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Conflict of interest: No Introduction and Objectives: Pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma, has shown efficacy in improving indirect markers of liver steatosis, inflammation, and fibrosis. It also addresses systemic and adipose tissue insulin resistance in patients with type 2 diabetes and metabolic dysfunction-associated fatty liver disease (MASLD). This study aims to evaluate whether sustained consumption of pioglitazone over 12-24 months can improve liver stiffness in individuals diagnosed with biopsy- proven metabolic dysfunction-associated steatohepatitis (MASH). Patients / Materials and Methods: Retrospective data from 56 MASLD patients who received pioglitazone treatment for 12-24 months (15-30 mg daily) were gathered from three public hospitals in Brazil. Vibration-controlled transient elastography [VCTE (FibroscanTM)] was performed before and after pioglitazone treatment as a non-invasive method to monitor disease progression. Additionally, a thorough analysis of both laboratory and clinical data was conducted. Results and Discussion: Most participants were female (63%, n = 35) and obese (BMI 31.1 ± 5.2) with a mean age of 58.4 ± 11.4 years. Initially, participants mostly had hypertension (71%, n = 40) and type II diabetes (61%, n = 34). During the second evaluation, the number of subjects with dyslipidemia and statin use increased. Initially, the liver stiffness measurement (LSM) median was 8.1 kPa (Min: 2.8; Max: 35.3) and 7.2 kPa (Min: 3.5; Max: 32.9) at the second evaluation. Prolonged pioglitazone treatment demonstrated LSM attenuation in 63% of cases (n = 35), resulting in an absolute reduction ranging from 0.1 to 14.4 kPa and a relative reduction ranging from 1.25% to 40.8%. Further analysis comparing the group with improved versus the group with worsened liver stiffness showed a decrease in the CAP parameter, FAST score, and levels of ALT, AST, GGT, TG, and ferritin. Conclusions: The administration of pioglitazone for 12 to 24 months effectively reduced hepatic inflammation and enhanced VCTE parameters in 63% of cases.

Objectives: There is a lack of large studies on long-COVID symptoms with symptoms measurements before the onset of COVID-19. Therefore, long-COVID is still poorly defined. Methods: The Norwegian COVID-19 Cohort Study is a population-based, open cohort of adult participants (aged 18-96 years) from Norway. From March 27, 2020, participants were recruited through social media, invitations, and nationwide media coverage. Fourteen somatic and cognitive symptoms were assessed at baseline and four follow-ups for up to 22 months. SARS-CoV-2 test status was obtained from a mandatory national registry or from self-report. Results: After follow-up, 15 737 participants had a SARS-CoV-2-positive test, 67 305 had a negative test, and 37 563 were still untested. Persistent symptoms reported more frequently by positive compared with negative participants one month after infection, were memory problems (3-6 months: adjusted odds ratio (aOR) = 6.8, CI = 5.7-8.1; >18 months: aOR = 9.4, CI = 4.1-22), and concentration problems (3-6 months: aOR = 4.1, CI = 3.5-4.7; >18 months: aOR = 4.4, CI = 2.0-9.7) as well fatigue, dyspnea, anosmia and dysgeusia. Conclusions: COVID-19 was associated with cognitive symptoms, anosmia, dysgeusia, dyspnea, and fatigue as well as worsening of overall health up to 22 months after a SARS-CoV-2 test, even when correcting for symptoms before the onset of COVID-19.

Abstract Background Previous studies assessed the prognostic effect of aspirin, statins, and metformin in breast cancer (BC) patients, with inconclusive results. Methods We performed a nationwide population-based cohort study to evaluate if post-diagnostic use of low-dose aspirin, statins, and metformin was associated with BC-specific survival. Women aged ≥ 50 years and diagnosed with BC in 2004–2017, who survived ≥ 12 months after diagnosis (follow-up started 12 months after diagnosis), were identified in the Cancer Registry of Norway. The Norwegian Prescription Database provided information on prescriptions. Multivariable Cox proportional hazard models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between post-diagnostic use and BC-specific survival, overall and by oestrogen receptor (ER) status. Results A total of 26,190 patients were included. Of these, 5324 (20%), 7591 (29%), and 1495 (6%) were post-diagnostic users of low-dose aspirin, statins, and metformin, respectively. The median follow-up was 6.1 years, and 2169 (8%) patients died from BC. HRs for use, compared to no use, were estimated at 0.96 (95% CI 0.85–1.08) for low-dose aspirin (ER+: HR = 0.97, 95% CI 0.83–1.13; ER−: HR = 0.97, 95% CI 0.73–1.29, p value for interaction = 0.562), 0.84 (95% CI 0.75–0.94) for statins (ER+: HR = 0.95, 95% CI 0.82–1.09; ER−: HR = 0.77, 95% CI 0.60–1.00, p value for interaction = 0.259), and 0.70 (95% CI 0.51–0.96) for metformin (compared to use of non-metformin antidiabetics) (ER+: HR = 0.67, 95% CI 0.45–1.01; ER−: HR = 1.62, 95% CI 0.72–3.62, p value for interaction = 0.077). Conclusion We found evidence supporting an association between post-diagnostic use of statins and metformin and survival, in patients with BC. Our findings indicate potential differences according to ER status.

Background This study investigated the effects of weight loss during follow-up on cardiovascular outcomes in a type 2 diabetes cohort and tested interactions with clinical and laboratory variables, particularly physical activity, that could impact the associations. Methods Relative weight changes were assessed in 651 individuals with type 2 diabetes and categorized as ≥5% loss,

Abstract Background Neoadjuvant chemotherapy (NAC) before radical cystectomy is associated with pathological downstaging (DS) and improved overall survival (OS) in patients with muscle-invasive bladder cancer (MIBC). Population-based studies have not unequivocally shown improved survival. The aim of this population-based study was to evaluate the effect of NAC on DS and OS in Norwegian patients with MIBC. Methods Patients in the Cancer Registry of Norway undergoing radical cystectomy (2008–2015) with or without NAC diagnosed with MIBC between 2008 and 2012 were included. Follow-up data were available until 31 December 2019. Logistic regression estimated the odds of DS with NAC, and a Cox model investigated the effect of DS on OS. Cox models, a mediator analysis and an instrumental variable approach were used to investigate the effect of NAC on OS. Results A total of 575 patients were included. NAC was administered to 82 (14%) patients. Compared to cystectomy only, NAC increased the proportion (43% vs. 22%) and the odds of DS (OR 2.51, CI 1.37–4.60, p = 0.003). Independent of NAC, the proportion of pN0 was higher in patients with DS (89% vs. 60%) and DS yielded a 78% mortality risk reduction (HR 0.22, CI 0.15–0.34, p = 1.9∙10–12), compared to patients without DS. We did not find an association between NAC and OS, neither by Cox regression (HR 1.16, CI 0.80–1.68, p = 0.417) nor by an instrumental variable approach (HR = 0.56, CI = 0.07–4.57, p = 0.586). The mediation analysis (p = 0.026) confirmed an indirect effect of NAC on OS through DS. Limitations include limited information of the primary tumour, details of NAC treatment and treatment indications. Conclusions NAC increases the probability of DS and is indirectly associated to OS. DS is related to the absence of regional lymph node metastases and is associated with an OS benefit. Improved staging and biomarkers are needed to identify patients most likely to achieve DS and to benefit from NAC.

Oncolytic viral immunotherapies are agents which can directly kill tumor cells and activate an immune response. Oncolytic viruses (OVs) range from native/unmodified viruses to genetically modified, attenuated viruses with the capacity to preferentially replicate in and kill tumors, leaving normal tissue unharmed. Talimogene laherparepvec (T-VEC) is the only OV approved for patient use in the United States; however, during the last 20 years, there have been a substantial number of clinical trials using OV immunotherapies across a broad range of cancers. Like T-VEC, many OV immunotherapies in clinical development are based on the herpes simplex virus type 1 (HSV-1), with genetic modifications for tumor selectivity, safety, and immunogenicity. Despite these modifications, HSV-1 OV immunotherapies are often treated with the same biosafety guidelines as the wild-type virus, potentially leading to reduced patient access and logistical hurdles for treatment centers, including community treatment centers and small group or private practices, and healthcare workers. Despite the lack of real-world evidence documenting possible transmission to close contacts, and in the setting of shedding and biodistribution analyses for T-VEC demonstrating limited infectivity and low risk of spread to healthcare workers, barriers to treatment with OV immunotherapies remain. With comprehensive information and educational programs, our hope is that updated biosafety guidance on OV immunotherapies will reduce logistical hurdles to ensure that patients have access to these innovative and potentially life-saving medicines across treatment settings. This work reviews a comprehensive collection of data in conjunction with the opinions of the authors based on their clinical experience to provide the suggested framework and key considerations for implementing biosafety protocols for OV immunotherapies, namely T-VEC, the only approved agent to date.

IntroductionFeto-maternal cell transfer during pregnancy is called microchimerism (Mc). Its persistence in respective hosts is increasingly studied as to its potential role in immune tolerance, autoimmunity, cancer, and degenerative diseases. Murine models with transgenic reporter genes, heterozygously carried by the mother, allow maternal Mc tracking in wild-type (WT) offspring. However, as gestation in mice is multi-embryonic, an exchange of cells between fetuses carrying the same reporter gene as their mother and negative WT littermate, named littermate Mc (LMc), can occur and be confounded with the maternal source. We propose here to evaluate LMc contribution in mice.MethodsTo avoid the maternal confounding source of Mc, transgenic males, heterozygous for a reporter gene, here, the human leukocyte antigen DRB1*04 (DR4+/−), were crossed with WT females (DR4−/−). DR4+/− LMc was specifically quantified by HLA-DR4 quantitative PCR, i) in utero in main organs from 15 DR4−/− fetuses from three litters of 11, nine, and five; and ii) after birth in two litters of eight pups: in two DR4−/− stillborns and four DR4−/− adult mice.ResultsAt embryonic stages, DR4−/− fetuses having one or two nearby DR4+/− littermates in the same uterine horn were almost seven times more frequently positive for DR4− microchimerism in their organs (p = 0.01) and had quantitatively more LMc (p = 0.009) than those without nearby DR4+/− littermates. Furthermore, LMc persists at birth and into adulthood with interindividual heterogeneity.ConclusionsThis study identifies heterogeneity for LMc acquisition according to in utero position and different interpretation of previously published results on maternal Mc in mice.

Abstract Background The prognostic importance of changes in aortic stiffness for the occurrence of adverse cardiovascular outcomes and mortality has never been investigated in patients with type 2 diabetes. We aimed to evaluate it in a cohort of 417 patients. Methods Changes in aortic stiffness were assessed by 2 carotid-femoral pulse wave velocity (CF-PWV) measurements performed over a 4-year period. Multivariable Cox analysis examined the associations between changes in CF-PWV, evaluated as a continuous variable with splines and as categorical ones (quartiles and stable/reduction/increase subgroups), and the occurrence of total cardiovascular events (CVEs), major adverse CVEs (MACEs), and all-cause and cardiovascular mortality. Results Over a median follow-up of 8.2 years after the 2nd CF-PWV measurement, there were 101 total CVEs (85 MACEs) and 135 all-cause deaths (64 cardiovascular). As a continuous variable, the lowest risk nadir was at -2.5%/year of CF-PWV change, with significantly higher risks of mortality associated with CF-PWV increases, but no excess risks at extremes of CF-PWV reduction. Otherwise, in categorical analyses, patients in the 1st quartile (greatest CF-PWV reductions) had excess risks of all-cause and cardiovascular mortality (hazard ratios [HRs]: 2.0–2.7), whereas patients in 3rd quartile had higher risks of all outcomes (HRs: 2.0–3.2), in relation to the lowest risk 2nd quartile subgroup. Patients in the 4th quartile had higher risks of all-cause mortality. Categorization as stable/reduction/increase subgroups was confirmatory, with higher risks at greater reductions (HRs: 1.7–3.3) and at greater increases in CF-PWV (HRs: 1.9–3.4), in relation to those with stable CF-PWV. Conclusions Changes in aortic stiffness, mainly increases and possibly also extreme reductions, are predictors of adverse cardiovascular outcomes and mortality in individuals with type 2 diabetes.

BackgroundThe wide availability of mobile phones has made it easy to disseminate health-related information and make it accessible. With gamification, mobile apps can nudge people to make informed health choices, including attending cervical cancer screening. ObjectiveThis matched retrospective cohort study examined the association between exposure to the FightHPV mobile app gamified educational content and having a cervical exam in the following year. MethodsWomen aged 20 to 69 years who signed an electronic consent form after downloading the FightHPV app in 2017 (intervention group) were matched 1:6 with women of the same age and with the same screening history (reference group) in 2015. To estimate the impact of exposure to the FightHPV app, we estimated cumulative incidence and hazard ratios (HRs) with 95% CIs. We used data from the Norwegian Cervical Cancer Screening Program database and Statistics Norway to determine screening participation and outcomes, respectively. ResultsWe matched 3860 women in the control group to 658 women in the intervention group; 6 months after enrollment, 29.6% (195/658) of the women in the intervention group and 15.21% (587/3860) of those in the reference group underwent a cervical exam (P

In recent decades, the breeding populations of the yellow-legged gull Larus michahellis (YLG) have increased significantly, primarily due to the increase in open refuse dumps and discards from fisheries. Portugal’s largest YLG breeding colony is located on Berlenga Island, where population numbers have been monitored since 1974. The population grew exponentially until 1994, prompting the implementation of population control measures, including culling adult birds and eggs. A long-term data base including number of breeding birds (since 1974), breeding parameters (since 2002), and trophic niches (using stable isotopes since 2011) of YLG breeding on Berlenga was related with oceanographic parameters, fish landings and quantity of urban waste. Trophic ecology showed strong relationships with oceanographic parameters (wNAO, Chl-a, and SST) and fisheries landings (the 10 most frequently consumed species by the YLG, traded at fish auctions in the main fishing harbour nearby). The results indicated significant relationships between reproductive performance and fisheries landings, particularly with demersal species that gulls primarily access through fisheries discards. However, population control measures played a pivotal role in stabilising and even reducing the population, despite sporadic events of poor oceanographic productivity in the past decade having a consistent impact on the reduction in breeding individuals.

Abstract Background Liver stiffness measurement (LSM, which reflects fibrosis) and controlled attenuation parameter (CAP, which reflects steatosis), two parameters derived from hepatic transient elastography (TE), have scarcely been evaluated as predictors of cardiovascular complications and mortality in individuals with type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). Methods Four hundred type 2 diabetic patients with NAFLD had TE examination (by Fibroscan®) performed at baseline. Multivariate Cox analyses evaluated the associations between TE parameters and the occurrence of cardiovascular events (CVEs) and mortality. TE parameters were assessed as continuous variables and dichotomized at low/high values reflecting advanced liver fibrosis (LSM > 9.6 kPa) and severe steatosis (CAP > 296 or > 330 dB/m). Improvements in risk discrimination were assessed by C-statistic and by the relative Integrated Discrimination Improvement (IDI) index. Results During a median follow-up of 5.5 years, 85 patients died (40 from cardiovascular causes), and 69 had a CVE. As continuous variables, an increasing LSM was a risk marker for total CVEs (hazard ratio [HR]: 1.05; 95% CI: 1.01–1.08) and all-cause mortality (HR: 1.04; 95% CI: 1.01–1.07); whereas an increasing CAP was a protective factor for both outcomes (HR: 0.93; 95% CI: 0.89–0.98; and HR: 0.92; 95% CI: 0.88–0.97; respectively). As dichotomized variables, a high LSM remained a risk marker of adverse outcomes (with HRs ranging from 2.5 to 3.0) and a high CAP was protective (with HRs from 0.3 to 0.5). The subgroup of individuals with low-LSM/high-CAP had the lowest risks while the opposite subgroup with high-LSM/low-CAP had the highest risks. Both LSM and CAP improved risk discrimination, with increases in C-statistics up to 0.037 and IDIs up to 52%. Conclusions Measured by hepatic TE, advanced liver fibrosis is a risk marker and severe steatosis is a protective factor for cardiovascular complications and mortality in individuals with type 2 diabetes and NAFLD.

Leon A.M. Berge, Tom K. Grimsrud, Ronnie Babigumira, Nathalie C. Støer, Nita K. Shala, Marit B. Veierød and Jo S. Stenehjem: Cancer epidemiology in practice: Working notes on cancer history-based selection and censoring

Abstract Background The prognostic importance of several hematological parameters has been scarcely investigated in type 2 diabetes. So, we aimed to evaluate their prognostic importance for development of complications in a cohort of type 2 diabetes. Methods In a prospective study, 689 individuals with type 2 diabetes had blood red cell, platelet and leukocyte parameters obtained at baseline. Multivariate Cox analyses examined the associations between several hematological parameters (including neutrophyl-to-lymphocyte, lymphocyte-to-monocyte, platelet-to-lymphocyte, and monocyte-to-HDL ratios) and the occurrence of microvascular (retina, renal and peripheral neuropathy) and cardiovascular complications (total cardiovascular events [CVEs], and major adverse CVEs [MACEs]), and all-cause and cardiovascular mortality. Improvements in risk discrimination were assessed by C-statistics and Integrated Discrimination Improvement (IDI) index. Results During a median follow-up of 10.5 years, 212 patients had a CVE (174 MACEs), 264 patients died (131 cardiovascular deaths); 206 had a renal, 161 a retinopathy and 179 patients had a neuropathy outcome. In multivariate-adjusted analyses, the lymphocytes count and lymphocyte-to-monocyte ratio were protective (hazard ratios [HRs]: 0.77 and 0.72, respectively), whereas the neutrophyl-to-lymphocyte and platelet-to-lymphocyte ratios were associated with increased risks (HRs: 1.19 and 1.17) for all-cause mortality. For cardiovascular mortality, the monocytes count, the neutrophyl-to-lymphocyte and monocyte-to-HDL ratios were associated with increased risks and the lymphocyte-to-monocyte ratio was protective. Higher lymphocyte-to-monocyte ratio was protective for renal failure outcome. However, none of them improved risk discrimination. Conclusions Low lymphocytes count and leukocyte ratios that mainly included lymphocytes were predictors of macrovascular complications and mortality in individuals with type 2 diabetes. However, they did not improve risk prediction over traditional risk factors.

Abstract Background Von Willebrand Factor (VWF) multimers are cleaved into smaller and less coagulant forms by the metalloprotease ADAMTS13. The aim of this study was to investigate the association between VWF and ADAMTS13 and mortality in dialysis patients. Methods We prospectively followed 956 dialysis patients. VWF levels and ADAMTS13 activity were measured. Cox proportional hazard analyses were used to calculate hazard ratios (HRs) with 95 % confidence intervals (CIs) to investigate the association between quartiles of VWF levels and ADAMTS13 activity and all-cause mortality. HRs were adjusted for age, sex, body mass index, cardiovascular disease, dialysis modality, primary kidney disease, use of antithrombotic medication, systolic blood pressure, albumin, C-reactive protein and residual GFR. Results Of the 956 dialysis patients, 288 dialysis patients died within three years (mortality rate 151 per 1000 person-years). The highest quartile of VWF as compared with lower levels of VWF was associated with a 1.4-fold (95 %CI 1.1–1.8) increased mortality risk after adjustment. The lowest quartile of ADAMTS13 activity as compared with other quartiles was associated with a 1.3-fold (95 %CI 1.0-1.7) increased mortality risk after adjustment. The combination of the highest VWF quartile and lowest ADAMTS13 quartile was associated with a 2.0-fold (95 %CI 1.3-3.0) increased mortality risk as compared with the combination of the lowest VWF quartile and highest ADAMTS13 quartile. Conclusions High VWF levels and low ADAMTS13 activity were associated with increased mortality risks in dialysis patients.

Abstract Background The prognostic importance of non-traditional risk factors for peripheral artery disease (PAD) development/progression is scarcely studied in diabetes. We investigated if carotid intima-media thickness (CIMT) and carotid-femoral pulse wave velocity (cf-PWV) added prognostic information beyond traditional cardiovascular risk markers for PAD outcomes. Methods Ankle-brachial index (ABI) was measured at baseline and after a median of 91 months of follow-up in 681 individuals with type 2 diabetes. Multivariate Cox regressions examined the associations between the candidate variables and the outcome. PAD development/progression was defined by a reduction in ABI ≥ 0.15 (to a level

The atypical chemokine receptor 3 (ACKR3) is known to regulate cell migration, but the underlying mechanisms are unclear. Here, the authors show, from an interactome analysis, ACKR3 association with the gap junction protein Connexin 43 in vivo and ACKR3-mediated inhibition of astrocyte gap junctional communication.

ABSTRACT: We analyzed 77 Salmonella spp. strains, from which 20 were isolated from broilers (cloacal swabs) and 57 from chickens from slaughterhouses under federal inspection. The following serotypes were identified: Salmonella Saint Paul (29), Salmonella Heidelberg (27), Salmonella Anatum (9), Salmonella Cerro (5), Salmonella Senftenberg (5), Salmonella enterica (O: 4,5) (1) and Salmonella enterica (O: 9.12) (1). Fifteen strains (19.5%) were resistant to enrofloxacin, six (7.8%) to ciprofloxacin, and 26 (33.8%) to nalidixic acid in the Disk Diffusion Test. The fifteen enrofloxacin resistant strains were selected for the PCR to detect the genes gyrA, gyrB, parC, and parE, and genetic sequencing to identify mutations in these genes. Five strains (33.3%) had point mutations in the gyrA gene, and one (6.7%) presented a point mutation in the parC gene. None of the 15 strains had mutations in the gyrB and parE genes, and none had more than one mutation in the gyrA gene or the other genes. The presence of point mutations in the strains studied corroborates with the phenotypic resistance observed to nalidixic acid. However, it did not explain the resistance to fluoroquinolones found in the 15 strains. Other mechanisms may be related to the fluoroquinolones resistance, highlighting the need for additional mutation screening.

Abstract Background The change in two measurements of a continuous outcome can be modelled directly with a linear regression model, or indirectly with a random effects model (REM) of the individual measurements. These methods are susceptible to model misspecifications, which are commonly addressed by applying monotonic transformations (e.g., Box-Cox transformation) to the outcomes. However, transforming the outcomes complicates the data analysis, especially when variable selection is involved. We propose a robust alternative through a novel application of the conditional probit (cprobit) model. Methods The cprobit model analyzes the ordered outcomes within each subject, making the estimate invariant to monotonic transformation on the outcome. By scaling the estimate from the cprobit model, we obtain the exposure effect on the change in the observed or Box-Cox transformed outcome, pending the adequacy of the normality assumption on the raw or transformed scale. Results Using simulated data, we demonstrated a similar good performance of the cprobit model and REM with and without transformation, except for some bias from both methods when the Box-Cox transformation was applied to scenarios with small sample size and strong effects. Only the cprobit model was robust to skewed subject-specific intercept terms when a Box-Cox transformation was used. Using two real datasets from the breast cancer and inpatient glycemic variability studies which utilize electronic medical records, we illustrated the application of our proposed robust approach as a seamless three-step workflow that facilitates the use of Box-Cox transformation to address non-normality with a common underlying model. Conclusions The cprobit model provides a seamless and robust inference on the change in continuous outcomes, and its three-step workflow is implemented in an R package for easy accessibility.

Abstract Background The prognostic importance of an increased visit-to-visit blood pressure variability (BP-VVV) for the future development of micro- and macrovascular complications in type 2 diabetes has been scarcely investigated and is largely unsettled. We aimed to evaluate it in a prospective long-term follow-up study with 632 individuals with type 2 diabetes. Methods BP-VVV parameters (systolic and diastolic standard deviations [SD] and variation coefficients) were measured during the first 24-months. Multivariate Cox analysis, adjusted for risk factors and mean BP levels, examined the associations between BP-VVV and the occurrence of microvascular (retinopathy, microalbuminuria, renal function deterioration, peripheral neuropathy) and macrovascular complications (total cardiovascular events [CVEs], major adverse CVEs [MACE] and cardiovascular and all-cause mortality). Improvement in risk discrimination was assessed by the C-statistic and integrated discrimination improvement (IDI) index. Results Over a median follow-up of 11.3 years, 162 patients had a CVE (132 MACE), and 212 patients died (95 from cardiovascular diseases); 153 newly-developed or worsened diabetic retinopathy, 193 achieved the renal composite outcome (121 newly-developed microalbuminuria and 95 deteriorated renal function), and 171 newly-developed or worsened peripheral neuropathy. Systolic BP-VVV was an independent predictor of MACE (hazard ratio: 1.25, 95% CI 1.03–1.51 for a 1-SD increase in 24-month SD), but not of total CVEs, cardiovascular and all-cause mortality, and of any microvascular outcome. However, no BP-VVV parameter significantly improved cardiovascular risk discrimination (increase in C-statistic 0.001, relative IDI 0.9%). Conclusions Systolic BP-VVV was an independent predictor of MACE, but it did not improve cardiovascular risk stratification. The goal of anti-hypertensive treatment in patients with type 2 diabetes shall remain in controlling mean BP levels, not on decreasing their visit-to-visit variability.

The critical immunological event in rheumatoid arthritis (RA) is the production of antibodies to citrullinated proteins (ACPAs), ie proteins on which arginines have been transformed into citrullines by peptidyl arginine deiminases (PAD). In C3H mice, immunization with PAD4 triggers the production of ACPAs. Here, we developed a peptide array to analyze the fine specificity of anti-citrullinated peptide antibodies and used it to characterize the ACPA response after hPAD4 immunization in mice expressing different H-2 haplotypes. Sera from C3H, DBA/2, BALB/c and C57BL/6 mice immunized with human PAD4 (hPAD4) or control-matched mice immunized with phosphate buffered saline (PBS) were used to screen peptide arrays containing 169 peptides from collagen, filaggrin, EBNA, proteoglycan, enolase, alpha and beta fibrinogen, histon and vimentin. Human PAD4 immunization induced antibodies directed against numerous citrullinated peptides from fibrinogen, histon 4 and vimentin. Most peptides were recognized under their arginine and citrullinated forms. DBA/2 and BALB/c mice (H-2d) had the lowest anti-citrullinated peptide IgG responses. C3H (H-2k) and BL6 mice (H-2b) had the highest anti-citrullinated peptide IgG responses. The newly developed peptide array allows us to characterize the ACPA production after hPAD4 immunization in mice on the H-2d, H-2k or H-2b backgrounds. This sensitive tool will be useful for further studies on mice for prevention of ACPA production by PAD tolerization.

Abstracts: Background: During pregnancy a feto-maternal exchange of cells through the placenta conducts to maternal microchimerism (Mc) in the child and fetal Mc in the mother. Because of this bidirectional traffic of cells, pregnant women have also acquired maternal cells in utero from their mother and could transfer grandmaternal (GdM) cells to their child through the maternal bloodstream during pregnancy. Thus, cord blood (CB) samples could theoretically carry GdMMc. Nevertheless this has never been demonstrated. Methods: Using Human Leukocyte Antigen (HLA)-specific quantitative PCR assays on three-generation families, we were able to test 28 CB samples from healthy primigravid women for GdMMc in whole blood (WB) and isolated cells (PBMC, T, B, granulocytes, stem cells). Findings: Five CB samples (18%) had GdMMc which could not be confounded with maternal source, with quantities 100 fold lower than maternal Mc in WB and PBMC. Risk of aneuploidies and/or related invasive prenatal procedures significantly correlated with the presence of GdMMc in CB (p=0.024). Significantly decreased HLA compatibility was observed in three-generation families from CB samples carrying GdMMc (p=0.019). Interpretation: Transgenerational transfer of cells could have implications in immunology and evolution. Further analyses will be necessary to evaluate whether GdMMc in CB is a passive or immunologically active transfer and whether invasive prenatal procedures could trigger GdMMc. Funding: Provence-Alpes-Côte d'Azur APEX grant # 2012_06549E, 2012_11786F and 2014_03978) and the Foundation for Medical Research (FRM Grant #ING20140129045).

Background and Aims: It remains unknown whether advanced liver fibrosis is associated with a higher risk of cardiovascular complications in patients with type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD).The aim was to evaluate the prognostic value of transient elastography (TE) by Fibroscan® as a predictor of mortality and cardiovascular outcomes in T2DM. Methods: On a prospective study, T2DM patients with no other cause of liver disease except NAFLD underwent TE at baseline and were followed-up for the evaluation of outcomes, including all-cause mortality and occurrence of any cardiovascular event. The associations between TE scores and outcomes were evaluated by Cox regressions adjusted for other potential confounders. Liver stiffness measurement (LSM) and Controlled attenuation parameter (CAP) were evaluated as continuous variables and categorized as advanced fibrosis if LSM >7.9 or 7.2 kPa (M-XL probe) and severe steatosis if CAP>296 dB/m. Results: 403 T2DM patients were included (64% female, mean age of 64±10 yrs) and followed for 94 months until March 2020. 395(98%) of the TE examinations were successful. At baseline, 104(26%) individuals had advanced fibrosis and 150(38%) had severe steatosis. During follow-up, 55(14%) patients died, 55(14%) had cardiovascular events, including 35 with coronary artery disease (CAD). As continuous variables, LSM (↑1 kPa) predicted all-cause mortality (HR 1.05, 95%CI 1.01-1.08,p=0.020) and CAP (↑20 dB/m) was associated with a significant reduced risk of cardiovascular mortality (HR 0.80,95%CI 0.66-0.96,p=0.014) and CAD (HR 0.81,95%CI 0.70-0.95,p=0.007). Increased LSM was associated with a significant 98% excess mortality risk (p=0.025), whereas a higher CAP was associated with a borderline 49% reduced risk (p=0.08). Conclusion: Advanced fibrosis is associated with all-cause mortality, independent of other potential risk factors. Severe steatosis could have some effect in the reduction of cardiovascular mortality and CAD events. Transient elastography may be useful to improve stratification risk of T2DM patients.

Introduction and objectives: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. It is often related to metabolic syndrome, presenting an increased risk of advanced liver disease and cardiovascular-related death. In some etiologies of chronic liver disease, thrombocytopenia has been associated not only with advanced stages of fibrosis but also with autoimmune disease. In NAFLD, however, its prevalence and related factors are still unknown. The aim of this study is to evaluate the prevalence of thrombocytopenia in NAFLD patients without cirrhosis and to investigate its related risk factors. Patients and methods: This was a retrospective study carried out in two tertiary hospitals in the South and Southeast regions of Brazil. Patients diagnosed with NAFLD by liver biopsy were included. Those with other causes of liver disease and/or cirrhosis were excluded. For analysis, patients were divided into two groups, with and without thrombocytopenia. Data was analyzed using a significance level of 5%. Results: 441 non-cirrhotic patients with NAFLD (evaluated by liver biopsy) were included in the study. The prevalence of thrombocytopenia was 3.2% (14/441 patients). In the comparative analysis between groups, thrombocytopenia was associated with male sex (p = 0.007) and level of hemoglobin (p = 0.023). Conclusion: Thrombocytopenia is an infrequent event in NAFLD patients without cirrhosis and is related with male sex and higher hemoglobin levels.

Abstract Background Although criticisms regarding the dichotomisation of continuous variables are well known, applying logit model to dichotomised outcomes is the convention because the odds ratios are easily obtained and they approximate the relative risks (RRs) for rare events. Methods To avoid dichotomisation when estimating RR, the marginal standardisation method that transforms estimates from logit or probit model to RR estimate is extended to include estimates from linear model in the transformation. We conducted a simulation study to compare the statistical properties of the estimates from: (i) marginal standardisation method between models for continuous (i.e., linear model) and dichotomised outcomes (i.e., logit or probit model), and (ii) marginal standardisation method and distributional approach (i.e., marginal mean method) applied to linear model. We also compared the diagnostic test for probit, logit and linear models. For the real dataset analysis, we applied these analytical approaches to assess the management of inpatient hyperglycaemia in a pilot intervention study. Results Although the RR estimates from the marginal standardisation method were generally unbiased for all models in the simulation study, the marginal standardisation method for linear model provided estimates with higher precision and power than logit or probit model, especially when the baseline risks were at the extremes. When comparing approaches that avoid dichotomisation, RR estimates from these approaches had comparable performance. Assessing the assumption of error distribution was less powerful for logit or probit model via link test when compared with diagnostic test for linear model. After accounting for multiple thresholds representing varying levels of severity in hyperglycaemia, marginal standardisation method for linear model provided stronger evidence of reduced hyperglycaemia risk after intervention in the real dataset analysis although the RR estimates were similar across various approaches. Conclusions When compared with approaches that do not avoid dichotomisation, the RR estimated from linear model is more precise and powerful, and the diagnostic test from linear model is more powerful in detecting mis-specified error distributional assumption than the diagnostic test from logit or probit model. Our work describes and assesses the methods available to analyse data involving studies of continuous outcomes with binary representations.

Introduction and aim: The gold-standard for fibrosis diagnosis in non-alcoholic fatty liver disease (NAFLD) is liver biopsy, despite its invasive approach, sampling limitations and variability among observers. The objective was to validate the performance of non-invasive methods (Fibroscan™; APRI, FIB4 and NAFLD score) comparing with liver biopsy in the evaluation of liver fibrosis in patients with NAFLD. Material and methods: NAFLD patients ≥18 years of age who were submitted to liver biopsy were included and evaluated at two reference tertiary hospitals in Brazil with transient hepatic elastography (THE) assessment through Fibroscan™, APRI, FIB4 and NAFLD scores were determined. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values for the diagnosis of advanced fibrosis were calculated to evaluate the performance of these non-invasive methods in NAFLD patients, adopting liver biopsy as the gold standard. Results: A total of 104 patients were studied. At three different cutoff values (7.9, 8.7 and 9.6 kPa) THE presented the highest sensitivity values (95%, 90% and 85% respectively), and the highest NPV (98%, 96.4% and 95.1% respectively) for the diagnosis of advanced fibrosis. It also presented the highest AUROC (0.87; CI 95% 0.78–0.97). Conclusion: When compared to the gold standard, transient hepatic elastography presented the best performance for the diagnosis and exclusion of advanced fibrosis in patients with NAFLD, overcoming APRI, FIB4 and NAFLD score.

BackgroundCord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition.Patients and MethodsHere we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status.ResultsMMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or –DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc.ConclusionsWe have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.

Abstract Background We introduce BPG, a framework for generating publication-quality, highly-customizable plots in the R statistical environment. Results This open-source package includes multiple methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it suitable for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for integration with computational pipelines. Conclusion BPG provides a new approach for linking interactive and scripted data visualization and is available at http://labs.oicr.on.ca/boutros-lab/software/bpg or via CRAN at https://cran.r-project.org/web/packages/BoutrosLab.plotting.general

Abstract Background The prognostic importance of carotid atherosclerosis in individuals with diabetes is unsettled. We aimed to evaluate the relationships between parameters of carotid atherosclerosis and the future occurrence of micro- and cardiovascular complications in individuals with type 2 diabetes. Methods Ultrasonographic parameters of carotid atherosclerosis, intima-media thickness (CIMT) and plaques, were measured at baseline in 478 participants who were followed-up for a median of 10.8 years. Multivariate Cox analysis was used to examine the associations between carotid parameters and the occurrence of microvascular (retinopathy, renal, and peripheral neuropathy) and cardiovascular complications (total cardiovascular events [CVEs] and cardiovascular mortality), and all-cause mortality. The improvement in risk stratification was assessed by using the C-statistic and the integrated discrimination improvement (IDI) index. Results During follow-up, 116 individuals had a CVE and 115 individuals died (56 from cardiovascular diseases); 131 newly-developed or worsened diabetic retinopathy, 156 achieved the renal composite outcome (94 newly developed microalbuminuria and 78 deteriorated renal function), and 83 newly-developed or worsened peripheral neuropathy. CIMT, either analysed as a continuous or as a categorical variable, and presence of plaques predicted CVEs occurrence and renal outcomes, but not mortality or other microvascular complications. Individuals with an increased CIMT and plaques had a 1.5- to 1.8-fold increased risk of CVEs and a 1.6-fold higher risk of renal outcome. CIMT and plaques modestly improved cardiovascular risk discrimination over classic risk factors, with IDIs ranging from 7.8 to 8.4%; but more markedly improved renal risk discrimination, with IDIs from 14.8 to 18.5%. Conclusions Carotid atherosclerosis parameters predicted cardiovascular and renal outcomes, and improved renal risk stratification. Ultrasonographic carotid imaging may be useful in type 2 diabetes management.

Accurate and actionable biomarkers that integrate diverse molecular, functional and clinical information hold great promise in precision medicine. Here, the authors develop SIMMS, a method for pathway-based cross-disease biomarker discovery.

Abstract Background The National Comprehensive Cancer Network guidelines for Merkel cell carcinoma recommend performance of the sentinel lymph node biopsy in all patients with clinically negative nodal disease for staging and treatment. Nevertheless, sentinel lymph node biopsy in the periocular region is debated as tumors are typically smaller and lymphatic variability can make performance procedurally problematic. Case presentation We present a case of a Caucasian patient in their seventies who presented with a 1.0 cm periocular Merkel cell carcinoma, who underwent Mohs surgery with a Tenzel flap repair, that was found to have a positive sentinel lymph node biopsy, but who, despite parotidectomy, selective neck dissection, and radiation, succumbed to the disease. Conclusions Evidence in both the site-specific and non-specific literature demonstrates: (1) Worsening prognosis with extent of lymph node burden, (2) improvements in our abilities to perform lymphoscintigraphy, (3) locoregional and distant metastatic disease in patients with tumor sizes ≤1 cm, and (4) significant rates of sentinel lymph node positivity in patients with tumor sizes ≤1 cm. Our case supports that sentinel lymph node biopsy should be considered in all clinically nodal negative periocular Merkel cell carcinoma, regardless of size, and despite limited site-specific studies on the subject.

Abstract Diabetic retinopathy (DR) is a chronic microvascular complication associated a worse prognosis. We aimed to evaluate the predictors of development/progression of DR in a cohort of 544 high-risk patients with type 2 diabetes who had annual ophthalmologic examinations over a median follow-up of 6 years. Ambulatory blood pressure (BP) monitoring and aortic stiffness by carotid-femoral pulse wave velocity were performed. Multivariate Cox survival analysis examined the independent predictors of development or progression of DR. During follow-up, 156 patients either newly-developed or worsened DR. Patients who developed/progressed DR had longer diabetes duration, higher ambulatory and clinic BP levels, higher aortic stiffness, and poorer glycemic control than patients who did not developed/progressed DR. After adjustments for baseline retinopathy prevalence, age and sex, a longer diabetes duration (p

Summary: Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs. : Molecular Genetics; Model Organism Subject Areas: Molecular Genetics, Model Organism

Cultured meat, in particular beef, is an emerging food technology potentially challenged by issues of consumer acceptance. To understand drivers of consumer acceptance as well as sensory perception of cultured meat, we investigated the effect of information content on participants' acceptance of cultured meat in a tasting context. Hundred ninety-three citizens from the Netherlands participated, divided across three age and sex-matched groups which each received information on either societal benefits, personal benefits or information on the quality and taste of cultured meat. They filled out a questionnaire and tasted two pieces of hamburger, labeled 'conventional' or 'cultured', although both pieces were in fact conventional. Sensory analysis of both hamburgers was performed. We observed that provision of information and the tasting experience increased acceptance of cultured meat and that information on personal benefits of cultured meat increased acceptance more than information on quality and taste but not than societal benefits of cultured meat. Previous awareness of cultured meat was the best predictor of its acceptance. In contrast to previous studies, sex and social economic status were not associated with different acceptance rates. Surprisingly, 58% of the respondents were willing to pay a premium for cultured meat of, on average, 37% above the price of regular meat. All participants tasted the 'cultured' hamburger and evaluated its taste to be better than the conventional one in spite of the absence of an objective difference. This is the first acceptance study of cultured meat where participants were offered to eat and evaluate meat that was labeled 'cultured'. We conclude that having positive information importantly improves acceptance and willingness to taste and that the specific content of the information is of subordinate importance. Awareness of cultured meat is the best predictor of acceptance.

INTRODUCTION:Global surgery is a growing field studying the determinants of safe and affordable surgical care and advocating to gain the global health community's attention. In Cameroon, little is known about the level of knowledge and attitudes of students. Our survey aimed to describe the knowledge and attitudes of Cameroonian medical students towards global surgery. MATERIALS AND METHODS:We performed an anonymous online survey of final-year Cameroonian medical students. Mann-Whitney U test and Spearman correlation analysis were used for bivariate analysis, and the alpha value was set at 0.05. Odds ratios and their 95% confidence intervals were calculated. RESULTS:204 respondents with a mean age of 24.7 years (±2.0) participated in this study. 58.3% were male, 41.6% had previously heard or read about global surgery, 36.3% had taken part in a global surgery study, and 10.8% had attended a global surgery event. Mercy Ships was well known (46.5%), and most students believed that surgical interventions were more costly than medical treatments (75.0%). The mean score of the global surgery evaluation was 47.4% (±29.6%), and being able to recognize more global surgery organizations was correlated with having assumed multiple roles during global surgery studies (p = 0.008) and identifying more global surgery indicators (p = 0.04). Workforce, infrastructure, and funding were highlighted as the top priorities for the development of global surgery in Cameroon. CONCLUSION:Medical students are conscious of the importance of surgical care. They lack the opportunities to nurture their interest and should be taught global surgery concepts and skills.

[This corrects the article DOI: 10.1371/journal.pone.0231176.].

Background: Romania has had one of the highest rates of cervical cancer incidence and mortality in Europe for decades. Data on the high-risk human papillomavirus (hrHPV) prevalence within the Romanian population are crucial for cervical cancer intervention in high risk groups. The aim of this study was to determine the prevalence of hrHPV infection in Romania, identifying high-risk areas for cervical cancer prevention efforts. Methods: The target population of this study were women of all forms in Romania, including ethnic minorities, women from urban and rural areas, and women in various regions. Women with no history of precancerous or cancerous lesions were offered hrHPV screening. The specimens were tested with Hybrid Capture 2 (HC2) DNA test. Age-standardized hrHPV prevalence rates with 95% confidence intervals (CI) were estimated. Results: hrHPV results of 2060 women aged 18 to 70 years were analyzed. The highest hrHPV prevalence rates were observed among: Romanians (17.9%; 95 CI: 15.5–20.7%), Hungarians (16.6%; 95% CI: 13.1–20.8%), Russians (15.6%; 95% CI: 11.3–21.3%), women living in North (19.2%; 95% CI: 16.5–22.3%), and West regions (23.0%; 95 CI: 18.6–28.0%), and women living in urban areas (20.0%; 95 CI: 18.5–28.0%). hrHPV prevalence rates were lower for the Roma population (7.8%; 95% CI: 4.7–12.5%). Conclusions: These hrHPV prevalence rates in a high cervical cancer incidence country provide baseline information for targeted cervical cancer intervention strategies as well as a baseline to measure the impact of hrHPV vaccination in the future.

The negative impacts of meat consumption for animals, the environment, and human health are more pressing than ever. Although some evidence points to an ongoing reduction in meat consumption in Europe, consumers are overall unwilling to cut their meat consumption in a substantial way. The present study investigates dietary identities and perceptions of cultured meat in nationally representative samples from Germany (n = 1000) and France (n = 1000). Participants were recruited through an Ipsos panel to answer an online survey, which included questions about their current and intended consumption of conventional meat, as well as questions about their opinions of cultured meat. We find that, whilst rates of vegetarianism were relatively low in France, unrestricted meat-eaters were a minority in Germany, and concern for animal welfare was the most common reason given for meat reduction. Substantial markets for cultured meat exist in both countries, although German consumers are significantly more open to the concept than the French. Strikingly, cultured meat acceptance is significantly higher amongst agricultural and meat workers, indicating that those who are closest to existing meat production methods are most likely to prefer alternatives. We found some evidence that pro-cultured meat messages, which focus on antibiotic resistance and food safety, are significantly more persuasive than those that focus on animals or the environment. Furthermore, consumers project that they would be significantly more likely to consume cultured meat that does not contain genetically modified ingredients. Overall, we find substantially large markets for cultured meat in Germany and France, and identify some potential ways to further increase acceptance in these markets. We conclude by highlighting the most promising markets for cultured meat, and highlighting a lack of antibiotics as a potentially persuasive message about cultured meat.

Women with scleroderma (SSc) maintain significantly higher quantities of persisting fetal microchimerism (FMc) from complete or incomplete pregnancies in their peripheral blood compared to healthy women. The non-classical class-I human leukocyte antigen (HLA) molecule HLA-G plays a pivotal role for the implantation and maintenance of pregnancy and has often been investigated in offspring from women with pregnancy complications. However data show that maternal HLA-G polymorphisms as well as maternal soluble HLA-G (sHLA-G) expression could influence pregnancy outcome. Here, we aimed to investigate the underlying role of maternal sHLA-G expression and HLA-G polymorphisms on the persistence of FMc. We measured sHLA-G levels by enzyme linked immunosorbent assay in plasma samples from 88 healthy women and 74 women with SSc. Male Mc was quantified by DYS14 real-time PCR in blood samples from 58 women who had previously given birth to at least one male child. Furthermore, eight HLA-G 5′URR/3′UTR polymorphisms, previously described as influencing HLA-G expression, were performed on DNA samples from 96 healthy women and 106 women with SSc. Peripheral sHLA-G was at lower concentration in plasma from SSc (76.2 ± 48.3 IU/mL) compared to healthy women (117.5 ± 60.1 IU/mL, p

Resumo Estudos têm revelado que a resistência às quinolonas em cepas de Campylobacter está relacionada à presença da mutação Treonina-86 para Isoleucina. Com o objetivo de investigar a presença dessa mutação em cepas de Campylobacter sensíveis e resistentes à ciprofloxacina e enrofloxacina, o conteúdo cecal de 80 frangos de corte de criação orgânica, abatidos sob Serviço de Inspeção Estadual (S.I.E.) do Estado do Rio de Janeiro, foram coletados e investigados para a presença de Campylobacter. A determinação da resistência à ciprofloxacina e enrofloxacina foi feita pela técnica de difusão em disco e de diluição em ágar para determinação da Concentração Inibitória Mínima (CIM). A detecção da mutação na Região Determinante de Resistencia às Quinolonas (RDRQ) no gene gyrA foi realizada através de sequenciamento. Campylobacter foi isolado a partir de 100% das amostras avaliadas, sendo 68,75% correspondente à C. jejuni e 31,25% à C. coli. No teste de difusão em disco, 100% das cepas foram resistentes à ciprofloxacina e 56,25% das cepas foram resistentes à enrofloxacina. No teste de diluição em ágar, todas as cepas foram resistentes à ciprofloxacina apresentando CIM variando de ≥ 16-64μg/mL, e resistência ou resistência intermediaria à enrofloxacina foi detectada em 42,50% (CIM ≥ 4-32μg/mL) e 38,75% (CIM = 2μg/mL) das cepas, respectivamente. A mutação Tre-86-Ile, foi observada em 100% das cepas analisadas. Além dessa mutação, foram observadas outras mutações não silenciosas (Val-73-Glu, Ser-114-Leu, Val-88-Asp, Ala-75-Asp, Ser-119-Gli, Arg-79-Lis) e mutações silenciosas (His-81-His, Ser-119-Ser, Ala-120-Ala, Fen-99-Fen, Ala-122-Ala, Gli-74-Gli, Ile-77-Ile, Ala-91-Ala, Leu-92-Leu, Val-93-Val, Ile-106-Ile, Tre-107-Tre, Gli-113-Gli, Ile-115-Ile, Gli-110-Gli). A observação de que cepas sensíveis à enrofloxacina pelos testes fenotípicos apresentavam a substituição Tre-86 para Ile sugere que outros mecanismos podem contribuir para a resistência à enrofloxacina em Campylobacter.

Objectives. To investigate treatment adherence in patients with type 2 diabetes and to evaluate its associated factors. Methods. The Summary of Diabetes Self-Care Activities (SDSCA) questionnaire was used to assess treatment adherence. Good adherence was defined as ≥5 days a week in each SDSCA item. Pain, emotional, and physical domains of the SF-36 quality of life questionnaire and the Canadian Occupational Performance Measure (COPM) were also evaluated. Multivariable logistic regressions explored the independent correlates of good general adherence and of specific items of the SDSCA (diet, exercise, and medications). Results. Good adherence was 93.5% for medication use, 59.3% for foot care, 56.1% for blood glucose monitoring, 29.2% for diet, and 22.5% for exercise. Patients with general good adherence had lower BMI, better serum lipid profile, higher values of functional capacity, emotional and pain domains of SF-36, better occupational performance, and lower prevalence of pain or limitation in the upper and lower limbs than patients with worse adherence. The variables associated with good adherence were younger age, lower BMI, presence of macrovascular complications, better occupational performance and emotional domain of SF-36, and higher HDL cholesterol levels. The presence of pain/limitation in the upper limbs was associated with worse adherence. Good medication adherence was associated with longer diabetes duration, lower BMI, and lower HbA1c levels. Higher values of pain and emotional domains of the SF-36 and lower BMI were related to better exercise and diet adherence, while the presence of peripheral neuropathy and joint pain/limitation were associated with worse exercise adherence. Conclusions. Emotional and physical performances are important determinants of good diabetic treatment adherence. Good adherence has beneficial impact on BMI, lipid, and glycemic control.

The swift clearance of apoptotic cells (ACs) (efferocytosis) by phagocytes is a critical event during development of all multicellular organisms. It is achieved through phagocytosis by professional or amateur phagocytes. Failure in this process can lead to the development of inflammatory autoimmune or neurodegenerative diseases. AC clearance has been conserved throughout evolution, although many details in its mechanisms remain to be explored. It has been studied in the context of mammalian macrophages, and in the nematode Caenorhabditis elegans, which lacks “professional” phagocytes such as macrophages, but in which other cell types can engulf apoptotic corpses. In Drosophila melanogaster, ACs are engulfed by macrophages, glial, and epithelial cells. Drosophila macrophages perform similar functions to those of mammalian macrophages. They are professional phagocytes that participate in phagocytosis of ACs and pathogens. Study of AC clearance in Drosophila has identified some key elements, like the receptors Croquemort and Draper, promoting Drosophila as a suitable model to genetically dissect this process. In this review, we survey recent works of AC clearance pathways in Drosophila, and discuss the physiological outcomes and consequences of this process.

Catecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine- and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response.

This study aimed to evaluate the agronomical and environmental aspects in corn, soil characteristics and leachate resulting from the material combination of swine wastewater and mineral fertilizer in the long run. The experimental area has a history of swine wastewater application for experiments conducted since 2006, this being the seventh crop cycle in the area. Four doses of swine wastewater (0; 100; 200 and 300 m³ ha-1) associated with presence and absence of mineral fertilizer were applied, consisting of eight treatments. The results obtained were subjected to variance analysis and means compared by Tukey test at 5% significance. We conclude that the swine wastewater can be partially used as an alternative fertilizer, since the P and Mn are complemented with specific fertilization. There must be carefulness about Fe+2, Cu+2 and Zn+2 accumulation in soil, and also to the concentrations of Fe+2, Cu+2, Zn+2, NO3- + NO2- and Na+ in leachate which exceeded the limits at the highest SWW applied levels.

BackgroundThe prognostic value of C‐reactive protein (CRP) is controversial in type 2 diabetes mellitus. We aimed to assess it in a cohort of high cardiovascular risk diabetic patients. Methods and ResultsCRP was measured at baseline and during the second year of follow‐up in 616 patients. The primary end points were a composite of total fatal and nonfatal cardiovascular events (CVEs), major CVEs, and all‐cause and cardiovascular mortalities. Association between baseline and second‐year CRP with end points were evaluated by multivariable Cox survival analyses. Baseline median CRP was 2.8 mg/L (interquartile range: 1.2–6.0 mg/L), and 47.8% of the patients either increased or persisted with high CRP levels during the first 2 years of follow‐up. After a median follow‐up of 8.4 years, 131 total CVEs occurred (89 major CVEs), and 129 patients died (53 of cardiovascular causes). Baseline and second‐year CRP, analyzed as a continuous variable and dichotomized at >3.0 mg/L, were significantly associated with total and major CVEs occurrence (with adjusted hazard ratios between 1.22 and 1.34 for increments of 1‐SD log of continuous CRP, and between 1.47 and 1.89 for dichotomized CRP), but not with mortality. Additionally, increasing CRP levels or persisting with high levels were associated with a 1.84 (95% CI: 1.10–3.06) excess risk of major CVEs, independent of baseline CRP values. ConclusionsBaseline and serial changes in CRP levels provide cardiovascular risk prediction independent of standard risk factors and glycemic control, and may be useful to refine cardiovascular risk stratification in high‐risk patients with type 2 diabetes mellitus.

Phagocytosis is an ancient mechanism central to both tissue homeostasis and immune defense. Both the identity of the receptors that mediate bacterial phagocytosis and the nature of the interactions between phagocytosis and other defense mechanisms remain elusive. Here, we report that Croquemort (Crq), a Drosophila member of the CD36 family of scavenger receptors, is required for microbial phagocytosis and efficient bacterial clearance. Flies mutant for crq are susceptible to environmental microbes during development and succumb to a variety of microbial infections as adults. Crq acts parallel to the Toll and Imd pathways to eliminate bacteria via phagocytosis. crq mutant flies exhibit enhanced and prolonged immune and cytokine induction accompanied by premature gut dysplasia and decreased lifespan. The chronic state of immune activation in crq mutant flies is further regulated by negative regulators of the Imd pathway. Altogether, our data demonstrate that Crq plays a key role in maintaining immune and organismal homeostasis.