Infectious bronchitis virus (IBV) is a vital pathogen in poultry farms, which can induce respiratory, nephropathogenic, oviduct, proventriculus, and intestinal diseases. Based on the phylogenetic classification of the full-length S1 gene, IBV isolates have been categorized into nine genotypes comprising 38 lineages. GI , GVI-1 and GVII-1 have been reported in China in the past 60 years. In this review, a brief history of IBV in China is described, and the current epidemic strains and licensed IBV vaccine strains, as well as IBV prevention and control strategies, are highlighted. In addition, this article presents unique viewpoints and recommendations for a more effective management of IBV. The recombinant Newcastle Disease virus (NDV) vector vaccine expressed S gene of IBV QX-like and 4/91 strains may be the dominant vaccine strains against NDV and IBV. Since the outbreak of Coronavirus Disease 2019 (COVID-19), there are more than 642 million confirmed cases and 6.6 million confirmed deaths caused by the coronavirus infection by December 7, 2022 in World Health Organization website. Since the first confirmed case was announced, the COVID-19 pandemic has lasted almost 3 years, and the virus has caused significant economic loss throughout the world , porcine epidemic diarrhea virus (PEDV), porcine Delta coronavirus (PDCoV), canine coronavirus (CCoV), feline coronavirus (FCoV) and avian infectious bronchitis virus (IBV) . AlthougIBV belongs to gamma group of coronavirus. Its complete genome is about 27.6 kb and composed of 5′-1a-1b-S-3a-3b-3c(E)-M-5a-5b-N-Poly(A)-3′ from 5′ end to 3′ end . A smallCoronaviridae by the International Committee on Nomenclature of Viruses ) and the Coronaviridae is currently divided into two genera Coronavirus and Cyclovirus based on the viral serological characteristics and nucleotide sequence differences. The common important coronavirus such as SARSV, IBV, TGEV and PEDV belongs to coronavirus genus.IB was first described in United States in 1931 as a respiratory disease of chickens . In 1965IBV has existed more than 60 years in China . In 1958S1 gene) may give rise to variants or new genotypes. Many SARS-CoV-2 variants emergence in the past 3 years told us the rapid mutation rate of all coronaviruses including IBV, which can induce the emergence of new virus types with the potential to cause disease  serotype isolation strain was isolated in France in 1985 and then entered the United Kingdom in the winter of 1990 , 37. SubTW genotype is an important gene type and the starts to become the most common genotype in mainland of China recently , 41. IBVTwo novel serotypes have been added into Genotype I IBV in China. The IBV strain ck/CH/LGX/111119 was found to be genetically and antigenically different from other known IBV types and designated as GI-28 based on the results of S1 sequence analysis and virus cross-neutralization tests. It is a nephropathogenic strain with a broader tissue tropism and may be a recombination of LX4 genotype (GI-19) and as-yet-unidentified IBVs . Then, mThe GVII-1 genotype was another novel IBV lineage in China. The GX-NN130021 strain was isolated from suspected IBV-positive 21 day-old diseased chickens, which were vaccinated by the H120 vaccine, in Guangxi province of China in 2013 and were designated as new-type I . An epidCurrently, vaccination inoculations with live attenuated or inactivated vaccines remain the most effective technique for protecting chickens from IBV infection in poultry farms around the globe. In addition to comprehensive biosafety epidemic prevention measures, it is crucial to develop appropriate immunization protocols to prevent and control the IBV infection. There are numerous types of IBV vaccinations in China, including live and inactivated vaccines as well as single and multiple combined vaccines. Moreover, there are also many types of IBV vaccine strains, such as Mass, QX-like and 4/91-like types.All available commercial IBV vaccines were summarized in Although there are numerous vaccines available, there are numerous predominant IBV serotype strains that compromise the vaccine’s effectiveness. Different IBV serotypes only induce limited cross-protective immunity between different serotypes, thus vaccination immunization failures are often reported. Therefore, the key to preventing IBV infection is timely knowledge of the precise local epidemic strain and selection of the appropriate IBV serotype vaccination.Houttuynia cordata (Saururaceae) had more than 90% inhibition rate against IBV infection in cells and more than 50% protection rate in SPF chickens against IBV challenge  vaccine, viral vector vaccine and subunit vaccine against IBV infection might be the future research direction.in vitro, laying the groundwork for further in vitro mRNA research , fowl adenovirus (FAdV), herpesvirus of turkey (HVT), have been reported. Each virus vector vaccine has its own advantage. Among NDV, FAdV and HVT virus vector, NDV might be the best viral vector for IBV in three aspects. Firstly, IBV and NDV are vaccinated at the same time in most cases in poultry farm, that is why all registered IBV combined vaccines contain NDV vaccine, of which most are the LaSota strain . Comparehaemophilus parasuis combination vaccine. Moreover, there is a recombinant subunit vaccination for sheep against Chlymidia psittaci. Overall, many difficulties need to be overcome to develop IBV subunit vaccine. A sustained and stable immune response cannot be produced by the purified pathogen protein generated in vitro due to the difficulty in maintaining stable protein structural epitopes. The immune response typically only affects humoral immunity and is unable to activate cellular immunity. Consequently, the number of doses of subunit vaccination increases, which indirectly raises the labor expenses of chicken farms. Moreover, the production method of protein extraction and purification directly raises the production cost of subunit vaccination. Therefore, the subunit vaccine, which is arduous and time-consuming, is not the optimal method for generating a novel IBV vaccine.At the present, the only genetic engineering subunit vaccine for poultry is infectious bursal disease virus subunit vaccine in China. Several registered subunit vaccinations are available for pigs, including swine fever, porcine circovirus type 2, streptococcus and Respiratory mucosal immunity plays an important role in preventing respiratory diseases, especially coronavirus infection. An aerosolized adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) has been evaluated in a clinical trial and displayed good neutralizing antibody responses . Meanwhiin ovo vaccination may be available immunization route. In ovo vaccination is performed at 18 day-old embryonated egg in broiler eggs coinciding with transfer of eggs from setter to hatcher compartment and it is adapted as a common practice worldwide now. Unlike coarse spray or water vaccination, in ovo vaccination ensures that each chicken can be immunized with precise, homogeneous distribution of vaccinations. More importantly, in ovo vaccination can induce early immune responses of the day-old chickens against circulating pathogens than post-hatch vaccination does. Therefore, in ovo vaccine is available against IBV combined with NDV, though there are some problems to be considered, including the virulence of vaccine strains on eggs, potential maternal antibody interference and immunosuppression observed with some vaccines , Guangxi Science and Technology Base and Talent Special (2021AC19372) and scientific research startup funds of Guangxi University.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.


Plasmodium falciparum  infection outcomes in Ghanaian children.Naturally acquired immunity to malaria may involve different immune mechanisms working in concert, however, their respective contributions and potential antigenic targets have not been clearly established. Here, we assessed the roles of opsonic phagocytosis and antibody-mediated merozoite growth inhibition in P. falciparum antigen-specific IgG of plasma samples from children  were measured at baseline prior to the malaria seasons in southern Ghana. The children were then actively and passively followed up for febrile malaria and asymptomatic P. falciparum infection detection in a 50-week longitudinal cohort. P. falciparum infection outcome was modelled as a function of the measured immune parameters while accounting for important demographic factors.The levels of merozoite opsonic phagocytosis, growth inhibition activities and six PfRh2a correlated with growth inhibition. Notably, IgG antibodies against RON4 correlated with both assays.High plasma activity of opsonic phagocytosis , and growth inhibition  were individually associated with protection against febrile malaria. There was no evidence of correlation  between the two assays. IgG antibodies against MSPDBL1 correlated with opsonic phagocytosis (OP) while IgG against Opsonic phagocytosis and growth inhibition are protective immune mechanisms against malaria that may be acting independently to confer overall protection. Vaccines incorporating RON4 may benefit from both immune mechanisms. Plasmodium falciparum febrile malaria, albeit not completely understood, may collectively account for several averted clinical malaria episodes in endemic populations. It has been established that naturally acquired immunity (NAI) against malaria is non-sterile and depends on persistent low-level parasitaemia and develops after repeated exposure to different parasite variants  supplemented with 1% L-glutamine , 50 µg/ml gentamycin , 25 mM HEPES (Sigma Aldrich), 50 mg/L hypoxanthine, and 10% heat-inactivated type O+ human serum. Cultures were incubated at 37 °C with a gas mixture of 5% O2, 2% CO2, balanced with N2  and used in subsequent assays.Isolation of intact merozoites was performed as has been described elsewhere . BrieflyoC in a humidified chamber with 5% CO2 gas. Cell numbers were maintained at 1×106 cells/mL. Cells were counted, resuspended, and aliquoted at 1x104 cells/150 µl/well for the opsonic phagocytosis assay.THP1 cells were maintained in THP1 medium at 375 merozoites were stained with 100 μg/ml ethidium bromide and opsonized with 50 μl heat-inactivated (HI) plasma (diluted 1:50) for 30 minutes at room temperature. THP1 cells (1x104) were co-incubated with opsonized merozoites in a U bottom 96-well plate pre-coated with 150 μl/well FCS. The plate was incubated for about 40 minutes in a humidified incubator at 37oC supplemented with 5% CO2 gas. Phagocytosis was stopped by spinning the plate at 500xg in a pre-chilled centrifuge for 5 minutes at 4oC. The plate was washed twice with cold PBS supplemented with 2% BSA. Cells were fixed with 2% paraformaldehyde in PBS/2% BSA and counted using a Beckman Coulter cytometer . Data were expressed as phagocytosis index, defined as the percentage of THP-1 cells with ingested merozoites relative to the THP1 cells with no merozoites.Opsonic phagocytosis assay was performed as described , 17. BrioC with a gas mixture of 5% O2, 2% CO2, balanced with N2. After approximately 12 hours of incubation, cultures were centrifuged at 500xg for 5 minutes in a pre-chilled centrifuge at 4oC to pellet the cells and washed with cold filtered 1xPBS. Cells were stained with 0.2x SYBR Green I nucleic acid stain (Sigma Aldrich) for 10 minutes and washed twice by spinning at 2500 rpm for 8 minutes. About 200 μl of cold PBS was added and the cells acquired with Accuri C6 Plus flow cytometer (Becton Dickinson), set to acquire 50,000 events. Growth inhibition was calculated and expressed as a percentage inhibition index using the formula: 100 − . Test well is the well with test antibodies and parasitized RBCs, and control well represents the parasitized RBCs without antibodies.Growth inhibition assay was performed as previously described . BrieflyThe gating strategy was adopted from Muh et al. , where sTo test the sensitivity of the assay to detect different samples at different concentrations, two sets of pooled plasma samples were diluted 10-fold from undiluted plasma to 1000x dilution and added to the culture in 96-well plates. Pool A was normal pooled plasma and pool B was HI pooled plasma. Both plasma pools demonstrated concentration (dilution)-dependent growth inhibitory effects on NF54 Plasmodium parasite  and incubated for 1hour at RT on a plate shaker set at 300rpm. Each plate had a standard reference curve obtained by a 3-fold serial dilution of pooled hyperimmune plasma. Also included on each plate were controls (Positive and Negative) added at 100µl/well. Total IgG was detected for an hour using 100µl/well of HRP conjugated goat anti-human IgG  diluted at 1:2,000. The assay was developed with 3,3',5,5' Tetramethylbenzidine for 15minutes and optical density read at 450nm. The standard reference curve was used to convert OD values to antibody units using an Excel-based 4-parameter logistic curve-fitting application .The antigens, MSPDBL1, EBA140RIII-V, RALP-1, eviously  and the eviously . BrieflyAll the three data sets of opsonic phagocytosis, growth inhibition, and IgG antibody data were normalized respectively, where the mean of the positive control of each assay output was averaged across replicates for each assay plate. The test sample on each plate was divided by the mean positive control of the plate and multiplied by the total mean positive control for all the plates to obtain the adjusted value for the sample using the formula:P. falciparum parasites detected by microscopy at any time during the 50 weeks were categorized as the exposed group and all those without parasites throughout the observation period were considered as unexposed. Though children above 5 years have been observed to have sub-microscopic infection  at any time during the 50 week follow up, or were asymptomatic throughout the same period. Participants with parasites at least one-time point but no febrile symptoms were considered protected, and those with at least one episode of febrile malaria during the follow-up period were considered susceptible.Participants with nfection , the golAll analyses were done separately for the age groups (< 5 years old and > 5 years old). Baseline characteristics were summarized using means and standard deviations for continuous variables and counts and frequencies for categorical variables. Associations between OP and the odds of protection against febrile malaria were assessed using multiple logistic regression adjusted for age. These analyses were repeated for growth inhibition.To generate antibody breadth score, antibody levels were grouped based on quartiles and assigned 0 for the lowest quartile, 1 for the second quartile, 2 for the third quartile, and 3 for the highest group. The scores for each individual were summed up for the 6 antigens with 18 being the highest breadth and the least being zero (0). An association of breadth with protection was assessed using logistic regression analysis adjusting for age. The associations between breadth score and the immune mechanisms were assessed by simple linear regressions separately.A multiple linear regression analysis adjusted for age was used to determine the correlation between the immune mechanisms (OP and growth inhibition) and antigen-specific total IgG antibody levels. Similarly, multiple linear regression was also used to explore the correlation between OP and growth inhibition. The level of statistical significance was set at 5%. All analyses were performed using R .P. falciparum parasite at least once during the follow-up period as confirmed by microscopy (exposed group)  between January 2016 and January 2017. A total of 848 children completed the 50-week longitudinal follow up period. At the end of the study, 238 children were infected with The OP and growth inhibition activities of baseline plasma samples were compared between children who had baseline parasitaemia and those without baseline parasitaemia separately. Children with baseline parasitaemia had a significantly higher OP activity  than those without baseline parasitaemia  = 0.85, 95% CI = 0.78-0.92; p<0.001]. Similarly, OP activity was associated with protection from febrile malaria . When the children were grouped into two based on age, there was no difference in the OP activity between protected and susceptible children (p=0.078) in the younger age group (Below 5 years). However, for those above five years, OP activity was higher in the protected children (33.11 ± 1.15 %) compared to the susceptible children   available for analysis in the growth inhibition assay due to insufficient plasma samples. Plasma from protected children above 5 years had higher growth inhibition activity (34.19 ± 2.95 %) compared to susceptible children  in the same age group while no significant difference was observed in those below 5 years (p=0.53) (PfRh2a (p=0.04), and RON4 (p=0.004) significantly increased with growth inhibition activity  between the OP and growth inhibition activities. The breadth of total IgG responses was assessed for associations with age, malaria and the two mechanisms. Higher total IgG breadth was associated with age  and protection  against febrile malaria. Also, total IgG breadth positively correlated with only opsonic phagocytosis (p=0.033) in those above five years, but not growth inhibition (p<0.05) (In this study, we found both merozoite OP and growth inhibition activity of plasma samples obtained from children at baseline to be associated with protection against malaria in the ensuing transmission season. In addition, the breadth of total IgG responses against a panel of 6 merozoite antigens was associated with protection and increased with both OP and growth inhibition activities. There was no evidence of correlation between OP and growth inhibition, suggesting these could be contributing independently to the protection against malaria observed in the study population. Notably, RON4 specific antibody levels was significantly associated with both mechanisms.The mechanisms and targets of antibody-mediated immunity to malaria are still under investigation and some studies have found OP , 17, ADROf the 6 recombinant antigens tested, higher antibody levels to MSPDBL1 and RON4 correlated with reduced risk of febrile malaria. Anti-MSPDBL1 antibodies have been found in other studies to confer protection by inhibition of parasite growth and also by opsonization of merozoites , 28. PfRThere may be several other malaria antigen-specific antibodies in the plasma samples used that may be involved in the OP and growth inhibition mechanisms which have not been measured here which is a limitation. Another possible limitation is the impact of these mechanisms on different strains of parasites circulating in the population which we have not assessed here. However, we believe the plasma used in the assays have wider antibody breadth since a study in Papua New Guinea found similar OP activity in both field and laboratory strains indicating that opsonization of merozoites is strain transcending .In conclusion, the study found both opsonic phagocytosis and growth inhibition mechanisms to independently mediate protection from febrile malaria in this cohort of Ghanaian children. The study also shows the importance of total IgG responses to RON4 in both OP and growth inhibition mechanisms suggesting that vaccines based on this antigen may trigger both and perhaps contribute to enhanced efficacy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Conceived and designed the experiments: EK-B, DD, MT, KAK and BA. Performed the experiments: EK-B, RT, EO-Y, and SS. Analyzed the data: EK-B, BA, TG, and SF. Contributed reagents/materials/analysis tools: SS, TG and MT. Wrote the manuscript: EKB, KAK, FKNA, BA, and MT. All authors contributed to the article and approved the submitted version.


Fall injuries caused by icy road conditions are a prevalent public health problem during winters in Sweden, especially in older populations. To combat this problem, many Swedish municipalities have distributed ice cleats to older adults. While previous research has shown promising results, there is a lack of comprehensive empirical data on the effectiveness of ice cleat distribution. We address this gap by investigating the impact of these distribution programmes on ice-related fall injuries among older adults.We combined survey data on ice cleat distribution in Swedish municipalities with injury data from the Swedish National Patient Register (NPR). The survey was used to identify municipalities that have distributed ice cleats to older adults at some point between 2001 and 2019. Data from NPR were used to identify municipality-level data on patients who have been treated for injuries related to snow and ice. We used a triple differences design—a generalisation of difference in differences—that compared ice-related fall injury rates before and after intervention in 73 treatment and 200 control municipalities, with unexposed age groups serving as within-municipality controls.We estimate that the average ice cleat distribution programmes reduced ice-related fall injury rates by −0.24 (95% CI −0.49 to 0.02) per 1000 person-winters. The impact estimate was larger in municipalities that distributed more ice cleats (−0.38 (95% CI −0.76 to –0.09)). No similar patterns were found for fall injuries unrelated to snow and ice.Our results suggest that ice cleat distribution can decrease the incidence of ice-related injuries among older adults. Ice cleat distribution may be a cost-effective way to reduce the burden of ice-related fall injuries, but comprehensive impact evaluations are lacking.We perform a comprehensive, quasi-experimental evaluation of 73 municipal ice cleat distribution programmes targeting older adults in Sweden. Our primary estimate suggests that ice-related injury incidence was 8.2% lower on average after ice cleat distribution.Our study reaffirms previous evidence suggesting that distributing ice cleats to older adults may be an effective prevention measure in settings affected by snow and ice.Fall injuries that occur outdoors are often associated with environmental risk factors such as snow and ice, which are very common during winter in Nordic countries. As a result, ice-related fall injuries are a prevalent public health problem in Sweden.Over the past decade, about 25% of Sweden’s 290 municipalities have distributed and offered ice cleats to older citizens to help combat the seasonal rise in ice-related fall injuries that typically occur during the Swedish winters. Previous research suggests that exposure to these programmes is associated with greater ice cleat use among older adults, especially in municipalities with high distribution rates per citizen.et al.In June 2019, we sent an electronic survey to all municipalities in Sweden (n=290) to collect data on ice cleats distribution programmes, with non-responding municipalities receiving up to 4 reminders . In the survey, we asked if the municipality had ever distributed ice cleats. If they answered yes, we collected data on implementation dates, targeted age groups, programme costs and how many ice cleats they distributed. Further details about the intervention data collection can be found in Holmberg We used municipality-level data from the Swedish National Patient Register (NPR)To assess the risk of bias, we also collected corresponding data on the number of patients with external cause codes W01–W18  as a negative control outcome.We used a triple differences designFor the statistical analysis, we constructed a panel dataset stratified by municipality, time, and eligible and ineligible age groups. We defined time intervals in winter periods  by aggregating data from 1 October to 30 April, as these months approximately capture the period at risk for ice-related injuries in Sweden see . The stu10.1136/ip-2022-044808.supp1Supplementary dataet al,The programmes were implemented in different years . It was recently discovered that two-way fixed effects models—the models typically applied in difference-in-differences studies—may be biased with this data structure.The imputation-based method estimates the impact by first fitting a fixed effects regression model to not-yet-treated observations . It then uses the estimated model to impute the expected counterfactual postperiod injury rates in all programme municipalities. It then calculates winter-and-municipality-specific impact estimates by taking the difference between the observed injury rates and the imputed counterfactual rates, and finally averages the estimates across programme municipalities and postintervention time points to estimate average effects. We performed the analysis in Stata V.17 (StataCorp), using the DID_IMPUTATION module,et al.et al,In our primary analysis, we aimed to estimate the average intention-to-treat effectet al,The validity of difference-in-differences analyses relies on the parallel trend assumption, which, in essence, means that the groups must have followed the same trend on the outcome in a counterfactual scenario without ice cleat programmes.No patients or members of the public were involved in the design of the study.We received a response from 228 (78.6%) out of the 290 municipalities invited to answer our survey . Of thesAll 73 municipalities provided the essential programme data required for our intention-to-treat analysis . Seven mOur analysis is based on data from 132 465 patients treated for injuries due to an ice-related fall  may also allow for a more precise estimation of the longevity of the impact.As expected, our results suggest that greater reach leads to greater impact. In a previous process evaluation, we found that the strongest determinant of high reach was simply how many ice cleats the municipality purchasedDistributing ice cleats may be a useful and cost-effective complement to winter road maintenance for reducing the incidence of ice-related fall injuries among older adults.


Introduction: Lag screw cut-out is a serious complication of dynamic hip screw fixation of trochanteric hip fractures. The lag screw position has been acknowledged as one of the important factors affecting the lag screw cut-out. We propose a modification of the Tip Apex Distance (TAD) and hypothesise that it could improve the reliability of predicting lag screws cut-out in these injuries. Materials and Methods: A retrospective study was conducted for hip fracture entries in the period from Jan 2018 to July 2022. A hundred and nine patients were suitable for the final analysis. The modified TAD was measured in millimetres based on the sum of the traditional TAD in the lateral view and the net value of two distances in the AP view, the first distance is from the tip of the lag screw to the opposite point on the femoral head along the axis of the lag screw while the second distance is from that point to the femoral head apex. The first distance is a positive value, whereas the second distance is positive if the lag screw is superior and negative if inferior. A receiver operating characteristic curve was used to evaluate the reliability of the different parameters assessing the lag screw position within the femoral head. Results: Reduction quality, fracture pattern as per the AO/OTA classification, TAD, Calcar Referenced TAD, Axis Blade Angle, Parker’s ration in the AP view, Cleveland Zone 1, and modified TAD were statistically associated with lag screw cut-out. Among the tested parameters, the modified TAD had 90.1% sensitivity and 90.9% specificity for lag screw cut-out at a cut-off value of 25 mm with a P-value < 0.001. Conclusion: The modified TAD had the highest reliability in the prediction of lag screw cut-out. A value ≤ 25 mm could potentially protect against lag screw cut-out in trochanteric hip fractures. Implant failure following surgical treatment of trochanteric hip fractures is a devastating problem which leads to a significant increase in morbidity and mortality . SeveralTip Apex Distance (TAD) is one of the important parameters predicting lag screw cut-out and subsequent implant failure , 8. BiomThe rationale of this study is to propose a modification of the TAD that could give more precise information about the lag screw insertion depth as well as direction, aiming to improve the reliability of predicting lag screw cut-out in these patients.The current study was conducted in a retrospective manner. The study was registered with the Research Ethical Committee with a reference number “MD-277-2020”.The inclusion criteria were:Elderly patients > 60 years old.Pertrochanteric fracture; 31 A1, and A2 fractures according to the AO/OTA classification, underwent Dynamic Hip Screw (DHS) fixation.Completion of 12 months follow up.Standard Hip radiographs.The exclusion criteria were:Patients with neurological problems.Loss of follow-up information.The sample size was calculated using the Power Analysis and Sample Size (PASS) software program version 11.0.10  with 0.05 alpha error, confidence interval of 0.95 and study power of 0.90. The minimum required number of patients was 102. Hip fracture entries for the period from Jan 2018 to Jul 2022 were reviewed by generating a query from the hospital database. The initial search generated 921 entries with hip fractures, of which 598 were intracapsular fractures, and 323 extracapsular fractures. Of the extracapsular fractures, 147 were treated with cepahlomedullary nails (CMN) and 176 had DHS. Thirty six patients had non-standard intra-operative radiographs, while 31 patients had early mortality or lost follow-up, hence were excluded. This left 109 patients suitable for the final analysis.The recorded data included age, gender, pre-injury mobility status, comorbidities, abbreviated mental test score (AMTS), and time to operation.Pre-operative X-rays were analysed for the fracture type according to AO/OTA classification , reductiReduction quality in the X-rays was classified into three grades; good, acceptable, and poor, based on a method developed by Baumgaertner et al. . The TADThe modified TAD was measured in millimetres (mm) based on the net value of two distances in the AP view added to the traditional TAD in the LAT view. In the AP view, the first distance is from the tip of the lag screw (point A) to the opposite point on the femoral head along the axis of the lag screw (point B). The second distance is from point B to the femoral head apex (point C). The first distance (AB) is always a positive value, but the second distance (BC) value depends on the location of the lag screw within the femoral head in relation to the head central axis. If the lag screw is inferior, then the second distance (BC) is a negative value. If the lag screw is superior, the second distance (BC) is a positive value. If the lag screw is central within the femoral head along the central axis of the neck, there is only one distance from the tip of the lag screw to the femoral head apex. This means if the lag screw is inferior within the femoral head, the net value of the novel parameter would be less than another screw which has the same distance from the femoral head apex yet is superior. This has been depicted in Two candidates reviewed the post-operative x-rays at 6 weeks, 3, 6 and 12 months. Mechanical complications were recorded, and any discrepancy was resolved by consensus amongst all authors. The collected data were analysed to investigate the sensitivity and specificity of the studied parameters in detecting lag screw cut-out.2) test was performed. An exact test was used when the expected frequency is less than 5 [P-values less than 0.05 were considered statistically significant [The statistical work was done by a statistician. Data were coded and entered using the statistical package for the Social Sciences (SPSS) version 28 . Data were summarized using mean, standard deviation, median, minimum and maximum in quantitative data, and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the non-parametric Mann–Whitney test . For coms than 5 . Receivenificant .Age distributionP-value of 0.067.Patients had a mean age of 81.61 years with a standard deviation (SD) ± 9.02 years. The Median was 83.00. There was no statistically significant relation between age and lag screw cut-out with a Gender distributionThere were 18.3% males and 81.7% females in the study group. There was no statistically significant relation between gender and lag screw cut-out.Pre-injury Mobility status45.9% of patients used to mobilise independently prior to the injury, 27.5% using one aid, 11.9% using a frame, and 14.7% only indoors. There was no statistically significant relation between the mobility status and lag screw cut-out with a slightly higher rate in the frame group.Co-morbiditiesP-value 0.044.Hypertension was prevalent in 47.7% of patients, cardiac problems in 31.2%, diabetes mellitus in 17.4%, and dementia in 22.9% of patients. The presence of diabetes mellitus (DM) has a statistically significant relation with mortality AMTS at presentationPatients with AMTS ≤ 7/10 at presentation represented 29.4% of the study group, while 48% had AMTS 10/10.Patients’ demographicsPeri-operative outcomesTime to operationP-value 0.152), however, 50% of patients who had their operation at 72 h died in the early post-op period. The reasons reported for the delay in the surgical intervention were awaiting theatre space, awaiting medical optimisation, or delay because of the COVID pandemic.The time between diagnosis and operation was ≤ 24 hours (h) in 80.7% of patients, 48 h in 13.8%, and 72 h in 5.5% of patients. Longer time to operation between 24 and 48 h was not associated with a statistically significant increase in mortality  patients, 10 of them were recognised in the first 3 months postoperatively. Other surgical complications occurred in 4 (3.7%) patients. This included femoral head AVN 2.7% (3 patients) and medialisation with screw breakage 0.9% (1 patient). Revision surgery was done in 13 patients (11.9%) in total. Revision surgery was in the form of total hip replacement 8.4% (9 patients) and proximal femoral replacement 3.6% (4 Patients).Lag screw position within the femoral head is one of the important factors influencing lag screw failure in trochanteric hip fractures . SeveralAlthough the modified TAD had about 2% better sensitivity and specificity than the TAD in this study, we believe a bigger cohort of patients is required to conclude more precise results. We also suggest that future research, involving a greater number of lag screw cut-outs and fractures fixed with CMN, shall be conducted to provide robust conclusions.Baumgaertner et al. populated the idea of TAD in the nineties as a predictive measure of lag screw cut-out. In their study of 198 cases, no complications of lag screw cut-out were noted in any patient with a TAD less than 25 mm. However, in patients with a TAD of more than 30 mm, 27% of patients experienced lag screw cut-out. They concluded that TAD of more than 30 mm increased the risk of cut-out and should be avoided , 13.In an interesting biomechanical study, Kane et al. evaluated the mechanical stability of the central lag screw position in the AP and LAT views in comparison with AP inferior and LAT central lag screw positions in instrumented models. There was no difference in the biomechanical strength between both groups. They suggested similar clinical results in patients treated with AP inferior LAT centre lag screw construct, although they have a TAD of more than 25 mm .In an attempt to provide an objective assessment of the lag screw direction and evaluate its relation to the cut-out, Mao et al. proposed a new parameter, ABA. They concluded a threshold value for lag screw cut-out of −10 degrees. However, their method does not account for lag screw insertion depth, therefore they suggested using both ABA and TAD or Cal TAD as predictive methods for lag screw cut-out .To summarise, the TAD takes into consideration the lag screw insertion depth. However, the TAD cannot solve the direction issue, therefore two lag screws with the same TAD value could have distinctly different positions within the femoral head . The CalThe theoretical advantage of the modified TAD is that it combines the lag screw insertion depth and direction in one method. Superior and inferior screws would not have the same value if they have the same insertion depth contrary to the TAD. It also retains the advantage of a central lag screw in the AP plane. The Cal TAD theoretically gives an advantage to the inferior lag screws over the central ones which have not been proved correct in many studies , 19. TheThe strength of the current study, besides proposing a modification of the TAD, comes from several aspects. This study has particularly focused on trochanteric fractures in the elderly >60 years old as this cohort of patients has a different bone quality compared with the younger population, which explains why most patients had SOI grade 3 or 4. The follow-up time was 12 months, longer than other similar studies. The inclusion of trochanteric fracture 31A1 and 31A2 fractures and the exclusion of other patterns such as 31B and 31A3 improves the reliability of the result because these fractures have other independent risk factors for lag screw cut-out.The modified TAD shows the highest combined sensitivity (90.1%) and specificity (90.9%) compared with other parameters predicting lag screw failure in patients with trochanteric hip fractures. Modified TAD value ≤ 25 mm reflects a potentially stable lag screw position within the femoral head with a low possibility of lag screw cut-out. Hence, we recommend the use of the modified TAD as a predictive measure for lag screw cut-out.


Coenonympha pamphilus was generally the species that revealed the highest concentrations of all the ten trace metals at the sites closer to the industrial area. It is known that C. pamphilus is a sedentary species and that its host plants are the Poaceae, capable of accumulating high quantities of metals in their rhizosphere region, thus providing the link with soil contamination. Therefore, monitoring the metal concentration levels in butterflies might be a good indicator and a control tool of environmental quality, specifically in areas affected by high anthropogenic pollution loads linked to a specific source.Anthropogenic trace metal contamination has significantly increased and has caused many hazardous consequences for the ecosystems and human health. The Terni basin valley  shows a heavy load of pollutants from industrial activities, while the characteristic orography structure of the valley favours air stagnation, thus limiting air pollution dispersal. The present study conducted in 2014 aimed to determine the concentration of ten metals in five species of butterflies at nine sites in the Terni valley along a 21-km-long transect, including both relatively pristine and industrial areas. At sites where soil contamination was high for a given metal, such as for chromium as in the case of site 4 (the closest to the steel plant) and for lead as in the case of site 2 (contaminated by a firing range), higher levels of contamination were observed in the tissues of butterflies. We found a correlation between soil contamination and the concentration of Cr, Al and Sr in the tissues of some species of butterflies. The sensitivity to contamination differed among the five species; in particular, The online version contains supplementary material available at 10.1007/s11356-023-28930-x. In the last decades, technological and industrial development has led to the widespread dispersal of anthropogenic trace elements with a significant impact on ecosystems and human health Järup . Their iDetection and quantification of trace element contamination in the environment are always challenging ), defoliators ) and silk producers ).Most of the studies on metals in Lepidoptera are mainly focused on species of economic interests, such as agricultural pests  the biological stages of the lepidopteran life cycle (from egg to adult) often show different values of metal accumulation  as bioindicators of trace metal levels, relating the concentration in their tissue with environmental contamination. We additionally propose using different butterfly species occurring in the study area. The choice of butterflies as environmental quality bioindicators is based on several factors. Many common species of butterflies are present in both natural, semi-natural and anthropized habitats, representing regional biodiversity , a current hot spot for environmental studies, due to its heavy load of pollutants by industrial activities further promoted by its orographic conditions favouring air stagnation and pollutant accumulation , an area densely populated  and Pieris rapae ; two species of Nymphalidae: Coenonympha pamphilus  and Lasiommata megera  and one species of Lycaenidae: Polyommatus icarus .Five species of butterflies , belonging to three different families, were sampled: two species of Pieridae: C. pamphilus and L. megera, showing a tendency to poor mobility, in particular C. pamphilus , which is adopted for the populations that are not at risk of extinction in the short or medium term  life cycle, and because of their polyphagous larvae  IBC . Some ofL. megera; 5.4, P. napi; 6, P. rapae; 11.8, P. icarus; 12, C. pamphilus), and the average weight of each individual amounted to 18.70, 17.23, 15.15, 8.26, 7.18 mg (d.w.) for L. megera, P. napi, P. rapae, P. icarus and C. pamphilus, respectively.An entomological net (35 cm diameter) made up of fine meshes was used to collect specimens to minimize the damage to the butterflies. The species were identified through butterfly-watching, that is, the observation of the specimens during the flight. All the samples were brought to the laboratory, stored at − 20 °C, and a more accurate identification was further conducted with the help of taxonomic keys IBC . Lepidop3 ultrapure 65% and 2 mL of H2O2 ultrapure 30% were added to each sample, and the mixture was heated up to 170 °C in the MARS 5 microwave system (CEM Corporation) for about 30 min , and ultrapure water (18.2 MΩ), 8 mL of HNO−1). Instrument parameters were optimized for best sensitivity in the whole mass range and minimum oxides (< 1.5%) and double charges (< 1.5%).Inductively coupled plasma mass spectrometry (ICP-MS) technique was used to measure the metal concentration in mineralized butterfly samples using the triple quadrupole Agilent 8900 ICP-MS-QQQ instrument. The ICP-MS was equipped with an SPS-4 autosampler (Agilent), a cyclonic spray chamber temperature controlled at 2.7 °C, a quartz torch and Ni cones. The acquisition was performed at 1550 W of plasma RF power in Kinetic Energy Discrimination (KED) — general purpose mode, using He as the collision gas  was also used to correct for changes in ICP-MS operating conditions and sample-specific matrix effects. Limit of detection (LOD) was estimated using the slopes of the calibration lines constructed with certified standard solutions of the examined elements . LOD values are reported in Table −1). The % RSDs obtained from the repeatability test of the metal solutions were defined as good (0.1–10.0% range).Ten trace elements  have been quantitatively determined. To this aim, the instrument was calibrated with standard solutions , obtaining a calibration curve for each metal with the least-squares method . A certified Internal standard mix solution for ICP-MS systems  independently from the metabolic pattern of each element in the various butterfly species.AF = [metal in the tissues]/[metal in the soil]) were calculated for nine trace elements  as the ratio between the trace element concentration in butterflies and the trace element concentration in soil. The AF was not calculated for Cd due to the absence of the corresponding soil data. Soil contamination can influence metal presence in the host plants of butterfly larvae, thus affecting the relative bioaccumulation in adult butterfly tissues. In order to check for trends in the ten elemental concentrations in the five species at the nine sampling sites, the first two axes of a principal component analysis (PCA) were analysed.Assimilation factors , Ni with 8.44 mg kg−1 (site 5), Cu with 45.78 mg kg−1 (site 5), Zn with 392.45 mg kg−1 (site 5) and Al with 246.22 mg kg−1 (site 4).This butterfly species was not found at sites 2 and 8. A total of 84 specimens were found at the remaining seven sites. In the three sites closer to the steel plant , this species, which is characterized by the lowest mobility among the five species collected, revealed the highest concentrations of all the trace elements (except for Cd) with Cr, Fe and Al peaking at site 4 and Pb, Ni, Cu, Zn and Mn at site 5, respectively. Among all the studied species, −1 (site 2), and Sr, with 9.22 mg kg−1 (site 8). The highest concentrations of Cr and Zn in L. megera were observed at site 4, close to the steel plant, where these elements are also very abundant in soils , Cd with 1.78 mg kg−1 (site 6) and Fe with 250.56 mg kg−1 (site 4).The butterfly was not found at sites 1 and 2. A total of 72 specimens were found at the remaining seven sites. In the three sites closest to the steel plant, the highest concentrations in The trace element concentration level of the soil observed in the study area  in all the species of butterflies showed average values for metals lower than 1 except Zn. AF calculation was not performed for Cd, because it was not measured in the soil Tab. .χ2 = 4.38, p = 0.36). P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum concentrations of Al respectively at site 6 (26.37 mg kg−1), at site 4 (94.80 mg kg−1), at site 3 (22.18 mg kg−1), at site 4 (246.22 mg kg−1) and at site 8 (10.27 mg kg−1). Al showed the maximum concentration value in the soil at site 8 (1.13 * 105 mg kg−1) and is ubiquitous in soils.No statistical differences were observed in concentration values among the species  between medians. P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum concentrations of Cd respectively at site 9 (0.10 mg kg−1), at site 6 (1.78 mg kg−1), at site 7 (0.54 mg kg−1), at site 9 (0.39 mg kg−1) and at site 2 (0.04 mg kg−1). Therefore, diffuse contamination was observed in the study area. Unfortunately, Cd data was not available for the corresponding soil samples.The Kruskal–Wallis test revealed highly significant differences  with noteworthy differences between P. icarus versus C. pamphilus and P. rapae (Wilcoxon test — p < 0.05). All five butterfly species, P. icarus, P. rapae, P. napi, C. pamphilus and L. megera, exhibited the maximum levels of chromium concentration at site 4, with values of 2.19, 3.86, 2.91, 6.13 and 3.43 mg kg−1, respectively. These data are in excellent agreement with the data of metals in soil, which showed, precisely at site 4, a particularly high concentration of Cr (444 mg kg−1).The Kruskal–Wallis test revealed significant differences . P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum concentrations of copper respectively at site 5 (28.10 mg kg−1), at site 7 (37.42 mg kg−1), at site 7 (35.57 mg kg−1), at site 5 (45.78 mg kg−1) and at site 2 (5.28 mg kg−1). The maximum concentration in the soil was observed at site 3 (140.00 mg kg−1).No significant differences were found among the species . P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum concentrations of Fe respectively at site 5 (127.27 mg kg−1), at site 4 (250.56 mg kg−1), at site 4 (174.59 mg kg−1), at site 4 (243.24 mg kg−1) and at site 8 (100.11 mg kg−1).No significant difference was found among the species .In the study area, the butterfly contamination tended to be higher at sites 4 and 5; however, surprisingly, in the soil of these sites, the maximum levels of this metal were not recorded; indeed, Fe showed the maximum concentrations in the soil at site 1  with marked different medians between L. megera versus P. napi (Wilcoxon test — p < 0.01), and of both these species versus the other species (Wilcoxon test — p < 0.05). P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum manganese concentrations, respectively at site 5 (16.35 mg kg−1), at site 4 (22.44 mg kg−1), at site 9 (31.64 mg kg−1), at site 5 (21.72 mg kg−1) and at site 2 (121.11 mg kg−1). Mn showed in the soil the maximum concentration at site 1 (3.6 * 103 mg kg−1).The Kruskal–Wallis test showed highly significant differences  between the medians of C. pamphilus and L. megera showing significantly higher concentrations than the other species (Wilcoxon test — p < 0.05). P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum concentrations of Ni respectively at site 7 (2.74 mg kg−1), at site 4 (1.02 mg kg−1), at site 9 (3.49 mg kg−1), at site 5 (8.44 mg kg−1) and at site 2 (5.28 mg kg−1). Ni showed in the soil the maximum concentration at site 1 (133.00 mg kg−1).The Kruskal–Wallis test revealed highly significant differences . All the butterflies found at site 2, P. icarus, P. napi and L. megera, showed the maximum concentration levels of lead at this site, respectively, 0.60, 4.07 and 2.82 mg kg−1. These data are in excellent agreement with lead occurrence in soil, which showed a peculiarly high content of Pb (296.00 mg kg−1) at this site. Butterflies not detected at site 2, P. rapae and C. pamphilus, showed the maximum Pb concentration at site 4 (5.51 mg kg−1) and at site 5 (2.39 mg kg−1), respectively.No significant differences were found among the species for this element  among the species, with the exception between C. pamphilus versus P. icarus and P. napi versus P. rapae (Wilcoxon test — p > 0.05). L. megera, C. pamphilus, P. icarus, P. napi and P. rapae showed the maximum concentrations of Sr respectively at site 8 (9.22 mg kg−1), at site 5 (8.16 mg kg−1), at site 3 (3.95 mg kg−1), at site 9 (2.56 mg kg−1) and at site 7 (1.86 mg kg−1). For strontium in the study area, there was a tendency of a less localized contamination. Sr showed in the soil the maximum concentration at site 5 (373 mg kg−1).The Kruskal–Wallis test indicated highly significant differences . P. icarus, P. rapae, P. napi, C. pamphilus and L. megera showed the maximum concentrations of Zn respectively at site 7 (204.39 mg kg−1), at site 7 (207.26 mg kg−1), at site 4 (201.70 mg kg−1), at site 5 (392.45 mg kg−1) and at site 4 (369.29 mg kg−1). Zn showed the maximum soil concentration at site 2 (245.00 mg kg−1). Only for Zn it was observed that the assimilation factor in all species of butterflies showed average values higher than 1, between 1.3 (P. napi) and 2.0 (C. pamphilus).No significant differences were found among the species .The correlation between trace metal concentrations in soil and in butterfly species  Fig. -S18 showapi Fig. , betweenrus Fig. , and betlus Fig. . In addiP. rapae, P. icarus, and C. pamphilus and site 6 for P. rapae and P. icarus. A negative spatial association between metals was observed between site 3 and P. rapae and C. pamphilus; between site 8 and P. icarus, P. rapae, and L. megera and between site 9 and C. pamphilus. PC2 axis (28.3%) showed an association between butterflies and metals characterized by a positive relationship between P. rapae and P. napi and Cd and a negative relationship between L. megera with Sr, Ni and Mn.Principal component analysis Fig.  of the 1Apis mellifera Linnaeus, 1758) plays a major role in studies concerning the bioindication of environmental pollution. Bees, used in apiculture, are reliable bioindicators of environmental contamination, showing clear patterns of contaminant accumulation in their tissues and in beehive products  in the present study were detected for Al (246.22), Cr (6.13), Cu (45.78), Ni (8.44) and Zn (392.45) in C. pamphilus; Mn (121.11) and Sr (9.22) in L. megera and Cd (1.78), Fe (250.56) and Pb (5.51) in P. rapae  and its translocation in the soil–mulberry–silkworm food chain. Lead concentrations in adults Lepidoptera showed the same trend of the lead content in the leaves of the plants of which the larvae fed and consequently in the soil . However, the lead levels in adult butterflies were much lower than those in the larvae. High concentrations of lead were found instead in the faeces of the larvae and in the pupal envelope.Besides, Bagatto and Shorthouse  did not From the analysis of the trace element concentration in the present study, we observed that the butterfly species examined are to be considered good bioindicators for chromium. In sites where the soil contamination was high for Cr, consequently, there were higher contamination levels in butterfly tissues, while a clear decrease of Cr concentration in butterflies at increasing distances from site 4, both toward site 1 and site 9, is observed Fig. .In the soil of site 4, Cr showed a concentration about 13 times higher than the crustal background , unlike for Cr, the relationship between soil and butterflies are inconsistent for sites with soil concentrations below 100 mg kg−1 .From the analysis of the regression lines, confidence intervals (95%) for intercept and slope showed, in absolute value, a decreasing sequence among the species: era Tab. a-e, thusP. icarus, P. napi and L. megera) and in soil. Pb reveals further relative maxima at sites 4 and 5 only for those species of butterflies that were not found at site 2 (P. rapae and C. pamphilus). Interestingly, in the soil, Pb showed a concentration about 17 times higher than the crustal background  and the closest sites to the industrial area. Cr showed a strong association to PC1 and no association with PC2, confirming that Cr in butterflies is controlled exclusively by the anthropogenic gradient. The anthropogenic origin of contamination is less evident for Al, Cu, Pb and Fe. On the other hand, not all butterfly species responded to the contamination in the same way when trace element concentrations were not particularly high, in fact, PC2 axis showed a relative trend of sensitivity of butterfly species to various trace elements .C. pamphilus, which always showed the highest concentration values for all the ten metals at sites 4 and 5. In addition, these values were the maximum values of contamination among all butterfly species for Cr and Al at site 4, and for Ni, Cu and Zn at site 5. This species was not found at site 2; therefore, the peculiar lead contamination caused by the firing range at this site could not be detected. C. pamphilus is a very common species, ubiquitous and with the lowest mobility among the species examined; its widespread presence is due to the fact that the plants of the family Poaceae, the host plants of its larvae, are very common in all the habitat types. The Poaceae are plants capable of accumulating high amounts of metals in the rhizosphere Below is the link to the electronic supplementary material.


Nowadays, usable plastic materials with defined properties are created by blending additives into the base polymer. This is the main task of compounding on co-rotating twin-screw extruders. The thermal and mechanical stress occurring in the process leads to a mostly irreversible damage to the material. Consequently, the properties of the polymer melt and the subsequent product are affected. The material degradation of polypropylene (PP) on a 28 mm twin-screw extruder has already been studied and modeled at Kunststofftechnik Paderborn. In this work, the transferability of the previous results to other machine sizes and polypropylene compounds were investigated experimentally. Therefore, pure polypropylene was processed with screw diameters of 25 mm and 45 mm. Furthermore, polypropylene compounds with titanium dioxide as well as carbon fibers were considered on a 28 mm extruder. In the course of the evaluation of the pure polypropylene, the melt flow rates of the samples were measured and the molar masses were calculated on this basis. The compounds were analyzed by gel permeation chromatography. As in the previous investigations, high rotational speeds, low throughputs and high melt temperatures lead to a higher material degradation. In addition, it is illustrated that the previously developed model for the calculation of material degradation is generally able to predict the degradation even for different machine sizes by adjusting the process coefficients. In summary, this article shows that compounders can use the recommendations for action and the calculation model for the material degradation of polypropylene, irrespective of the machine size, to design processes that are gentle on the material. To produce usable plastics, the properties of the materials must be specifically modified. This is conducted during compounding on co-rotating twin-screw extruders. Additives such as fillers, fibers or colors are mixed with the base polymer. Besides the desired melting and homogenization of the components, the thermal and mechanical stress in the extruder causes mostly irreversible damage to the material. The occurring damage mechanisms can be divided into purely thermal, thermal–oxidative, thermal–mechanical and hydrolytic degradation. These reactions usually run simultaneously and lead to different types of damage. Mainly, chain scission results in a reduction in the molar mass and a change in the molar mass distribution. This influences the rheological properties of the melt in the process as well as the mechanical properties such as the tensile strength of the end product. Moreover, color or odor changes may occur ,2,3.The evaluation of the material degradation caused by the compounding process, which is usually only possible retrospectively, is associated with a high economic and time expenditure, since a suitable operating point must first be detected using a try and error procedure. This problem led to the objective of a research project carried out at Kunststofftechnik Paderborn (KTP). It pursues the development of recommendations for gentle processing as well as mathematical modeling to describe the material degradation of market-dominating polypropylene on the co-rotating twin-screw extruder.The literature already contains several studies on the material degradation of PP on single- or twin-screw extruders. Pongratz , for exaIn previous research projects at the KTP, the material degradation of polyolefins on the single-screw extruder ,11,12 anWithin the research project on the material degradation of PP on the twin-screw extruder, basic investigations were first carried out ,16. Two The left side of the equation contains the weight-average molar mass to be calculated, 2) and carbon fibers (CF). The measuring methods used to determine the material degradation are then presented, as well as recommendations for gentle processing. In addition, it is illustrated that the introduced calculation model is also generally transferable to varying plant sizes.This article explains the further investigations to verify the previously developed recommendations for gentle processing and the applicability of the model to varying machine sizes and PP compounds. For this purpose, the following section first explains the tests carried out on pure polypropylene on a 25 mm and 45 mm extruder, as well as studies on compounds with titanium dioxide  and 45 mm (Barmag Saurer BTS-PET-045/40D) were used to investigate the transferability of the developed degradation model to other machine sizes. The PP compounds were processed on a ZE 28 BluePower twin-screw extruder from KraussMaffei with a screw diameter of 28 mm. Since the screw configuration can be assumed to have a small influence on the material degradation according to the findings of the previous studies, only one screw configuration was considered for each setup . The conGravimetric feeding and a water bath were used for all experimental investigations. The cooled extrudates were granulated with the help of a strand pelletizer. A comprehensive examination of the influence of the process parameters’ rotational speed, and the throughput and barrel temperature on material degradation was ensured by full-factorial test designs. Three different rotational speeds and three different throughputs were varied for each investigation according to To assess the effects of the temperature configuration on material degradation, low, medium and high barrel temperatures were considered in each investigation . While tAs already described in section one, processing on the twin-screw extruder leads to molar mass degradation and thus to a reduction in melt viscosity. Knowledge of the quickly measurable and evaluable melt flow rate as a dimension for the viscosity therefore allows conclusions about the occurred material degradation. For this reason, the melt mass flow-rate (MFR) and the melt volume flow-rate (MVR) of the taken granulate samples were measured according to  using a For the evaluation of the preliminary basic investigations carried out on the 28 mm extruder, the measured MFR values were converted into weight-average molar masses by using a model developed by Bremner et al. . To veriSince the calculation with Equation (2) is only suitable for pure polymers, the PP compounds were analyzed by GPC. This provides the advantage that the molar mass distribution is determined in addition to the weight-average molar mass. Essentially, the measurement conditions corresponded to those of the previously performed verification.The results of the further investigations largely correspond to those of the basic investigations on the 28 mm extruder. An increase in screw speed with otherwise constant parameters generally increases material degradation on all twin-screw extruder sizes. The reasons for this are the higher mechanical stress due to the increasing shear rates and the associated increased temperature of the material, which, for example, outweigh the material-protecting effect of the reducing residence time . In addi2 compound (a–c) and the PP/CF compound (d–f) are shown for different rotational speeds, throughputs and mass temperatures. The chain scissions caused by the processing on the twin-screw extruder leads to a shift of the distribution curves into the range of lower molar mass. Higher stress on the material due to high rotational speeds, low throughputs or high melt temperatures results in increased chain scissions and thus in a more significant displacement of the molar mass distribution.The effect of the individual process parameters on the PP compounds is illustrated in As explained, an increase in speed causes a higher shear stress, which in turn causes a higher mechanical degradation. Here, a deformation of the bond angles and the bond distance and consequently a breakage of the molecular chain occurs. Due to the structural viscosity of PP, the higher shear stress also causes the chains to stretch and align more, so that less energy is required for the flow. This reduces the effects of mechanical damage when the rotational speed is steadily increased. The higher the shear stress and the resulting dissipation, the more the melt heats up. This heating primarily causes chain scissions through the purely thermal and especially the thermal-oxidative degradation of the material. In terms of the pure thermal degradation, PP is relatively stable. However, it reacts easily with oxygen and radicals and therefore has only a low resistance to thermal-oxidative degradation. The thermal-oxidative degradation proceeds autocatalytically, meaning the initially slow reaction is accelerated by the formation of hydroperoxides and their decomposition into radicals. In addition, the thermal-oxidative degradation is accelerated by primary radicals which are formed during mechanical degradation. The increase in rotational speed also reduces the residence time of the material in the extruder, which is gentle on the material. Since the damaging effects associated with the increase in rotational speed dominate, an overall rise in material degradation takes place ,23.With increased throughput comes a reduction in the residence time of the material in the extruder. This particularly reduces the mechanical degradation. The higher filling level in the channel also has a positive effect. In combination with the shorter residence time, it leads to a lower melt temperature. As a result, the purely thermal and thermal-oxidative degradation is reduced. The latter is also decreased; due to a high filling level, less oxygen is available for the oxidation of the melt. Accordingly, the higher the throughput, the lower the material degradation. Raising the barrel temperature leads to a higher mass temperature. This in turn boosts the purely thermal as well as the thermal-oxidative degradation. At the same time, a higher mass temperature is accompanied by a lower viscosity, which reduces the mechanical degradation. However, overall, there is a clear increase in material degradation. In general, the material degradation is increased by high rotational speeds, low throughputs and high melt temperatures.w at the particular operating point and the weight-average initial molar mass Mw,0. If no material degradation occurred, this molar mass quotient equals one. The higher the material degradation, the more the molar mass quotient approaches zero. For pure PP on the 25 mm extruder, all modeled values show an upward deviation according to The result of the modeling with Equation (1) is the ratio of the reduced weight-average molar mass MAn adjustment of the process coefficients of the model according to 2 and PP/CF compounds were produced on a 28 mm twin-screw extruder. For the pure PP, the melt flow rates were measured and the molar masses were calculated. Since the calculation of the molar mass from the MFR is only suitable for pure polymers, the compounds were analyzed by GPC. The results correspond to the findings obtained for the 28 mm extruder and to the conclusions in the literature [From the previously conducted investigations on the material degradation of PP on a 28 mm twin-screw extruder, recommendations for gentle processing have already been derived. Additionally, a mathematical model for describing the material degradation was developed and implemented in the SIGMA simulation software. In this work, further experimental investigations were carried out to ensure the transferability of the previous results to other machine sizes as well as PP compounds. On the one hand, pure PP was processed on a 25 mm and a 45 mm twin-screw extruder. On the other hand, PP/TiO


RAPTOR (specific inhibition of mTOR Complex 1), RICTOR (inhibition of mTOR Complex 2), or DEPTOR (activates both mTOR Complexes). Subsequently, HepG2 cells, a well-established model for human fetal hepatocytes, were cultured in CM from PHT cells, and IGFBP-1 secretion and phosphorylation were determined. CM from PHT cells with either mTORC1 or mTORC2 inhibition caused the marked hyperphosphorylation of IGFBP-1 in HepG2 cells as determined by 2D-immunoblotting while Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) identified increased dually phosphorylated Ser169 + Ser174. Furthermore, using the same samples, PRM-MS identified multiple CK2 peptides coimmunoprecipitated with IGFBP-1 and greater CK2 autophosphorylation, indicating the activation of CK2, a key enzyme mediating IGFBP-1 phosphorylation. Increased IGFBP-1 phosphorylation inhibited IGF-1 function, as determined by the reduced IGF-1R autophosphorylation. Conversely, CM from PHT cells with mTOR activation decreased IGFBP-1 phosphorylation. CM from non-trophoblast cells with mTORC1 or mTORC2 inhibition had no effect on HepG2 IGFBP-1 phosphorylation. Placental mTOR signaling may regulate fetal growth by the remote control of fetal liver IGFBP-1 phosphorylation.The mechanisms mediating the restricted growth in intrauterine growth restriction (IUGR) remain to be fully established. Mechanistic target of rapamycin (mTOR) signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental function. Increased secretion and the phosphorylation of fetal liver IGFBP-1 are known to markedly decrease the bioavailability of IGF-1, a major fetal growth factor. We hypothesized that an inhibition of trophoblast mTOR increases liver IGFBP-1 secretion and phosphorylation. We collected conditioned media (CM) from cultured primary human trophoblast (PHT) cells with a silenced  Intrauterine growth restriction (IUGR) is associated with functional changes in multiple fetal tissues, including skeletal muscle, the islet, and the liver ,2,3. TheEmerging evidence shows that factors secreted by the placenta are critical for normal fetal organ development. Using Stable Isotope Labelling by Amino Acids in Cell Culture, we have identified a wide array of secreted proteins in the conditioned media of a Primary Human Trophoblast (PHT) cell culture . MoreovemTOR regulates cellular metabolism, growth, and proliferation ,11. PlacInsulin-like growth factor 1 (IGF-1) is a key regulator of fetal growth ,35,36, aRAPTOR ; RICTOR, a key component of mTOR Complex 2 ; or DEP domain-containing mTOR-interacting protein DEPTOR (leading to the activation of both mTORC1 and mTORC2 Complexes). Subsequently, HepG2 cells  from PHT cells in a culture . To expeatocytes ,51) wereRAPTOR siRNA) moderately, but significantly, increased IGFBP-1 secretion in HepG2 cells  or RICTOR (inhibition of mTORC2) both showed marked hyperphosphorylation of IGFBP-1 secreted from HepG2 cells compared to the control (non-coding siRNA). The 2-D immunoblots are displayed in RAPTOR siRNA (RICTOR showed significantly increased phosphorylation (spots 2–5), showing that the incubation of HepG2 cells in CM from PHT cells with mTORC1 inhibition and incubation with CM from PHT cells mTORC2 inhibition resulted in similar patterns of IGFBP-1 hyperphsophorylation. We propose that the marked shift in the IGFBP-1 phosphoisoform profile to the left is caused by an increased net protein charge (pI) due to an increased phosphorylation level.We validated the effects of the CM from PHT cells with OR siRNA B exhibitOR siRNA C showed OR siRNA B,C, RICTTo determine the site-specific IGFBP-1 phosphorylation, we performed immunoblotting with antibodies previously validated to recognize pSer101, pSer119, and pSer169 ,52. As sTo determine relative levels of dual IGFBP-1 phosphorylation at pSer169 and pSer174, we performed PRM-MS, as no antibodies were available to assess this modification. Following co-immunoprecipitation, PRM-MS readily detected unmodified and dual pSer169/pSer174 IGFBP-1 peptides A–C. In aWe then assessed casein kinase CK2, which has been shown to be the major kinase phosphorylating IGFBP-1 in HepG2 cells ,54. FromRAPTOR, or RICTOR siRNA was determined (n = 3) , termined A by asseRAPTOR  (Ser473(mTORC2).  C and pAKmTORC2). D.RAPTOR or RICTOR was silenced. As shown in To explore to what extent the marked effects of PHT cell CM on HepG2 cell IGFBP-1 phosphorylation is specific to trophoblast cells, we examined the impact of CM collected from renal carcinoma cells in which The mechanisms mediating the restricted growth in IUGR are poorly understood. mTOR signaling functions as a placental nutrient sensor, indirectly influencing fetal growth by regulating placental functions like protein synthesis, nutrient transport, and oxidative phosphorylation. In this study, we provide evidence to support the model  that plaIGFBP-1 is the predominant IGF-1 binding protein in fetal life, and phosphorylation of the protein increases its affinity for IGF-1 6–10-fold  and makeRAPTOR and RICTOR siRNA-treated PHT cells caused marked shifts in spots toward the acidic pH. These observations indicate that the phosphorylated amino acid residues contribute to the IGFBP-1 net protein charge showing pronounced increases in IGFBP-1 phosphorylation.The 2-D immunoblotting based on isoelectric points of IGFBP-1 demonstrates that CM from both Using targeted multiple reaction monitoring-mass spectrometry (MRM-MS) for relative quantification of IGFBP-1 phosphorylation, we have previously shown Ser174 phosphorylation singly and/or combined with Ser169 is an mTOR-sensitive IGFBP-1 phosphorylation potentially involved in IGF-1 binding . FurtherRICTOR silencing involves Ser174 phosphorylation combined with Ser169. Based on molecular modeling and mapping the proximity for pSer174 binding with IGF-1, we earlier proposed that phosphorylation at residue Ser174 likely affects the affinity of the IGFBP-1 to bind IGF-1  and peptides post-translationally modified at specific sites by phosphorylation. In addition, CK2 peptides co-immunoprecipitation (co-IP) with IGFBP-1 were identified with PRM-MS and tested for their activation status via autophosphorylation.4) HCO3, pH 7.8) and then digested with 10 ng of sequencing grade trypsin  overnight at 37 °C. Half of each sample was then diluted with 2X Asp-N reaction buffer  and digested with 10 ng of Asp-N  overnight at 37 °C. The resultant peptides were desalted with C18-ZipTip , dried by SpeedVac, and resuspended in 20 μL MS-grade water containing 0.1% formic acid. PRM-MS was performed at the John L. Holmes Mass Spectrometry Facility at the University of Ottawa. Briefly, peptides were chromatographically separated on a Thermo Scientific™ Acclaim PepMap™ 100 precolumn  and a Thermo Scientific™ Acclaim™ PepMap™ RSLC C18 column  using an EASY nLC II System (Thermo Fisher Scientific). First, the peptides were loaded onto the columns for 100 min at a flow rate of 0.25 μL/min. Separation occurred using multiple linear gradients of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) solutions . Next, eluted peptides were directly electrosprayed  into a Q-Exactive Plus hybrid quadrupole-orbitrap mass spectrometer, where PRM scanning was performed and guided by an isolation list  developed in Skyline software HPGQEYNVR-COOH) and unmodified CK2 internal peptide (NH2-DVNTHRPREYWDYESHVVEWGNQ-COOH). To quantify levels of CK2 and PKC co-immunoprecipitated with IGFBP-1, the total peak area for multiple peptides along the CK2 and PKC protein sequences were summed for each sample and normalized to that of the control.Chromatographs depict transitions ions detected for a given parent ion , where the retention time specifies the elution of the parent ion from the column just prior to MS. To quantify IGFBP-1 dual pS169/pS174 phosphorylation, the total peak area was derived using Skyline software and normalized to that of an unmodified IGFBP-1 internal peptide (NHRAPTOR-silenced (inhibition of mTORC1), or RICTOR-silenced (inhibition of mTORC2) CM from PHT cells containing equal concentrations of IGFBP-1 (250 ng IGFBP-1) were buffer-exchanged to P6 cell media using Amicon Ultra-0.5 mL Centrifugal Filter Units  per manufacturer’s instructions. As illustrated in To examine whether the alterations in IGFBP-1 phosphorylation functionally alter IGF-1 receptor (IGF-1R) autophosphorylation, P6 cells  were used. P6 cells are immortalized mouse embryonic fibroblast cells that overexpress human IGF-1R but do not express IGF-1 . We usedRAPTOR-silenced (inhibition of mTORC1), or RICTOR-silenced (inhibition of mTORC2) PHT cells were analyzed for CK2 activity using P32 as described previously [CM collected from HepG2 cells treated with CM from control (non-coding siRNA), eviously ,59,80. Ieviously .RAPTOR-silenced (inhibition of mTORC1), and RICTOR-silenced (inhibition of mTOR C2) PHT cells were used for the quantitative measurement of IGF-1 using human IGF-1 ELISA kit (Abcam). IGF-1 was also measured in CM from PHT cells with either control, RAPTOR-, or RICTOR-silenced PHT cells before treatment of HepG2 cells.HepG2 cells treated for 24 h with CM collected from PHT cells either control (non-coding siRNA), RAPTOR and RICTOR knockdown according to the manufacturer’s protocol. Samples of CM were harvested at 72 h following transfection. A non-targeting scramble siRNA pool was used as negative control (Thermo Fisher) [Renal carcinoma A498 cells were obtained from the ATCC and maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum at 37 °C in a humidified 5% CO2 atmosphere. Cells were transfected with pooled siRNA reagent (Thermo Fisher) with the Amaxa Nucleofector system for  Fisher) .p < 0.05.Data were analyzed using GraphPad Prism 5 . Three replicates were analyzed for each treatment condition, including the control treatment. For each quantified protein, the mean density of the control sample bands was assigned an arbitrary value of 1, and averaged densitometry values for each treatment were expressed relative to this mean. We employed a one-way analysis of variance with Dunnett’s Multiple Comparison Post-Test and expressed results as the mean ± SEM. Significance was accepted at *


However, MCh-induced AMPK activation is calcium-dependent and mediated by CaMKK1 and CaMKK2 isoforms. In contrast, PD 102807-induced signaling is calcium-independent and mediated by the atypical subtype protein kinase C-iota and the CaMKK1 (but not CaMKK2) isoform. Both MCh- and PD 102807-induced AMPK activation involve the AMPK α1 isoform. PD 102807-induced AMPK α1 (but not AMPK α2) isoform activation mediates inhibition of the mammalian target of rapamycin complex 1 (mTORC1) in ASM cells, as demonstrated by increased Raptor (regulatory-associated protein of mTOR) phosphorylation as well as inhibition of phospho-S6 protein and serum response element–luciferase activity. The mTORC1 inhibitor rapamycin and the AMPK activator metformin both mimic the ability of PD 102807 to attenuate transforming growth factor-β–induced α-smooth muscle actin expression (a marker of hypercontractile ASM). These data indicate that PD 102807 transduces a signaling pathway (AMPK-mediated mTORC1 inhibition) qualitatively distinct from canonical M3 mAChR signaling to prevent pathogenic remodeling of ASM, thus demonstrating PD 102807 is a biased M3 mAChR ligand with therapeutic potential for the management of obstructive airway disease.Airway smooth muscle (ASM) cells attain a hypercontractile phenotype during obstructive airway diseases. We recently identified a biased M3 muscarinic acetylcholine receptor (mAChR) ligand, PD 102807, that induces GRK-/arrestin-dependent AMP-activated protein kinase (AMPK) activation to inhibit transforming growth factor-β–induced hypercontractile ASM phenotype. Conversely, the balanced mAChR agonist, methacholine (MCh), activates AMPK yet does not regulate ASM phenotype. In the current study, we demonstrate that PD 102807- and MCh-induced AMPK activation both depend on Ca M3 muscarinic acetylcholine receptors (mAChRs) are crucial regulators of airway smooth muscle (ASM) tone. Increased M3 mAChR signaling is also a key driver of pathogenesis in asthma and COPD, including increased ASM tone and airway obstruction , inflammWe recently identified PD 102807—an established M4 mAChR antagonist , 21—as avia either canonical (Gq-dependent) or biased (GRK/arrestin-dependent) signaling pathways (A and B) or small interfering RNA (siRNA)-mediated knockdown  did not decrease M3-mediated AMPK phosphorylation induced by PD 102807 or MCh. Interestingly, siRNA-mediated knockdown of STK11 led to an 80% increase of PD 102807-induced phospho-AMPK (p-AMPK) signal. To assess the role of CaMKK in M3-mediated phosphorylation of AMPK, we used the small molecule inhibitor of CaMKK, STO-609. Pretreatment of human ASM cells with STO-609 inhibited both PD 102807- and MCh-induced p-AMPK, by ∼77% and ∼83%, respectively (A and B). To assess the role of CaMKK isoforms in M3-mediated AMPK phosphorylation, we used siRNA to selectively knock down either CaMKK [CaMKK1 (aka CaMKKα) or CaMKK2 (aka CaMKKβ)] and assessed phosphorylation of AMPK as well as phosphorylation of the AMPK substrate acetyl-CoA carboxylase (ACC), as a measure of AMPK activation. Knockdown of CaMKK1 inhibited PD 102807-induced phosphorylation of AMPK by ∼81% and its downstream target ACC by ∼50% (C). Knockdown of CaMKK2 did not affect PD 102807-induced activation of AMPK signaling. Knockdown of either isoform reduced the MCh-induced p-AMPK and p-ACC (D), although knockdown of CaMKK2 was more effective at abrogating MCh-induced p-AMPK (∼96% inhibition) compared to CaMKK1 knockdown (∼68% inhibition). Similarly, MCh-induced p-ACC was fully inhibited by CaMKK2 knockdown, whereas CaMKK1 knockdown resulted in a ∼50% inhibition (D). These findings suggest CaMKK isoforms play a major role in M3-mediated AMPK activation, whereas there is no discernible role for STK11/LKB1 or TAK1. Further, biased ligand PD 102807 differs from balanced ligand MCh in terms of CaMKK-mediated activation of AMPK signaling in ASM cells.We previously demonstrated that stimulation of human ASM cells with M3 mAChR ligands results in AMPK activation pathways . In ordepathways , A and Bn STK11; C did notectively , A and B by ∼50% C. Knockdnd p-ACC D, althouhibition D. These via different mechanisms and with different kinetic profiles. While the MCh-induced p-AMPK is mediated by Gq, the PD 102807-induced p-AMPK is mediated by GRK/arrestin (A and B).Our previous studies have shown that PD 102807 and MCh both induce AMPK phosphorylation, albeit arrestin . In addiarrestin , A and B2+] 0 mM), PD 102807 induced a 4.5-fold increase of AMPK phosphorylation. With [Ca2+] clamped at 0.2, 2, or 20 mM, PD 102807 induced approximately a 3.5- to 4-fold increase of p-AMPK. Under calcium-free conditions ([Ca2+] 0 mM), MCh failed to induce p-AMPK whereas increasing calcium concentrations led to increases in MCh-induced p-AMPK; 0.2, 2, and 20 mM calcium resulted in a 1.5-, 2-, and 5-fold increase of p-AMPK, respectively (C). Collectively, these data indicate that PD 102807-induced p-AMPK is independent of calcium, whereas MCh-induced p-AMPK requires calcium.In order to determine whether AMPK activation requires intracellular calcium, we permeabilized the cells with β-escin and clamped the calcium concentrations at 0, 0.2, 2, and 20 mM. Cells were then stimulated with PD 102807 or MCh to determine the magnitude of p-AMPK induction at each calcium concentration. Under calcium-free conditions (. Maximal Ca2+ increase was induced by stimulating cells with the calcium ionophore ionomycin (1 μM) and was used to normalize agonist-induced Ca2+ response in each experiment.Human ASM cells were plated onto 96-well plates and loaded with 2 μM Fluo-4 acetoxymethyl (BD Biosciences) and probenecid for 1 h according to manufacturer’s instructions. Indicated final concentrations of agonists were added by an automated pipetting system [FlexStation 3 (Molecular Devices)] in duplicate. The calcium-bound Fluo-4 dye fluorescence emission was detected at 525 nm using excitation wavelength of 485 nm by Flexstation 3 and SoftMax Pro 7 software (Molecular Devices), as described previously . MeasureStable expression of a reporter construct for SRE-luciferase was established in hTERT ASM and HEK 293 cells using Cignal Lenti luciferase reporter viral particles (SA Biosciences). Expression of SRE-Luc was maintained using puromycin (2 μg/ml). Cells were serum-deprived for 8 h and then stimulated for 18 h and harvested in passive lysis buffer. Luminescence was determined after adding firefly luciferase substrate reagent using a microplate luminometer.t test was used where appropriate, for comparisons of means of two groups. A value of p < 0.05 was considered significant to reject the null hypothesis.Data analysis was performed using GraphPad (GraphPad Software), and data are expressed as mean ± SD. Group comparisons were performed using a one-way ANOVA followed by Bonferroni’s multiple comparison test. Two-tailed All the data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.