cross-sectional studies . This scThis part of the research was conducted independently by two authors (M.R. and D.M.); if any discrepancy occurred, a decision was made by the third author (J.D.).Searching was conducted in two databases (Web of Science and Scopus); after the duplicates were eliminated, 2904 articles qualified for the first screening . An initFinally, eight articles were included in the review. Two described studies took place in India, one in Iraq, one in Japan, one in Saudi Arabia, and three in Poland. One was published in 2016, one in 2017, two in 2018, two in 2020, and two in 2021 . Oral caThree out of eight review articles discussed the problem of oral hygiene in multimorbid hemodialyzed patients and were analyzed. The simplified Oral Hygiene Index by Greene and Vermilion (sOHI) was used as a research tool in each of them; additionally, Trzcionka et al.  assessedResearchers from Japan  determinSwapna et al.  checked periodontitis the condition where CAL > 1 mm. They also mentioned that they analyzed gingiva recessions, the depth of periodontal pockets, and teeth mobility and furcation involvement; however, they did not present the results of their analysis. They also measured the papilla Bleeding Index by Muhlemann, which they used for the assessment of gingival status (gingivitis).Dande et al.  in theirTrzcionka et al. , in ordeThe oral mucosa status of hemodialyzed patients was examined by Swapna et al. , Dande eResearchers from Poland examined saliva parameters with the usage of the salivary flow rate (stimulated), the buffer capacity of saliva, and its pH. They noted the presence of the following: ulcerations, white and red patches, malformations, candidiasis, ecchymosis, herpes, a geographic tongue, a fissured tongue, the smell of acetone, trauma-related lesions or signs of operations, the overgrowth of gingiva, burning mouth syndrome or pain.Swapna et al.  examinedThe problem of Candida infections was analyzed by three groups of researchers; however the group from Poland analyzed only the presence of mucosal pathologies, while two other groups assessed Candida colonization on the basics of a microbial examination. We decided to include the results of Trzcionka et al.  in the pAyinampudi et al. , in ordeAl-Sarray et al.  gatheredThe results of Ayinampudi et al.  and Al-SThe quality assessment of the eight studies included in the review was conducted using the Newcastle\u2013Ottawa Scale. It was performed independently by two researchers (M.R. and D.M.), and the discrepancies were solved by the most experienced member of the research team (J.D.) .Ayinampudi et al. Dande et al. Al-Sarray et al. Trzcionka et al. Trzcionka et al. Trzcionka et al. Naruishi et al. Swapna et al. To present the results of the assessment, the following numbers were given to particular articles:Articles written by Naruishi et al.  and SwapThe aim of our investigation was to establish evidence of the impact of hypertension and diabetes mellitus on the oral status of hemodialyzed patients who are a major part of our society. It seems important to provide them with appropriate dental care that nowadays seems to be inadequate.The problem of oral findings in people diagnosed with general diseases is widely discussed in the available literature ,29,30,31The presented review discusses oral findings in hemodialyzed patients diagnosed with diabetes mellitus and/or hypertension published after 2012; however, this issue was assessed earlier. In articles presented before 2018, no information on the influence of hypertension in hemodialyzed patients or on their oral status was found. However, researchers agree that the oral cavity condition of patients diagnosed with end-stage chronic kidney disease and hemodialyzed is worse than in healthy ones ,34,35,36The comparability of the examined and control groups between studies included in the review was not satisfactory; in fact, they were impossible to compare directly. The authors decided to prepare the review as an introduction for future research and to improve the methodology of future studies. Our expectations were focused on finding studies that were comparable to our past studies in terms of patient group selection (based on diseases the patient was diagnosed with) and looking for standards in the clinical studies discussing.Two publications published before 2018 discussed the oral health status of adult hemodialyzed patients diagnosed with diabetes mellitus ,36. Terap = 0.015), which is similar to the findings of the Polish examiners [p = 0.044), while Trzcionka et al. did not note any differences [Swapna et al. conducted an oral cavity status assessment in 97 hemodialyzed patients in Bhimavaram Hospital, dividing them into non-diabetic and type 2 diabetic groups . They asxaminers . While axaminers . Out of xaminers . Swapna xaminers  and Trzcxaminers  also assferences .A detailed analysis of the correlations and bilateral dependency between the oral cavity condition and the general condition of the human organism led to the conclusions emphasized by the authors ,23 that The articles included in the review were written by researchers outside Europe, even though more and more people from Europe also suffer due to multimorbidity and are diagnosed with end-stage chronic kidney disease, hypertension, and diabetes mellitus. We realize that the proper analysis of health in that group of patients demands interdisciplinary cooperation ,21,25.We had faced a few problems. First of all, a part of our research team was previously engaged in the examination of hemodialyzed patients, which might have caused their bias . That is why among the authors, there were other researchers who had never before dealt with the oral status of hemodialyzed patients, including D.M\u2014a researcher with hardly any experience\u2014, M.R\u2014highly experienced in the field of dentistry\u2014and J.D.\u2014an expert in the field of nephrology. The researcher with the least experience (deliberately) was asked to search the databases. A comparison of the results obtained by her with the results obtained by the person who was familiar with the topic resulted in an agreement among the reviewers, which assessed was with the use of Cohen\u2019s Kappa coefficient as moderate (0.42). The differences between the reviewers were then checked by the third author, who was familiar with the topic of the study. The presented results proved that in order to identify the available literature regarding any specific issue, it must be checked if the researchers are familiar with the issue and perfectly understand the inclusion and exclusion criteria.The risk of bias assessment is a very important aspect of systematic reviews ,38. The There is still a limited number of publications discussing the oral status of hemodialyzed patients diagnosed with hypertension.It is crucial to analyze a wide range of articles  in order to prepare a high-quality review. There are hardly any articles combining the systematic review and presentation of results, while in our opinion, this kind of article is the most effective if there are no plans for long-term and multi-stage studies. If multi-stage research is planned, a good systematic review can be a source of information on how to properly prepare the methodology of the study.There is a necessity to properly organize the research team (the gradation of the experience and engagement in the assessed topic).The knowledge provided in the included review studies confirms that coexisting diseases (diabetes mellitus) influence the oral cavity status of hemodialyzed patients, causing the deterioration of periodontal status, hygiene, and saliva parameters and making the risk of Candida infections occurrence higher. These facts confirm the necessity for multimorbid patients to be taken care of by an interdisciplinary team of specialists."}
+{"text": "Study objectives: While zolpidem is considered as an example of a gender effect on drug response, there is insufficient evidence to reach a consensus. This study aimed to investigate gender differences in adverse events (AEs) of zolpidem.Methods: We estimated the difference between the reporting odds ratios (RORs) calculated in gender subgroups for the AEs signals detected in data mining using 2015\u20132019 Korea voluntary adverse drug events reporting system (KAERS) data. Different reporting risk by gender was evaluated by using the log RORs being significantly different by gender at the 5% significance level and the 95% confidence intervals of the gender ROR.Results: A total of 94 AE signals were detected. Among these, 35 signals showed significant disparities by gender at the 5% level or were detected only in one gender. When categorized by similarity of AEs, parasomnia including somnambulism and paroniria, and cardiovascular disorders including coronary thrombosis had higher reporting risks in women. Men were more likely to report cognitive disorders such as delirium, insomnia related disorders, and movement disorders. Among all AEs with gender differences in reporting risk, the difference in somnambulism was the most consistent and substantial.Conclusion: For several AEs associated with zolpidem, gender-based reporting disparities were evident. Notably, women exhibited a higher susbeptibility to somnambulism, potentially serious adverse effects of zolpidem. This underscores the need for further investigation into the underlying factors influencing these gender-specific reporting patterns. Zolpidem is a major hypnotic agent that selectively targets the \u03b3-aminobutyric acid type A (GABAA) receptor. Due to the short acting effect and general tolerability, it has been a preferred choice among prescribers and patients for treating insomnia . With thHowever, there is an argument that these regulatory actions lack a confirmative clinical evidence . AccordiGender is rarely taken into account in the majority of mental health studies . For insAs such, although zolpidem is cited as an example of a gender effect on drug response, there is no consensus in both regulatory authorities worldwide and healthcare professionals due to insufficient scientific evidence. This study aimed to explore gender differences in adverse effects related to zolpidem using Korea\u2019s voluntary adverse events reporting data.https://open.drugsafe.or.kr/original/invitation.jsp) websites host the KAERS datasets (The Korea Adverse Event Reporting System (KAERS) was retrospectively observed as the data source for the analysis, which covered the period between January 2015 and December 2019. The Korea Institute of Drug Safety and Risk Management (KIDS) established the automated AE reporting system known as KAERS in 2012. Both databases contain voluntarily submitted AE reports from consumers, healthcare professionals, 27 local pharmacovigilance centers, and marketing authorization holders, most of which are pharmaceutical firms. Reports from all types of reporters were included in the analysis. Each case contains data on the patient\u2019s age, sex, administration date of zolpidem, type, and symptom of AEs, and patient outcomes without identifying any particular individuals. The international drug monitoring program operated by the WHO-Uppsala Monitoring Center is compatible with the KAERS database. The Anatomical Therapeutic Chemical Classification System (ATC Code) was utilized to record the drug names, and the World Health Organization- Adverse Reaction Terminology (WHO-ART)\u2019s preferred terms (PTs) were use to code the adverse events (AEs). The KIDS  , reportiWe investigated the ROR of adverse drug reactions (ADRs) grouped by ADR type, which can be difficult for reporters to distinguish due to their similarity, or can be grouped together by a common characteristic, such as parasomnia , which it-test and Chi-square test were applied for continuous variables and categorical variables, respectively to examine the differences in the basic demographic and AE reporting data by gender. Serious AEs refers to any of the following: 1) death or a life-threatening condition, 2) hospitalization or prolongation of existing hospitalization, 3) persistent or significant disability/incapacity, 4) congenital anomaly/birth defect, 5) any other medically important condition that requires medical intervention, such as drug dependence or abuse, or a blood disorder, etc.To explore the gender differences, each analysis was performed separately by gender. The For each signal detected, we calculated the frequency and ROR with 95% confidence intervals for each gender subgroup. The difference between the two odds ratios was estimated as the difference between the logarithms of the two RORs. We evaluated different reporting risks by gender using the log reporting odds ratios (RORs), with statistical significance determined at the 5% level, and the 95% confidence intervals of the gender ROR were also considered.In a secondary analysis, we defined control group as patients exposed to benzodiazepine anxiolytic/hypnotic drugs, then the RORs were calculated. We expected the size (ROR) of signals detected in this analysis to generally be smaller than those in the primary analysis due to the similar mechanisms of action on the nervous system. Therefore, the number of signals detected was anticipated to decrease significantly. However, if we detected gender differences in the signals and reporting risk in this analysis, it would support the robustness of the primary study results. Benzodiazepine derivatives included all drugs in the WHO ATC N05BA category.In another secondary analysis, RORs were calculated by gender for ADR categories only for suspected drugs. In KAERS, \u201csuspected drugs\u201d are drugs suspected to have caused the adverse reaction in question, while other drugs are classified as \u201cconcomitant drugs.\u201dAll data were analyzed using the SAS statistical application program .The dataset consisted of 1,016,161 reports, with 599,311 reports from women and 416,850 from men. The number of drug-adverse event combinations was 3,524,587, with 11,341 AE reports associated with zolpidem, 5,791 occurring in women and 5,550 in men. In the reports containing zolpidem, 476 types of AEs were observed in women, and 468 in men. Out of the 2,442 reports that provided information on the daily dosage of zolpidem, there was no significant difference between women and men in the administered dosage, which was 8.55 and 8.54\u00a0mg, respectively. Among all reports, the proportion of serious AEs was lower in women than in men (7.37% vs. 10.28%), while the proportion of serious AEs associated with zolpidem use was similar between women and men (25.20% vs. 24.49%). The majority of reporters (96.49%) were healthcare professionals, such as doctors, pharmacists, and nurses .In total, 94 PT signals were detected. p = 0.057) had higher RORs in men. Parasomnia showed the largest gender difference, with a higher ROR in women than in men . Somnambulism had the highest ROR among both genders, with higher reporting risk in women   and cognitive disorders   . In the < 0.001) .This study aimed to investigate gender differences in AEs associated with zolpidem using voluntary AE reporting data in Korea. Due to potential differences in reporting behavior of adverse drug reactions between genders , and thep = 0.057) were dominant in men. Parasomnia had the largest gender difference in ROR among all PT-level AEs.When comparing by gender, we found that the distribution of AEs was different, with 38% of AEs in PT level (36 out of 94) having different reporting risks by gender. Somnambulism had a significantly higher ROR in women than in men, while delirium, hyperkinesia, anxiety, and depression were higher in men. Categorizing PTs according to the similarity of AEs revealed that parasomnia and cardiovascular disorders had a higher risk of reporting in women, while insomnia related disorders, cognitive disorders, and movement disorders  as a symptom, this study found that somnambulism was reported three times more frequently in women than in men, and had the largest gender difference in ROR among all PT-level AEs. Although there is limited research on whether the risk of complex sleep behaviors due to zolpidem is related to gender, previous case reports and a systematic review are consistent with our findings, suggesting that women may be more at risk . HoweverAlthough these examples are exceptional, a review of post-2000 literature has demonstrated that out of five cases of homicide related to zolpidem use among patients with mood or anxiety disorders , three wThere have been several studies on the association between zolpidem use and cardiovascular or cerebrovascular risks , but theIn our study, a total of 68 cases of cardiovascular AEs associated with zolpidem were reported, of which 42 cases were reported in women, and the gender difference in log ROR was significant at 0.62. Besides, although the frequencies were low, signals of coronary thrombosis and myocarditis were detected only in women. In the secondary analysis, which set users of benzodiazepine anxiolytics/hypnotics as the non-exposed control group, heart failure showed the largest gender difference in ROR which is in line with the primary analysis. On the contrary, only two cases of cardiovascular disorders were reported in the secondary analysis \u2161, which focused on suspected cases only. This is most likely because the reporter was unsure whether the cardiovascular reactions were caused by the adverse effects of zolpidem and did not mark them as suspected drug reactions. The estimates from voluntary AE reports do not allow for the confirmation of causality or association. Additionally, insomnia itself is known to be a risk factor for heart failure or myocardial infarction . Due to post hoc clinical trial of chronic nightly zolpidem, there was no gender difference in rebound insomnia, which differed from the results of our study (In our study, men were more likely to report insomnia, hyperkinesia, or aggressive reaction than women. Although the frequency and size of the difference are varied, these AEs seem to share a common possibility of being related to a rebound effect from zolpidem with a short half-life . While tur study . MovemenIn case of cognitive disorders or delirium, both primary and two secondary studies showed higher reporting risk in men. To determine whether there are gender-specific vulnerabilities in the cognitive function issues brought on by zolpidem use, more research may be required.To our knowledge, this study is the first to investigate overall gender differences in the risk of reporting AEs of zolpidem using national voluntary AE reporting data. The use of vast amounts of national data over the last 5\u00a0years would have yielded reliable findings. Second, by comparing the RORs that were not dependent on the size of drug use and reporting behavior for each gender, we were able to make a reasonable comparison of gender differences in reporting risk. A number of studies have shown a higher frequency or rate of AEs reported in women . HoweverOur study has several limitations. First, owing to the inherent limitations of data source and the signal detection methodology, causal inferences were not possible. This study underscores the need for future research to investigate potential factors such as concomitant medications, comorbidities, pharmacokinetics, dose variability, and alcohol intake that may contribute to gender disparities in adverse effects. Secondly, the data quality is inconsistent and underreporting is prevalent, especially concerning AEs like complex sleep behaviors, where concerns about discontinuing prescriptions or embarrassment may lead to underreporting. Thirdly, although the average daily prescription dose was similar for each gender, it remains unknown whether the dose variability affects the individual AEs signal discrepancies by gender. Furthermore, only 2,442 out of 11,341 reports contained dose information. Lastly, using data limited to Korean population makes it challenging to directly compare with studies involving other populations, particularly regarding genetic diversity and pharmacogenetics.The analysis of real-world data showed that reporting of AEs among zolpidem users was different by gender. This gender imbalance was pronounced in some of AEs such as parasomnia including somnambulism and cardiovascular disorders which is dominant in women, while cognitive disorders and insomnia are more frequent in men. Specifically, women demonstrated a greater vulnerability to somnambulism, which is a potentially severe adverse effect of zolpidem. The results support the need for more comprehensive clinical research on gender differences related to zolpidem in the future."
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