To determine the prevalence of enteric infections in Aboriginal children aged 0–2 years using conventional and molecular diagnostic techniques and to explore associations between the presence of pathogens and child growth.n = 62) residing in a remote community in Northern Australia, conducted from July 24th - October 30th 2017. Stool samples were analysed for organisms by microscopy , and by qualitative PCR for viruses, bacteria and parasites and serology for Strongyloides-specific IgG. Child growth (height and weight) was measured and z scores calculated according to WHO growth standards.Cross-sectional analysis of Aboriginal children (Cryptosporidium hominis/parvum (8.2%). Non-pathogenic organisms were detected in 22.5% of children. Ten percent of children had diarrhea at the time of stool collection. Infection with two or more pathogens was negatively associated with height for age z scores , as was carriage of the non-pathogen Blastocystis hominis .Nearly 60% of children had evidence for at least one enteric pathogen in their stool (37/62). The highest burden of infection was with adenovirus/sapovirus (22.9%), followed by astrovirus (9.8%) and Infants and toddlers living in this remote Northern Australian Aboriginal community had a high burden of enteric pathogens and non-pathogens. The association between carriage of pathogens/non-pathogens with impaired child growth in the critical first 1000 days of life has implications for healthy child growth and development and warrants further investigation. These findings have relevance for many other First Nations Communities that face many of the same challenges with regard to poverty, infections, and malnutrition.The online version contains supplementary material available at 10.1186/s12879-020-05685-1. Shigella sp.and Giardia lamblia have been associated with a significantly higher risk of stunting . The wealth index score was grouped into tertiles from tertile 1 (poorest) to tertile 3 (richest) .Following enrolment into the study each child received a general health check by a paediatrician. This included clinical history of diarrhea at presentation , and clinical examination for signs of infection  . If signPrimary carers were instructed to deliver soiled nappies to the study field laboratory or telephone the research team who collected them within two hours. Fresh stool was examined as soon as possible by direct microscopy and Kato-Katz, and participants with enteric infections were referred to the health clinic for further management. Of the remaining sample, an aliquot was stored in 10 ml of sodium acetate formalin (SAF) solution (Bioline), and another frozen at − 20 °C for PCR analysis. Stool samples collected into SAF were sent to the Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, Melbourne, Australia for testing using the formalin ethyl acetate (FEA) concentration method . StainedAscaris lumbricoides, Necator americanus, Ancylostoma ceylanicum, Ancylostoma duodenale (“pan-hookworm”), Trichuris trichiura and Strongyloides stercoralis. In addition, DNA was extracted from 0.2 g of fresh stool sample on the Freedom Evo 100® (Tecan. Swithzerland) robotics system using the NucleoMagR Virus Kit  as per manufacturer’s instructions. Prior to DNA extraction on the Freedom Evo 100®, a processing step of mechanical lysis as described by Mu, et al [Hymenolepis species (re-classified as Rodentolepis species), Enterocytozoon/Encephalitozoon species, Taenia species, Strongyloides species, Ascaris species, Trichuris trichiura, Ancylostoma species and Necator americanus. The limits of detection of the assay for the selected targets is reported to range between 17 and 93 copies/10uL by the manufacturer, [Blastocystis hominis, Entamoeba coli, Iodamoeba butschlii, Chilomastix mesnili.DNA was extracted from stool samples using the MolBio Powersoil® kit  for faecal pathogens and soil-transmitted helminths. DNA concentration was measured using Quant-iT™ dsDNA HS assay kit  and normalized to 1 ng/μl in UltraPure DNase/RNase-free distilled water (Invitrogen). DNA samples (10 μl) were tested using the Tandem- Plex™ Fecal Pathogens M kit  as described by the manufacturer Bacteria and viruses tested for are listed in Supplementary u, et al  was perfacturer,  but is lStrongyloides infections were diagnosed serologically using a commercially available ELISA that detects IgG antibodies against NIE, a recombinant protein from S. stercoralis, following kit manufacturer’s instructions . Positive and negative controls were tested in duplicate and patient serum samples tested singly. Control sera and sera from each subject were diluted to 1/100 using sample diluent buffer. It should be noted the kit manufacturer does not provide a cut- off value. As stated by the manufacturer the ‘cut-off value will vary depending on the test population and geographical location where the kit is being used.’ We calculated a cut-off value using ROC analysis of 98 sera collected in Australia. The results are reported qualitatively [tatively .Data was analyzed using StataIC, version 14 . Categorical data are presented as percentages with frequency, and continuous data are presented as mean and standard deviation (SD). Multivariate linear (height for age z scores) and logistic regression (stunting) was performed to examine the association between prevalence of enteric infection and child growth outcomes. Unadjusted and adjusted estimates of mean differences or odds ratios, and their 95% confidence intervals (CI) are presented. Potential confounding factors were selected a priori. All variables were included and adjusted for in the final model.We enrolled 70 of the ~ 100 children aged between 0 and 2 years resident in the community. Stool samples were collected from 62 (88.5%) children. In the sample of children who had stool samples collected, more than half of mothers/carers (51.6%) were educated to year 11 level. The prevalence of maternal underweight was 23%, overweight 19.4%, and obesity 8.1%. A smoker resided in 87.1% of households, and the average number of residents per household was six. Traditional medicine was used by 70.9% of families. The majority of children (87.1%) were currently breastfed. Food insecurity was common with 58.1% of households reporting running out of food in the previous two weeks. Almost all women (98.4%) received government benefits. Baseline characteristics are shown in Table The prevalence of enteric infections is presented in Table Cryptosporidium, Shigella, Salmonella, Giardia and Enterobius vermicularis. The most commonly detected pathogen was adenovirus/sapovirus , followed by astrovirus (6/61) and Cryptosporidium hominis/parvum (5/61). No cases of strongyloidiasis infection were detected by stool testing or serology (53 serology samples tested). No cases of hookworm were detected. Non-pathogens were detected in 14/62 (22.5%) of children, and in 10 cases coexisted with a pathogen. Ninety two percent of pathogen carriage was asymptomatic, with no reports of diarrhea or fever on clinical history or examination. Of the seven children who presented with diarrhea at the time of stool collection, pathogens were identified in four stool samples and included adenovirus, Giardia lamblia, Rodentolepsis nana (R.nana), Trichuris trichiura, and Dientamoeba fragilis and Blastocystis hominis.Of the 24 children who had an enteric viral infection, eleven (47.8%) were co -infected with another pathogen, including Blastocystis alone was also associated with reduced height for age z scores .Univariate and multivariate regression analyses to determine associations between pathogen and non-pathogen carriage and growth outcomes are presented in Table Cryptosporidium, and infections with two or more pathogens was associated with impaired linear growth.To our knowledge, this is the largest survey of young children aged 0–2 years in a remote Aboriginal community to document the prevalence of enteric infections and impact on child growth outcomes, and the first to compare different methodologies for identification of pathogens in this remote setting. We found that almost 60% of young children were infected with at least one enteric pathogen, most commonly adenovirus/sapovirus and Ancylostoma. duodenale  (9%), T.trichiura (36%), R.nana (9%) and Entamoeba spp (9%). in < 24 months old children (n = 11), and S. stercoralis (27%), R. nana (17%), Giardia duodenalis (13%), Entamoeba spp. (10%), A. duodenale (13%), and T.trichiura (87%) in children aged 2–4 years 11 months (n = 30) between July 1994 to October 1996 [S.stercoralis (3% on microscopy (n = 29), 9% on agar plate culture (n = 23), 8% on PCR [n = 38) [T.trichiura (14% on microscopy) and R.nana (7% on microscopy) (n = 29) [T. trichiura and R. nana positive were reduced. This reflects the implementation of public health measures  in this area that were introduced for soil transmitted helminths , leading to a reduction in S. stercoralis and hookworm prevalence [There is a paucity of data on the prevalence of enteric pathogen infections in infants and toddlers living in remote Aboriginal communities. We found only two studies in a remote Aboriginal community in the Northern Territory which documented the presence of intestinal parasites in 0–2 year old children . One stuber 1996 . Anotherdren n = , and S. rcoralis %, R. nandren n = , and S. dren n = , and S. evalence . Anotherevalence . SpecifiBlastocystis hominis was associated with impaired linear growth (lower height for age z scores). This confirms that pathogens not causing diarrhoea may still cause significant morbidity in resource- constrained settings, and efforts towards improving environmental conditions including improvements in clean drinking water, housing standards and hygiene and sanitation are critical.There is growing evidence of the importance of pathogens in a child’s gut, even when asymptomatic. This “pathobiome” is thought to be causal in the development of ‘environmental enteropathy’, with associated chronic intestinal inflammation, villous blunting and intestinal leakage and malabsorption, leading to a cycle of malnutrition, micronutrient deficiencies, and growth impairment . ChildhoB.hominis is usually considered a non-pathogen but in some instances has been reported as a pathogen [B. hominis and lower height for age z scores. This has been documented previously, with infection with B. hominis shown to be associated with lower anthropometric indexes in young children in Turkey [B.hominis [B. hominis in stool may also represent exposure to organisms spread by the faecal-oral route [pathogen . We obsen Turkey . These fn Turkey . Poor nu.hominis .. The fial route .E. coli) remaining in the top five causes of diarrhea identified by the original microbiological work up [Our study also provides important evidence on the application of molecular diagnostics to identify enteric pathogen burden. Use of PCR has previously been shown to substantially alter previous estimates of pathogen prevalence. Re-analysis of specimens from the MAL-ED cohort study using quantitative PCR demonstrated a predominance of viral causes of diarrhea  with only two pathogens , stool samples were not collected from mothers or carers of the child and hygiene practices being assessed only by questionnaire and not by direct observation ; and we only asked about diarrheal infections in the last 24 h.We found a high prevalence of enteric pathogen and non-pathogen carriage in Australian Aboriginal infants and toddlers and a significant association between infection with multiple pathogens and child growth (impaired height for age z scores). These findings suggest that environmental contamination in remote Northern communities where extreme heat and humidity is common, may contribute to the high rates of childhood growth impairment and stunting reported,  with heaAdditional file 1.Additional file 2.


To study incidence, time trends, and outcomes of serious infections in systemic sclerosis (SSc).We used the 1998–2016 US National Inpatient Sample data. We examined the epidemiology, time trends, and outcomes of five serious infections (opportunistic infections (OI), skin and soft tissue infections (SSTI), urinary tract infection (UTI), pneumonia, and sepsis/bacteremia) in hospitalized people with SSc. We performed multivariable-adjusted logistic regression analyses to analyze independent association of factors with healthcare utilization  and in-hospital mortality.There were 49,904,955 hospitalizations with serious infections in people without SSc and 61,615 in those with SSc. During 1998–2016, the most common serious infections in SSc were pneumonia (45%), sepsis (32%), SSTI (19%), UTI (3%), and OI (3%). In 2013–2014, sepsis surpassed pneumonia as the most common serious infection; by 2015–2016, sepsis was 1.8 times more common than pneumonia. Over the study period, hospital charges increased, while length of hospital stay and in-hospital mortality decreased, overall and for each serious infection. Multivariable-adjusted analyses showed that sepsis, age ≥ 80 years, and Deyo-Charlson score ≥ 2 were associated with significantly higher odds of healthcare utilization and in-hospital mortality, and Medicare or Medicaid insurance payer, Northeast location, urban teaching or non-teaching hospital, and medium or large hospital bed size with significantly higher odds of healthcare utilization.Outcomes in people with SSc hospitalized with serious infections have improved over time, except higher hospital charges. Identification of factors associated with higher healthcare utilization and in-hospital mortality allows for developing interventions to improve outcomes. Sepsis and pneumonia are the most common serious infections in systemic sclerosis.Over a 19-year study period, the length of hospital stay and in-hospital mortality decreased, overall and for each serious infection, in people with systemic sclerosis.A diagnosis of sepsis (relative to other serious infections), age ≥ 80 years, and Deyo-Charlson score ≥ 2 were associated with significantly higher odds of healthcare utilization and in-hospital mortality in systemic sclerosis patients hospitalized with a serious infection.Systemic sclerosis (SSc) is a multisystem autoimmune disease, associated with high morbidity and mortality  and freqOur study cohort included five, common serious infection hospitalizations in people with SSc in the US NIS 1998–2016 sample. The NIS is a 20% stratified sample of discharge records from all participating community hospitals from all participating states [We identified five types of serious infections based on the presence of International Classification of Diseases, Ninth Revision, Clinical Modification ICD-9-CM) codes in the primary diagnosis position for hospitalization: 1) opportunistic infections ; (2) skin and soft tissue infections ; (3) urinary tract infection ; (4) pneumonia ; and (5) sepsis/bacteremia (038.xx and 790.7), as previously [-CM codes opportunWe adjusted each regression model for covariates/confounders, including age, sex, race, serious infection type , median household income, the insurance payer, hospital characteristics , and Deyo-Charlson comorbidity index, a validated measure of medical comorbidity that includes 17 comorbidities , based on the presence of ICD-9-CM codes at index admission , with hiWe examined healthcare utilization and in-hospital mortality, and the details are as follows: (1) health care utilization: total hospital charges above the median for each calendar year; the length of hospital stay above the median of 3 days; and discharge to non-home settings (rehabilitation or an inpatient facility); and (2) in-hospital mortality.t test, as appropriate. Rates were calculated per 100,000 NIS claims. We analyzed time trends in rate of each serious infection using Cochran Armitage test. We performed multivariable-adjusted logistic regression analyses for each study outcome, adjusting for all covariates listed in the section above. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. We used SAS 9.3  for all analyses. We considered a p value < 0.05 to be statistically significant, which corresponds to a 95% CI that excludes unity.We followed the survey analysis procedures that account for the weights, clusters, and strata as defined in NIS, including the modified weights with the change in sampling in 2012. We compared the summary statistics using chi-square or Student’s There were 49,904,955 hospitalizations with serious infections in people without SSc and 61,615 in those with SSc. The average age of patients with SSc with a serious infection was 61.4 years , were more likely to be female (52% vs. 84%), or have Deyo-Charlson score of 2 or more  and a longer median hospital stay (3.7 vs. 4.4 days); were more likely to have hospital stay > 3 days (59% vs. 66%), hospital charges above the median (75% vs. 79%), or to be discharged home (74% vs. 83%); and had a higher in-hospital mortality (6.2% vs. 9%)  and sepsis 32%), followed by SSTI (19%), UTI (3%), and OI (3%; Appendix 2%, folloThe median length of hospital stay over the study period was the highest for OI hospitalizations at 6.1 days and the lowest for UTI at 2.8 days Appendix . Serious.We noted a significant increase in the overall frequency of sepsis, and possibly OI, SSTI, and UTI Appendix . In compBy 2013–2014, sepsis surpassed pneumonia as the most common serious infection in people with SSc, and by 2015–2016, sepsis accounted for 1.8 times as many hospitalized serious infections as pneumonia (Appendix We found a 3.6-fold increase in the overall mean hospital charges for composite serious infection in SSc patients from $23,152 in 1998–2000 to $87,095 in 2015–2016 (Appendix Multivariable-adjusted analyses showed that compared to sepsis, other infections were significantly associated with lower healthcare utilization and mortality; age ≥ 80 years and Deyo-Charlson score ≥ 2 were also significantly associated with higher healthcare utilization and mortality Table . MedicarIn this national study of people with SSc hospitalized with serious infection, we found that compared to patients hospitalized with serious infection without SSc, people with SSc had higher healthcare utilization and in-hospital mortality. In people with SSc, hospital charges and the length of hospital stay were the highest for OI and/or sepsis and the lowest for UTI.The frequency and rate of sepsis increased over time. Sepsis surpassed pneumonia as the most common serious infection in SSc in 2013–14. By 2015–2016, sepsis was twice as common as pneumonia. This is an important observation. This trend in SSc cohort may reflect the increase in hospitalizations with sepsis in the general population ; and/or We noted a reduction in the in-hospital mortality from 10.3% in 1998–2000 to 7.8% in 2015–2016 for composite serious infection in people with SSc. Not surprisingly, the largest reductions were noted for the serious infections with the highest mortality in 1998–2000. We noted large reductions of in-hospital mortality for OI, 21.2 to 7.7%; sepsis, 22.5 to 13.2%; and pneumonia, 9.8 to 2.8%, from 1998–2000 to 2015–2016, respectively. To our knowledge, these are important novel findings in a SSc cohort hospitalized with serious infections. Survival rates in SSc have improved over time , which oWe noted a 3.6-fold increase in the mean hospital charges with a 9% concomitant decrease in the mean/median hospital stay for serious infections in SSc patients, from 1998–2000 to 2015–2016. These trends over time are consistent with the general trends in the overall NIS cohort. The reduction in median hospital stay was the greatest for OI, from 9.5 to 5.8 days, and minimal for sepsis, from 5.5 to 5.3 days. In 2015–2016, mean hospital charges for SSTI were similar to that of pneumonia in people with SSc, which may be partially related to associated Raynaud’s disease and digital ulcers.Multivariable-adjusted analyses showed lower odds for healthcare utilization and in-hospital mortality for all serious infections compared to sepsis. The reduction in odds was 28–62% for healthcare utilization and 54–98% for in-hospital mortality. These important differences separate sepsis from other serious infections in SSc, with regard to outcomes.Several other factors were also independently associated with poorer healthcare utilization and in-hospital mortality outcomes. A higher Deyo-Charlson score ≥ 2 was associated with higher healthcare utilization and in-hospital mortality, and odds were increased by 37–54%. Our finding validates findings from another study that showed that diabetes, anxiety, and depression increased in-hospital mortality in hospitalized SSc patients , and extOur finding of an independent association of unmodifiable factors such as older age, Medicare or Medicaid insurance payer, Northeast location, urban teaching or non-teaching status, and medium or large hospital bed size with higher odds of healthcare utilization can help in a better understanding of associated healthcare utilization.Our study has several limitations. Misclassification bias is possible, since we used the ICD-9-CM or ICD-10-CM codes to identify people with SSc and infections. However, this bias may be minimal since the diagnostic codes for SSc –9 have bThe strengths of our study are the use of national US data that can produce national estimates of mortality, charges and healthcare utilization, the inclusion of several covariates and confounders in regression analyses resulting in robust estimates of association, and inclusion of a large sample size.In conclusion, we found important differences in hospitalized serious infection patients between SSc vs. non-SSc. In the USA, the most common serious infection in hospitalized SSc patients was pneumonia followed by sepsis; sepsis was the most common in the most recent study period. We noted a significant increase in the rate of sepsis, and possibly OI and UTI. Hospital charges increased, and the duration of hospital stay and in-hospital mortality decreased from 1998–2000 to 2015–2016. We identified several modifiable and non-modifiable independent risk factors for poorer outcomes that can help policymakers and spark new interventions for improving outcomes.Additional file 1 : Appendix 1. ICD-10-CM codes for serious infections. Appendix 2. Characteristics of people with serious infection in cohorts with versus without systemic sclerosis. Appendix 3. Characteristics of patients with each hospitalized serious infection as primary diagnosis in people with systemic sclerosis as a secondary diagnosis. Appendix 4. Frequency of serious infections in people with systemic sclerosis over time. Appendix 5. Serious Infection Rate in the general NIS cohort per 100,000 NIS population. Appendix 6. Rate of serious infections in people with systemic sclerosis over time using two denominators. Appendix 7. Contrast between the first and the last study periods, 1998-2000 versus 2015-2016 for healthcare utilization and mortality outcomes in people with systemic sclerosis. Appendix 8. Time-trends in the length of hospital stay and total hospital charges across all study periods, 1998-2000 from 2015-2016 in people with systemic sclerosis hospitalized with each serious infection


Brain metastases (BM) are the most common brain tumors in adults, and it remains a major complication in cancer patients. Exosomes or extracellular vesicles (EV) and integrins contribute to the development of BM, and exosomal integrins have been shown to determine organotropic metastasis. To our knowledge, this is the first clinical evidence demonstrating that high plasma EV integrin β3 level is associated with worse overall survival and intracranial control in BM patients undergoing whole brain radiotherapy, and supports the determination of organotropic metastases by exosomal integrins in the clinical setting. As utilization of whole brain radiotherapy for BM gradually declines in favor of local therapy such as neurosurgery and stereotactic radiosurgery (SRS), intracranial and distant brain control remain relevant because of the improved survival. Circulating EV integrin β3 may serve as a novel biomarker in predicting the progression of BM and survival.p = 0.04) following WBRT. Multivariate regression analysis also showed a higher cumulative incidence of intracranial failure . In conclusion, circulating EV integrin β3 levels correlated with survival and intracranial control of patients with lung cancer after WBRT for BM. This supports that EV integrin β3 mediates a brain-tropic metastasis pattern, and may serve as a novel prognostic biomarker for BM.Brain metastasis (BM) is a major problem in patients with cancer. Exosomes or extracellular vesicles (EV) and integrins contribute to the development of BM, and exosomal integrins have been shown to determine organotropic metastasis. We hypothesized that circulating EV integrins are able to influence the failure patterns and outcomes in patients treated for BM. We prospectively enrolled 75 lung cancer patients with BM who received whole brain radiotherapy (WBRT). We isolated and quantified their circulating EV integrins, and analyzed the association of EV integrins with clinical factors, survival, and intracranial/extracranial failure. Circulating EV integrin levels were independent of age, sex, histology, number of BM, or graded prognostic assessment score. Age, histology, and graded prognostic assessment score correlated with survival. Patients with higher levels of circulating EV integrin β3 had worse overall survival (hazard ratio: 1.15 per 1 ng/mL increase;  Brain metastases (BM) are the most common brain tumors in adults, and it is estimated that >20% of patients with cancer will develop BM . As a reThe prognosis of patients with BM depends on various factors, including age, performance status, primary disease control, presence of extracranial metastases, and treatment status ,8. In adCancer metastasis involves changes in cell–cell contact and cell–extracellular matrix adhesion . IntegriExosomes or extracellular vesicles (EV) are small membrane vesicles (30–150 nm) containing functional biomolecules  that can be transferred horizontally to recipient cells . It has Between March 2015 and July 2019, 75 patients with BM originating from primary lung cancer were enrolled and received whole brain radiotherapy. The patient characteristics are summarized in p = 0.003) (p = 0.005) (p = 0.123).The median OS, IC-PFS, and EC-PFS for the entire cohort were 14 months , 7.9 months (95% CI: 7.1–8.7 months), and 5.1 months (95% CI: 4.1–6.1 months), respectively . Patient= 0.003) A. In add= 0.005) B. There p = 0.002). Higher GPA scores were also associated with better IC-PFS , and there was a trend toward worse IC-PFS with advancing age . In contrast, EC-PFS was longer in patients with adenocarcinoma  and younger patients , but only trended toward better EC-PFS with higher GPA scores. The survival outcomes did not differ in relation to sex, number of BM, or presence of leptomeningeal carcinomatosis  of particular interest. The median expression level of integrin β3 was 1.668 ng/mL , and the median expression levels of integrin β6 were 2.024 ng/mL . The medians of circulating EV integrin β3 and integrin β6 concentrations did not differ in relation to histology, number of BM, GPA score , age, orp = 0.04). The maximally selected log-rank statistics suggested that the optimal cut-point was 1.818 ng/mL, and the resultant high integrin β3 level group (>1.818 ng/mL) demonstrated shorter OS compared with the lower integrin β3 level group (≤1.818 ng/mL) . The univariate analysis of prognostic factors associated with OS were shown in High levels of circulating EV integrin β3 correlated significantly with worse OS . The univariate competing risk analysis of prognostic factors associated with intracranial failure were shown in p = 0.037), while the GPA score demonstrated correlation with the competing cause , with age and histology exhibiting a non-significant trend  and EV integrin levels has not been established yet; however, evidence supporting integrin transfer via tumor cell-derived EV is growing ,25,26. SNature, Hoshino et al. demonstrated that the tumor exosome integrins α6β4/α6β1 and integrin αvβ5 were associated with lung and liver metastasis, respectively, while integrin β3 was present in exosomes isolated from brain-tropic cells [The positive correlation between increasing EV integrin β3 levels and early intracranial failure after WBRT attested to the tendency of BM to occur in patients with high EV integrin β3 levels. Accumulating evidence on exosome-mediated organ-specific conditioning has shed light on Stephen Paget’s “seed-and-soil” hypothesis , which wic cells . Anotheric cells . Our resic cells , highligFurthermore, our study demonstrates that increasing circulating EV integrin β3 levels are associated with worse survival. The median survival of 14 months was in line with that noted in earlier cohorts, considering the distribution of GPA and the extent of BM requiring WBRT . HoweverIn contrast, EV integrin β6 levels were not associated with either a specific failure pattern or survival in our study. The upregulation of integrin ανβ6 expression is associated with poor prognosis in many types of cancer . HoweverAs utilization of WBRT for limited BM gradually declines in favor of local therapies such as neurosurgery and stereotactic radiosurgery (SRS), intracranial and distant brain control remain relevant because of the improved survival times, and have become a concern without the use of WBRT ,36. A seThe potential limitations of our study include the following: (1) The absence of pathological specimens to determine the relationship between the EV integrin levels and cellular expression of integrins makes its source a matter of debate; (2) insufficient data to analyze changes in EV integrin levels upon recurrence; and (3) possible inconsistencies in EV isolation and integrin quantification, which may be inherent as standardized protocols and reference levels were lacking at the time of assessment, thus limiting generalizability.All patients were pathologically and/or cytologically diagnosed with radiographic evidence of BM, and provided informed consent at the time of enrollment. The present study cohort were treated with WBRT at the discretion of the attending radiation oncologist. In accordance with contemporary guidelines, radiotherapy with 30 Gy in 10 fractions was delivered with a linear accelerator using 6 MV photons. Two-/three-dimensional or conformal volumetric-modulated radiation therapy techniques with or without hippocampal avoidance were applied. Patient and tumor characteristics, including disease-specific GPA /lung-molTM Midi Columns  according to the protocol provided by the manufacturer. The circulating EV integrins β3 and β6 were subsequently measured with an enzyme-linked immunosorbent assay (ELISA) using a commercially available human protein sandwich enzyme immunoassay kit . Concentrations of integrins in EV were determined against a standard curve.The EV were isolated from 1mL of stored plasma by size exclusion purification using Exo-SpinOverall survival (OS) was measured from the day of enrollment to death. Intracranial progression-free survival (IC-PFS) and extracranial progression-free survival (EC-PFS) were calculated from enrollment to death or until radiographic evidence of progression and/or recurrence of BM or leptomeningeal carcinomatosis, and extracranial progression, respectively. Patients without a known date of progression were censored at the time of the last follow-up. Correlations between the medians of circulating EV integrin concentration of subgroups were assessed with the Kruskal–Wallis test. The correlations between survival and EV integrin concentrations were assessed with univariate Cox proportional hazard regression. Extreme outliers were identified using Grubb’s method , and remThis prospective study identified circulating EV integrin β3 as a potential novel BM-specific biomarker to predict the outcomes after WBRT in patients with lung cancer. Patients with high circulating EV integrin β3 levels have worse intracranial control and poor survival outcomes, independent of the graded prognostic assessment for lung cancer using molecular markers. The translational evidence suggests that EV abundant in integrin β3 are brain-tropic in lung cancer. This finding requires validation in an independent cohort.


Pocillopora acuta from two sites with differing distance to the mainland (Kusu (closer to the mainland) and Raffles Lighthouse (further from the mainland)) to heat stress through differential expression analysis of target genes and quantification of photophysiological metrics. We then examined how these attributes were regulated after the stressor was removed to assess the recovery potential of P. acuta. The fragments that were subjected to heat stress (2 °C above ambient levels) generally exhibited significant reduction in their endosymbiont densities, but the extent of recovery following stress removal varied depending on natal site and colony. There were minimal changes in chl a concentration and maximum quantum yield  to maximum fluorescence (Fm)) in heat-stressed corals, suggesting that the algal endosymbionts’ Photosystem II was not severely compromised. Significant changes in gene expression levels of selected genes of interest (GOI) were observed following heat exposure and stress removal among sites and colonies, including Actin, calcium/calmodulin-dependent protein kinase type IV (Camk4), kinesin-like protein (KIF9), and small heat shock protein 16.1 (Hsp16.1). The most responsive GOIs were Actin, a major component of the cytoskeleton, and the adaptive immune-related Camk4 which both showed significant reduction following heat exposure and subsequent upregulation during the recovery phase. Our findings clearly demonstrate specific responses of P. acuta in both photophysiological attributes and gene expression levels, suggesting differential capacity of P. acuta corals to tolerate heat stress depending on the colony, so that certain colonies may be more resilient than others. The ability of corals to withstand changes in their surroundings is a critical survival mechanism for coping with environmental stress. While many studies have examined responses of the coral holobiont to stressful conditions, its capacity to reverse responses and recover when the stressor is removed is not well-understood. In this study, we investigated among-colony responses of  Coral reefs are faced with severe degradation due to anthropogenic activities and the progression of climate change is exacerbating these stresses even further . PerhapsThe ability to constantly sense and exhibit flexibility to environmental changes is important for all organisms to maintain cellular homeostasis . It is ePorites astreoides from different populations—inshore versus offshore—a positive correlation between cellular stress response genes and measurement of fitness  was found for inshore coral fragments, suggesting enhanced plasticity is beneficial for this population of corals . For each sample, complementary DNA (cDNA) was immediately prepared from the amount of total RNA equivalent to 1 µg, using a one-tube format of Bio-Rad iScript RT supermix for RT-qPCR (reverse transcription quantitative polymerase chain reaction). Reaction setup was composed of iScript RT supermix (4 µL), RNA template , and nuclease-free water (variable), with final volume of 20 µL. Incubation of reaction mix was performed in a Labcycler  following the manufacturer’s protocol: priming for 5 min at 25 °C, RT for 20 min at 46 °C, and RT inactivation for 1 min at 95 °C.P. acuta following heat exposure [The selected genes of interest (GOIs) and internal control genes (ICGs) were derived from an RNA-Seq experiment conducted on exposure  as beingexposure . The fulhttps://www.ncbi.nlm.nih.gov/tools/primer-blast/, June 2017), specifying “Pocillopora acuta” in the target organism field. Primer parameters include GC content = 50–60%; Tm (melting temperature) = Min 58 °C, Max 60 °C, Tm difference = 1 °C; PCR product size = Min 100, Max 1000. Forward primer sequences for each gene were searched in the consensus gene sequence used for the primer design. The reverse primer sequences were reversed using an online reverse primer tool  and similarly searched against the consensus sequences.Transcript sequences from Poquita-Du et al.  that matThe specificity of each primer pair for each GOI was verified by PCR using the GoTaq Green Master Mix. PCR reaction steps were (1) denaturation: 95 °C for 45 s (2) annealing: start at ~55 °C, increasing in increments of 1 °C to annealing temperature (60 °C) for 45 s; (3) extension: 72–74 °C for 5 min, repeated for 30 cycles. Gel electrophoresis was performed to check for successful amplification of target regions. Presence of gDNA contamination was also assessed using a no-RT control.P. acuta cDNA covering two orders of magnitude of template amount (0.078–5 ng) using qPCR . Calculations of efficiencies  needed to correct for amplification efficiencies per primer were done using MCMC.qpcr package in R developed by Matz et al. (2013) [Efficiencies of primers were determined by amplifying a series of dilutions of . (2013) . The funRT-qPCRs (15 µL volume per reaction) were performed in Bio-Rad CFX96 using SsoAdvanced inhibitor-tolerant SYBR Green Supermix following the manufacturer’s protocol  repeated for 40 cycles. For each RT-qPCR run, three wells  were prepared for each cDNA sample in a 96-well hard-shell low-profile PCR plates with two wells each for no template and no-RT controls. Melting curve analysis  of the amplification product obtained was performed to further validate specificity of each primer. To control for variations in expressions of genes due to differences in RNA concentration of each sample, amount of cDNA template was standardized to ~10 ng of cDNA for every reaction mix.a concentration (continuous data) and generalized linear mixed model for endosymbiont density (count data) using lme4 package in R [To examine the effects of “colony”, “site”, “treatment”, and “period” on coral photophysiological performances, data were fitted with linear mixed models for Fv/Fm and chl age in R . The fixn = 96 samples from six colonies × two treatments × two periods × four replicates) and technical (three RT-qPCR reactions per sample) replicates for each GOI. Following the Bayesian methodology outlined by Matz et al. (2013) [Actin was designated as an ICG, it was found to be differentially expressed following heat stress and, therefore, no longer utilized as an internal control here and the model was run in the “naive” form . While specification of control genes can sharpen estimates of model parameters, this was not critical as all normalizations can be performed within the model [Data obtained from RT-qPCRs expressed as “cycle of quantification values”  were collated and sorted for subsequent analysis. The data contained raw Cq values from all biological (. (2013) , the Cq . (2013) ,49,50,51he model .Each gene profile was examined to determine (1) treatment responses, (2) whether changes of gene expressions for corals previously exposed to heat occurred following the recovery period, (3) among-site variation in responses, and (4) influence of colony. The model contained fixed effects: “treatment”, “period”, “site”, and “colony”; a global fixed effect: “RNA quantity” (14.6–200 ng/µL) and a random effect: “sample replicate” that denotes individual cDNA preparations to account for unequal cDNA template loading between samples as a form of normalization.P. acuta corals were significantly influenced by treatment , period , site , and the interactions between these factors .Endosymbiont densities of  factors . Howevera concentration in response to heat across periods and sites. However, there were significant differences in response among colonies and sites   (There were no significant changes for average chl  0.0004) . While t= 0.016) ; Table 4Actin, Camk4, GAS8, Hsp16.1, and KIF9) showed reliable amplification. Efficiency of amplification was high, within the range of acceptable values of 1.49–2.1 ) [Among the nine candidate genes, only five ( (2011))   mean = −2.035; pMCMC < 0.001) . While the overall changes in expression levels of Hsp16.1 and KIF9 following heat exposure were not significant, there were colony-specific differences, particularly for G2 . Further, corals which were previously exposed to heat showed upregulation of Camk4 during the recovery period, but this change was colony-specific with significant changes for G2  and G3 . Expression levels of GAS8 did not show any significant changes following heat stress and recovery among colonies and between sites.The main effect of heat on expression levels of all genes examined was significant for only < 0.001) . FollowiP. acuta corals from two sites with differing distances to the mainland (Kusu (closer to the mainland) and Raffles Lighthouse (further from the mainland)) responded to heat stress and recover from it. Results showed that water temperature affected the photophysiological performances of P. acuta corals, after both the heat stress and recovery periods. Heat-induced differences in endosymbiont density between sites and among colonies were significant, as were changes in average chl a concentration. Both treatment and experimental period influenced Fv/Fm and responses were similar across sites and colonies. Significant changes in expression levels of selected GOIs were also detected, however, the changes were mainly colony-specific for most GOIs examined except for GAS8. The most responsive GOIs were Actin, a major component of the cytoskeleton and the adaptive immune-related gene Camk4. Both genes exhibited downregulation following exposure to heat and colony-specific changes in heat-stressed corals upon removal of the stressor. Findings here underscore the capacity of P. acuta corals to achieve homeostasis under sub-optimal conditions and recover from perturbations by a combination of photophysiological regulation and orchestration of gene expression.Our experiment tested how different colonies of a concentration [Pocillopora colonies exposed to heat stress . Ind. Inda coess  rather than the effective antennae-absorption involving chlorophyll a [P. acuta’s thermal tolerance and capacity to recover when conditions are improved. This can be attributed partly to the hosted endosymbionts which are known to be composed mainly of Durusdinium (previously Symbiodinium clade D) [Pocillopora as one of the least susceptible genera [There were subsequent changes in endosymbiont densities for the heat-stressed corals after the stressor was removed but, again, there was variation in their recovery responses depending on colony and natal site. While there were significant changes in endosymbiont densities for most coral colonies, none of them showed visible changes in terms of tissue pigmentation (pers. obs). Furthermore, a small change in chl  to heat , suggestophyll a . Howeverophyll a . These rclade D) ,59, a tye genera ,60.Actin was observed following heat exposure. Considering this is a major cytoskeletal component involved in growth, downregulation of this gene could be indicative of growth inhibition in response to heat stress. This pattern of Actin has previously been observed in similar experiments by Kenkel et al. (2011) [Actin was initially tested as a control gene in this experiment based on its unresponsiveness to heat in our previous transcriptome-wide study of P. acuta [Actin here indicates that its expression pattern may vary widely in this species. The gene’s involvement in coral stress response needs to be further validated. Furthermore, KIF9, which is a kinesin-like protein linked to extracellular disassembly also showed changes in expression level following heat exposure. The extracellular calcifying matrix is the site of calcification [KIF9 are likely associated with regulation of the calcification process, which is controlled by exogenous factors such as temperature [Actin and KIF9 expression levels and growth indices in corals following exposure to heat stress.While increased thermal tolerance is advantageous for coral survival, elevated temperature is often associated with growth impairment in corals as normal calcification physiology may be compromised ,62,63,64. (2011)  and DeSa. (2011) . We noteP. acuta . Howeverfication ,67, so cperature ,69,70,71Pocillopora damicornis (a close relative to P. acuta) revealed the occurrence of lineage-specific genes that are associated with the immune response pathway, indicating that this species may have evolved unique immune strategies to cope with changes in the surrounding environment [Camk4 expression levels which were then reversed significantly following removal of heat for some specific colonies (G2 and G3), particularly those from Kusu. The pattern of Camk4 during the recovery period appears to correspond to the site, suggesting a potential influence of natal site on coral responses to heat and its subsequent recovery. Kusu generally has a more variable environment compared to Raffles Lighthouse as it typically experiences strong tidal currents [Camk4 is a calcium/calmodulin-dependent protein kinase which is associated to adaptive immune response [Camk4 which is activated by presence of Ca2+/calmodulin ions [P. acuta’s acclimatization response. It is interesting to note that the involvement of Ca2+ homeostasis disruption associated with thermal stress had been previously reported in cnidarians [Camk4 in coral stress response. While it is unclear whether or not corals have an adaptive immune response, the recent report on the genome of P. damicornis by Cunning et al. (2018) [As most corals are sessile, the capacity to constantly sense their environment and respond specifically to a distinct stressor via adaptive immune responses is critical for their survival. Recent research on the genome of ironment . Here, ecurrents  and othecurrents  that maycurrents . Camk4 iresponse  and has response . The demresponse , so the lin ions , is poteidarians ,78,79 wh. (2018)  highlighHsp16.1  was upregulated following heat stress and subsequently downregulated for the recovery period. Conversely, in our study, exposure of P. acuta corals to heat showed slight increases of Hsp16.1, however, the change was only significant for G2. Interestingly, the expression levels of Hsp16.1 in heat-stressed corals appeared to increase following the recovery period and this was more pronounced for corals from Kusu compared to Raffles Lighthouse. These changes, however, were not significant. Nevertheless, the observations suggest a likely influence of natal site on the expression pattern of this gene similar to Camk4 discussed above. While the relationship between Hsp16.1 level and the water temperature profile of natal sites was not explored in this study, the pattern observed for this gene in response to heat stress corroborates previous observations [Hsp16.1 expression even when the stressor was removed, it is unclear what role this gene serves during a heat stress response.Heat-shock proteins ,80 in corvations ,28,38,81P. acuta’s response to heat stress and its capacity to recover. These differences are likely due to genotypic variability between the colonies examined and reflect the potential role this variation has for the resilience of this species in the face of climate change. It is worth mentioning that P. acuta corals found in Singapore exhibit variation in branching morphologies: compact and thin [Pocillopora species [The main findings here indicate strong intercolonial variability in and thin . It has and thin . This di species ,83,84,85P. acuta corals in response to changing thermal conditions which, however, depended largely on the coral colony. It is important to note that the underlying factors driving these differences remain unclear and warrant further investigation. In particular, as gene expression is followed by post-transcriptional changes [Taken together, findings presented here highlight significant regulation of photophysiological attributes and gene expression of  changes ,86,87,88 changes ,90 in or


We recently published the results of the AVASTEM study, in which we explored the impact of the addition of an angiogenesis inhibitor (bevacizumab) to standard pre-operative chemotherapy for breast cancer. In this work, we aimed to identify biological parameters correlated to prognosis and treatment efficacy. To do so we explored if circulating tumor cells (CTCs), that are cells released by the tumor and that can be detected in the bloodstream of a patient; can predict the outcome of the women treated in this study. We also analyzed if bevacizumab concentration in the blood during treatment was associated with outcome, i.e., response to treatment and survival. We observed that CTCs detection before treatment initiation is associated with survival but was not correlated to local response to treatment. We moreover found that CTC status after three weeks of chemotherapy was not correlated to outcome as well as bevacizumab levels before and during treatment. However, bevacizumab concentration tended to be associated with an increase of hematological toxicities during the study. We thus show in this work that CTCs detection at baseline is prognostic for patients with breast cancer receiving a pre-operative chemotherapy-bevacizumab combination, and that this prognostic value was independent of tumor response.p = 0.001, log-rank test). Similar results were observed for RFS with 5 y-RFS of 78% vs. 44% , p = 0.017, log-rank test). However, CTC status at baseline was not predictive of pCR (p = 0.74). CTC status after one cycle was not a significant prognostic factor ; p = 0.68 for OS and HR = 2.76; 95%CI (0.60–12.61); p = 0.17 for RFS, log-rank test). Bevacizumab serum levels could not predict pCR and survival. PK values were not associated with treatment-related toxicities. In conclusion, CTCs detection at baseline is a prognostic marker for breast cancer receiving a neoadjuvant chemotherapy-bevacizumab combination independently of tumor response.The phase II AVASTEM trial explored the impact of chemotherapy-bevacizumab combination on breast cancer stem cells in the neoadjuvant setting. We aimed to identify biological features associated with preoperative chemotherapy efficacy and prognosis by analyses of circulating tumor cells (CTCs) and bevacizumab pharmacokinetics (PK). The main objective was to assess the prognostic  and predictive  values of CTCs (CellSearch technology) and bevacizumab PK (ELISA). Seventy-five patients were included. Out of them 50 received bevacizumab-chemotherapy and 25 received chemotherapy alone. CTC results were available for 60 patients and PK data for 29 patients in the experimental arm. The absence of CTC at inclusion was correlated to better outcome. Five-years overall survival (OS) was 91% for CTC-negative patients vs. 54% for CTC-positive cases ,  According to the ECIS -positive tumors, and cytotoxic chemotherapy. Neoadjuvant chemotherapy (systemic chemotherapy before breast and lymph nodes surgery) aims to downstage the tumor to enable breast-conserving surgery, evaluate the effectiveness of new systemic therapy or new therapeutic schemes, and to eradicate micro metastases and to reduce the risk of distant recurrence ,2. Most Addition of bevacizumab to these regimens has been shown to increase pathological response rates  but ther® system which is the only assay that has received FDA approval [Circulating tumor cells (CTC) are released by the main tumor mass and may be at the origin of metastases spreading . They haapproval . CTC detapproval ,9. CTC dapproval ,11. Moreapproval . No dataDespite bevacizumab pharmacokinetics characteristics have been showed to be associated with treatment efficacy in the metastatic setting , its impWe previously published the main results of the AVASTEM trial (NCT01190345), which showed that addition of bevacizumab did not modify the stem cells rates in comparison to standard neoadjuvant chemotherapy . The curAmong the patients included in the AVASTEM trial, treatment groups were well balanced in terms of age, menopausal status, tumor stage, and axillary lymph node involvement .p = 0.05 and 33% vs. 7% p = 0.02, respectively, We obtained CTCs data at inclusion for 60 patients, including 38 who received bevacizumab and 22 who received chemotherapy alone. Among them, percentages of positive axillary lymph node and De novo metastatic patients were significantly different between CTC positive and negative subgroups : twenty five patients at inclusion , 27 after cycles 1–2 , and for 22 after end of treatment .Demographic and clinical factors for the 60 patients with at least one CTC measure and the 29 patients in experimental arm with at least one PK measure are also presented in p = 0.77). After one cycle (sample collected at C2D1), CTCs were still observed in five of 56 patients . After one cycle of treatment, one of the 15 patients with pCR status had still detectable CTCs versus four of the 39 with RD .CTC positivity at baseline was not correlated to pCR achievement . Responsp = 0.001, log-rank test). This survival gain was not observed when analyzing CTC status after one cycle of treatment. p = 0.68, log-rank test).The absence of CTC at inclusion was associated with better outcome. p = 0.017, log-rank test). CTC status after one cycle was not a significant prognostic factor in our set. p = 0.17, log-rank test).Regarding non-De Novo metastatic patients, p = 0.426. As expected, bevacizumab levels were higher in the experimental arm after treatment initiation and after end of treatment, with a median of 111.2 µg/mL (range 43.0–640.9) and 74.2 µg/mL (1.0–956.5), respectively  neither RFS (p = 0.617 at baseline and p = 0.291 after the end of treatment).Measurements of bevacizumab were not correlated to either OS (p = 0.895) but tent to be correlated to hematological adverse events (p = 0.065 for FN and p = 0.084 for anemia), (Anemia was notified for 13 of 50 patients (26%) in the bevacizumab arm. Seventeen patients (34%) had been admitted with febrile neutropenia (FN), and 14 (28%) were diagnosed with bevacizumab-induced hypertension (BIH). Bevacizumab levels after one to two cycles were not correlated to occurrence of BIH (anemia), .In this retrospective ancillary analysis of the randomized phase II AVASTEM trial, we aimed to find biological markers that may help to predict efficacy and response to neoadjuvant chemotherapy in breast cancer.p = 0.001, log-rank test) and RFS . However, CTC status after treatment initiation (after one cycle) was not a prognostic factor in our set (p = 0.68 for OS and p = 0.17 for RFS). The low number of patients who were still CTC-positive after the first cycle of treatment may be an explanation of this absence of correlation. However, we cannot rule out that CTC measurement after one cycle of therapy is not an adequate surrogate marker of long-term efficacy and may not affect survival. Moreover, treatment modality (here bevacizumab vs. no bevacizumab) may also have modified the prognostic value of CTC status. It has been recently shown that treatment modalities can indeed influence the prognostic impact of clinical and biological features [We found that the absence of CTCs before treatment initiation was a prognostic factor associated with better OS . CTC status after one cycle of chemotherapy was also not predictive of response . The lack of significant predictive value of CTCs has already be described for patients receiving neoadjuvant treatments combining chemotherapy and HER2-inhibitors (trastuzumab and lapatinib) in the NeoALLTO trial [Despite its capacity to anticipate prognosis of patients with early breast cancer, CTC count predictive value is still not proven. Pathological complete response was not correlated neither to the status of CTCs nor to the PK values in our set. CTC-positivity rate at baseline was similar for patients who achieved pCR and for those with RD  and previously published PK data . Averagep = 0.065 and p = 0.086, respectively). Bevacizumab has been described to enhance hematological toxicities. Grade three or higher hematological adverse events  were indeed more frequent in the bevacizumab arm of the AVASTEM study, as well of other randomized trials [Another hypothesis we addressed was that PK may be correlated to toxicity. We focused our analysis on three major adverse events under chemotherapy-bevacizumab regimen: anemia, febrile neutropenia, and hypertension. A quarter to a third of the patients included in our set experimented each of these toxicities . We observed no correlation between bevacizumab blood concentration after treatment initiation and BIH. Hematological toxicities (FN and anemia) tent to occur more frequently in patients with high bevacizumab exposure  would have been of interest to improve the understanding of relationships between drug exposure and efficacy or safety; and correlation between PK and hematological adverse events would probably be significant with more patients included in these analyses. However, as this PK/efficacy comparison was a retrospective ancillary of the AVASTEM trial, we were not able to collect more samples.This study is a retrospective ancillary analysis of an open-label, randomized (2:1), multicenter, phase II trial with chemotherapy with or without bevacizumab combination for the treatment of primary breast cancer candidate to receive preoperative chemotherapy. Its primary objective is to evaluate the effect of circulating tumor cells (CTC) and pharmacokinetics (PK) of bevacizumab on pathological complete response rates (pCR). Secondary objectives are correlation of CTC and kinetics at baseline and after one cycle of treatment on relapse-free survival (RFS) and overall survival (OS). As an exploratory analysis, PK data were compared to incidence of the main adverse events observed with chemotherapy and bevacizumab combination: anemia, febrile neutropenia, and high blood pressure.Blood samples available from the 75 patients included in the AVASTEM randomized controlled trial  were analyzed. Inclusion criteria and treatments received have been previously detailed [Collection of CTC was done at baseline and after one cycle of treatment. For the PK, sample collection was done at baseline, after one or two cycles of chemotherapy, and after the eighth cycle.® system  within 96 h by experienced technicians as previously described [CTC were detected from 7.5 mL blood samples using the Cellsearchescribed . The CTCBevacizumab was provided by Abreos Biosciences Inc. and antibodies were obtained from the UCSD Moores Cancer Center Pharmacy.All peptides were synthesized as C-terminal free acids and N-terminal free amines with an additional C-terminal GGG-Lys(biotin) for assay immobilization .ELISA Protocol. All incubations were conducted at room temperature on an orbital shaker unless otherwise noted. All washes were performed manually.2O for 1 h. Wells were washed five times with 200 µL TBST. Wells were then blocked with 200 µL of 5% BSA in TBST for 1 h to prevent nonspecific binding. Wells were washed five times with TBST. Clinical Samples were diluted in 2.5% BSA/TBST to a final concentration of 0.2% and incubated on the plate for 1 h. Nonselective serum component were washed away 5 times with TBST. Bound bevacizumab was detected with goat anti-human Fc IgG-HRP  diluted 1:5000 in BSA/TBST. Wells were incubated with 100 µL of diluted anti-IgG-HRP for 30 min and then washed nine times with TBST. Ultra TMB substrate (Thermo Fisher) was added to the wells (100 µL) and allowed to develop for 1–2 min shielded from light. The reaction was stopped with 100 µL of 1 M H2SO4  Optical density (OD) was read at 450 nm on a EnVision Xcite Multilabel S/N  and data were analyzed with GraphPad Prism v5 or v7 software .Wells of a NeutrAvidin-coated 96-well plate or 8-well strips  were coated with 100 µL of biotinylated peptide at 1 µg/mL in Hyclone HEach sample was run in duplicate and the resulting OD values were averaged. Reported bevacizumab concentrations were interpolated from the averaged ODs using a 4PL fit to a calibrator dilution series run on each plate. Calibrators used for this assay were made by spiking concentrations of bevacizumab into 0.2% commercial pooled human serum. These nominal concentrations were 500, 250, 125, 62.5, 31.25, 15.625, 7.8, 3.9, and 0 ng/mL.Quality Control (QC) samples were included in the assays performed to confirm accuracy of concentration measurements. These samples were made by spiking bevacizumab into commercial 0.2% pooled human serum, as with the calibrators. The nominal spiked concentrations were 100, 50, and 20 ng/mL.Categorical variables  were described using counts and frequencies, and quantitative variables  were described using medians and ranges.Pathological complete response (pCR) was based on the ypT0/is pN0 definition. Patients without pCR or with disease progression during neoadjuvant treatment were considered as patients with residual disease (RD). CTC levels were compared to response and treatment arm using Fisher’s exact test. PK values were compared according to treatment arms and, for bevacizumab arm only, according to adverse events occurrence and response using the Wilcoxon rank sum test.® software version 9.4 .Survival analyzes were conducted on all patients for OS and on non-De Novo metastatic (M0) patients for RFS. Survival probability of outcomes was considered between the measurement of interest variable  and the occurrence of interest event: death from any cause for OS and disease recurrence or death for RFS. Patients without any event were censored at the date of last contact. Survival curves were estimated using the Kaplan–Meier method and compared using the Log-rank test. Hazard ratios were provided with their bilateral 95% confidence interval (95%CI) and Wald’s test for significance. All tests were two-sided. The level of statistical significance was set at α = 0.05. Statistical analyses were carried out with the SASCTC detection and bevacizumab had no correlation to pCR for breast cancer patients included in the AVASTEM trial. The absence of CTCs at inclusion was associated with improved survival. Bevacizumab concentrations under treatment or after treatment completion were not correlated to survival and treatment-related adverse events. Larger prospective data are warranted to implement CTC use in routine practice for prognosis assessment of patients with breasts cancer treated in the neoadjuvant setting.


Computed tomography confirmed recurrence as PVT aggravation was reported in 43 patients, with ≥50% PVT volume reduction following anticoagulation therapy. In 43 patients, recurrence significantly correlated with the pretreatment PVT volume (P = 0.019). Factors influencing recurrence included a Child–Pugh score >8 (P = 0.049) and fibrosis index ≤7.0 based on four factors (FIB-4) (P = 0.048). Moreover, the relationship between recurrence and correlating factors showed that 15 patients who received warfarin experienced recurrence more often when Child–Pugh scores were >8 (P = 0.023), regardless of maintenance treatment. For patients who did not receive warfarin, a PVT volume ≥3.0 mL significantly influenced recurrence (P = 0.039). Therefore, the platelet count influences the PVT volume. The pretreatment PVT volume correlated with recurrence after anticoagulation therapy. According to the Kaplan–Meier curve, risk factors for PVT recurrence after anticoagulation therapy included Child–Pugh scores >8 and FIB-4 ≤7.0. Therefore, the FIB-4 is a unique factor that shows trends opposing other liver function markers.This retrospective study investigated factors influencing the portal vein thrombosis (PVT) volume and recurrence in 52 cirrhosis patients with PVT from November 2008 to September 2018. All patients were treated with danaparoid sodium with or without additional antithrombin III. Blood platelet counts significantly correlated with the PVT volume (r Portal vein thrombosis (PVT) is defined as the presence of a thrombus in the lumen of the vein that causes complete or partial obstruction of portal venous blood flow. If a thrombus is present, then a cavernoma is evidence of chronic thrombosis. Extrahepatic portal vein obstruction includes occlusion of the portal vein, with or without thrombosis, and cavernoma formation, with or without portal hypertension .PVT can occur in various clinical situations such as cirrhosis, myeloproliferative disease, cancer, and infection . Common Generally, patients treated with anticoagulants have significantly higher rates of PVT recanalization than patients not treated with anticoagulants. Furthermore, PVT recanalization rates were 71% with anticoagulants and 42% without anticoagulants. Complete PVT recanalization rates were 53% with anticoagulants and 33% without anticoagulants. Additionally, the PVT progression rate decreased significantly for patients using anticoagulants compared to those not using anticoagulants. There was no difference in the bleeding rates of patients using and not using anticoagulants .Danaparoid sodium is an effective anticoagulant that inhibits activated factor Xa. An important feature of this agent is the low rate of cross-reactivity with antibodies associated with immune-mediated heparin-induced thrombocytopenia . This agPreviously, we reported danaparoid sodium therapy with and without AT-III anticoagulation therapy for PVT in cirrhosis patients ; in that2 = 0.17; P < 0.01). However, other factors, such as the Child–Pugh score, ALBI score, FIB-4 index, plasma AT-III level, plasma fibrinogen degradation products (FDP) level, plasma D dimer (DD) level, serum albumin level, and plasma prothrombin time (PT) activity, revealed no significant differences  was defined as a reduction of ≥75% in the volume of PVT. Ineffective group (n = 24) was considered if the reduction in the PVT volume was <75%. An analysis was performed in those two groups to investigate the correlation factors that affected treatment efficacy. However, this analysis also indicated no significant difference in the correlation factors: the Child–Pugh score, ALBI score, FIB-4 index, plasma AT-III level (this date has been presented in our previous report ), plasmaP = 0.019)  .P = 0.017) limited to the effective group , history of varices treatment, and previous PVT (including thrombosis in the MPV) showed no significant differences. A summary of these results is presented in ve group .4 (n = 19) or <8.0 × 104 (n = 24); PVT volume ≥3.0 mL (n = 20) or <3.0 mL (n = 23); history of splenectomy or PSE present (n = 7) or absent (n = 36); history of varices treatment present (n = 20) or absent (n = 23); and HCC diagnosis present (n = 16) or absent (n = 27). The Kaplan–Meier curve was used to analyze the recurrence event for each correlation factor.To investigate the recurrence events among the 43 patients, we divided the correlation factors into two groups. The cut-off values for both groups were decided based on the receiver-operating characteristic (ROC) curve and the median. The following factors were below: Child–Pugh score ≤8 (n = 28) or >8 (n = 15); ALBI ≤−1.7 (n = 20) or >−1.7 (n = 23); FIB-4 ≤ 7.0 (n = 20) or >7.0 (n = 23); plasma AT-III ≥60 (n = 22) or <60 (n = 21); blood platelet count ≥8.0 × 10P = 0.049), and in the group with FIB-4 ≤7.0 than in the group with FIB-4 >7.0 . These outcomes were statistically significant. Other correlation factors revealed no significant differences . However, there was no significant difference between the groups of patients with and without maintenance treatment. A PVT volume ≥3.0 mL (n = 7) significantly predicted recurrence  when patients did not receive maintenance treatment. Nonetheless, there was no significant difference in the PVT volume of the group of patients with maintenance treatment. Other correlation factors revealed no statistical significance (The group of patients with maintenance treatment experienced more recurrence events when the Child–Pugh scores were >8 (n = 5) than when the Child–Pugh scores were ≤8 (n = 10); this difference was statistically significant . Other factors did not show a significant relationship. Thus, we considered the possibility of a correlation between blood platelet count and PVT volume. Some reports indicated that the increased risk of PVT in cirrhosis is associated with decreased liver function (Child–Pugh score B/C) [According to the analysis, the PVT volume correlated significantly only with blood platelet count, although the correlation was very weak (ore B/C) , and incore B/C) . Howeverore B/C) .9/L) than in the non-PVT group  after laparoscopic splenectomy [However, for cirrhosis patients who had undergone splenectomy, the incidence of PVT after surgery was 37.7% (49/130), and an increased platelet count has been described as a risk factor . Anotherenectomy . Anotherenectomy . FurtherBased on these reports, low platelet levels are usually related to PVT because they can reflect low liver function and/or the presence of portal hypertension, causing splenomegaly. However, a drastic increase in platelets such as after splenectomy or with the use of TPO agonists is also a risk factor for PVT.Although the platelet count may influence the PVT volume, it does not influence the PVT reduction after anticoagulation treatment. To our knowledge, no report has investigated the relationship between the PVT volume and platelet count.Factors that contributed to therapeutic efficacy such as sustained liver function (Child–Pugh class A) have been previously reported ; howeverIt is vital to investigate predictive factors when anticoagulant drugs are used to treat PVT. One factor affecting recurrence after anticoagulation therapy is the pretreatment PVT volume. As previously stated, there is no obvious factor that predicts PVT recurrence. However, it is reasonable to conclude that a large PVT volume tends to lead to recurrence. A diagnosis of HCC, previous splenectomy or PSE, previous treatment of varices, previous PVT including thrombosis of the MPV, and decreased liver function did not affect the recurrence of PVT.P < 0.05). Although the PVT volume affected recurrence after anticoagulation treatment in this study, our investigation of groups with PVT ≥3.0 mL and with PVT <3.0 mL using the Kaplan–Meier curve could not, unfortunately, prove a significant difference. However, another investigation using the Kaplan–Meier curve showed a significant difference between groups with PVT ≥3.0 mL and PVT <3.0 mL without maintenance treatment.According to the Kaplan–Meier curve for PVT recurrence after anticoagulation therapy, Child–Pugh scores >8 and the FIB-4 index ≤7.0 were considered recurrence factors (P = 0.023) with maintenance treatment, and a PVT volume ≥3.0 mL was a significant factor for recurrence (P = 0.039) without maintenance treatment.FIB-4 was a novel factor. The FIB-4 index has been known to be correlated with the stages of liver fibrosis ,32,33. TThe prevention of PVT recurrence using anticoagulant agents was effective, leading to significant reductions in the risk of PVT development and liver decompensation, marked improvements in liver function and Child–Pugh score, and improvements in overall survival . As discSeveral limitations existed in this study. First, this was a retrospective study; therefore, there may have been some confounding factors because there was no randomization. Second, four patients had exceptionally larger PVT volumes (≥20 mL) compared to others. Two of four patients had a recent treatment history of splenectomy or radiofrequency ablation and three of four patients had a post-treatment history of varices, which suggested high portal hypertension. As such, the large PVT volumes would have influenced the results of this study. Third, some patients had the benefit of AT-III, but others did not. We conducted a second analysis to confirm whether there was a difference in relation to factors and PVT volume, reduction, and recurrence. According to the results, the Child–Pugh score and the ALBI score were significantly higher in the group of patients treated with combination therapy than in the group treated with monotherapy. This result indicated that liver function was lower in patients with combination therapy before treatment. As PVT volume/reduction/recurrence, there were no significant differences in PVT volume .Further research of the treatment regime for combined AT-III and danaparoid is required. We believe that our analysis is novel and has never been reported before. We hope that additional large-scale, long-term prospective studies will verify our results and clarify whether large PVT volumes affect liver function and prognosis, and the usefulness of the FIB-4 index to determine recurrence after anticoagulant therapy.We previously reported the aims and protocol of this study . This reAll PVT patients were treated with danaparoid sodium with AT-III (combination therapy) or without AT-III (monotherapy) at Kanazawa University Hospital. Combination therapy was performed from November 2008 to September 2011; monotherapy was performed from October 2011 to March 2014. Therefore, from April 2014 to September 2018, we performed combination therapy for patients with AT-III ≤70% and monotherapy for those with AT-III >70% .All patients with PVT included in this study received an intravenous injection of 1250 units of danaparoid sodium  twice daily for 2 weeks. This was the protocol treatment for PVT. Patients in the combination therapy group received an additional intravenous infusion of AT-III  at a dose of 1500 units/day from days 1 to 5 and from days 8 to 12 .Maintenance treatment using warfarin after combination therapy or monotherapy was performed in certain patients. Drug indication was decided by the attending doctor taking into consideration the status of PVT, bleeding tendency, blood platelet count, coagulation markers, other comorbidities, and prognosis.Recurrence was defined as the aggravation of PVT confirmed by computed tomography (CT) after anticoagulation therapy. Forty-three patients whose PVT volumes were reduced by ≥50% were selected for analysis because it was difficult to evaluate the recurrence of PVT in cases with <50% reduction. Patients’ medical records were reviewed and their demographic, clinical, laboratory, and imaging data were collected. All laboratory data were obtained on the first day±1day when combination therapy or monotherapy was administered.The Institutional Review Board (IRB) of Kanazawa University Hospital approved the study’s treatment strategy and study protocol (no. 2016–096). The study was conducted according to the Declaration of Helsinki. The requirement for obtaining informed consent from the patients was waived by the IRB because of the study’s retrospective nature.PVT was confirmed by contrast-enhanced CT (CECT). We traced the thrombus on an axial CECT image and calculated the volume of the thrombus by using a three-dimensional image analysis system . The measurement was performed by either a radiologist or a gastroenterologist.CECT involved two stages: before anticoagulation therapy and between days 13 and 18 after treatment. PVT volume reductions were based on the following calculation: reduction of PVT (%) = {(PVT volume pretreatment − PVT volume post-treatment)/(PVT volume pretreatment)} × 100.2 test or Fisher exact test when appropriate. The Student’s t-test or Mann–Whitney U test was used for continuous variables. Recurrence rates were analyzed using the Kaplan–Meier method with the log-rank test. All P values were two-tailed, and P < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism software 6.0 . Categorical variables were compared using the χDuring this analysis, we demonstrated that the PVT volume was significantly related to the platelet count and that the pretreatment PVT volume affected recurrence following anticoagulation treatment.Child–Pugh scores >8 and the FIB-4 index ≤7.0 were factors that predicted recurrence after anticoagulation treatment. The FIB-4 index is a unique factor that shows trends opposing those of other liver function markers. Additionally, maintenance treatment was recommended if the liver function was good (Child–Pugh score of <8) and the pretreatment PVT volume was large (PVT volume ≥3.0 mL).


Trachypithecus auratus) is considered a strict forest primate, and little is known about populations living in savannah. In 2017–2018, we investigated the density and distribution of Javan lutung in Baluran National Park, Indonesia. We conducted ad libitum follows and line transect distance sampling with habitat suitability analysis of Javan lutung.Research of many mammal species tends to focus on single habitats, reducing knowledge of ecological flexibility. The Javan lutung (− 2 (95% CI 7.91–28.08), and estimated population size was 3727 individuals (95% CI 1979 – 7019). Long-tailed macaque (Macaca fascicularis) habitat suitability was the main driver of lutung habitat suitability as the probability of lutung occurrence increased greatly with macaque habitat suitability. Distance to roads, and distance to secondary forest had a negative relationship with lutung occurrence. Lutung habitat suitability decreased with increasing elevation, however, Mt Baluran and the primary forest on Mt Baluran was under-sampled due to treacherous conditions. Follows of six focus groups revealed considerable use of savannah, with terrestrial travel. The follows also revealed polyspecific associations with long-tailed macaques through shared sleeping sites and inter-specific vocalisations.Estimated density was 14.91 individuals kmOur study provides new knowledge on the general ecology of Javan lutung, such as use of savannah habitats, underlining our need to branch out in our study sites to understand the flexibility and adaptability of our study species. Another undocumented behaviour is the polyspecific association with long-tailed macaques. We encourage more research on this subject. Pan troglodytes) for example, are often categorised by their forest behaviour, although their behaviour in savannah habitats differs greatly , we decided to include the mountain in our estimates. We placed the grid randomly in respect to habitats and wildlife distribution , but ens− 1, finishing one transect trip (outbound or return) within 4 h with observations never exceeding 15 min [All transects were 4.5 km long with at least 2.0 km between adjacent transects totalling 189.0 km for outbound and return trip. However, we were only able to walk 160.65 km due to inaccessibility  in our analysis to avoid double counts. The analysis was conducted in Distance 7.1 . For cal Mt Ijen . We coulp < 0.05 and lowest χ2/df accumulated for χ2 tests half-normal key detection function with 2 cosine adjustments was chosen [We right-truncated our data to increase robustness in estimating detection function, excluding all observations beyond 90 m . We inves chosen , 24, 46.n = 5) included elevation (m), slope (°), aspect (radians), hill-shade (radians) and terrain ruggedness (index), derived from a digital elevation model of the study area. We generated a raster layer using ESRI shapefiles, that included 13 major vegetation/habitat types found in the census region including: rice fields, livestock fields, teak plantations, dwarf forests, evergreen forest, shrub forest, primary forest, secondary forest, mangroves, acacia forest, savannah, restored savannah, and beach. Instead of using categorical classes for each habitat/vegetation type, and to capture potential edge effects in habitat suitability, we generated new rasters (15 × 15 m pixel size). We did this by calculating for each raster cell the Euclidean distance (km) to the nearest cell with a given vegetation type (n = 13). Euclidean distance to (km) paved roads, human settlements, trails, and rivers found in the census region were included in the eight last raster layers.Shapefiles containing vector layers provided by BNP, updated using Google Earth enabled us to create a map of BNP in QGIS 2.18.19, in which vector layers from the LTDS census were inserted. Using the raster package in R , we geneOnce all raster layers were created, we excluded collinear raster layers by calculating the Variance Inflation Factor (VIF), then excluding the one with highest VIF and repeating this process until only layers with a VIF < 2 were remaining . With thN = 62) comprised projected waypoints of observations from both the outbound and return trip of each transect from the LTDS population census. All non-correlated environmental raster layers were used to extract the values of environmental conditions for presence points.We used the SDM package in R for modelling and mapping of habitat suitability, and employed the MaxEnt algorithm . PresencModel accuracy of MaxEnt was assessed by calculating the area under the curve of the receiver operating characteristic . An AUAdditional file 1:. Figure S1. the Javan lutung in Baluran National Park.Additional file 2:. Figure S2. savannah in rainy season with Baluran Mountain in the background in Baluran National Park.Additional file 3:. Figure S3. Long-tailed macaques in Baluran National Park.Additional file 4: Appendix Fig. 1. Response curves for the six variables with lowest importance (out of the ten included covariates) as based on the MaxEnt algorithm.


HRF was the strongest predictor of future PA (β = 0.353), and also predicted PC (β = 0.336) and MC (β = 0.163). Object-control MC predicted future PA (β = 0.192). Reciprocal relationships existed between object-control MC and PA, and between object-control MC and PC. HRF was the strongest predictor of PA. Object-control MC also predicted PA. PA promotion strategies should target the development of HRF and object-control MC in primary school to reduce the decline in PA frequently observed after the school transition.Physical activity (PA) decreases with age. The school transition is noted for significant changes in PA behaviour. Motor competence (MC), health-related fitness (HRF), and perceived competence (PC) are generally positively associated with PA. The aim of this study was to examine longitudinal cross-lagged relationships between PA, MC, HRF, and PC across the school transition from final year of primary school to first year of second-level school in Irish youth. PA (accelerometery), object-control and locomotor MC (TGMD-III), PC , and HRF  were measured in final year of primary school (6th class) and first year of second-level school (1st year). In the sixth class, 261 participants  were tested. In first year, 291 participants  were tested. In total, 220 participants were involved in the study at both timepoints. Cross-lagged regression in AMOS23, using full information maximum likelihood estimation, was conducted to test reciprocal and predictive pathways between variables. The full cross-lagged model showed acceptable fit (χ Physical activity (PA) is associated with positive health outcomes in youth ,2, but gNumerous factors are associated with youth engagement in PA ,13. A seMC is generally used to describe goal-directed movement requiring coordination of the body . An impoHRF, a proposed mediator in Stodden et al.’s (2008)  model, gPC refers to an individual’s ability to effectively master a task  and, likIndividual pathways outlined in Stodden et al.’s (2008)  conceptuData were collected from participants in the 6th class of primary school (January–April 2017), and one year later in 1st year of second-level school (January–May 2018). Participant recruitment began with contacting second-level schools which fit the criteria of having a maximum of three main feeder primary schools. Two second-level school principals consented for their schools to be involved in the study. Following this, the main feeder primary schools for both consenting second-level schools were contacted. Six primary school principals (three feeder schools for each second-level school) provided consent. Parental consent and participant assent were obtained in the 6th class of primary school for each participating student. At baseline, 261 participants were tested . Of these, 41 participants were not available for testing in 1st year of second-level school as they did not transfer into the catchment area schools selected for data collection. In 1st year, 291 participants  were tested. The sample in 1st year included an additional 71 students who had not attended the feeder primary schools. These additional participants provided assent, and parental consent, prior to data collection in 1st year. In total, 335 participants were involved in the study for at least one timepoint, and 220 of these participated at both timepoints. Ethical approval for all measures was obtained from the authors’ institutional ethics committee .Actigraph  accelerometers , set to capture data in 10-s epochs, were used to measure PA . ParticiMC was assessed using skills selected from the 3rd edition of the Test of Gross Motor Development  was usedceptions . At bothceptions .HRF was measured using five tests . A previous confirmatory factor analysis (CFA) showed these five tests give a fair representation of HRF in youth . Protoco2 (33) = 22.05, p = 0.93) and 1st year (Little’s MCAR χ2 (37) = 52.28, p = 0.05) indicating that missing data was MCAR [Due to the longitudinal study design, a missing data analysis was conducted. All variables had some degree of missingness at both timepoints. In 6th class 220 participants (65%) had missing data on at least one variable. In 1st year 295 participants (88%) had missing data on at least one variable. A large proportion of this missing data was due to accelerometer non-compliance. Excluding MVPA, 68% of participants in 6th class had complete data, while 34% of participants had complete data in 1st year. Little’s “missing completely at random” (MCAR) test was conducted to determine the nature of missingness for data at each timepoint. Little’s MCAR test was insignificant for data in 6th class  were conducted in AMOS version 23 to create latent variables for locomotor MC, object-control MC, PAC, and HRF. First, the measurement models for each latent variable in 6th class were tested. All items tested for each variable were included in the initial CFA’s. Following statistical and theoretical analyses, catch was removed from the object-control MC latent variable due to both its relatively low loading , and tht-tests were conducted to analyse sex differences for each variable in 6th class, and in 1st year. Effect sizes were calculated to determine the magnitude of any differences.Mean values for each variable at each time point were calculated and independent samples 2) test, the Comparative Fit Index (CFI), the Normed Fit Index (NFI), and the Root Mean Square Error of Approximation (RMSEA). A statistically insignificant χ2 indicates a good fitting model, although a statistically significant χ2 need not necessarily signify poor model fit, given that large samples have been shown to give erroneous χ2 results [A cross-lagged regression model was specified consisting of four latent  and one observed (MVPA) variable, each measured in 6th class and again in 1st year. Full panel analyses in AMOS, using maximum likelihood estimation, was used to test all hypotheses simultaneously. The model included autoregressive paths for each variable over time and cross-lagged paths from each variable in 6th class to each other variable in 1st year. As measurement invariance was satisfied the full cross-lagged regression model was specified with observed variables to allow for a more parsimonious model fit . Variabl results . CFI and results ,67, whil results . RMSEA a results . In mode results .2) difference test was conducted on the full model to identify any sex differences in pathways. A significant χ2 test indicates variance between sexes for model fit [Using the multigroup analysis, a Chi-square (χodel fit . The tabodel fit .d)  . Accordi2 = 69.12, df = 8, p < 0.01, NFI = 0.93, CFI = 0.94, RMSEA = 0.15 (CI: 0.12–0.19)). Pathways with standardised regression coefficients greater than 0.10 are shown in Analysis of the full cross-lagged model showed a reasonable fit (χEach variable in sixth class significantly and positively predicted itself in first year . HRF andHRF was a moderate and significant predictor of MVPA (β = 0.353) and PAC (β = 0.336) in first year, and significantly predicted first year locomotor MC (β = 0.163), though this association was small. Other than the associations between sixth class HRF and first year MVPA and PAC, all other pathways from sixth class to first year variables were small (0.10–0.29). Reciprocal relationships between object-control MC and MVPA, and object-control MC and PAC were found. In addition, object-control MC in sixth class was a significant predictor of HRF (β = 0.125) and locomotor MC (β = 0.165) in first year. Locomotor MC in sixth class negatively predicted MVPA in first year, though this association was small and insignificant (β = −0.175). PAC in sixth class was a predictor of locomotor MC in first year (β = 0.100).For variance in first-year variables, the overall model predicted 81% variance in HRF, 61% variance in PAC, 44% variance in object-control MC, 39% variance in locomotor MC, and 34% variance in MVPA. Excluding autoregressive pathways, HRF was the only 6th class variable to exhibit moderate associations with multiple variables in 1st year. Object-control MC also contributed to a variance in multiple factors in first year, but these associations were small.2 difference test . Inspection of the critical ratios table showed differences in regression coefficients for several pathways in the model   found thSubstantial differences in some of the pathways depicted in the model were seen between males and females in the current study. For males, MVPA in sixth class was a strong predictor of first year MVPA, whereas for females this pathway was virtually non-existent. This supports previous research nationally  and inteWhile MVPA was less stable in females than males across the transition, the opposite was found for object-control MC, indicating that object-control MC is less likely to change in females compared to males across the school transition. A longitudinal study in Irish youth reported that, between the ages of 10 and 18, females were more likely to drop out of organised sport and less likely to take up a new sport compared to males . PopularContrary to expectation, sixth class MVPA negatively predicted first year MC for females. In comparison, sixth class MVPA did not have a substantial effect on first year MC for males. MC has been identified as an important factor for social acceptance within peer groups for males . For femThat being said, HRF in sixth class was a positive predictor of MC for females in first year. HRF in females, and males, is highly stable over time. In comparison, MVPA for females is highly unstable. This lack of stability in MVPA compared to HRF for females may in part explain how MVPA could negatively predict MC while HRF positively predicts MC. Notwithstanding the significant mean change in MVPA in females across the school transition, significant intra-individual change is also occurring, as indicated by the low stability of MVPA. Thus, females who were highly active in sixth class may be low active in first year and vice versa. This may then have a knock-on effect on their execution of MC in first year. In comparison, HRF in females is much more stable indicating that fitness levels attained by sixth class of primary school carry through into first year of second-level school. In addition, compared to MVPA which is a singular variable, HRF is a multifaceted construct ,88. HaviThis is the first study to explore the predictive pathways between all of the variables depicted in Stodden et al.’s (2008)  model acThe findings from the current study point to the importance of HRF for future PA engagement in adolescents. Whereas MC is commonly positioned as one of the primary predictors of PA, this study found HRF, measured as a composite, to be more important in predicting PA across the transition from primary to second-level school. Object-control proficiency, as a component of MC, was however found to have a positive effect on PA, and other factors shown to be associated with positive PA behaviour. From this perspective it would seem that developing HRF and object-control MC in primary school may be crucial factors to consider as we strive to address the frequently reported decrease in PA observed across the school transition.