Adolescent sexual debut and non-consensual sex have been linked to higher sexual risk and STI infection in adulthood among men who have sex with men (MSM) in high-income countries. This study aimed to examine adolescent and non-consensual anal sexual debut among Chinese MSM and to evaluate factors associated with adolescent sexual debut and non-consensual anal sex.A cross-sectional study was conducted recently among Chinese men assigned male sex at birth, ≥18 years old, and who had ever engaged in anal sex with a man. Participants answered questions regarding socio-demographics, condomless sex, age at anal sexual debut with a man, and whether the first anal sex was consensual. Factors associated with an adolescent sexual debut (< 18 years old) and non-consensual sex at sexual debut were evaluated. We defined adolescent sexual debut as having anal sex with another man at 17 years old or younger, and the participants were asked whether their first male-to-male anal sex was non-consensual.Overall, 2031 eligible men completed the survey. The mean age of sexual debut was 20.7 (SD = 4.3) years old. 17.6% (358/2031) of men reported adolescent sexual debut, and 5.0% (101/2031) reported a non-consensual sexual debut. The adolescent sexual debut was associated with having more male sexual partners  and condomless anal sex in the last three months . MSM whose sexual debut was non-consensual were more likely to have condomless anal sex , and to have reported an adolescent sexual debut .Many Chinese MSM reported adolescent sexual debut and non-consensual sex, both of which are associated with sexual risk behaviors and drive STI transmission. These findings highlight the need for designing tailored interventions for MSM who experienced adolescent sexual debut and non-consensual sex at debut. Adolescent sexual debut and non-consensual anal sex among men who have sex with men (MSM) may contribute to adulthood high-risk behaviors and sexually transmitted infection (STI) transmission , 2. PrevDue to social and cultural pressures of the local environment, MSM in China are often subject to stigma and social discrimination , 11. As China provides a strong environment for this study. First, with the popularization of gay dating apps , researcThis study aimed to examine adolescent and non-consensual sexual debut among Chinese MSM and to evaluate factors associated with adolescent sexual debut and non-consensual anal sex.This study is a secondary analysis of baseline data from a stepped-wedge randomized controlled trial (RCT). The study evaluated the efficacy of a crowdsourced intervention in promoting HIV testing among MSM in eight Chinese cities , while oThe nationwide, online, cross-sectional study was conducted by the University of North Carolina Project-China in July 2016. We collaborated with a gay partner seeking internet company (Blued) to recruit participants from eight cities in China . Blued is the largest MSM social networking mobile phone application (app) in China, with more than 40 million registers. MSM usually use this platform for socialization, seeking HIV care services, and dating. While most MSM in China uses this platform, the frequency users tend to be young, well-educated, and with high income.http://www.sojump.com), a widely used online survey platform in China [For recruitment, banner advertisements linking to the online survey were sent to registered Blued users in the eight cities. Participants who clicked on the survey link were directed to the online survey hosted by Sojump , marital status , education , Household registration , and annual income . Every participant was asked to self-report their sexual orientation , whether they disclosed their sexual orientation to non-male sexual partners (yes or no), and whether they self-identified as a transgender woman (yes or no).Participants answered whether they had a stable male partner (yes or no), a casual male partner (yes or no), a stable female partner (yes or no), and a casual female partner (yes or no) in the last three months. We also asked the number of male partners they had in the last three months (continuous), whether they engaged in anal sex with a stable male partner in the last three months (yes or no), whether they engaged in anal sex with a casual male partner in the last three months (yes or no), whether they engaged in condomless sex with a man in the last three months (yes or no), and the type of the last partner .Survey questions also covered HIV testing history. Participants answered questions on whether they had ever tested for HIV (yes or no) and the results from their last HIV test .With regard to male-to-male sexual debut, the participants were asked the age at which they first engaged in anal sex with a man. We defined adolescent sexual debut as having anal sex with another man at 17 years old or younger, as 18 years old was considered to be adult in China, and having sex before 18 was considered to have sex early in China . In addiDescriptive analysis was used to present the distribution of socio-demographic characteristics and HIV-related behaviors of the participants. Univariate and multivariable logistic regressions were used to evaluate factors associated with the adolescent sexual debut and non-consensual anal sex at sexual debut, while adolescent sexual debut and non-consensual anal sex at sexual debut were treated as outcomes, to make the data analysis easier. Age, residence, educational level, and annual income were adjusted in the multivariable logistic regressions. All data analysis was completed using SAS 9.4 .Overall, all the 2031 MSM who finished the baseline survey were included in the current study. Their median age was 26.3 (SD = 6.3) years old, while about three-quarters of participants were less than 30 years old (75.8%). In addition, the majority of the participants had household registration in the two study provinces (68.0%), never married (85.4%), and had attended at least some college (66.6%) (Table About 5% of the participants (5.1%) identified as transgender women. Close to three-quarters of the participants self-identified themselves as gay (72.5%), and about two-thirds of the participants reported that they ever disclosed their sexual orientation to others beyond their partners (68.0%). Overall, two-thirds of the participants reported having a regular male partner in the last three months (66.4%), and about two-fifths of the participants reported having a casual male partner in the last three months (40.7%).Over 60% of the participants self-reported that they had ever tested for HIV (63.9%). About half of the participants reported that they did not know their HIV status . Of those who ever tested for HIV, 5.9% reported that they were living with HIV (62/1048).p < 0.001 for each comparison), respectively.The median and mean ages of reported sexual debut were close, so we only reported mean age in the current study. Overall, 17.6% (358/2031) of men reported adolescent sexual debut. The mean ages at anal sexual debut for participants in the age groups of < 20, 20–29, 30–39 and 40 or above were 17.1 (SD = 1.4), 19.8 (SD = 2.8), 23.2 (SD = 4.4) and 29.7 (SD = 7.5) years old (p < 0.001), respectively. The proportion of participants experiencing non-consensual sex at anal sexual debut with another man was higher in those whose sexual debut happened before age 18 (10.1%), as compared to those for whom it happened at 18 or later  .In addition, 5.0% of participants self-reported that they experienced non-consensual sex at anal sexual debut (101/2031). The mean ages at anal sexual debut for participants who experienced and who did not experience non-consensual sex were 15.8 (SD = 1.5) and 21.8 (SD = 4.0) years old  compared to men who did not disclose their sexual orientation. In addition, men reporting adolescent sexual debut with a male tended to have two or more sexual partners in the last three months , were more likely to engage in condomless anal sex with a male partner in the last three months , and to report that their most recent sexual partner being a casual partner   and engaged in condomless anal sex with a male partner in the last three months . In addition, men whose sexual debut with a male was non-consensual were more likely to have ever tested for HIV  Table .Table 3Adolescent sexual debut and non-consensual sex at sexual debut with a male were closely correlated with subsequent condomless anal sex and among Chinese MSM. Our findings indicate that 5% of MSM in the study experienced a non-consensual sexual debut. This study adds to the current literature by providing data on sexual debut and non-consensual sex at sexual debut with a male and identifying the correlates of adolescent sexual debut and non-consensual sex among Chinese MSM, and it is a rare example of research on adolescent and non-consensual anal sexual debut among MSM in a low- and middle- income countries (LMICs) context.We found that about 18% of the participants’ experienced adolescent anal sexual debut, and at the mean age of 20.7 years old. The mean age at anal sexual debut among the participants in our study was similar to two studies in China, , 14 HoweWe found that about 5% of the participants reported a non-consensual anal sexual debut. The proportion of non-consensual anal sex in our study was lower than in a previous small study from China . This prWe also found that about 10% of men with adolescent anal sexual debut also reported non-consensual anal sex. Due to the limited literature on this topic, we did not find any similar study on this topic. However, the strong association between adolescent sexual debut and non-consensual sex in our study suggests that some young gay men are being assaulted or coerced. One potential reason for this phenomenon is that there is a lack of legal protection for gay men, and in the event of a same-sex assault, the perpetrators were rarely held accountable, and victims remain unprotected in China. Thus, strategies such as providing community support and protective services  to people who experienced non-consensual sexual debut would be important. Legal aid and psychological counseling would also be useful, especially in China where the current criminal law does not recognize men being victims of rape .We found that men who reported both adolescent and non-consensual sexual debut were more likely to engage in condomless anal sex. This finding is consistent with the limited literature on adolescent sexual debut , 17, 31.Our study has several limitations. First, the cross-sectional nature of this study means that all the correlations identified in this study should be inferred with caution. Second, we did not collect information on violence, mental health, alcohol, and substance abuse from the participants, while these variables are considered to be highly correlated with adolescent sexual debut and non-consensual sex. Third, as an online survey conducted in eight Chinese cities, our results cannot represent the general situation among Chinese MSM, as the online recruited participants tended to be young and well-educated. Fourth, as all data collected were self-reported, and since same-sex sexual activity is stigmatized in China, the rates of adolescent sexual debut and coerced sex may be underestimated in China, and social desirability bias may be present. However, we anticipate that this bias is small since the survey was online and no face-to-face meeting was involved. Regardless, our study provided useful information for a glimpse of the whole picture of adolescent sexual debut and non-consensual sex among Chinese MSM.A substantial proportion of Chinese MSM reported adolescent anal sexual debut, and 5% of MSM reported non-consensual anal sex at sexual debut. In addition, both adolescent sexual debut and non-consensual sex were associated with current risk behaviors such as condomless sex. These findings highlight the need for evidence-based sex education and intervention to protect the sexual autonomy of young men in the rising male-to-male sex culture in contemporary China. The findings of this study also have implications for expanding research about sexuality among youth. For example, studies evaluating the lifetime and recent non-consensual sex events, law or legal support needs, and health service needs would be very useful for providing tailored services for MSM.


Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is challenging health systems all over the world. Cancer patients have a higher risk of being infected by SARS-Cov-2 and higher coronavirus disease 2019 (COVID-19) severity and mortality. Up to date, there were no data about COVID-19 in patients with thyroid cancer (TCs). The aim of the study was to describe the prevalence of COVID-19 in a well-characterized series of TC patients evaluated for the persistence of the neoplastic disease from March to September 2020; as secondary objective, we looked for the COVID-19 disease severity in a subgroup of multimetastatic TC patients.We evaluated 1464 patients affected by persistent TC: 67 patients who were taking multikinase inhibitors (MKIs) and 1397 under active surveillance for a persistent but stable disease. During the clinical evaluation, all patients were specifically investigated about a positive history of Sars-Cov-2 infection.SARS-Cov-2 infection was identified in 4/1464 (0.3%) cases of patients affected by TC. We identified three cases among patients under active surveillance (0.2%), and one case among patients treated with MKI systemic therapy . This patient was taking vandetanib for metastatic medullary thyroid cancer (MTC), when he came to our attention referring severe fatigue, dyspnea for light physical activities. He presented a mild COVID-19 and he received exclusively supportive care. After a multidisciplinary consultation, we decided against the discontinuation of vandetanib. After 2 months from the infection, he did not present any signs of active infection, and the MTC metastatic disease was stable.We showed that COVID-19 is not more frequent in TC patients than in general population, although a relatively higher prevalence in the group of TC patients treated with MKIs. A single patient with advanced TC and SARS-Cov-2 infection during MKIs treatment had a mild COVID-19 and did not require the discontinuation of MKI therapy. In cases of more severe COVID-19, an accurate evaluation from a multidisciplinary team would consider risks and benefits in taking the decision to continue or stop MKI treatment. On March 11, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) infection outbreak was declared as pandemic by the World Health Organization. As of November 25, 2020, more than 59 million of infected patients have been registered globally, and more than 1,390,000 patients passed away after virus infection disease, now known as SARS-Cov-2. According to the most recent data, coronavirus disease 2019 (COVID-19) presents a mortality rate of 1.38% [Cancer patients are vulnerable patients, mostly due to their multiple comorbidities. Fowler et al.  have recAccording to their “frail” status, neoplastic patients demonstrated to have higher risk of being infected by SARS-Cov-2 . MoreoveAfter 6 months from the begin of COVID-19 pandemic in Italy, we investigated the impact of COVID-19 in patients with thyroid cancer (TC) with particular regard to those with an advanced and multimetastatic disease who were taking multikinase inhibitors (MKIs) as systemic antineoplastic therapy. Main aim of this study was to describe the prevalence of COVID-19 in our series of biochemical and structural persistent TC patients evaluated at the endocrine-oncology unit of our University Hospital from March to September 2020; additionally, we described the COVID-19 disease severity in a subgroup of multimetastatic TC patients who would be theoretically at higher risk to develop medical complication from COVID-19. In particular, we concentrated our attention on those metastatic patients treated with MKIs to analyze the possible interactions between MKIs and anti-COVID-19 drugs.Thank to this survey, we discovered 4/1464 (0.3%) cases of patients affected by TC who had SARS-Cov-2 infection. We identified three cases among patients under active surveillance, and one case among patients treated with MKI systemic therapy . The three patients with TC under active surveillance developed a mild COVID-19 and none of them required hospitalization. No worsening of biochemical and/or structural neoplastic disease was observed after COVID-19.Epidemiological, clinical, and pathological features of patients with metastatic TC who were taking MKIs are summarized in Table Most of the patients were taking lenvatinib (40.3%) or vandetanib (34.7%), while only a few patients were taking cabozantinib (5.6%), selpercatinib (11.1%), or sunitinib (1.4%). At October 2020, the median duration of MKIs treatment was 32 months.On May 7, 2020, after 18 months of vandetanib treatment (200 mg/day), a 64-year-old man affected by an MTC with multiple metastases in cervical and mediastinal lymph nodes, lungs, liver, and bone came to our attention reporting severe fatigue, dyspnea for light physical activities, and a remarkable loss of weight (−6 Kg in 2 months). Otherwise, he denied fever, cough, and further symptoms. At physical examination, he presented only tachypnea. He also reported that, 2 months before, his mother had got SARS-Cov-2 infection and she passed away, after 1 month of hospitalization.− 20.7 mmol/L) and optimal O2 saturation (96.9%). As supposed, nasopharyngeal swab was positive for SARS-Cov-2 infection. Computer tomography (CT) scan documented the appearance of interstitial thickening of the right lung, in addition to the well-known multiple lung metastasis  [During the observational period, 4 of our 67 advanced TC patients died for disease progression but no one died for COVID-19. About COVID-19 mortality in cancer patients, in a cohort of 44,672 of patients, the history of neoplastic disease was described as a significant higher risk of mortality (RR = 0.006) . However= 0.006) . In our Different kinds of cancer as well as different metastatic sites seemed to produce a different impact on the severity of COVID-19. Patients with hematologic cancers , with lung cancer, and with lung metastasis had higher risk of death, intensive care unit (ICU) admission, severe symptoms, and use of invasive mechanical ventilation . MoreoveSince patients with active/progressive disease present higher mortality, the impact of anticancer therapies on COVID-19 mortality came out to the attention and it was addressed by many reports and questioned by many clinicians. For this reason, a multidisciplinary meeting was performed to decide if to continue or not the vandetanib therapy in our COVID-19 affected patient. In a recent article by Lee et al. , 22% of In order to investigate if it could be reasonably safe to continue MKI in our patient during COVID-19 at same or potentially lower dosage, especially if the COVID-19 would become worse, we evaluated the presence of adverse effects (AEs) in common between drugs that had been used against COVID-19 and vandetanib. By performing this evaluation, we took the opportunity to expand this analysis to the other MKIs used in the clinical practice for the treatment of the different types of TC: cabozantinib, lenvatinib, selpercatinib, and sorafenib –24 Tabl, using aThe interactions inducing QT prolongation between vandetanib and chloroquine/hydroxychloroquine, azithromycin, or lopinavir/ritonavir should induce the clinicians to stop vandetanib treatment. Likewise, sorafenib should be stopped in case of chloroquine/hydroxychloroquine therapy. As general rule, caution about QT prolongation must be had in case of treatment with all MKIs whenever an anti-COVID-19 treatment must be started by referring to a recent consensus . If it iThe other possible drug interaction is concerning CYP3A4 inhibition that could decrease the MKI metabolism and increase the risk of AEs. Cabozantinib therapy should be stopped in case of treatment with lopinavir/ritonavir or with nelfinavir, in order to avoid the increase of AEs risk . SimilarBetween March and September 2020, a total of 1464 patients affected by TC were evaluated at the Endocrine Unit of the University Hospital of Pisa. According to the most recent guidelines , 30, we During the clinical evaluation, all patients were specifically investigated about a positive history of SARS-Cov-2 infection . Moreover, on October 2020 we reassessed all the 67 patients taking TKIs by phone questionnaire.This study shows that COVID-19 is not more frequent in TC patients than in general population, although a relatively higher prevalence was found in the restricted group of TC patients treated with MKI. The single case of advanced TC with COVID-19 during MKIs treatment had a mild COVID-19 and did not require the suspension of the systemic antineoplastic therapy. A more severe COVID-19 disease might require a MKI-reduced dosage or suspension; however, the decision is certainly difficult, because the risk of a rapid progression of the malignancy would be very high, and require an accurate evaluation from a multidisciplinary team who would consider all risks and benefits in taking the decision. According to our clinical experience with MKIs, if possible, the reduction of the daily dosage should be preferred respect to the withdrawal if anti-COVID-19 drugs are needed.


Sus scrofa) in eastern Australia. Ballistic testing revealed that lead-based and lead-free bullets achieved similar performance in precision and muzzle kinetic energy (E0) levels . An aerial shooting trial was conducted with wild pigs shot with one type of lead-based and one type of lead-free bullets under identical conditions. Observations were made from 859 shooting events (n = 430 and 429 respectively), with a sub-set of pigs examined via gross post-mortem (n = 100 and 108 respectively), and a further sub-set examined via radiography (n = 94 and 101 respectively). The mean number of bullets fired per pig killed did not differ greatly between lead-based and lead-free bullets respectively (4.09 vs 3.91), nor did the mean number of bullet wound tracts in each animal via post-mortem inspection (3.29 vs 2.98). However, radiography revealed a higher average number of fragments per animal (median >300 vs median = 55) and a broader distribution of fragments with lead-based bullets. Our results suggest that lead-based and lead-free bullets are similarly effective for aerial shooting of wild pigs, but that the bullet types behave differently, with lead-based bullets displaying a higher degree of fragmentation. These results suggest that aerial shooting may be a particularly important contributor to scavenging wildlife being exposed to lead and that investigation of lead-free bullets for this use should continue.In response to the health threats posed by toxic lead to humans, scavenging wildlife and the environment, there is currently a focus on transitioning from lead-based to lead-free bullets for shooting of wild animals. We compared efficiency metrics and terminal ballistic performance for lead-based and lead-free (non-lead) bullets for aerial shooting of wild pigs ( In response to the health threats posed by toxic lead to humans and scavenging wildlife, there is currently a focus on transitioning from lead-based to lead-free (non-lead) bullets for shooting  of wild animals , 2. ConcSus scrofa, known as wild pigs, feral pigs, feral swine, wild boars or wild hogs [Aerial (helicopter) shooting  is a professional wildlife control method used to kill millions of wild animals of at least 23 species worldwide every year . Aerial ild hogs –13. An iild hogs . Lead exild hogs –16.Wild pigs have become a growing focus for wildlife management in many parts of the world. They are considered a pest species across much of their range globally and cause damage to natural and anthropogenic ecosystems . Their iCanis latrans) in the western United States [® cartridges under conditions typical of aerial shooting in Australia. We did not aim to test lead-based and lead-free bullets in general.To our knowledge, no published studies have assessed the efficacy of lead-free bullets for aerial shooting or the lead exposure risks for scavenging wildlife associated with using lead-based bullets for this purpose. However, risks to scavenging raptors have been discussed from the use of other aerial shooting methods, e.g. use of lead shot for the control of coyotes  ballistic testing using inanimate targets, and (ii) a study of aerial shooting outcomes.® cartridges: 1) lead-based Speer® 130 gr non-bonded core hollow-point bullets , and 2) lead-free copper-based Controlled Chaos® 115 grain hollow-point ammunition . Lead-based bullets were of a frangible (‘varmint’) lead-core design typically associated with extensive fragmentation [Our ballistics trials were conducted on a public shooting range near Nowra, NSW, eastern Australia, in October 2018. Ballistic tests were performed on a recently developed non-lead bullet of appropriate size and design for wild pigs and the results were compared to lead-based bullets traditionally used and approved by the Feral Animal Aerial Shooting Team (FAAST) of the NSW National Parks and Wildlife Service  for aerientation . Lead-frentation . The desentation , 35. We ® chronograph  (K) at the level of the muzzle (E0) [Two five-shot groups were measured to assess precision, with group size calculated according to , 36. A Curi, US)  was usedzle (E0)  for 10 sOur aerial shooting trial was conducted on conservation estate (Gwydir Wetlands State Conservation Area) near Moree, New South Wales, eastern Australia  in May 2020. The reserve had a semi-tropical climate; vegetation was characteristic of diverse floodplain wetland communities in the Murray-Darling Basin drainage division .® helicopter . Two shooters were used, and both were professional aerial marksmen accredited under the FAAST program [® SCAR-H LB semi-automatic rifle  chambered in .308 Winchester® with an Aimpoint® CompM4 red-dot sight without magnification , zeroed at 30 m.Helicopter shooting operations targeted all observed wild pigs, adhering to the current Australian national model standard operating procedure  and Civi program . All shoWe were unable to collect ante-mortem (before death) data from wild pigs that were shot. This was due to inherent difficulties associated with examining animals soon after shooting when using an aerial platform , 8 and pin situ as per standard practice during aerial shooting [Our post-mortem sampling strategy and observation procedures were similar to those described in : we usedshooting .Post-mortem examinations were performed on freshly shot carcasses to minimize artefactual post-mortem change. The observer performing post-mortem examinations (ALP) was a veterinarian experienced in collecting data from wildlife shooting programs. We conducted a post-mortem categorization of the location of bullet wound tracts  following methods used in other published aerial shooting studies , 8. Dete® Slate Plus portable digital radiography system  set at 80 kVp and 2.5 mAs and a wireless 14” x 17” (36 x 43 cm) digital plate [We then used radiography to assess the terminal ballistic performance of bullets in animal tissues. Radiography is an accepted method in wildlife studies for detecting lead particles , 47, 48.” x 17” 3 x 43 cm ” x 17” 3 x 43 cm al plate . Number Muzzle kinetic energy was calculated from bullet mass and estimated muzzle velocity for each shot fired during the ballistics trial. Mean muzzle velocity and kinetic energy for the two bullet types were compared using linear models implemented in JAGS  called vFor the aerial shooting trial, the mean number of shots fired per wild pig killed was calculated after each shooting run and differences between bullet types were compared using a linear model implemented in JAGS, as described above. The cost per pig killed was calculated within the model as the product of the number of shots and the cost of each bullet type. From post-mortem examinations, mean body mass of pigs shot with each bullet type was estimated using a linear model, as above, and the mean number of bullet wound tracts was estimated using a linear model with Poisson distribution and log link function. From radiography, the number of bullet fragments detected in each pig were counted manually and rounded to the nearest 10, with a maximum count value of 300. The median number of fragments produced by each bullet type was estimated using a linear model with Poisson distribution and log link function. The mean width of bullet fragment fields for each bullet type was estimated using a linear model. The percentage of pigs receiving shots in each of the anatomical zones  and the shot accuracy (percentage of shots hitting a pig) were analyzed using binomial linear models with logit link function. Estimates are reported as back-transformed posterior means or medians, depending on the skew of the posterior distribution, and 95% credible intervals (CrI).The research was approved by Murdoch University Animal Ethics Committee (O3103/19). The individuals shown in figures in this manuscript have given written informed consent (as outlined in PLOS consent forms) to publish these case details.The details of the bullets and their performance are shown in We shot a total of 859 pigs over five days, 430 with lead-based bullets and 429 with lead-free bullets. Of these, post-mortem examinations were performed on 100 and 108 pigs respectively. Of these, radiography data of diagnostic quality was derived from 94 and 101 pigs respectively.For nine shooting runs, lead-based bullets were used exclusively, and for ten shooting runs, lead-free bullets were used exclusively. An average of 0.18  more shots were fired per pig during runs in which lead-based bullets were used  than during those in which lead-free bullets were used . However, the probability that the difference between bullet types was > 0 was only 65%. The mean number of bullet wound tracts detected via post-mortem was slightly greater for pigs shot with lead-based bullets  than for pigs shot with lead-free bullets  . There was a 90% probability that the number of wound tracts per pig was greater for lead-based bullets than for lead-free bullets. Based on these figures, 80% of lead-based bullets and 76% of lead-free bullets struck the intended target. Shooters reported identical satisfaction scores (3 out of 5) for 100% of shooting runs, regardless of bullet type used.Mean body mass of shot pigs was almost identical for those shot with lead-based bullets  and lead-free bullets  . The median probabilities of bullets striking the thorax, abdomen, cranium and limbs were similar for both bullet types, but the odds of a pig being struck in the neck were 2.67 times greater  , during Radiography results indicated that metallic fragments were visible in all pigs. For 95.7% of pigs shot with lead-based bullets, >300 visible bullet fragments were visible, while 89.1% of pigs shot with lead-free bullets had <100 visible fragments . RadiogrFragment size varied, with average fragment size being classed as small (< 1 mm)  for 100%The costs of purchasing lead-based and lead-free ammunition was AUD$1.31 and AUD$1.80 per cartridge, respectively. The mean cost per pig killed wild pig was AUD$5.36 for lead-based bullets  and AUD$7.04 for lead-free bullets , a difference of AUD$1.68  per pig.Our results demonstrated that fragmenting lead-free bullets produced comparable efficacy to lead-based bullets for the aerial shooting of wild pigs. To our knowledge, this is the first study to report the use of lead-free bullets for aerial shooting. Accuracy and precision were similar for both bullet types, as was muzzle kinetic energy, despite the difference in bullet weight (130 gr vs. 115 gr), owing to the higher muzzle velocity of the lead-free bullets. Costs were comparable with lead-free cartridges ~27% more expensive than lead-based cartridges, but this difference ($1.47 per cartridge), while considerable, is relatively minor in the context of operating costs for an aerial shooting program . RadiogrCapreolus capreolus) in the United Kingdom [Cervus elaphus/canadensis) in the US [Alces alces) in Scandinavia [Several recent studies have shown comparable outcomes for lead-based and lead-free centerfire ammunition for ground-based shooting of large mammal species. These studies have evaluated shooting of large ungulates such as roe deer . FourthRusa unicolor) [Capra hircus) and fallow deer (Dama dama) [We do not suggest that the results of the present study are indicative of lead-free performance for aerial shooting of all species. Further studies are required to assess the efficacy of lead-free bullets for aerial shooting of larger species (e.g. sambar deer (nicolor) ) and thema dama) .The main arguments for the use of lead-free ammunition are to prevent harm to scavenging wildlife through lead exposure, to human consumers of game meat  and to pAlces alces) hunting [We argue that aerial shooting of smaller ungulate species (< 150 kg) is likely to pose the greatest threat of harmful lead exposure to scavenging wildlife of all shooting methods for several reasons. First, frangible (‘varmint’) but relatively heavy (~130 gr) lead-core (not bonded) bullets are used. Bonded lead-core bullets tend to be used for larger ungulates and lose less of their mass to fragmentation when compared to lead-core bullets, e.g. 10–24% and 18–27% respectively for moose  [Lead fragments from aerial shooting programs in Australia pose risks to raptors such as wedge-tailed eagles (a audax) –66. If ta audax)  is takena audax) . For an a audax)  suggest a audax) , estimata audax) .There is also the possibility that the large, sharp and hard (compared to lead) copper fragments produced by the lead-free bullets could cause mechanical  damage to the gastro-intestinal tracts of scavenging animals. While the potential for copper toxicity has been assessed for lead-free bullets , we are Our results suggest that lead-based and lead-free bullets are similarly effective for aerial shooting of wild pigs, but that the bullet types behave differently, with lead-based bullets displaying a higher degree of fragmentation. Aerial shooting may be a particularly important contributor to scavenging wildlife being exposed to lead and investigation of lead-free bullets for this use should continue. Our results emphasize the importance of considering aerial shooting in discussions of transitioning to lead-free bullets.S1 Data(CSV)Click here for additional data file.S2 Data(CSV)Click here for additional data file.S3 Data(CSV)Click here for additional data file.S4 Data(CSV)Click here for additional data file.


The genotype/alleles rs2295080-G, rs11121704-C, rs1057079-C and rs73105013-T associated with an increased, whereas rs2295080-TT, rs2295080-T, rs11121704-TT, rs1057079-TT, rs1057079-T, rs573775-AA and rs73105013-C with a decreased occurrence of wet AMD. In addition, the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were a positively correlated cumulative number of anti-VEGF injections in 2 years. Therefore, variability in autophagy genes may have an impact on the risk of wet AMD occurrence and the efficacy of anti-VEGF treatment.Age-related macular degeneration is an eye disease that is the main cause of legal blindness in the elderly in developed countries. Despite this, its pathogenesis is not completely known, and many genetic, epigenetic, environmental and lifestyle factors may be involved. Vision loss in age-related macular degeneration (AMD) is usually consequence of the occurrence of its wet (neovascular) form that is targeted in the clinic by anti-VEGF  treatment. The wet form of AMD is associated with the accumulation of cellular waste in the retinal pigment epithelium, which is removed by autophagy and the proteosomal degradation system. In the present work, we searched for the association between genotypes and alleles of single nucleotide polymorphisms (SNPs) of autophagy-related genes and wet AMD occurrence in a cohort of Finnish patients undergoing anti-VEGF therapy and controls. Additionally, the correlation between treatment efficacy and genotypes was investigated. Overall, 225 wet AMD patients and 161 controls were enrolled in this study. Ten SNPs  in the  Age-related macular degeneration (AMD) is the most common cause of permanent sharp and color vision loss in the elderly which has a significant socioeconomic burden on patients and their caregivers and societies . The AMDMost commonly, AMD starts in its dry form, which may progress to wet (exudative) AMD in 10–20% of cases ,10. DespThe cellular pathogenesis of AMD includes impaired proteolytic clearance in the macular retinal pigment epithelium (RPE). In the normal visual cycle, RPE cells are constantly exposed to light and phagocytose the lipid-rich outer photoreceptor segments. This combined with high metabolic activity results in an enhanced oxidative stress . ChronicATG5 and ATG7, was associated with an AMD-like phenotype in mouse RPE cells [Autophagy is a lysosomal clearance mechanism to degrade damaged, aged or unneeded proteins in cells. Detailed autophagy signaling has been reviewed elsewhere . BrieflyPE cells . This phPE cells ,21,24,25PE cells . HoweverPE cells ,20,21,26Since most of the autophagy studies for AMD pathology have been done in cell culture and animal models, we wanted to unravel how the single nucleotide polymorphisms (SNPs) of autophagy genes have been associated with wet AMD and the outcomes of anti-VEGF treatments in a Finnish case-control cohort.pro re nata (PRN) were applied in the follow-up and treatments of wet AMD patients [The study population consisted of 225 wet AMD patients and 161 controls . The meapatients .Clinical data included blood pressure, anti-cholesterol, anti-coagulant and anti-platelet drugs. Body mass index (BMI) and smoking history were also examined .Ethics Committee of the Kuopio University Hospital (42/2014) has approved the study and the tenets of the Declaration of Helsinki were followed. All participants signed an informed consent form.g centrifugation at 20 °C for 15 min. DNA was extracted from the peripheral blood leucocytes with QiAamp DNA Blood Midi kit  according to the manufacturer’s instructions. The quality and the amount of the extracted genomic DNA were measured with NanoDrop Microvolume spectrophotometer model ND-1000 .Some 10 mL aliquots of whole blood were taken into a 3 mL tube containing EDTA, shaken gently 10 times, and kept at room temperature for 30–60 min prior to 3200× The public domain of the single-nucleotide polymorphism database at the National Center for Biotechnology Information and the related literature to identify potentially functional polymorphisms in autophagy genes were used for SNP selections. The selected polymorphisms have known distributions in various European populations. SNP selection favored those with a minor allele frequency no less than 3%. Moreover, we selected polymorphisms at different positions in the relevant genes. There were polymorphisms located directly in the core promoter and thousands base pair upstream from the transition start site. We selected both exonic and intronic locations as well as the 3′ untranslated region. This diversity in the locations of SNPs within genes ensures their potential various functionality. Genotyping was performed using TaqMan SNP Genotyping Assays   accordint test or Mann–Whitney U test was used. In some bivariate and multivariate analyses, we used the approach of resampling with replacement  to make sure that the revealed differences were not detected by a pure chance.The descriptive data are shown as the mean ± SD or as the number of patients in each category. The normality of the studied group was verified with the Shapiro–Wilk test, the homogeneity of variance was checked with Brown–Forsythe test. Accordingly, either the unpaired Student’s d-jackknife technique with the patient group as the modelled class. This was intended to overcome any possible bias related to relatively low sample sizes. The goodness-of-fit of logistic regression models showing a significant discrimination between controls and patients was estimated with the Hosmer–Lemeshow test. The analysis of collected data was done in Statistica 12 , SigmaPlot 11.0 , Resampling Stats Add-in for Excel v.4  and StudSize3.02 .An unconditional multiple logistic regression model was used to calculate the associations between the studied polymorphisms and the occurrence of a disease (with or without stratification for sex). The results are shown as odds ratios (ORs) with a 95% confidence interval (±95% CI). In addition, the significant outcomes were further validated with the use of two approaches: the bootstrap-boosted multiple logistic regression  and the cross-validated logistic regression that corresponds to the mTOR, ATG5, ULK1, MAP1LC3A, and SQSTM1) SNPs cover the genes and 10 kbp flanking regions both up- and downstream (p < 0.05) differences were detected in genes that showed agreement with Hardy–Weinberg equilibrium (HWE) (p < 0.05) departed from the HWE disequilibrium.Autophagy-related genes (wnstream . Based ownstream . The totum (HWE) . HoweverAs shown in Since the amount of anti-VEGF intravitreal injections varies between patients and with personalized demands, we were curious to analyze the connections of autophagy gene SNPs and the amount of cumulative injections during two years of treatment. Our results showed that the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were associated with two years of a cumulative number of injections (0.13 < R < 0.14 Spearman’s correlation coefficient) .AMD is a multifactorial, multi-compartmental complex eye disease. The importance of genetic factors and their variability were confirmed in many studies (reviewed in ). HowevemTOR gene loci rs2295080, rs11121704 and rs1057079 indicated OR 0.6 protection against wet AMD. The ATG5 rs573775 A allele and the LC3 rs731105013 C allele SNPs showed a similar trend in the prevention of wet AMD. These SNP associations revealed that autophagy cascaded from the autophagy initiation to the flux termination and may have a regulatory role in the development of wet AMD. The rs73105013 T allele might be a useful genotyping marker in deeper wet AMD risk analysis. Interestingly, the mTOR rs2295080 G allele associated with both an increased wet AMD and weaker anti-VEGF treatment response once the cumulative amount of injections for two years was calculated. According to the Finnish wet AMD guidelines, a pro re nata protocol was used for anti-VEGF injections [mTOR rs1057079 T allele had a protective sign to develop wet AMD, but was associated with a weaker anti-VEGF treatment response, as it was related to the number of given injections. Similar observations were made for the protective genotype of rs800292 variant of the complement H (CFH) gene with a poor anatomical response [CFH, ARMS2, and VEGFA was associated with a younger age of AMD onset in combination with a poor response rate to anti-VEGF treatment [C10orf88 and UNC93B1 genes associated with a worse response to anti-VEGF therapy [Since AMD is strongly associated with disturbed proteostasis ,23, we tjections . To showresponse . A cumulreatment . A limit therapy . Rare vaCFH Y402H at-risk genotypes [ADIPOR1 variant was associated with advanced AMD in the Finnish population [Numerous AMD-related risk factors have been documented, but the evidence and strength of association is variable depending on ethnicity and study design. Our Finnish cohort shows that smoking history and the use of anti-cholesterol drugs are associated with wet AMD. One systemic review and meta-analysis showed moderate and consistent risk associations with higher BMI, history of cardiovascular disease, and hypertension . A recenenotypes . In numeenotypes . Our resenotypes . In our enotypes ,37. In apulation .An increased BMI is found to be associated with the increased chance of developing AMD in some studies, while some other studies show no association . We did The number of injections, regimens and patient compliance changes between patients. We used Finnish national guidelines for modified PRN which allows a different anti-VEGF drugs and injection regimen. By identifying the prognostic biomarkers of AMD that could be used to select the individual drug treatment and follow-up protocol, the quality and safety of treatment and patient compliance could be improved. This would also more precisely allocate available resources, especially as the need for AMD patients care increases as the population ages. Variability in autophagy-related genes in the Finnish population may moderately contribute to wet AMD pathogenesis and therapy. The Finnish population has reduced diversity and homogeneity, which is beneficiary for gene mapping studies. Since we used RT-PCR-based SNP genotyping, further work should include the whole-genome sequencing of the samples with subsequent analysis and public deposition of the acquired datasets. We believed that autophagy is one important target to develop pharmacogenetics and personalized medicine for AMD.Autophagy genes are associated with wet AMD and the efficacy of anti-VEGF treatment in a Finnish Case-Control Study.


E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for  Enterobacteriacea family, involves recruiting omptin proteases to cleave AMPs and making them ineffective. Omptins are a family of outer membrane proteases that share 40–80% amino acid sequence similarity and a highly conserved active site . Basically, the contribution from the autoproteolyzed fragment is removed when calculating the refolding efficiency. For the RC-31 and RC-10 samples, the refolding efficiency was now close to ~60% and 20%, respectively. However, for the RC-31 samples, the refolding efficiency varied between 50–70% for different batches (data not shown) and was difficult to control using the protocol adopted from Kramer et al. (2000) [Although the initial refolding efficiency of ~69% and 32% for the RC-31 and RC-10 samples, respectively, were based on the relative intensities of the 33.5 and 27 kDa bands for the non-heated samples in . (2000) . DespiteLPS has previously been shown to be essential for OmpT’s activity ,37,38,39To explore the role of LPS in OmpT autoproteolysis, OmpT IBs solubilized in 8 M urea were refolded in a buffer containing 31 mM detergent (RC-31) along with excess LPS (4.5 mg/mL) or no LPS. For the sake of convenience, we refer to these two refolding conditions as RC-31(LPS) and RC-31 to differentiate between samples refolded in the presence of excess or no externally added LPS, respectively. The RC-31(LPS) and RC-31 samples collected at three different intervals over a 72 h period and run on an SDS-PAGE exhibited an almost identical band intensity profile . The intMALDI-TOF experiments were undertaken to confirm that the substrate specificity of OmpT (RC-31(LPS) samples) for the dibasic sites was preserved , in agreE. coli that shares some structural similarity with OmpT, and has a known LPS-binding motif [138, Arg175, Lys226, Glu136, and Tyr134 on OmpT  or all five (mt5OmpTLPS) of the predicted LPS binding residues modified were expressed and purified . Together with additional structural data focusing on the OmpT–LPS interaction, we should be able to gain better insights into the role and mechanism of LPS in activating OmpT in vitro and in vivo. Moreover, the previously proposed  putative


Since the 1970s outcome of aplastic anemia (AA) patients has improved significantly due to the introduction of immunosuppressive therapy (IST) and allogeneic hematopoietic transplantation (HCT). However, patients may suffer from persistent disease, relapse, clonal evolution, graft-versus-host disease and other late effects. Here, we analyse very long-term outcome of all AA patients at our institution comparing not only survival, but also response status and complications.Patient charts of all 302 AA patients treated between 1973 and 2017 at the University Hospital Basel, Switzerland, were retrospectively analysed.First line treatment was IST in 226 (75%) and HCT in 76 (25%) patients. Overall survival at 30 years was similar in patients treated initially by HCT and IST (44% (±14%), and 40% (± 9%) respectively, with better results in more recent years. Partial and no response occurred more frequently after IST, relapse incidence after IST was 24 %, whereas non-engraftment and graft failure was documented in 15 patients (19 %) after HCT. Clonal evolution to myelodysplastic syndrome / acute myeloid leukemia was 16 % at 25 years in IST patients, 1.3 % in HCT patients, iron overload  and cardiovascular events  occured significantly more often in IST than HCT treated patients. The majority of long-term survivors, 96% of those alive at 25 years, were in complete remission at last follow up, irrespective of the initial treatment modality.Very long term survivors after AA are those with stable hematopoietic recovery. Aplastic anemia (AA) is a rare hematological disease characterized by bone marrow failure causing pancytopenia with symptoms of bleeding, anemia and infections. Originally a fatal condition, outcome has improved significantly since the 1970s, particularly due to introduction of immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and hematopoietic cell transplantation (HCT) as the major first line treatment strategies . The staIn this single-center study, we analysed very long-term outcome of all patients with AA treated since 1973 by first-line HCT or IST, comparing not only survival, but also long-term response status and complications.This retrospective cohort study included 302 patients diagnosed with AA who received HCT or IST as first-line therapy from 1973 to 2017 at the University Hospital Basel, Switzerland. Data included demographic information, pre-treatment blood values, date, type, source and protocols for therapy, response to therapy, status at last follow-up, causes of death and type of complications. Given the long observation period, many diagnostic methods had changed over time. Follow up was completed by 2019. Patients were followed at our center. Additional follow-up informations were obtained from referring centers.Diagnosis and severity were defined by the Camitta Criteria . For sevA total of 76 25 %) patients received HCT as first line treatment, the first HCT was performed in 1973. Twenty nine patients 10%) received HCT as a second- or third- line treatment for relapse or non-response to immunosuppression. Treatment protocols and drug preparations changed over time; the majority of patients were conditioned with cyclophosphamide. After 1994 ATG (Thymoglobulin) was added. Fludarabine was given additionally since 2000 for older patients. Irradiation  was used in a minority of patients . GvHD prophylaxis consisted of methotrexate, combined with cyclosporine since 1979. Bone marrow was used as a primary source of stem cells , less frequently peripheral stem cells  or cord blood . Mainly matched-related donor HCT was performed . Unrelated donor HCT was mostly second line treatment (n=24); 9 patients had unrelated donor HCT as part of first line treatment . A total of 226 (75 %) patients received IST as first line therapy, of which the first course of ATG-based immunosuppression was given in 1973. Treatment protocols and drugs preparations changed over time: From 1976 to 1991 Lymphoser Berna , from 1992 to 2006 Lymphoglobuline Merieux  and since 2007 ATGAM  was used as equine antithymocyte globulin. Besides, thirteen patients were treated with rabbit ATG (Thymoglobulin). From 1973 to 1981 thirty-two patients received haploidentical bone marrow infusion in addition to ATG, as previously described . In none5 % patie% receiveResponse was classified according to NIH criteria . CompletRelapse in the HCT group is defined as primary non-engraftment or late graft failure. In patients treated by IST relapse is defined as decreasing blood count counts requiring transfusions or administration of another course of IST respectively performing HCT in patients with a prior response (PR or CR) .Patients were evaluated regarding the following complications: clonal evolution to MDS/AML, secondary lymphoma and solid tumor, disease-related complications such as infections , hemorrhage and thromboembolic events, therapy-related complications such as iron overload, cardiovascular events, endocrine dysfunctions, aseptic osteonecrosis and GvHD. Acute (aGvHD) and chronic GvHD (cGvHD) were diagnosed and graded according to published criteria , 17. In Cause of death was subdivided in six categories: disease-related (infection or hemorrhage), treatment-related , clonal evolution (MDS or AML), secondary malignancy (solid tumor or lymphoma), other (no association to the disease) and unknown.> partial remission even after several lines of treatment at the time of last treatment; e.g. a patient with IST, relapsing after response with HCT as second line treatment with long term survival with response, is considered a current event free survivor. Survival probabilities were estimated using the Kaplan-Meier estimator. The log-rank test was used to compare survival between patient groups. Cumulative incidence was used for relapse, secondary clonal complications and GvHD, with death of other causes as competing events, significance testing was by Fine and Gray. Data was analysed in the IBM SPSS Statistics 22 and STATA v15 for cumulative incidence.The chi-square test was used for comparisons of categorial and the Mann-Whitney U-test for continuous variables among groups. OS was defined as time to death, event free survival (EFS) as time to death, time to relapse in responding patients or time to secondary clonal events. We defined current event free survival (cEFS) as the probability of being alive and in Median follow up of surviving patients was 17 years (range: 1-41 years). Median age at diagnosis was 24 years (1-80 years), 18 years (1-66 years) in patients treated by HCT compared to 27 years (IQR 1-80 years) in the IST group. Sixty-nine patients (22.8%) were diagnosed with NSAA, 142 patients (47%) with SAA, 79 patients (26.2%) with VSAA and 9 (3%) with inherited bone marrow failure . Additional patient characteristics are shown in Table First line treatment was IST in 226 patients (75%), HCT in 76 (25%) of patients. Many patients had second line and subsequent lines of treatment, e.g. IST was second line treatment in 70 (61%) and HCT in 18 (16%); some patients had HCT as third  or subsequent line of treatment. Overall, 103 of the 302 patients had a HCT during their treatment course. The third most common second or subsequent line of treatment was the use of eltrombopag . Other treatment included growth factors (n=18), cyclosporine as single agent treatment (n=6) and eculizumab (n=2) to treat clinical paroxysmal nocturnal hemoglobinuria (PNH).Figure Figure Figure Figure Bleeding and infection were common , but its occurrence did not differ significantly between the two treatment groups Table .Table 3CIron overload was significantly more common in patients treated by IST  as well as cardiovascular events . Acute GvHD Grade 2-4 occurred in 36.8% of the transplanted patients, of which 46.4% resolved over time. Fifty-five patients (72.4%) were at risk for cGvHD, of those 25 (32.9 %) developed cGvHD, of which 44% resolved.Secondary malignancy, mostly solid tumors but also a few cases with lymphoma, occurred in 10.5% of patients in the HCT group and in 7.5% of patients in the IST group (p=0.41).During follow up 145 patients (48%) died Figure . There wIn this retrospective study we analysed long-term outcome of AA patients receiving first-line treatment with HCT or IST at our center since 1973. Considering the extensive study period over several decades and a median follow-up time of surviving patients of 17 years, we could gain insights on very long term survival of AA patients, therapy response and complications.By this, we can report five major findings from our cohort: First, most deaths occured early and response obtained either through HCT or IST tended to be stable after five years, particularly in the second and third decade after treatment. Second, a considerable proportion of patients became long term responders and remained in remission long-term, with 96% of survivors at 25 years after first line treatment being in complete remission. HCT patients reached CR earlier whereas NR and PR was observed more often in IST patients, characterizing patients at risk for relapse. Since blood counts in IST treated patients more often do not return to normal and long-term cyclosporine maintenance therapy is required in a subgroup of patients , explainThis study is unique in the very long term follow-up of AA patients treated since the 1970s. Although treatment strategies - particularly supportive care - has changed and outcome has considerable improved since the 1990s, this series can answer additional questions about long term prospects of these patients since the 1970s, given that a considerable propotion of patients is diagnosed at a young age. Over many years of follow-up the rate of severe late complications occuring in this population is overall moderate and focuses on very specific causes:Clonal evolution to MDS/AML was seen more often in patients treated by IST than by HCT, with a rate (10 – 15 %) not higher than published previously , 9, but Next to clonal evolution, IST as well as HCT treated patients showed an elevated risk for solid tumors , 9 emphaAlso the screening for iron overload and cardiovascular events in IST patients has not been addressed and compared with HCT patients so far. The increased rate of iron overload in our cohort could be explained by higher transfusion loads in IST patients, since response to therapy is often delayed and patients remain transfusion dependent for months after ATG. In addition, IST patients are at higher risk of relapse (11), leading to additional transfusions. At last, effective and tolerable iron chelation therapy had been introduced in recent decades  and may With respect to cardiovascular events, it is well-known that these occur at a higher rate in HCT patients than in the normal population . HoweverOverall, the higher complication rate in IST patients  emphasizes the need for outcome registries and consensus recommendations on the post-IST follow up care in AA patients and validate them thereafter.Many studies have compared first line treatment with IST to HCT as an initial strategy , 6, 24. Several trials also have reported on the overall response to IST and HCT in AA , 25, 26.Several limitations of the study merit consideration. The diagnosis criteria for AA have changed through the decades although the Camitta criteria had been defined already in the 1970 . NeverthIn conclusion, the majority of AA survivors experience a good long-term survival and regain hematopoiesis irrespective of treatment modality, but patients treated with IST suffer from more complications and incomplete treatment response. To improve the outcome of IST patients consensus recommendations on post-IST follow-up care are urgently needed.


Suboptimal circulating vitamin B12, folate and homocysteine concentrations during fetal life seem to be associated with cardiometabolic health at school-age. We examined whether fetal exposure to lower circulating vitamin B12 and folate concentrations and higher circulating homocysteine concentrations is also associated with early signs of atherosclerosis at school-age.This study among 3826 school-age children and their mothers was embedded in the Generation R Study, a population-based prospective cohort study from early pregnancy onwards. We examined the associations of early-pregnancy and cord blood serum total and active B12 and plasma folate and homocysteine concentrations with common carotid artery intima-media thickness and distensibility in the children aged ten years.As compared to normal early-pregnancy serum total B12 concentrations (≥145 pmol/L), low serum total B12 concentrations (<145 pmol/L) were associated with higher carotid intima-media thickness in the children at school-age (difference 0.09 standard deviations score (SDS); 95% confidence interval (CI): 0.01, 0.16). As compared to normal early-pregnancy plasma folate concentrations (≥8 nmol/L), low plasma folate concentrations (<8 nmol/L) were associated with lower carotid disten-sibility in the children at school-age . Early-pregnancy circulating total and active B12, folate and homocysteine concentrations measured continuously were not associated with carotid intima-media thickness or carotid distensibility in the children at school-age. One SDS higher plasma homocysteine concentrations measured in cord blood at birth was associated with a −0.05 SDS  lower carotid distensibility at school-age. Cord blood total and active B12 and folate concentrations were not associated with carotid intima-media thickness or carotid distensibility at school-age.Circulating total B12, folate and homocysteine concentrations during fetal life seem to be associated with markers of subclinical atherosclerosis at school-age. Further studies need to examine the causality and mechanisms underlying these associations. AutopFolate, vitamin B12 and homocysteine are micronutrients that interact in the one-carbon metabolism, which is crucial for cellular growth and differentiation, nucleic acid synthesis and DNA methylation ,17. VitaWe hypothesized that higher circulating vitamin B12 and folate concentrations and lower circulating homocysteine concentrations during fetal development are associated with higher carotid intima-media thickness and lower carotid distensibility at school-age. In a population-based study among 3826 mother–child pairs, we examined the associations of these one-carbon metabolism markers, sampled in early pregnancy and in cord blood at birth, with common carotid artery intima-media thickness and distensibility in children aged ten years.22.1This study was embedded in the Generation R Study, a population-based prospective cohort study from fetal life onwards in the city of Rotterdam, the Netherlands . The Med2.2As described previously, maternal venous blood samples were drawn in early pregnancy  and cord blood samples were taken by midwives  . EDTA se2.3−1*10−3) as main outcomes of interest. In a reproducibility study performed among 47 subjects, the interobserver and intraobserver intraclass correlation coefficient were greater than 0.85 for carotid distensibility and 0.94 for carotid intima-media thickness.When children visited the research facility at age ten years, we measured intima-media thickness and distensibility three times at both common carotid arteries (n = 5746) using the Logiq E9  device. Children were in the supine position, with the head tilted slightly away from the transducer. The common carotid artery was identified in a longitudinal plane, ~10 mm proximal from the carotid bifurcation. We obtained six recordings that ideally included multiple heart cycles. The analyses were performed offline and semi-automatically, using the application Carotid Studio ). For each recording, at all R-waves of the simultaneous ECG, carotid intima-media thickness was computed at the far wall as the average distance between lumen-intima and media-adventitia borders. The average carotid intima-media thickness of all frames of the acquired image sequence was computed. The distensibility coefficient, or distensibility, was defined as the relative change in lumen area during systole for a given pressure change. Lumen diameter was computed as the average distance between the far and near media-adventitia interfaces, for each frame of the acquired image sequence. Distension was calculated as the difference between the maximal (diastolic) and minimal (systolic) lumen diameter. Per recording, the average distension and diameter values were used to compute the average carotid distensibility. During these offline analyses, we excluded 516 and 704 children, without any carotid intimamedia thickness or carotid distensibility measurement, respectively. Reasons were no appropriate recording, insufficient quality of the recording, recording of the heart only, or no blood pressure measurement available to calculate carotid distensibility. Further data processing for the remaining 5230 and 5042 children with carotid intima-media thickness and carotid distensibility data, respectively, was performed using R . We excluded 9 children with unreliable low or high carotid distensibility values. All 5230 and 5033 children with at least one successful carotid intima-media thickness or carotid distensibility measurement were included in the current study. We used the overall mean carotid intima-media thickness (mm) and carotid distensibility (kPa2.4We constructed a directed acyclic diagram . Potenti2.5First, we performed a non-response analysis by comparing subject characteristics with and without any carotid intima-media thickness or carotid distensibility measurement, using Student’s t-tests, Mann–Whitney tests and Chi-square tests. Second, we examined the associations of early pregnancy and cord blood total and active B12, folate and homocysteine concentrations with the overall mean carotid intima-media thickness and carotid distensibility using multivariable linear regression models. We also explored associations of folic acid supplementation and of dichotomized maternal folate and total B12 concentrations with carotid intima-media thickness and carotid distensibility. To compare effect estimates, we analyzed continuous exposures and outcomes in SDS, after natural log transformation of carotid distensibility, which had a skewed distribution. Basic models were adjusted for sex, child age and gestational age at blood sampling. Main models were additionally adjusted for maternal age, education, parity, pre-pregnancy body mass index, smoking and alcohol consumption during pregnancy and child ethnicity. We performed several sensitivity analyses. First, we explored whether birth weight SDS, and body mass index SDS and blood pressure at the same age as the carotid artery measurements substantially changed the results obtained using the main models. Birth weight and body mass index were considered as potential mediators. We considered blood pressure to be involved in the same pathway as the outcomes under investigation. Only in case of associations with P < 0.05 in the main models, we additionally adjusted for circulating concentrations of other one-carbon metabolism markers to see whether associations were specific to the respective exposure under investigation. Second, as early signs of atherosclerosis seem to depend on ethnicity, we assessed whether the effect estimates of the associations were similar in the homogeneous subgroup of children from Dutch ethnic ancestry . Third, 33.13.2Cord blood total and active B12 and folate concentrations sampled at birth were not associated with carotid intima-media thickness or carotid distensibility at school-age . However3.34Results from our population-based prospective cohort study from early pregnancy onwards suggest that low maternal early-pregnancy total B12 concentrations are associated with higher carotid intima-media thickness in the children at school-age. In addition, low maternal early-pregnancy folate concentrations seem to be associated with lower carotid distensibility in the children at school-age. Higher cord blood homocysteine concentrations sampled at birth were associated with lower carotid distensibility at school-age.High carotid intima-media thickness and low carotid distensibility are early signs of atherosclerosis, which may partly originate from the earliest phase of life ,5,9–15. We observed that low maternal early-pregnancy total B12 status was associated with higher carotid intima-media thickness in the children at school-age. Vitamin B12 is needed to convert methyltetrahydrofolate and homocysteine into tetrahydrofolate and methionine ,17. TetrTo the best of our knowledge, the current study is the first that assessed the associations of one-carbon metabolism markers during pregnancy with early signs of atherosclerosis in the children.A few previous studies examined the associations of one-carbon metabolism markers with carotid intima-media thickness in adults. A study among 818 Canadians reported positive associations of circulating homocysteine but not vitamin B12 or folate concentrations with carotid intima-media thickness . A studyin utero with childhood health [The associations of one-carbon metabolism markers during fetal life with carotid intima-media thickness and carotid distensibility at school-age may be explained by the positive association between homocysteine and atherothrombogenesis . This ind health . The cycin utero, our findings may not be generalizable to the general population. We constructed a directed acyclic diagram in order to visualize relationships between exposures, outcomes and covariates. Using this approach, we attempted to minimize bias in the effect estimates of the models by selecting the relevant covariates only for inclusion in the models. Still, residual confounding might be in issue in the observed associations, as in any observational study.Strengths of this study are the fact that it was set in a large observational birth cohort and the availability of repeated measurements of one-carbon metabolism markers, including active B12. This study also has some weaknesses. We had no information on circulating concentrations of one-carbon metabolism markers available at school-age. The ranges of carotid intima-media thickness and carotid distensibility were small, but seem comparable with other studies among healthy children of similar age ,42. Impo5Our findings suggest that circulating total B12, folate and homocysteine concentrations during fetal life may be associated with markers of subclinical atherosclerosis at school-age. The causality and mechanisms underlying these associations need further study.Supplementary data


Breast cancer-related lymphedema (BCRL) is one of the most common complications of breast cancer treatments, which may be exacerbated by obesity. Dysbiosis may negatively impact the management of obesity and lymphedema by increasing inflammation. The objective of this study was to assess the effects of supplementation with synbiotics on inflammatory markers, serum leptin concentration and edema volume in overweight and obese BCRL women following a low-calorie diet (LCD). In a randomized double-blind controlled clinical trial, 88 breast cancer survivors with lymphedema were supplemented once a day for 10 weeks with either a synbiotic or a placebo capsule. Both groups were under a low-calorie diet (LCD). At the end of the study, synbiotic supplementation resulted in a significant reduction in leptin (P=0.003) and TNF-α (P=0.039) between the study groups. Besides, edema volume was significantly reduced within the synbiotic group after the intervention. We did not observe any significant effects of the synbiotic supplementation in hs-CRP, and IL-1β between the study groups . Conclusively, synbiotic supplementation along with an LCD program in breast cancer survivors with lymphedema had beneficial effects on the concentration of serum inflammatory markers and edema volume.  Breast cancer (BC) is an important health concern in women worldwide . Due toBreast cancer survivors may experience lymphedema complications for months or years after cancer treatment . BreastLymphatic injury due to cancer treatments leads to the disruption of lymph flow. Lymphatic fluid stasis may cause increased inflammatory mediators such as TNF-α, fat deposition and progressive fibrosis , which We are aware of no study that has examined the effect of synbiotic supplementation on inflammatory profiles and edema volume in BCRL. The current study was therefore performed to investigate the effects of synbiotics on the inflammation status and edema volume of breast cancer survivors with lymphedema following a low-calorie diet. The result of this research may provide new strategies for lymphedema management.Women with BC related lymphedema who were treated by complex decongestive therapy (CDT) were recruited in this randomized double-blind controlled clinical trial. Lymphedema therapy was carried out in two phases, including an intensive phase that is performed in the clinic and a maintenance phase which is achieved by the patient and family at home to stabilize edema reduction. This study was conducted in the maintenance phase to assess the effect of the interventions on changing limb volume.Our study was approved by the Ethics Committee of Iran University of Medical Sciences and followed the Declaration of Helsinki and Good Clinical Practice guidelines. All participants signed a written informed consent before study enrollment. This research was registered at the Iranian Registry of Clinical Trials (IRCT2017092023861N7).2, stage 1 or 2 of lymphedema, completed surgery and adjuvant treatment completion (except hormone therapy/aromatase inhibitors) at least six months before enrollment. Patients with a history of probiotic supplements or anti-inflammatory drugs consumption and participating in weight loss programs in the last 6 months, were excluded from the study. Other exclusion criteria were included BC recurrence or metastasis, infection, smoking and alcohol consumption, autoimmune diseases, serious food allergies, mental illness, endocrine diseases including thyroid disorders, diabetes, gastrointestinal problems, taking multivitamin-mineral and omega-3 up to one month before the start of the study, continuous and heavy physical activity and history of diseases leading to edema such as heart, kidney, or liver failure.Eighty-eight women with unilateral arm lymphedema diagnosed by a lymph therapist were recruited to the study at Seyed Khandan Rehabilitation Clinic at Tehran, Iran from October 2017 to November 2018. Inclusion criteria were age between 18 and 65 years, BMI 25 - 40 kg/mParticipants were randomly assigned to the synbiotic supplementation group (n=44( or the placebo group (n=44). All participants received a low-calorie diet from the beginning to the end of the intervention.www.sealedenvelope.com). To apply the concealment in the randomization process, unique codes were assigned to the supplement boxes, which were generated by the software. Randomization and allocation were concealed from investigators, participants, and analyzers until the statistical analysis was completed.For randomization, permuted-block randomization with a block size of 4 was performed by the statistician consultant using the online site  per gr (CFU/g) beneficial bacteria such as Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium breve, Bifidobacterium longum, Streptococcus thermophiles, and 38.5 mg fructo-oligosaccharides. The placebo was a capsule similar in appearance, weight, smell, and packaging, containing lactose .The synbiotic supplements  contained 10The women in the synbiotic or placebo groups took a daily capsule for 10 weeks. They were asked to keep the study capsules refrigerated (between 2 and 7˚ C) throughout the study. They were also requested not to take any probiotic-containing food, probiotic yogurt or its products in 14 days before (2 week run-in period) and throughout the study. Compliance with consumption of capsules was monitored once a week through phone calls and confirmed by counting the capsules in face to face interviews after 5 weeks of intervention.2. Waist circumference was measured at the narrowest part of the torso. Demographic and clinical characteristics were collected by personal interview and the medical report. They were recorded in a checklist. Anthropometric indices were measured for all participants at baseline and after 10 weeks of intervention. Height and weight were measured without shoes and in light clothing using a digital scale . Body mass index (BMI) was calculated using formula weight in kg divided by height in mLymphedema volume was measured using the water displacement method . Edema volume was calculated as the volume difference between affected and unaffected arms in milliliters. All measurements conducted by an experienced examiner who was unaware of the study-group assignments.Nutrient intake of the participants at the beginning and end of the study, based on a 3-day food record (two weekdays and one weekend day), were calculated by Nutritionist IV software  modified for Iranian foods. Physical activity was assessed using an International Physical Activity Questionnaire (IPAQ) by interview . Blood samples were taken after 8-12 hour fasting at baseline and the end of the 10 weeks intervention. Serum samples were separated by centrifugation  at 3500 rpm for 10 min and stored at -80 °C until assayed. Serum concentrations of leptin, IL-1β, and TNF-α were measured using ELISA kits  and hs-CRP was measured by an immunoturbidimetric assay .The primary outcome of this study was TNF-α, since its effect had not been reported in similar studies. Based on the amount of Cohen's standard effect, assuming probability of a type I error of 5 % (α = 0.05) and a type II error of 20 %  and the Cohen standardized effect value of 0.65 (one effect at moderate level), also considering up to 10 % losses to follow-up, to detect the desired effect, the sample size was calculated 44 patients for each group.For continuous variables, the Shapiro-Wilk's test and a histogram were applied to ensure normality. All participants who were randomly assigned and completed an initial assessment were included in the final results by using an intention-to-treat analysis. For quantitative variables, the mean ± standard deviation or the median (first quartile - third quartile) and for qualitative variables, frequency (percent) was used.To compare quantitative outcomes between the study groups, an independent samples t-test  or a non-parametric Mann-Whitney test  was used. Changes of outcomes within each group after the intervention compared to baseline values were assessed by a paired sample t-test or a non-parametric Wilcoxon rank signed test. Chi-square test or Fisher's exact test was used to compare qualitative factors  between the study groups. Delta (Δ) was used to show the differences in the variables before and after the intervention. We used ANCOVA to compare post-intervention outcomes between the study groups by adjustment on baseline values of each outcome and baseline BMI as the covariate. Spearman's rank correlation coefficient analysis was performed to evaluate the relationship between changes in body weight/waist circumference and the blood factors/edema volume difference. The level of significance for all statistical analyses was p < 0.05. All statistical analyses were performed using SPSS version 24 . Non-parametric ANCOVA analysis was performed with R software (package 'sm' in R-3.5.1 for windows).Our approach for analyses can be considered as an intention-to-treat (ITT) analysis, since it includes every subject who was randomized according to randomized treatment assignment. It ignores noncompliance, protocol deviations, withdrawal and anything that happens after randomization.As demonstrated in the study flow diagram Figure 1, we recrNo side effects were reported following the supplementation of synbiotic in breast cancer survivors with lymphedema throughout the study.Baseline demographic and clinical characteristics of lymphedema patients have been presented in Table 1According to the 3-day food records obtained within each group before and after the intervention, there was no statistically significant difference in terms of energy, dietary macro- and micro-nutrient intakes between the study groups in baseline and after 10 weeks of intervention Table 2. However, both synbiotic and placebo groups showed a significant within-group reduction in daily energy intake , carbohydrate , protein , total fat , SFA , PUFA , MUFA , cholesterol , sodium  and beta-carotene  respectively Table 2.2), body fat percent (%), waist circumference (cm) and physical activity (min/week) at baseline and after 10 weeks in synbiotic and control groups are depicted in Table 32, -0.68 ± 0.90 kg/m2), body fat percent  and waist circumference changes , respectively. The mean values of weight (kg), BMI  min/week and 21.0  min/week in synbiotic and control groups respectively, P< 0.001]. The intention-to-treat (ITT) analysis did not show different results between the study groups.As shown in Table 4Baseline measures of inflammatory markers were not significantly different between the study groups, except hs-CRP . In the synbiotic group after 10 weeks of intervention, hs-CRP , IL-1β , and leptin  decreased significantly Table 4. InitialThe correlation of changes in serum hs-CRP with body weight  and waist circumference  alterations in the synbiotic group was statistically significant. Also, edema volume changes significantly correlated with body weight  and waist circumference  in the synbiotic group Table 6.This is the first interventional trial that investigates the possible beneficial role of synbiotic supplementation on inflammatory markers , serum leptin concentration, anthropometric parameters, and edema volume in BCRL patients. Our results revealed that synbiotic supplementation for 10 weeks reduced edema volume, the serum concentration of hs-CRP, IL-1β and leptin in overweight and obese patients with BCRL. Furthermore, the synbiotics group had significantly lower levels of serum leptin and TNF-α concentration compared to placebo after the intervention.A significant improvement was seen in anthropometric indices including body weight, BMI, fat percent and waist circumference within groups at the end of the intervention which is the predictable consequence of the low-calorie diet prescribed to all patients. The anthropometric changes were not significant between the study groups because the simultaneous occurrence of cancer, lymphedema and obesity increased the patient's inflammation  ProbiotIn different studies, the prognostic effect of high BMI in lymphedema incidence . Obesitbifidobacterium and lactobacillus in the form of probiotic yogurt, decreased serum IL-1β concentration in inflammatory bowel disease patients compared to control , but the non-significant differences of this variable between groups are probably due to the beneficial effects of the weight loss program on this cytokine, owing to the fact that a decrease in body fat during weight loss reduces the production of IL-1β . Shadnoal., 2013). No weiObesity leads to impairment and imbalance in gut microbiota called dysbiosis which weakens intestinal integrity accompanied by lipopolysaccharide (LPS)-related endotoxemia. LPS is a large molecule that exists in the cell wall of gram-negative bacteria. LPS stimulates the activation of the transcription nuclear factor-kappa B (NF-κB) by binding to toll-like receptor 4 (TLR4) in intestinal epithelial cells. NF-κB upregulates gene expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 which mediate a rise in hs-CRP, inflammation, and consequently edema . Our sual., 2017), which al., 2017). Leptinal., 2017; Zhou etal., 2017). Any faIn spite of the potential strengths of our study, such as using a probiotic intervention for the first time among BCRL patients, this study has some limitations. Although the synbiotics supplementation may affect fecal bacterial load, we could not assess this correlation because of budget limitations. Also, we did not consider a control group receiving placebo capsules without any low-calorie diet. So, we can not conclude if the weight reduction program had a synergistic effect along with the synbiotic capsules or not. Evaluation of the gene expression of inflammatory cytokines in future researches may provide valuable evidence in this field. In conclusion, 10 weeks of synbiotic supplementation along with an LCD program among BCRL patients resulted in a significant reduction in serum levels of TNF-α and leptin compared with the control group. Also, edema volume was significantly reduced within the synbiotic group after intervention. However, no significant differences were found in either of the anthropometric variables, IL-1β, and hs-CRP between the study groups. Weight reduction and synbiotic product consumption in the daily diet of lymphedema patients may be suggested as effective and safe interventions in lymphedema management.Shahpar Haghighat and Mitra Zarrati  contributed equally as corresponding authors.We would like to gratefully thank the participants for their support in the study. We are also thankful to Namdar laboratory and Zist Takhmir Company and all patients that participated in this study.This study was supported by Iran University of Medical Sciences (IUMS) with a 96-02-27-31433 grant number. The authors declare that they have no conflict of interest.