Performing total joint arthroplasty (TJA) in Parkinson’s disease (PD) patients may encounter a higher complication rate or worse functional outcomes compared with common patients. The relationship between PD and clinical outcomes after TJA is not fully understood., we used manual charts to investigate the clinical outcomes in 41 patients including 24 total hip arthroplasty (THA) patients (28 hips) and 18 total knee arthroplasty (TKA) patients (22 knees) with a diagnosis of PD from 2009 to 2016. The stage of PD was confirmed by Hoehn and Yahr scale. Prosthesis survivorship was estimated with revision for any reason as the endpoint.Retrospectivelyp < 0.05). Subgroup analysis revealed worse functional outcomes in mid- or end-stage PD patients. Sixteen short-term mild to moderate complications were noted. Two revisions were conducted for hip periprosthetic osteolysis and postoperative knee pain. The prosthesis survivorship at 60 months for TJA, total hip arthroplasty (THA), or total knee arthroplasty (TKA) was 91.6%, 94.1%, and 87.5%, respectively.All the clinical outcomes improved significantly (Patients with PD who underwent TJA would result in excellent pain relief and gain of function. However, patients at late-stage PD may suffer from functional loss. The effectiveness of TJA in patients with severe PD remains a concern. Physician should help delay the progression of PD which may optimize and stabilize the functional outcomes of TJA. Parkinson’s disease (PD) is the second most common neurodegenerative disease (after Alzheimer’s disease) with a life expectancy of 7–14 years . The motThe connection between PD and the potential requirement of TJA has been studied. Gait abnormality and instability mean a higher rate of fall of patients with PD. The study suggested that 60.5% of participants reported at least one fall, with 39% reporting recurrent falls . A relevPD is a progressive disease characterized by worsening of motor features . Hoehn aTherefore, this study aims to demonstrate the mid-term pain and functional outcomes of PD patients who underwent THA or TKA. Further subgroup analysis would be done to demonstrate the potential relationship between the stage of PD and the outcomes of patients.After the approval of the Institutional Review Board of West China Hospital, this retrospective study was conducted at West China Hospital, Sichuan University, which serves as a tertiary level center and the largest medical center in the southwest China. Patients with the diagnosis of PD who underwent primary total hip or knee arthroplasty between June 2009 and June 2016 were included in our study and established TKA and THA groups afterward. The informed consent was obtained. Patients who meet the following conditions would be excluded: (1) not primary joint arthroplasty, (2) patient with other neurological diseases, and (3) patient with severe or uncontrolled comorbidities.2 in average . More baseline demographics could be referred to in Table A total of 56 consecutive patients were primarily included in our study. Among them, seven patients were failed to follow-up due to the incorrect telephone numbers, four patients were excluded for incomplete chart filling, and four patients were excluded for not being primarily admitted. No demise happened during follow-up. Therefore, 41 patients including 24 THA patients (28 hips) and 18 TKA patients (22 knees) were finally enrolled in the study. One of the patients received both THA and TKA successively. The median duration of follow-up is 41.00 months (IQR 32.00–67.75) for all the candidates and, separately, 51.50 months (IQR 34.00–72.00) for THA patients and 38.00 months (IQR 29.75–50.75) for TKA patients. There were 14 males and 31 females with an average age of 67.86 years . The body mass index (BMI) was 22.73 kg/mThe collection of the clinical data and evaluation was conducted by two independent observers (X-R and ZY-L). Our patients would undergo routine follow-up at postoperatively 3 weeks, 6 weeks, 12 weeks, and 6 months and annually. And manual chart interview was adopted for evaluation of the preoperative and postoperative clinical outcomes. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)  and The Short Form (12) Health Survey scale (SF-12)  and mental component summary (MCS)) were used for all the candidates. Respectively, for THA patients, Harris Hip Score (HHS) was used to evaluate the pain, function, and deformity. Moreover, for TKA patients, Knee Society Score (KSS) and Knee Society Function Score (KSFS) were used in terms of stability, pain, deformity, and function evaluation. The multiple scales provided a more comprehensive assessment which would cover pain and function, mental and physical state, daily activity, quality of life, and symptoms. Other evaluation terms including the preoperative and postoperative range of motion (ROM) and the severity of PD was assessed by Hoehn and Yahr scale  . Patients at stage III to V were enrolled in the functional restriction group (group II). The clinical outcomes were compared between them.t test was used to compare the continuous variables while Pearson chi-square test or Fisher exact test was used to analyzing the qualitative comparative variables. For the prosthesis survival analysis, Kaplan-Meier was utilized with any reason as the endpoints for revisions. Spearman correlation analysis was adopted for demonstrating the relationship between ranked data and continuous data. The significance was set at p < 0.05. We used SPSS for Windows  to perform all the statistical analyses.Quantitative data were presented as means and standard deviations or as median with the interquartile ranges (IQR 25th and 75th percentiles). Qualitative data were presented with frequencies and percentages. Student’s p < 0.01). Moreover, in specific terms of pain , function , and deformity , the improvement was obvious as well.The outcomes with regard to hip or knee pain and functions were listed in Table p < 0.01). Pain  and stability  showed a significant rise while the ROM score  presented a slit elevation but is also statistically significant. KSFS revealed functional relief from 39.72 ± 6.52 to 58.06 ± 31.11 (p = 0.02). Further, WOMAC, SF-12, and ROM were reviewed, and all showed statistically significant improvement  and six knees (27.27%) showed 16 mild short-term complications . Also, for the THA group and TKA group respectively, the 60 months survivorship rate was 94.1% (95% CI 82.9–99.9%) and 87.5% (95% CI 64.6–99.9%)  and the WOMAC pain (p = 0.01) and function score (p < 0.01) revealed significant improvement in group II for the entire case series. Respectively, in the THA group, the HHS score (p = 0.03) and function score (p = 0.03), SF-12 score , and the WOMAC function score (p < 0.01) were statistically different between subgroups (Table p < 0.01), SF-12 , and the WOMAC pain (p = 0.03) and function score (p < 0.01) were significantly higher in group II. Furthermore, comparisons of the final clinical data with preoperative data were done in each group. Significant differences of all the data were seen in group I. However, in group II, there were no differences in HHS function score (p = 0.25) and KSFS (p = 0.50). The SF-12  and WOMAC function score  were not statistically different as well.The SF-12 score  and WOMAC function score  correlated with the H-Y stage. The HHS function score  in the THA group and KSFS  in the TKA group also demonstrate the same result. The scatter diagrams and the fitted lines are presented in Fig. Spearman correlation analysis was done to demonstrate the relationship between functional outcomes and the H-Y stage of the patient. For all the candidates, the SF-12 score  could be a challenge in functional regaining due to the high risk of fall , bone frWe find mild to moderate complications with acceptable rate happened in this case series. There was no evidence of DVT, embolism, or nerve palsy. Two of our patients suffered from transient delirium and were controlled with olanzapine, which confirmed that orthopedic surgery could induce a cognitive decline in PD patients . RecentlLower limb muscle weakness is common in both postoperative TJA , 30 and There are certain limitations to this study. First, the outcomes of our patients were based on conventional questionnaires. It would be more objective and reliable if a quantitative gait analysis were conducted for the patients. Second, we investigated both hip and knee prosthesis in one study. However, the evaluation forms were used correspondingly and all the data were presented in separate groups.Patients with PD who underwent TJA would result in excellent pain relief and gain of function. However, patients at late-stage PD may suffer from functional loss. The effectiveness of TJA in patients with severe PD remains concern. Physician should help delay the progression of PD which may optimize and stabilize the functional outcomes of TJA.


Prospective data are sparse for active commuting to work and body weight in Asia. We assessed the association of 5-year changes in commuting mode with body mass index (BMI) and the indicators of abdominal obesity in Japanese working adults.In this longitudinal study, we studied 29,758 participants  in Japan aged 30 to 64 years at baseline who underwent further health examination 5 years after the baseline examination. Changes in BMI were calculated from objectively measured body height and weight at baseline and follow-up examination. Visceral and subcutaneous fat areas and waist circumference measured by computed tomography scans were used as indicators for abdominal adiposity. Linear regression was applied to estimate the association of changes in commuting mode with the obesity outcomes.n = 16,087), the adjusted mean  of the BMI change over 5 years in kg/m2 were 0.27 (0.24 to 0.30) for maintained inactive commuting group (reference), 0.34 (0.30 to 0.38) for switching to inactive commuting group (P = 0.046), 0.20 (0.18 to 0.22) for switching to active commuting or public transportation group (P = 0.001), and 0.09 (0.06 to 0.13) for maintained active commuting or public transportation group (P < 0.001). Maintained inactive commuting tended to be associated with larger gain in abdominal adiposity indicators.Within the 5-year study period, adults who maintained inactive commuting gained weight, and compared with this group, adults who switched to inactive commuting had higher weight gain; conversely, adults who switched to active or public transportation commuting and especially those who maintained active or public transportation commuting experienced less weight gain. Subgroup analysis showed similar tendency across the different transitions of leisure-time exercise or occupational physical activity. For example, among adults who maintained no exercise (Switching from inactive commuting mode to more physically active commuting mode and maintaining active commuting can promote body weight control among working adults across different levels of occupational or leisure-time physical activity. The prevalence of obesity has substantially increased worldwide over 30 years , imposinAccumulating evidence suggest that active commuting may help body weight control, whereas prolonged car use may not. However, several points should be further considered. First, most data were derived from cross-sectional studies –18, whicHerein, we report the 5-year changes in commuting mode to work with simultaneous changes in objectively measured obesity indicators including BMI, waist circumference, and visceral and subcutaneous fat measured via computed tomography (CT) scans among adults with majority of men. We also investigated the commuting-BMI associations according to the leisure-time or occupational physical activity transitions, which may have an impact on the magnitude of BMI change.The J-ECOH Study is an ongoing collaborative study to elucidate the risk factors for non-communicable diseases among workers in Japan from more than 10 large-scale companies. We analysed longitudinal data of its subcohort (one major company) where data on primary commuting mode has been collected at the annual health checkup. This company is located in Hitachi city, Ibaraki prefecture, which is a suburban area of East Japan. The type of workplace is a mixture of office and factories. The details of the J-ECOH Study  and the n = 4326), lifestyle habits , and occupational factors  at baseline, leaving 30,749 workers. Some participants met two or more exclusion criteria. Three participants did not have data on body height at baseline, and it was imputed from the following health checkup data. Then, we additionally excluded 991 workers without data on commuting mode after 5-year follow-up, remaining 29,758 participants  aged 30 to 64 years (mean [SD]: 43.2 [8.2] years) for the main analysis. Of these, approximately 0.1 to 3.5% of them did not have 5-year follow-up data of covariates ; these missing data of covariates were imputed by using last observation carried forward method. Most of the missing data were replaced by 4-year follow-up data.A total of 38,329 participants  aged 30 to 64 years at baseline underwent a baseline health examination and a follow-up examination 5 years after baseline measurement. Of these, 478 participants who had a history of cancer or cardiovascular disease at baseline were excluded because these diseases may influence both commuting mode and BMI. Of the remaining 37,851 participants, we further excluded 7102 (18.6%) who did not have data on commuting mode  inactive commuting  and (2) active commuting  or public transportation (train or bus) , 30, 31.For sensitivity analysis, commuting mode during 1 to 4 years after the baseline period was categorized into the following three categories: persistent inactive commuting, intermittent, and persistent active commuting or public transport use.2. For the main outcome, we obtained the 5-year changes in BMI by subtracting the baseline BMI from the BMI obtained 5 years after baseline.The body height and weight were measured during annual health examinations, and the BMI of each participant was calculated using the formula kg/mn = 4322). Single slice imaging was performed at the umbilical level under fasting condition using a Redix Turbo CT scanner  and visceral fat area, subcutaneous fat area, and waist circumference were estimated by the PC software application fatPointer  as described elsewhere [Visceral and subcutaneous fat areas and waist circumference were measured in a subgroup of the present participants with additional measurements of these variables , work-related factors  and socioeconomic status (job position) of the participants were assessed using a questionnaire. Details of this questionnaire have been explained previously . The metWe calculated the means (SD) and number (%) to illustrate baseline characteristics of the participants according to the 5-year changes in commuting mode.2, continuous) as model 1. Then, we adjusted for all factors in model 2, namely, smoking transition , baseline alcohol use , 5-year changes in alcohol use , baseline sleep duration , changes in the sleep duration , baseline weekly exercise duration during leisure , 5-year changes in the exercise duration , occupational physical activity transition , job position transition , shift work transition , and overtime work transition . The estimated changes in BMI according to commuting mode were calculated from these models. Subgroup analysis was performed for leisure-time exercise transition  and occupational physical activity  in model 2.Multivariable linear regression was used to quantify the association of 5-year changes in commuting mode with simultaneous BMI changes. We treated maintained inactive commuting mode as reference. First, we adjusted for baseline variables including age , sex, and BMI  for missing data of exposures and covariates at baseline and follow-up. A total of 100 imputed data sets were generated. All analyses were performed on each imputed data set; the 100 estimates were combined into an overall estimate using the rules from Rubin. Second, we conducted an analysis considering the transitions in commuting mode during 1 to 4 years after the baseline. Lastly, we performed an analysis while treating public transport use as an independent category, separated from active commuting.p-values of less than 0.05 was considered as statistically significant. All analyses were performed using Stata 14.2 .We repeated the main analysis for other obesity outcomes  where the baseline obesity outcome was adjusted for in each analysis . Two-sided Compared with adults included in the main analysis , those excluded due to missing data (n  =  8093) had a higher proportion of women (13.3% vs. 22.5%) and walked less during commuting . They were less likely to engage in long overtime work and sedentary work and be in high job position . Compared with participants who maintained inactive commuting, the participants who switched from active commuting or public transport use to inactive commuting showed higher weight gain. In contrast, the participants who switched from inactive commuting to active commuting or public transport use had lower weight gain. Adults who maintained active commuting or public transportation showed no material weight change. This relationship did not substantially change after adjustment for many potential confounders (model 2); the greatest weight gain was observed in switching to inactive commuting group , whereas the least gain in maintained active commuting or public transportation group . A sensitivity analysis using MICE also showed the similar results  are shown in Table The associations to visceral adiposity  among 4322 participants who received visceral adiposity CT scans , and adults who switched from active or public transportation commuting at baseline to inactive commuting at follow-up had greater weight gain (0.24 kg/m2) than those who maintained inactive commuting. By contrast, those who switched to and those who maintained active commuting or public transportation showed less weight gain than those who maintaining inactive commuting . Prospective studies [The present analysis showed that adults who maintained inactive commuting gained weight over 5 years 0.19 kg/m kg/m2, a studies , 23 have studies . A recen studies . CollectIn the present data, adults who switched to inactive commuting tended to show greater weight gain compared with other groups. A UK study also reported a similar finding . The rea2 vs. approximately 24 to 27 kg/m2); the extent of weight change in Asians may be smaller than Caucasians. Another reason of smaller changes in BMI in the present study might be partly explained by the differences in the assessment of BMI. The present study used objectively measured body height and weight, whereas most of the previous studies used self-report [The extent of BMI change in the present study was smaller than those in the comparable longitudinal studies –21, 23. f-report , 21, 23.f-report  and accuf-report . Althougf-report . Given tOur novel finding is that the beneficial association of active or public transportation commuting with BMI was observed across different transitions of leisure-time exercise and occupational physical activity, while both leisure-time exercise and occupational physical activity were inversely associated with BMI over time. Similarly with the present findings, observational studies have shown that exercise was associated with weight reduction . Althougn = 320 to 330). Nonetheless, recent randomised controlled trials showed that active commuting by cycling reduced body fat mass compared with no intervention [In the present analysis for abdominal obesity  measured by CT scans, maintaining inactive commuting tended to show larger gain in these parameters than other commuting patterns. However, these changes did not reach significance and were somewhat different from those of BMI. The non-significant and less clear changes may be due to the small sample size with abdominal CT measurements in our data set when participants were divided according to commuting mode transition . Table S4. A sensitivity analysis for BMI with consideration of transitions in commuting mode during 1 to 4 years after baseline . Table S5. Associations of active commuting, public transport use, and inactive commuting with BMI. Table S6. Associations of active commuting, public transport use, and inactive commuting with BMI by leisure-time exercise transition. Table S7. Associations of active commuting, public transport use, and inactive commuting with BMI by occupational physical activity transition. Table S8. Changes in BMI over 5 years according to the transitions in time spent in walking to and from work among workers who persistently used public transport as main commuting mode Figure S1. Trajectories of non-adjusted average body mass index according to the changes in commuting mode.


To compare and characterize the gut microbiota in women of childbearing age from sub-Saharan Africa  and South Asia (India), in relation to dietary intakes.Women of childbearing age were recruited from rural DRC and India as part of the Women First (WF) preconception maternal nutrition trial. Findings presented include fecal 16S rRNA gene-based profiling of women in the WF trial from samples obtained at the time of randomization, prior to initiation of nutrition intervention and to conception.n = 117; India n = 100). Alpha diversity of the gut microbiota was higher in DRC than in India . The gut microbial community structure was not significantly affected by any demographical or environmental variables, such as maternal BMI, education, and water source. Prevotella, Succinivibrio, and Roseburia were at relatively high abundance without differences between sites. Bifidobacterium was higher in India (4.95 ± 1.0%) than DRC , as was Lactobacillus  and Faecalibacterium . Ruminococcus was higher in DRC (2.3 ± 0.7%) than in India  and was positively associated with consumption of flesh foods. Succinivibrio was positively associated with dairy intake in India and fish/insects in DRC. Faecalibacterium was positively associated with vitamin A-rich fruits and vegetables. Overall, these observations were consistent with India being primarily vegetarian with regular fermented dairy consumption and DRC regularly consuming animal-flesh foods.Stool samples were collected from 217 women (DRC Consumption of animal-flesh foods and fermented dairy foods were independently associated with the gut microbiota while demographic variables were not, suggesting that diet may have a stronger association with microbiota than demographic characteristics. Prevotella and lower abundance of Bacteroides, and also a lower risk of colon cancer .Women of childbearing age were recruited from rural DRC (Equateur Province) and India  as part of the WF preconception maternal nutrition trial , 2019. IQuestionnaires of demographic information were administered: cell phone (Yes/No), education (None vs. At least secondary), electricity (Yes/No), man-made floor (Yes/No), flush toilet (Yes/No), improved water (Yes/No), landline (Yes/No), motorcycle (Yes/No), fridge (Yes/No), worry no food (Yes/No), and open sewage near house (Yes/No). Improved water means the participant had access to filtered or treated water. Questionnaires were administered in the home by the local home visitor research assistant and were completed within 1 week of enrollment. A mobile assessment team obtained past medical history and height and weight, from which body mass index (BMI) was calculated.n = 117; India n = 100). A pre-labeled fecal bag, Ziploc bag, a black cryogenic pen, and a Styrofoam storage box containing ice or ice packs were provided to each participant. Stool was collected into the fecal bag using a sterile scoop and then placed into a second Ziploc bag. Participants then placed the bag into the Styrofoam storage container until picked up by the research team on the day the stool was passed. When receiving samples, the research team labeled the sample date and time of stool passage. The research team scooped about a teaspoon of stool and transferred the sample to a sterile stool storage tube with 3 ml RNAlaterTM , ensuring that the specimen was coated with RNAlaterTM and that the label was complete. The stool samples were then frozen at −20°C or colder. Samples were shipped to the University of Colorado on ice packs or at ambient temperature.A stool sample was collected from each participant  and then pooled. The amplicon pool was partially lyophilized to reduce its volume then purified and concentrated using a DNA Clean and Concentrator Kit . Pooled amplicons was quantified using a Qubit Fluorometer 2.0 . Illumina paired-end sequencing was performed following the manufacturer’s protocol on the MiSeq platform using a 600 cycle version 3 reagent kit and versions v2.4 of the MiSeq Control Software.Bacterial profiles were determined by broad-range amplification and sequence analysis of 16S rRNA genes following our previously described methods . In brieIllumina Miseq paired-end reads were aligned to human reference genome hg19 with bowtie2 and matching sequences discarded . As prevn = 50; India n = 50). In brief, two 24-h dietary recalls , permutation-based multivariate analysis of variance (PERMANOVA) tests were used to assess the associations between gut microbial community composition and demographical variables. Associations between the relative abundances of bacterial taxa and nutrients/food-groups were assessed by Spearman rank-order correlation tests. Results were visualized by plotting heatmaps of Spearman’s rho correlation coefficient using the heatmap.2 R function; hierarchical clustering and dendrograms were generated using the default parameters of heatmap.2/dist/hclust functions, using Euclidean distances. Nominal p-values not accounting for multiple testing are reported. Data were analyzed using R version 2.7.2 .Values are presented as mean ± SD for continuous variables. Alpha diversity indices measured for the gut microbiota were tested using the Mann–Whitney statistic. Beta diversity was calculated using weighted UniFrac distances. To compare between DRC and India for differences of the gut microbial profiles, non-parametric Mann–Whitney tests were used. n = 1732) and India (n = 1821). Stool samples were collected at baseline from a random sample of n = 217 women . Baseline characteristics are summarized in n = 50 from DRC and n = 50 from India became pregnant and had dietary intake data during first trimester.Recruitment in India started in January 2014 and enrollment was completed in August 2014. For DRC, the first participant was enrolled in April 2014 and enrollment was completed in September 2014. A total of 3810 women were screened and 3553 were enrolled in the WF trial from DRC , often complemented with caterpillars, termites, small fish, and/or peanuts in sparse amounts. Also prominent was the consumption of cassava, both its leaves and flour made from its root, with the addition of palm oil to most recipes. Fruit and vegetables were also significant elements of the diet. Rice was the main staple in India, with frequent consumption of jowar (sorghum) flour roti. In this primarily lacto-vegetarian population, a large variety of legumes were consumed in addition to many types of vegetable “bhaji,” whereas very few animal-flesh foods were eaten. The diets in India included multiple spices . Buffalo milk tea and yogurt curds were regular features of the diet in Indian women; dairy products were not in the diets of the participants from DRC.n = 50; India n = 50). Although BMI was similar between participants from DRC and India, women from India had a lower average body weight than women from DRC at both baseline  and first trimester . Due to the nature of different dietary intakes, DRC and India had different food groups. In DRC, the four most common food groups based on intakes (g/day) were starchy/staples  (1085 ± 570 g/day), vitamin A-rich fruits/vegetables (822 ± 542 g/day), legumes/ground nuts (143 ± 202 g/day), and fish/insects (122 ± 225 g/day). In India, the four most common food groups were staples  (1025 ± 376 g/day), non-fermented dairy (512 ± 266 g/day), pulses (260 ± 164 g/day), and non-vitamin A rich vegetables (117 ± 110 g/day) (0 g/day) .P = 6.58E-07) and Shannon diversity index . Beta diversity by weighted UniFrac at genus level showed that the gut communities across DRC women were more similar than among Indian women (P < 0.001). The average Good’s index of each sequence library was 99.87 and 99.88% for DRC and India, respectively, indicating the most biodiversity was captured in each library (participant) and the depth of sequencing was sufficient to represent the biodiversity in the specimens. Alpha diversity of the gut microbiota was higher in DRC than in India using the Chao1 richness index . At the family level , Lachnospiraceae , and Ruminococcaceae , none of which differed significantly between DRC and India. In contrast, Bifidobacteriaceae (phylum Actinobacteria) was significantly higher in India (5.5 ± 6.5%) than in DRC . At the genus level, Prevotella remained the only strain that was over 10% abundance . Some fiber fermenting strains Succinivibrio  and Roseburia  were at relatively high abundance without differences between DRC and India. Consistent with findings of Bifidobacteriaceae, the genus Bifidobacterium was higher in India (4.95 ± 1.0%) than in DRC , as were Lactobacillus  and Faecalibacterium . In contrast, Ruminococcus was higher in DRC (2.3 ± 0.7%) than in India . The Bacteroides/Prevotella ratio was 0.11 ± 0.50 and 0.23 ± 0.90 for DRC and India, respectively (P = 0.22).The three most abundant phyla were Firmicutes, Bacteroidetes, and Proteobacteria with average relative abundances at 51, 33, and 7%, respectively and there was no statistical difference between DRC and India . These tly level , the thrEscherichia-Shigella did not differ by site , but was higher in those who reported “No” nearby open sewage compared to those that reported “Yes” in India .The only demographical variable that was significantly associated with the gut microbiota composition was the categorical variable “open sewage.” This question asked the participants if they had open sewage near their house. All of the participants in DRC answered “No” while ∼50% in India answered “No” and ∼50% answered “Yes” . The genRuminococcus was positively associated with iron intake at both sites and with fiber intake in India. The potentially pathogenic genus Escherichia-Shigella was negatively associated with iron intake in India. Succinivibrio was positively associated with dairy intake in India and fish/insects in DRC (P < 0.05). In DRC, Ruminococcus was positively associated with flesh foods (P < 0.05) and Faecalibacterium was positively associated with vitamin A-rich fruits and vegetables. In India, Escherichia-Shigella was also negatively associated meat/fish intakes (P < 0.01) and positively associated with fermented dairy intakes (P < 0.01).Strong associations were noted between several dietary intake measures and the gut microbiota. Although the areas of our study in both DRC and India were both rural and low resourced regions, they had distinctive dietary patterns and geographical features. Various studies have shown that diet and geographical location are the two strongest determinants of the gut microbial community structure . One stuBifidobacterium and Lactobacillus are commonly used probiotics and were enriched in participants from India. Bifidobacterium is significantly higher in infants than in adults  in DRC was consistent with the fishing, farming and hunter-gatherer populations in South Cameroon (0.1–0.5%) who also have little or no access to dairy products   were typically homemade in an environment of relatively poor hygiene , it is plausible that the home-made fermented dairy was contaminated with Escherichia-Shigella.Both n adults  due to t species . Bifidob in Peru , as well in Peru . Indeed,products . Likewison (∼4%) . HoweverLachnospiraceae (20%), Prevotellaceae (29%), and Ruminococcaceae (18%). These findings were consistent with previous research in Hadza hunter-gatherers of Tanzania (Lachnospiraceae (10%), Prevotellaceae (6%), and Ruminococcaceae (34%) were also the three most abundant families. All three families include member species that can degrade complex polysaccharides and are considered potent short-chain fatty acid producers. The lack of direct association between fiber intake and these bacterial taxa may be due to the lag between collections of microbiota samples and diet records, which may have weakened some of the correlations between microbiota and dietary data, especially if diets changed in early pregnancy. Also, dietary data from two 1-day recalls may not be sensitive enough to link with individuals’ biomarkers, including the microbiota, although the monotony of the typical diets in these populations tends to mitigate that concern. Finally, because dietary data were available from only one-half of the participants in each site for this analysis, statistical power was reduced. However, the overall dietary patterns observed from the diet records still were consistent with agricultural communities. At the genus level, genera Ruminococcus has frequently been reported to be depleted in agriculturalist communities compared with urban communities . Prevotella is strongly associated with plants and fiber intakes and can be used to differentiate agriculturalist and urban communities  was low in both DRC and India, and the Bacteroides/Prevotella ratio was also low. A recent study .Consistent with previous research, we found that the family munities . Most urmunities . Bacteroteroides . In the nt study  demonstrThe richness of the gut microbiota  was higher in DRC than in India. Emerging research has shown that diet diversity is positively associated with richness within gut microbiota . In the To our knowledge, this is the first study to directly compare gut microbial composition and diversity in women of child-bearing age in DRC and India, and to link these with quantitative dietary intakes. Strengths of this study include a relatively large sample size, using repeat 24-h dietary recalls and addressing understudied populations. There are also several limitations. First, the 24-h dietary recall data were available for only a subgroup of participants and were obtained at the end of the first trimester of pregnancy, which may not reflect their habitual dietary intakes. Although mid-late pregnancy is associated with changes in the gut microbiota , the fecIn conclusion, diet is an influential determinant of the gut microbiota of women in DRC and India. The relationship between diet and gut microbiota was much stronger than demographic factors such as socioeconomic status. Consumption of plant-based foods, animal-flesh foods, and fermented dairy foods all showed evidence of associations with gut microbiota.https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA553183.The datasets generated for this study can be found in the NCBI ClinicalTrials.gov (NCT01883193).This study was approved by the Institutional Review Board at each participating site and registered at KH and NK conceived the original idea and designed the initial protocol. AT, AL, SG, SD, JW, and MS conducted the procedures at DRC and India sites. DF, DI, CR, and JK analyzed the stool samples and processed the microbiota data. RL analyzed the dietary data. MT and DF performed the statistical analysis. MT wrote the report with close collaboration with NK, DF, and AH, and with input from all authors. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Few studies have addressed mental illness-related discrimination in low-income countries, where the mental health treatment gap is highest. We aimed to evaluate the experience of discrimination among persons with severe mental illnesses (SMI) in Ethiopia, a low-income, rapidly urbanizing African country, and hypothesised that experienced discrimination would be higher among those living in a rural compared to an urban setting.The study was a cross-sectional survey of a community-ascertained sample of people with SMI who underwent confirmatory diagnostic interview. Experienced discrimination was measured using the Discrimination and Stigma Scale (DISC-12). Zero-inflated negative binomial regression was used to estimate the effect of place of residence  on discrimination, adjusted for potential confounders.2(1) = 4.80; p = 0.028), the police (χ2(1) =11.97; p = 0.001), in keeping a job (χ2(1) = 5.43; p = 0.020), and in safety (χ2(1) = 5.00; p = 0.025), and had a significantly higher DISC-12 score than those living in rural areas .Of the 300 study participants, 63.3% had experienced discrimination in the previous year, most commonly being avoided or shunned because of mental illness (38.5%). Urban residents were significantly more likely to have experienced unfair treatment from friends (χPersons with SMI living in urban settings report more experience of discrimination than their rural counterparts, which may reflect a downside of wider social opportunities in urban settings. Initiatives to expand access to mental health care should consider how social exclusion can be overcome in different settings. Stigma has been branded “the most basic cultural and moral barrier” to ending the global burden of mental illness . DiscrimNevertheless, there have been very few studies of discrimination in low-resource settings , 7 in geThe study was a cross-sectional survey of community-ascertained people with SMI in an Ethiopian district as part of the Programme for Improving Mental health carE (PRIME) study . PRIME wThe study took place in Sodo District, Gurage Zone, of the Southern Nations, Nationalities and Peoples’ Region (SNNPR), Ethiopia, between December 2014 and July 2015. The district is located 100 km from the capital city, Addis Ababa. Reflecting Ethiopia as a whole, 90% of the district inhabitants live rurally with the main sources of livelihood being farming and animal husbandry. The official language is Amharic. At the time of the study, the district had a population of approximately 165,000 with no mental health specialists. However, through PRIME, a district level mental health care plan was being implemented with the goal of expanding access to integrated primary mental health care .Figure Aged 18 years or older,Planning to continue living in the district for the next 12 months,Provided informed consent  or, if lacked capacity to consent, did not refuse and caregiver permission was obtained, andAble to understand Amharic.People with confirmed SMI and their caregivers were then recruited into the study if they met the following criteria:Those diagnosed with a mental, neurological and substance use (MNS) disorder prior to the study were not excluded.The sample size was powered for the primary objectives of the PRIME study: detection of a 20% reduction in symptom severity after 12 months, with 90% power, two-sided significance level of 5%, and an assumed attrition rate of 20% . The finThe primary outcome of the current study was discrimination experienced by people with SMI and the primary exposure was residence. Potential confounders were clinical diagnosis, symptom severity, disability, alcohol use, social support, poverty, age, sex, marital status, and education level. All assessments were administered directly to participants. Most participants had caregivers present during interviews. The caregivers were able to contribute to the responses so that the assessor had the most complete information available to them.Discrimination was assessed by lay interviewers using section 1 of the Discrimination and Stigma Scale (DISC-12), which asks about the frequency of negative experienced discrimination over the past year . ResponsResidence was self-reported as either urban or rural neighbourhood.Psychiatric nurse-administered measuresInformation on potential confounding variables was obtained from measures administered by  psychiatCategorised as affective psychosis , or primary psychotic disorder (schizophrenia and other non-affective psychotic disorders) based on the OPCRIT.(2)Lay data collector-administered measuresSymptom severity was determined by using the Brief Psychiatric Rating Scale - Expanded Version (BPRS-E) translated into Amharic . The BPRThe 36-item World Health Organization Disability Assessment Schedule (WHODAS-2.0) questionnaire measures difficulties performing activities over the last 30 days due to all health problems . The comThe Alcohol Use Disorders Identification Test (AUDIT) self-reported version measures alcohol use in the past 3 months . Total sThe Oslo 3-item Social Support Scale (OSSS) asks about ease of getting practical help, number of close acquaintances, and level of concern from others . ResponsCalculated based on indicators used in the 2011 Ethiopia Demographic and Health Survey . Using eThese variables were self-reported.Data management and analysis were done using STATA version 15.1 . CategorThe potential confounders were determined a priori, as described above, and were included in the adjusted model regardless of statistical significance.Coefficients are on a log scale; they have been exponentiated and presented as risk ratios for ease of interpretation. The risk ratio represents the increase in total discrimination score for a one-unit increase in explanatory variable.A total of 300 people with SMI were included in the analysis. The majority of participants were Orthodox Christians (90.0%), of Gurage ethnicity (94.7%), and resided in rural neighbourhoods (79.9%); 36.9% were unemployed. Primary psychotic disorders (schizophrenia and psychotic disorders) were the most common diagnoses (85.3%) Table .Table 1Two-thirds of the respondents reported experiencing discrimination (63.3%). There was some variation between individual discrimination items, with 38.3% experiencing being avoided or shunned by those aware of their mental condition, compared to 6.3% experiencing unfair treatment in their religious practices. The most common response to the DISC-12 discrimination questionnaire (‘how many times treated unfairly in the past year’) was “not at all” or “not applicable”, with 36.7% answering “not at all” or “not applicable” to all 17 items (Table χ2(1) =4.80; p = 0.028), the police (χ2(1) =11.97; p = 0.001), in keeping a job (χ2(1) =5.43; p = 0.020), and in safety (χ2(1) =5.00; p = 0.025). Approximately half (52.0%) of respondents reported at least “moderate” discrimination on one or more items and 29.9% reported “a lot” of discrimination on one or more items.Those from urban neighbourhoods experienced more discrimination on all items except unfair treatment by people in their neighbourhood and in dating or intimate relationships Fig . Urban rResidence was significantly associated with experienced discrimination: participants living in urban areas experienced 1.66 times the discrimination of those living in rural areas, after adjusting for potential confounders . Female gender , hazardous alcohol use , and disability  were also independently and significantly associated with discrimination in the adjusted model . Care is also needed when generalizing these results to other Ethiopian populations, as what is considered urban might differ between predominantly rural districts like Sodo, and the capital city of Addis Ababa, for example. Although contact coverage for PRIME’s district mental health care plan was high (81.7%), participants from rural areas were significantly less likely to attend the health centre than their urban counterparts and therefore less likely to be included in this sample [Strengths of the study include the community-based case ascertainment (which reduced the risk of selection bias that is present in facility-based studies), confirmatory diagnostic interviews performed by trained mental health specialists and clinical assessments of symptom severity. However, there were study limitations. There is evidence that the construct and manifestations of discrimination varies between countries , and thes sample ; howeverThere is some evidence that social contact and mass educational campaigns can reduce stigmatizing attitudes among the general public , 6. It mAmong persons with SMI in Sodo district, urban residence was associated with greater experienced discrimination. Overall, the reported levels of experienced discrimination were low considering the consistently high levels of stigma reported in the current literature. There may be distinct aspects of urban living in low-income countries, such as inadequate infrastructure and job opportunities, that should be further investigated as potential risk factors for discrimination. Policymakers seeking to expand access to mental health care should be aware of these distinct experiences and consider how social exclusion can be overcome indifferent settings.


Rumex obtusifolius L. in rabbits with hyperglycemia. The high content of total phenolic compounds, flavonoids, and tannins has been determined in this plant extract. Oral administration of ethanol extract showed significant effect on hyperglycemia, reducing fasting glucose levels , improving glucose tolerance, and increasing liver glycogen content  compared to the hyperglycemic control group. Furthermore, ethanol extract of R. obtusifolius reduced total cholesterol, low-density lipoprotein cholesterol levels, and vice versa increased high-density lipoprotein cholesterol levels and also decreased liver enzymes levels  compared with untreated group. These findings suggest that R. obtusifolius may have beneficial effects and should be supplement, as herbal remedy in the treatment of DM.Diabetes mellitus (DM) is a serious chronic metabolic disorder. Various diseases are being treated with medicinal plants and that is because of the less side effects of the current therapy. The diversity of plants in Armenia is due to the singularity of natural environment. However, biochemical activity of these plants has not been studied well. Thus, the goal was to investigate biochemical activity and antihyperglycemic properties of Dyslifunction –6. It isImmobilization stress can increase the blood glucose levels and the risk of cardiovascular diseases due to impaired endothelial function. Chronic immobilization stress leads to atherosclerosis, which has an endothelial dysfunction at early stages . The oxiThere are many drugs currently used in the treatment of diabetes. Some reports indicate that treatments with synthetic antidiabetic drugs have toxicity and cause adverse effects, such as hypoglycemia, gastrointestinal and liver problems . Seconda Rumex have been used traditionally either as edible plants or for the treatment of several diseases in many parts of the world [ Rumex species are important edible and medicinal plants used in Armenian traditional medicine. These plant species have been used in folk medicine for the treatment of various diseases and ailments, including hypertension, inflammation, and bacterial infections [ Rumex obtusifolius (RO) has been shown to have antibacterial activity [Armenian flora has large diversity of plants which might delay the development of diabetic complications and correct metabolic disorders. Thus, the identification of potent antidiabetic agents from natural sources, such as edible plants with minimum side effects for diabetic patients, is crucial. Plants belonging to the genushe world . Rumex sfections , 12. Rumactivity . Methanoactivity . HoweverThus, the goal was to investigate biochemical properties and antihyperglycemic properties of RO in rabbits with hyperglycemia.The seeds of the plant were harvested after maturation. Identification of plant was carried out at the Department of Botany and Mycology, Yerevan State University, Yerevan (Armenia). A voucher specimen (No ERCB13208) has been deposited in the herbarium of Yerevan State University. The dried seeds were extracted with 40% ethanol for 20 min at 60°C. Briefly, 270-280 mg dry matter in 6 ml of ethanol. The extract was filtered with Watman filter paper  and after cooling at room temperature was orally administrated.2CO3 were added to 0.5 mL sample extract (200 mg/mL concentration). The reaction mixture was incubated at 45°C for 40 min, and afterwards absorption was measured at 765 nm wavelength . Solvent was used, as blank. TPC of plant extracts was evaluated according to gallic acid calibration curve and expressed as micrograms of gallic acid equivalents per mg of dry extract (GAE/mg DW). Flavonoids were determined by the colorimetric method with AlCl3 [3, 0.05 mL 1 M Na-K tartrate, and 1.4 mL distilled water were added to 0.25 mL plant extract (200 mg/mL concentration). The mixture was incubated at room temperature for 30 min; then absorption was measured at 415 nm wavelength, using quercetin, as standard. The amount of flavonoids expressed as micrograms of quercetin equivalent per mg of dry extract (QE/mg DW). For quantify of total tannins 3 mL of 4% vanillin (methanol solution) and 1.5 mL of concentrated HCl were added to 50 μL plant extract (200 mg/ml) [The total phenolic content (TPC) of crude extracts was measured using Folin-Ciocalteu method . 2.5 mL th AlCl3 . 0.75 mL0 mg/ml) . The mixOryctolagus cuniculus domesticus) (1800-1900 g) were used in this study, which was authorized by the “International Recommendation on Carrying out of Biomedical Researches with use of Animals,” and the study plan has been approved by the National Center of Bioethics (Armenia). They were acclimatized for 1 week prior to experimentation. The animals were housed under standard environmental conditions (temperature 22±2°C in a light/dark cycle of 12 h) and had free access to food and water during the experimental period. Initial body weights were recorded one day before the start of experiments.Nine domestic male rabbits (Hyperglycemia was induced by immobilization stress in the rabbits during 21 days (5 h daily) , 8. TheyThe animals were randomly divided into three groups (n=9) as follows: group 1: normoglycemic, group 2: hyperglycemic control, putting immobilization, and group 3: hyperglycemic experimental, received RO seed extract (150 mg of extract per kg body weight (BW). This number of animals was chosen because they showed reliable reproducible results. Each rabbit was housed individually in a separate cage (0.90 × 0.60 × 0.40 m) with standard laboratory diet.The biochemical analysis was performed determining the serum level of glucose, TC, HDL- cholesterol, and LDL-cholesterol. All parameters were assayed using enzymatic kit. Serum glucose level (mmol/L) was determined using glucose test kit based on the glucose oxidase method . TC and th day of treatment an oral glucose tolerance test (OGTT) was carried out. Animals were fasted overnight before commencing of experiments. 20% glucose solution (2g/kg BW) was administrated a signal oral dose to all groups of rabbits. The blood glucose level was measured by portable glucometer . Blood samples were collected from aural vein at 0, 30, 60, 90, and 120 min after glucose loading. Glucose clearance was evaluated by calculating the area under the curve (AUC) of the glycemic profile.On the 10glucose) by trapezoid rule for the 120 min [Total glycemic responses to OGTT were calculated from respective areas under the curve for glucose  was <0.05. A difference of p < 0.05 or less in the mean values was considered as statistically significant.μg GAE/mg DW. Flavonoids and tannins contents were of 47.37±1.23 μg GAE/mg DW and of 23.93±0.26 CE/mg DW, respectively. It should be noted that there is no literature data about phytochemical composition of RO extracts; however, it is well known that plants of the genus are abandoned with anthraquinones, flavonoids, terpenoids, and carotenoids [TPC determination has shown a significant concentration of phenolics in RO extract of 327.2±0.33 otenoids ; detaileth day value.Antihyperglycemic activity of RO seeds ethanol extract was evaluated on hyperglycemic rabbit model. Fasting blood glucose levels in the hyperglycemic control (56.2%) and hyperglycemic + RO extract (48.0%) groups during the first day of immobilization (5 h) were significantly increased, compared to the normoglycemic group, p<0.05 . TherefoIt was observed that the seeds extract showed a significant effect on hyperglycemia compared to the hyperglycemic group . Blood gglucose value for glucose at 0 to 120 min was significantly increased in the untreated animals while glucose concentration was decreased to 52.6% (p<0.001) for treated group compared to the hyperglycemic control group.The area under the curve (AUC) is represented in The data showed that the TC and LDL-cholesterol levels in the hyperglycemic control group were significantly increased (68.8% and 61.9% respectively), TG level was increased (17.5%) compared to the normoglycemic group . After 2As β-cells, therefore, enhancing the impaired capacity of the liver to synthesize glycogen [Glycogen levels were depleted in both hyperglycemic control and hyperglycemic + RO groups  compared to the normoglycemic group (p<0.01) . Howeverglycogen , 23.The effect of ethanol extract of RO seeds on body weight in experimental rabbits was summarized in st century. The number of people with diabetes has been growing and causing increasing concerns in medical sphere and the public [Diabetes is a disease that affects many people in the 21β-cell failure are among the leading causes of type 2 diabetes [Hyperlipidemia is one of the major factors linked with hyperglycemia due to insulin deficiency during diabetes and correlated with carbohydrate metabolism. Insulin resistance and lack of insulin secretion due to pancreatic diabetes , 25.In this study, hyperglycemia in rabbits was induced by immobilization stress. The latter leads to disorder of the endocrine system, especially increase the blood glucose levels and lipid metabolism consequently to the increase of TG, LDL-cholesterol. and to the decrease HDL-cholesterol levels. Probably prolonged immobilization stress increases the risk of cardiovascular disease due to impaired endothelial function leads to development of atherosclerosis, which has an endothelial dysfunction at early stages , 8. Stevia rebaudiana that possess antihyperglycemic and antihyperlipidemic activities has hepatoprotective effect in hyperglycemia induced by immobilization stress in rabbits [ Rumex are widespread in the Armenian flora and are used for the prevention and treatment of various diseases; however antihyperglycemic properties of this plant have not been sufficiently studied.Previously, it was observed that aqueous extract of hydroponic rabbits . Wild sp Rumex species are used, as anti-inflammatory agents. The decoction of the seeds of R. obtusifolius is used for the treatment of coughs of all types, colds and bronchitis [ Rumex species are most frequently utilized plant parts as foods, while roots and seeds are applied preferably for the treatment of different diseases [ Rumex species possess different kinds of biological activities, especially antioxidant [In traditional medicine numerousonchitis , and renonchitis , and alsonchitis , 28. Mandiseases . Roots adiseases . Pharmacioxidant –31, antiioxidant , anti-inioxidant . Rumex species are rich in flavonoids and other phenolic compounds which have shown antioxidant properties [ Rumex crispus seeds [ R. obtusifolius revealed antibacterial activity and also it could verify its traditional use for the treatment of several skin diseases [Someoperties , 29, 31.diseases , 31. Rumex species may have considerable carcinogenic potential [ Rumex patientia roots has protective effect against the oxidative damage of lipids and DNA and restores AST, ALT, ALP, and bilirubin levels. The seeds of R. patientia have a hypoglycemic effect and improve serum lipid profile in regard to HDL- and LDL-cholesterol levels [Due to the high tannin content the roots of someotential . Methanootential , 13. Thiotential  reportedl levels . Rumex species and encourage their future use in modern medicine.Our findings showed that RO extract has a high concentration of phenolic compounds, flavonoids, and tannins (see the Results). These substances might be responsible for hypoglycemic effects of RO. Among the phenolic compounds, the flavonoids act as insulin secretagogues that may improve glucose uptake in peripheral tissues. Also the flavonoids may regulate the activity of rate-limiting enzymes involved in carbohydrate metabolism pathways . Tanninsst day demonstrated reduction in fasting glucose level , glycogen content, and body weights in hyperglycemia induced by immobilization stress in rabbits for 21 days. Our results showed that the rabbits in the hyperglycemic control group demonstrated increased blood glucose and lipids levels and loss of body weight compared to the normoglycemic group. It was noted that strong stressful pressure provokes hyperglycemia. However, the oral treatment with the RO extract at the 21evel see . This reondition . The bloondition . Our stuIt was observed that treatment with the seed extract of RO increased glucose tolerance compared to the untreated group of animal see . OGTT waAbnormal lipid metabolism leading to increasing of several serum parameters, including increase in TC, TG, and LDL-cholesterol levels, and reducing in HDL-cholesterol level are hyperglycemia indicators. They are included in increased risk of clinical diseases , 37, 38.In the current study a reduction in the TG, TC, and LDL-cholesterol after treatment with RO extract it was observed see . Hypolipβ-cells and secreted by exocytosis in response to different hormonal modulators [Insulin is stored in large dense core vesicles in the pancreatic dulators , 36, 40.dulators , 41, 42.β-cells, thereby increased liver glycogen synthesis [Our study has shown that as a result of immobilization, liver and muscle glycogen content were reduced which could be linked to an inactivation of glycogen synthase. However, our results indicated that administration of RO increased liver glycogen level see , compareynthesis , 23, 42.Liver enzymes  were investigated in this study as marker for hepatic diseases and hepatic damage. They are released into the blood stream after cell membrane damage . In the The body weight lowering is often associated with hyperglycemic conditions, as a result of insulin deficiency which produces degeneration of structural proteins and resulted in muscle wasting , 39. How R. obtusifolius seeds in the treatment of hyperglycemia. The extract revealed hypoglycemic activity, improved lipid profile and body weight, corrected liver enzymes activities, and restored liver and muscle glycogen levels in hyperglycemia induced by immobilization stress in rabbits.The present study has shown the efficacy and safety of the ethanol extract ofFurther investigations on the chemical compounds responsible for these effects should be performed to clarify the mechanisms of action.


Proteins were isolated and quantified using liquid chromatography-mass spectrometry, and protein expression in abdominal and femoral subcutaneous AT was compared. Moreover, correlations between fat cell size and the proteome of both AT depots were determined. In total, 651 proteins were identified, of which 22 proteins tended to be differentially expressed between abdominal and femoral AT after removal of blood protein signals (p < 0.05). Proteins involved in cell structure organization and energy metabolism were differently expressed between AT depots. Fat cell size, which was higher in femoral AT, was significantly correlated with ADH1B, POSTN and LCP1. These findings suggest that there are only slight differences in protein expression between abdominal and femoral subcutaneous AT. It remains to be determined whether these differences, as well as differences in protein activity, contribute to functional and/or morphological differences between these fat depots.Body fat distribution is an important determinant of cardiometabolic health. Lower-body adipose tissue (AT) has protective characteristics as compared to upper-body fat, but the underlying depot-differences remain to be elucidated. Here, we compared the proteome and morphology of abdominal and femoral AT. Paired biopsies from abdominal and femoral subcutaneous AT were taken from eight overweight/obese (BMI ≥ 28 kg/m Being a highly active metabolic and endocrine organ3, adipose tissue (AT) is involved in the regulation of many physiologic processes, like immune responses, energy balance, blood pressure regulation, and glucose homeostasis4. The expansion and remodeling of AT during excessive weight gain renders the tissue dysfunctional5. AT dysfunction in obesity is strongly linked to metabolic dysregulation and increased risk of cardiometabolic diseases6.Obesity is related to cardiometabolic disorders that contribute to increased morbidity and mortality8. Contrary to central obesity, accumulation of lower-body fat appears protective against metabolic derangements and hypertension9, and is associated with a reduced incidence of type 2 diabetes mellitus and cardiovascular disease when adiposity is comparable11. However, the underlying mechanisms for the differences in disease risk associated with a certain body fat distribution remain elusive. We have recently demonstrated that abdominal subcutaneous adipose tissue is characterized by smaller adipocytes and a distinct pattern of gene expression compared to femoral adipose tissue in overweight/obese women, which may contribute to functional differences between these fat depots12.In addition to total AT mass, the location where lipids are stored seems an important determinant of the cardiometabolic consequences13. Interestingly, for GFAT, the association between AT inflammation and BMI was weaker as compared to abdominal AT, which was confirmed by a lower secretion of interleukin-6 from lower-body AT. Moreover, markers of macrophage infiltration were not enriched in GFAT but increased in abdominal AT with obesity13.Omics methodology provides excellent opportunities to investigate putative differences between AT depots. Microarray analysis of gluteofemoral (GFAT) and abdominal AT revealed that expression of energy-generating metabolic genes was inversely, and of inflammatory genes was positively associated with obesity14. Furthermore, proteome differences in either subcutaneous or visceral AT in relation to BMI or metabolic health have been investigated in humans18. In this respect, it has previously been found that several proteins related to AT remodeling, including several keratin and annexin proteins, and proteins related to oxidative stress were more abundant in the abdominal AT of obese and overweight as compared to lean individuals, both in men and women16. Although structural and functional differences between visceral and subcutaneous adipose tissue may be more pronounced than differences between different subcutaneous AT depots, a direct comparison of the proteome of upper- and lower-body subcutaneous human AT has not been performed yet.To investigate AT depot-differences at a more functional level, proteomics analysis may be highly valuable. It has previously been shown that proteins related to metabolic processes such as glucose and lipid metabolism, lipid transport, protein synthesis, protein folding, response to stress and inflammation were differentially expressed in abdominal subcutaneous as compared to omental AT in humansIn the present study, we compared for the first time, to our knowledge, the proteome of abdominal and femoral subcutaneous AT in overweight and obese women with impaired glucose metabolism using untargeted quantitative liquid chromatography-mass spectrometry to obtain insights in the physiological differences between these subcutaneous AT depots in humans.2) women with an impaired fasting glucose (IFG: fasting plasma glucose 5.6–7.0 mmol/l) or impaired glucose tolerance (IGT: 2 h plasma glucose 7.8–11.1 mmol/l) participated in the present study. Exclusion criteria were smoking, cardiovascular disease, type 2 diabetes mellitus, liver or kidney disease, use of medication known to affect body weight and glucose metabolism, marked alcohol consumption . Furthermore, subjects had to be weight stable (weight change <3.0 kg) for at least three months prior to the start of the study. Subjects were asked to refrain from strenuous physical activity for at least two days before biopsies were collected and measurements were performed.Eight overweight and obese . Height was measured using a wall-mounted stadiometer . Waist (top of the iliac crest) and hip (widest portion of the buttocks) circumferences were measured. Body composition and body fat distribution were determined by DEXA .Abdominal and femoral subcutaneous AT biopsies (~1 g) were collected using needle aspiration under local anaesthesia (2% lidocaine), 6–8 cm lateral from the umbilicus and from the lateral site of the upper leg, respectively, after an overnight fast. Biopsies were immediately rinsed with sterile saline and visible blood vessels were removed with sterile tweezers. The tissue was snap-frozen in liquid nitrogen and stored at −80 °C until analysis.12.Histological sections were cut from paraffin-embedded AT, and stained with haematoxylin and eosin. Fat cell size was measured using digital images that were captured and analyzed using a Leica DFC320 digital camera  and software , as described beforeFrozen AT (~100 mg) was ground in a mortar with liquid nitrogen. Per microgram of grounded powder, 2 μl of 50 mM ammonium bicarbonate with 5 M urea was added to dissolve the powder. The solution was freeze-thawed in liquid nitrogen 3 times after which it was vortexed for 5 min. The homogenate was centrifuged at 20,000 g for 30 min at 10 °C. The supernatant was carefully collected and protein concentrations were determined with a Bradford-based protein assay . A control sample was prepared from a pool of 10 μl of each sample.19. Equal amounts of the 16 samples from 8 subjects  were combined and analyzed by LC-MS in two injections; each injection composed of a mixture of 8 samples from 4 subjects and the control.Samples were digested with Trypsin (Promega) and peptides from 100 µg protein were labelled with TMT isobaric mass tagging labelling reagent  according to the manufacturer’s protocol. Briefly, 100 μg of protein was used for each sample. The TMT labeling reagents were dissolved in 41 μl acetonitrile per vial. The reduced and alkylated samples and control were added to the TMT reagent vials. The reaction was incubated for 1 h at room temperature and quenched for 15 min by adding 8 μl of 5% hydroxylamine, as described previouslyA nanoflow HPLC instrument (Dionex ultimate 3000) was coupled on-line to a Q Exactive HF (Thermo Scientific) with a nano-electrospray Flex ion source (Proxeon). One μg of TMT labeled peptide mixture was loaded onto a C18-reversed phase column . The peptides were separated with a 150 min linear gradient of 4–50% in buffer A (100% water with 0.1% TFA) with buffer B (80% acetonitrile and 0.08% formic acid) at a flow rate of 300 nL/min.MS data was acquired using a data-dependent top-10 method, dynamically choosing the most abundant precursor ions from the survey scan (280–1400 m/z) in positive mode. Survey scans were acquired at a resolution of 60,000 and a maximum injection time of 120 ms. Dynamic exclusion duration was 30 s. Isolation of precursors was performed with a 1.8 m/z window and a maximum injection time of 200 ms. Resolution for HCD spectra was set to 30,000 and the Normalized Collision Energy was 30 eV. The under-fill ratio was defined as 1.0%. The instrument was run with peptide recognition mode enabled, but exclusion of singly charged and charge states of more than five.The MS data were searched using Proteome Discoverer 2.1 Sequest HT search engine (Thermo Scientific), against the UniProt human database. The false discovery rate (FDR) was set to 0.01 for proteins and peptides, which had to have a minimum length of 6 amino acids. The precursor mass tolerance was set at 10 ppm and the fragment tolerance at 0.02 Da. One miss-cleavage was tolerated, oxidation of methionine was set as a dynamic modification and carbamidomethylation of cysteines, TMT reagent adducts (+229.162932 Da) on lysine and peptide amino termini were set as fixed modifications.The MS-acquired data were first analyzed with Thermo Scientific Proteome Discoverer software version 2.1. Relative quantitation of peptides from mixed samples was extracted by comparing the signal to noise ratio (S/N) of the TMT reporter ions peak in the MS/MS spectrum. The S/N signal of multiple distinct peptides from each protein was summed to report the protein signal.The intra-run variation for each sample in the mix was normalized with the Proteome Discoverer software to get the total protein signal of each sample the same as the highest one in the run. The inter-run variation was normalized based on the identical control samples in each run, performed in R environment (Supplementary File 1).Then, low-quality proteins with identification Score Sequest HT <5 were removed from the data set.20, including all immunoglobulins. The final signal was the protein abundance in a blood protein-free AT sample.To reduce the influence of the blood protein contamination on the AT proteome, we retrieved information from the UniProt database to set up a blood protein exclusion list Final signal This final signal  was used in data analysis.The paired biopsies from abdominal and femoral subcutaneous adipose tissue from the same overweight/obese individuals (n = 8) were used to perform Western Blotting for periostin (POSTN) and annexin A2 (ANXA2), which appeared to be differentially expressed between abdominal and femoral adipose tissue using LC-MS methodology, to confirm the LC-MS results. Samples with equal amount of protein were run on a 12% SDS polyacrylamide gel , then transferred  to 0.45-mm nitrocellulose membranes. Total protein was stained with Ponceau S followed by destaining. Membranes were blocked in 5% non-fat dry milk power  in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Thereafter, the blots were incubated with the primary antibodies against Periostin  and AnnexinA2  in 5% NFDM-TBST overnight at 4 °C on a shaker. Then, the blots were washed three times for 10 min in TBST and incubated for 1 h with a 1:10,000 dilution of the horseradish peroxidase-conjugated secondary antibody (DAKO) in 5% NFDM-TBST. A CCD camera  was used to detect immunoreactive bands using chemiluminescent substrate . The quantification was performed with the program Quantity One version 4.6.5 (Bio-Rad). Ponceau S was used to standardize for the amount of protein loaded.For statistical analyses, missing values in proteome data were imputed with the half of the lowest positive signal in the dataset. Thereafter, data were log2 transformed. Two-sided paired Student’s t-test was used to test AT depot-differences for fat cell size and each protein detected by the LC-MS approach. False discovery rate (FDR) q-value was calculated to adjust proteomics data for multiple testing. Proteins with a p-value < 0.05 were regarded as differentially expressed, and were selected for further biological annotation and analysis.A heat-map was generated based on these proteins using their scaled data . The Euclidean method was used to compute distance, and the ward.D method was used to compute the hierarchical clusters of proteins.The association between fat cell size and protein expression was analyzed by Spearman’s rank correlation for both abdominal and femoral subcutaneous AT.The protein expression determined by Western blotting was analysed by one-sided non-parametric Wilcoxon signed-rank test to confirm the differences found with LC-MS.2-test for two dependent samples is the multivariate extension of the two-group paired Student’s t-test22. The proteome profile data of 610 proteins Log2 transformed expression values were first downscaled to 7 principle components  by multidimensional scaling, and then entered the Hotelling’s T2-test. A p-value < 0.05 was considered to be statistically significant. All statistical analyses were performed in R environment, version 3.4.2, with various packages .Hotelling’s T2) subjects included in the present study had an impaired glucose metabolism. Fat cell size was smaller in abdominal as compared to femoral subcutaneous AT   by multivariate Hotelling’s TComparison of protein expression revealed that 22 of the 610 identified AT proteins tended to be differentially expressed between both AT depots . More specific, protein expression of periostin (POSTN), myosin regulatory light chain 12B (MYL12B), gelsolin (GSN), tropomyosin alpha-4 chain (TPM4), actin cytoplasmic 1 (ACTB), and actin-binding protein lymphocyte cytosolic protein 1 (LCP1) was higher in femoral as compared to abdominal AT. Moreover, some proteins related to energy metabolism were expressed at a higher level in femoral than abdominal AT, including mitochondrial hydroxyacyl-coenzyme A dehydrogenase  and L-lactate dehydrogenase A chain (LDHA). In contrast, certain other proteins related to energy metabolism such as alcohol dehydrogense 1B (ADH1B), and cytochrome b5 (CYB5A), were expressed at a lower level in femoral versus abdominal AT. We also found a higher expression of protein synthesis complex component elongation factor 2 (EEF2) and redox superoxide dismutase (SOD1) in femoral AT. Furthermore, chaperone heat shock protein HSP 90-alpha (HSP90AA1), nitric oxide production related enzyme dimethylarginine dimethylaminohydrolase 2 (DDAH2), and glycogen synthesis enzyme UTP-glucose-1-phosphate uridylyltransferase (UGP2) were also expressed at a lower level in femoral as compared to abdominal subcutaneous AT.Western blot analyses was performed, using the paired biopsies from abdominal and femoral subcutaneous adipose tissue from the same overweight/obese individuals (n = 8), to confirm the adipose tissue depot-differences in protein expression of POSTN and ANXA2 found using LC-MS. In line with the reported LC-MS data , while fat cell size was positively correlated with LCP1 (p = 0.028) and POSTN (p = 0.028) in femoral AT Fig. .Figure 3The aim of the present study was to investigate differences in the proteome of abdominal and femoral subcutaneous AT in overweight and obese women with impaired glucose metabolism, and to examine the associations between fat cell size and the AT proteome, since this may yield mechanistic insight into functional differences between these AT depots. For this purpose, quantitative LC-MS analysis of paired biopsies was performed to identify and quantify proteins. Here, we identified and quantified 610 proteins, and demonstrated that no major differences exist between the proteome of abdominal and femoral subcutaneous AT following an overnight fast. Nevertheless, 22 proteins seemed to be differentially expressed between abdominal and femoral AT, and fat cell size was significantly correlated with ADH1B, POSTN and LCP1.23, the 22 differentially expressed proteins were related to ‘protein binding’ (8/22) and ‘catalytic activity’ (8/22) in the category ‘molecular function’. Moreover, in the category ‘biological processes’, the most prominent differences were related to ‘metabolic processes’ (13/22) and ‘cellular processes’ (11/22). In line with this global analysis, a more detailed examination of the expression of these proteins showed that differences in structural proteins may exist between abdominal and femoral AT, indicated by the lower abundance of the ECM protein POSTN and the cytoskeleton-related proteins MYL12B, TPM4, ACTB, GSN, TAGLN2, and LCP1 in abdominal as compared to femoral subcutaneous AT. Interestingly, POSTN was the only protein that showed consistent AT depot differences in all subjects, and also showed the most pronounced difference . According to mRNA expression data, POSTN is ubiquity expressed in most tissues, of which high expression levels were found in digestive organs/metabolic tissues, suggesting a relation with nutrient metabolism24. This protein functions as a cell adhesion component25, which may stimulate the maturation of ECM . POSTN is also highly expressed in collagen-rich connective tissue and has previously been associated with obesity26 and weight regain in females27. These findings suggest that POSTN may be involved in lipid storage in adipocytes, and repair and/or expansion of AT. POSTN is able to bind to integrins and can transduce external signals into cells via the focal adhesion kinase pathway28. Intracellularly, focal adhesions are attached to the actin filaments, which undergo a structural reorganization during the differentiation of adipocytes29. In this regard, both POSTN and ACTB may be involved in AT expandability or, in other words, the fat storage capacity of AT. Indeed, we found POSTN to be positively correlated with femoral but not abdominal fat cell size. In addition, the protein abundances of EEF2 and RNH1, a translation elongation factor and an inhibitor of mRNA turnover, were also lower in abdominal AT, which may be indicative of differences in protein synthesis accompanying tissue expansion. In agreement with these findings, fat cell size was significantly smaller in abdominal than femoral subcutaneous AT, as we have also reported previously12. The latter is in line with earlier studies showing larger femoral than abdominal adipocytes in obese men and women31. Adipocyte expansion by fat storage may lead to cell stress32. In this respect, a lower abundance of HSP90AA1 in femoral AT would be in line with a larger expansion capacity of femoral adipocytes, without being hampered by cell stress, although this remains to be elucidated. Altogether, the present proteome analysis may point towards key proteins involved in human AT remodelling.We found that, using the PANTHER library of protein (sub)families34. MYL12B is a component of myosin II, which can form contractile structures in connection with actin filaments35. Its phosphorylation triggers formation of myosin II filaments but also actin polymerization. Both TAGLN2 and LCP1 are proteins which regulate actin filament polymerization37. Notably, both of these proteins may be involved in the formation of immunological synapses between leukocytes and target cells39. Taken together, the differences in protein expression levels of structural proteins may indicate that differences exist in tissue structure between upper- and lower-body AT depots in humans, and suggest a different tendency for the interaction of immune cells inside AT. This is further strengthened by the positive correlation between fat cell size and LCP1 in femoral AT.Moreover, many of the proteins that had a lower abundance in abdominal subcutaneous AT are functionally related to actin filaments. GSN binds to the positive end of actin monomers and filaments, thereby preventing monomer exchange. TPM4 forms dimers, which interact with the actin filaments and controls the access of actin-associated proteins to the filaments40, was also expressed at a lower level in abdominal subcutaneous AT. Together, these findings suggest that these subcutaneous AT depots may differ in the level of oxidative stress. Moreover, the lower abundance in HADHA and LDHA proteins in abdominal versus femoral AT further suggests differences in metabolic activity between these AT depots, since these proteins are involved in mitochondrial beta-oxidation and anaerobic glycolysis, respectively. Furthermore, other metabolic enzymes (ADH1B and CYB5A) appeared to be higher expressed in abdominal AT, implying pathway-specific differences in the expression of proteins involved in metabolic activity between both fat depots. Interestingly, protein expression of ADH1B, of which gene expression is highest in AT as compared to other tissues24, was negatively correlated with abdominal and femoral fat cell size. Moreover, ADH1B has also been implicated in body weight regulation41, and was negatively correlated with waist circumference, BMI and fasting plasma insulin42. Taken together, it is tempting to suggest that ADH1B may be involved in AT expandability.Furthermore, we found that protein expression of cytoplasmic SOD1 was lower in abdominal than femoral AT. This protein plays a role in scavenging naturally occurring oxygen radicals. In accordance, RNH1, previously found to be implicated in protection against oxidative stressThe strength of the present study is that we, for the first time to our knowledge, compared the proteome of human abdominal and femoral subcutaneous AT. Moreover, the paired comparisons that were made between both subcutaneous AT depots for each individual further strengthen this study. Thus far, very few proteomic analyses of human whole-AT have been performed. Despite thorough cleaning, the AT biopsies still contained residual blood, which resulted in the identification of several blood proteins that accounted for ~20–30% of the total AT protein signal. Blood contamination is not surprising, since AT is a relative highly vascularized tissue. Unavoidable blood contamination might be one of the barriers to obtain a real ‘AT proteome’, but we adjusted for blood proteins in the present analyses. Furthermore, Western Blot analyses confirmed the adipose tissue depot-differences in protein expression of POSTN and, to a lesser extent, ANXA2 that were found using LC-MS. Noteworthy, the data obtained using Western blotting showed more variation between samples as compared to the LC-MS results. The latter suggests that the LC-MS methodology that we have employed in the present study is more sensitive to detect adipose tissue depot-differences in protein expression, which may partly be due to the adjustment for blood protein contamination.https://www.proteinatlas.org/humanproteome/adipose). Thus, our findings may reflect only part of the AT depot proteome differences. Furthermore, AT depot differences in protein abundance did not yield significant findings following adjustment for multiple testing in the present study, which might be due to limited power and/or subtle differences in protein expression between these AT depots. Therefore, the differences reported here should be confirmed in future studies, also taking into account that certain differences in protein expression between adipose tissue depots may be either more or less pronounced depending on the study population. Here, we studied adipose tissues from metabolically compromised individuals, because we expected this to increase potential differences between the depots. However, it might well be that a disturbed glucose metabolism masks any depot differences in protein expression that might exist among individuals with a different metabolic phenotype . Finally, AT biopsies were collected following an overnight fast. Therefore, it may well be that under challenged conditions such as after physical exercise, prolonged fasting or a dietary intervention, proteome differences are more outspoken. Noteworthy, protein abundance may not always be a valid surrogate for protein activity and, as such, our observations warrant follow-up research.A limitation of the present study is the relatively small number of participants. Furthermore, our proteomic analysis covered only a small amount of proteins of the AT proteome, based on human adipose tissue transcriptome data (In conclusion, comparison of human abdominal and femoral subcutaneous AT using non-targeted, quantitative proteomics revealed slight but specific differences in protein expression between these AT depots in overweight/obese women, and indicated that fat cell size was significantly correlated with ADH1B, POSTN and LCP1. These differences in protein expression may reflect depot-differences in adipocyte morphology, adipocyte expandability, immune cell interaction and energy metabolism. Importantly, it cannot be excluded that differences in protein activity, particularly under challenged conditions, contribute to divergent functioning of these AT depots.Supplementary file


Fusarium mycotoxins: deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), and zearalenone (ZEN) in two consecutive years to search the relationship between disease incidence and severity with mycotoxins found in control and inoculated grains and corresponding malt. In addition, deoxynivalenol-3-glucoside (D3G) in one-year research was measured. Tested wheat varieties showed infection scores of 3% (‘U1’ and ‘Sirban Prolifik’) to 79% (‘Golubica’) for Type I resistance evaluation. There were few moderately resistant varieties in view of their areas under the disease progress curve, which can be considered Type III resistance (‘Sirban Prolifik’ and ‘U1’). According to the data quantified by LC–MS/MS, DON decreased in infected malt in comparison to corresponding grain, while ZEN occurred only in infected malt samples. Both 3-AcDON and NIV increased in inoculated malt in comparison to corresponding grain, due to a combination of plant metabolism and de novo synthesis by molds during malting. Based on the results, we can draw a few conclusions: the resistance to Fusarium decreased quantified concentrations of DON; ZEN gets synthetized during malting; unregulated 3-AcDON and NIV increase during malting; more resistant varieties have converted DON to D3G more successfully. Modified mycotoxins should be also included to legislation, since they could be transformed back to the corresponding mycotoxins under food processing conditions or during digestion.Fusarium head blight (FHB) is one of the most destructive wheat fungal diseases, causing yield loss, quality reduction, and accumulation of mycotoxins. The aim of this research was to summarize the occurrence of major  Fusarium species, is one of the most important fungal diseases worldwide that affects the wheat (Triticum aestivum L.) by decreasing yield and quality [Fusarium species, such as F. poae, can produce NIV and other emerging mycotoxins, such as beauvericin, enniatins, and fusarin [Fusarium head blight (FHB), caused by several  quality ,2. A maj quality ,4, a typ quality ,6. The g quality . Due to  quality . The uti quality ,10,11. D quality . Type II quality : DON acc quality  and fusa quality , or fusa quality . Xu  is also reported due to the metabolism of the plant, and possible conversion to the DON through the hydrolysis or de-glycosylation process in vertebrates or food processing, which contributes to the overall DON exposure [In this research, authors report the comparison of resistant, moderately resistant, and susceptible varieties of wheat to the accumulation of exposure .For Type I FHB resistance, the fraction of plants showing disease symptoms ranged from 3% (‘Sirban Prolifik’ and ‘U1’) to 79% (‘Golubica’), scored at 26 days after inoculation (dai) . ‘SirbanFusarium species infection rates were 0, due to the weather conditions in the given years. In the last assessment (26 dai) for general resistance varieties, ‘Golubica’ had up to 71% diseased ears  for Type I resistance. Two resistant varieties (‘U1’ and ‘Sirban Prolifik’) kept FHB symptoms below 1%. The range of AUDPC for general FHB resistance in the 25 tested winter wheat varieties ranged from 2.7 (‘U1’) to 272.3 (‘Golubica’) AUDPC units , while no other Fusarium species were detected.For the control check, the non-inoculated wheat samples were taken, where the PC units . The Fusr = 0.91; p < 0.01 for Type I resistance and r = 0.82; p < 0.01 for the general resistance, respectively), FHB symptoms, and 3-acetyldeoxynivalenol (3-AcDON) contamination in inoculated wheat grains , as well as between FHB symptoms and DON in the corresponding malt samples , FHB symptoms and 3-AcDON in the corresponding malt samples , FHB symptoms, and ZEN  in the corresponding malt samples. FHB symptoms were statistically significantly correlated with NIV in the grain  and in the corresponding malt  , DON, and 3-AcDON in infected malt , and DON and NIV in infected malt . In infected malt, 3-AcDON showed a statistically high significant correlation with ZEN  and a statistically significant correlation with NIV   and DON contamination in inoculated wheat grains (ctively) . Signifi < 0.05) .r = 0.85; p < 0.01), and between 3-AcDON in infected grain and corresponding infected malt   .None of the control samples analyzed contained mycotoxins (DON and ZEN) at or above the EU maximum permitted levels set out in commission regulation (EC) No. 1881/2006 for unprocessed wheat . ControlD3G mycotoxin in naturally-contaminated (control) crops was below the limit of detection in wheat samples before and after malting . This myF. graminearum and F. culmorum (1:1) and measuring disease incidence and percentage of infected heads in the plots, which is a standard method for FHB evaluations [Triticum turgidum L. var. durum) samples were collected during two harvest seasons (2007 and 2008) showing Fusarium contamination, with infection levels ranging from 8 to 66%. For control check, the non-inoculated plots were taken, where the infection rate of Fusarium were 0 due to climate conditions in given years. In the last assessment (26 dai) for general resistance varieties, ‘Golubica’ had up to 71% Fusarium symptoms, similar to Type I resistance. Similar data for resistance was obtained both for Type I and general resistance. Twenty-five winter wheat varieties were evaluated in the field conditions for two components of FHB resistance during two consecutive years. The evaluation of Type I and general FHB resistance was carried out using spray inoculation with a mixture of luations . All winluations . Most valuations ,16. Alonluations , where 5Fusarium resistant. It is believed that breeding for resistance to FHB will increase resistance to DON accumulation in most cases, as confirmed by our research, which gives the significance to post-harvest mycotoxin testing. FHB severity and mycotoxins in the grain  and the malt  were highly correlated. Chrpová et al. (2007)  and LemmA highly positive relationship between DON in the grain and other mycotoxins in the grain and malt suggests that samples with high DON levels would likely produce malt with high mycotoxin levels, so resistant varieties should be a major safety factor in malt/beer production. Similarly, this was concluded by Schwarz et al. (2006)  and VáňoIn the present study, ‘U1’, ‘Sirban Prolifik’, ‘Apache’, and ‘Renan’ exhibited the highest level of general resistance and appeared to possess both Type I and Type III resistance due to lower amounts of DON in the infected grains, which was potentially a stimulation of higher epidemiological conditions. The same varieties had no D3G in the infected grain samples in the 2016/2017 vegetation season. Conversely, a few varieties  with moderate levels of FHB resistance in the field trials were susceptible to DON accumulation, indicating that their resistance was predominantly Type I and general, but was without Type III resistance. In our investigation, DON was found in a surprisingly low fraction on average in control, compared to the fraction of inoculated wheat samples, but the natural infection (control) depends mainly on the previous crops and the weather conditions during flowering of the wheat . A similThe maximum content of DON in unprocessed wheat, as defined in EU regulations (1250 µg/kg), was exceeded in 12 inoculated grain samples and in 6 inoculated malt samples. There was elevation of DON in all corresponding malt samples in comparison to the grain. In natural infection (control samples) there was an average level of DON 70.1 µg/kg. Higher mycotoxin infection was recorded in the research of Vrabcheva et al. (1996)  with theFusarium species include humidity, temperature, aerate, and substrate type [The reduction of DON in malt samples, in comparison to grain, indicates that DON content can be reduced or transformed during the commercial malting process, and there is even a possibility that, during steeping, the level of DON can sometimes decrease and is no longer detectable . The decate type . The presence of DON and ZEN derivatives can be a risk to food safety. Lorenz et al. (2019)  concludeF. graminearum prefers higher temperatures and high water activity for ZEN production, and such conditions are met during the malting. However, during favorable weather conditions, ZEN will also occur in grain wheat grain samples. According to Edwards (2011) [Fusarium species occurring at different climatic areas or different weather conditions in the year when research was done. ZEN was detected only in infected malt samples, which can be explained by the fact that contamination of grain is largely restricted to the outer layers of wheat grain, and therefore is partitioned after malting and by the large dependents on humidity and temperatures. This could result in higher concentrations of ZEN in malt products. In the research of Galaverna et al. (2009), a high increase of ZEN content was observed in the malted wheat in comparison with the raw material . Similars (2011) , more ths (2011)  detectedFusarium attack and its relationship with other mycotoxins before and after malting. D3G was present in all winter wheat varieties after malting of inoculated samples. Berthiller et al. (2005) [It is important to track the accumulation of masked mycotoxin, because, for example, D3G can represent a possible hazard to human and animal health. . The det. (2005)  found th. (2005) . In the . (2005) , 50% DON. (2005) . Lemmens. (2005)  got simi. (2005) , the pre. (2005) . Also, i. (2005) . In the . (2005)  the majoIn conclusion, our results suggest that (1) resistance to Fusarium decreases the median of quantified concentrations of DON; (2) ZEN gets synthetized during malting; (3) NIV will increase during malting; (4) more resistant varieties will convert DON to D3G more successfully. Fusarium graminearum  and F. culmorum  were sub-cultured on synthetic nutrient-poor agar (SNA) medium. After growing 10 days on SNA, the agar was cut into plugs and these were used for multiplication of spores in a liquid mung bean medium by the bubble breeding method [Fusarium species (1:1) were counted on a hemocytometer and suspensions were diluted or concentrated to reach the concentration of 1 × 105 mL−1. The spore suspensions were set to a concentration so that a single bottle of one strain contained a sufficient amount of suspension (>900 mL), which could be diluted in 100 L of water right before inoculation (100 mL per m2).Spore cultures of g method , or in ag method . The con2.The study was conducted at the Agricultural Institute Osijek in the east part of Croatia  in 2015/2016 and 2016/2017. In this site, soil is eutric cambisol with a pre-crop of soybean in both seasons. The standard fertilization (NPK) was applied. Meteorological data for vegetation seasons in Osijek were obtained from the Croatian Meteorological and Hydrological Service. The obtained climate data include average monthly precipitation and temperatures in 2015/5016 and 2016/2017. The sum of precipitation in the vegetation season was 595 and 482 mm, respectively, with average temperatures of 9.7 and 10.0 °C, respectively. The two treatments (control and Fusarium inoculated) were arranged in a randomized block design with two replications for each treatment. The area of each plot was 7.56 mThe inoculation was carried out at the time of flowering for twenty-five winter wheat varieties  and repeated two days later  Table 2Table 2. The wheat grains were dried to content below 14% and stored in the dark at 20 °C for six weeks to overcome seed dormancy. Afterwards, exactly 200 g of each variety was malted in an automated Joe White malting systems micro-malting unit  at the Agricultural Institute Osijek, Croatia. Samples were placed randomly within the micro-malting unit. The malting program consisted of a 37 h interrupted steep program , a 96 h germination program , and a 18 h kilning program . The moisture decreased from 14% moisture wheat to 4–6% moisture malt. Rootlets and seedlings were removed, and the finished malt was then stored in plastic containers with caps at –20 °C until mycotoxin analysis. Mycotoxin analysis on all samples was performed by LC-MS/MS according to Ren et al. (2007) . DON and−1 (the exact concentration may vary 2% depending on the exact concentration in the purchased mix), except for D3G, where the concentration range was: 5, 10, 20, 25, 50, 75, and 100 µL min−1. Validation parameters are summed up in The used method vas validated and fit for purpose. Some validation results were included in a previous publication . ConcentTo estimate disease progress, the AUDPC was used to combine multiple observations from five data points (different dates) into a single value . For cor