Previously, the nutritional contribution, environmental and financial costs of dairy products have been examined independently. Our aim was to determine the nutritional adequacy, financial cost and environmental impact of UK diets according to dairy content.n = 1655), dietary intakes assessed from 4-day estimated food diaries were organized into quartiles (Q) total grams of dairy  and analyzed using ANCOVA controlling for age, sex and energy intake with Bonferroni post hoc test for nutritional adequacy, Alternative Healthy Eating Index (AHEI-2010), environmental impact , financial cost, markers of health and cardio-metabolic diseases.In this cross-sectional study of adults (19–64 years) from the UK National Diet and Nutrition Survey years 1–4 (P < 0.0001) and AHEI-2010 (P < 0.0001) were significantly higher and systolic BP  was significantly lower for the higher-dairy diets , compared with diets containing lower dairy . Diets in Q4 had lower financial cost  and the greatest eutrophication potential, compared with Q1 . Yet the environmental (GHGE) and financial costs per unit nutrient  were lower in Q4 than Q1 .Nutritional adequacy, particularly for protein, calcium and iodine  contains supplementary material, which is available to authorized users. Global population growth, which is estimated to rise to > 9 billion by 2050, is placing greater demand on the planet’s finite natural resources. Estimates suggest that world food demand will increase at an average rate of 1.1% annually between now and 2050 [The manufacture of food impacts on the environment through, for example, the production of greenhouse gas emissions (GHGE) such as carbon dioxide, methane and nitrous oxide, and the use of land as well as water resources. The environmental impact arises at all stages in the life cycle from the processes of agricultural production, processing, transport, storage, cooking, through to disposal of waste . Estimat12, respectively [In the UK, consumption of milk and dairy products has changed substantially over the previous decades. Since the 1970s, milk consumption by adults has fallen from around 2.5 L per person per week to 1.5 L per person per week . Moreoveectively .Milk and dairy products also contribute around 27% of saturated fat intake in the UK diet . HoweverTherefore, when assessing the role of dairy products in sustainable diets, it is important to consider not only the environmental impact, but also the nutritional contribution dairy products make to the diet, together with other health beneficial functionality.The aim of this study was to determine the associations between UK diets containing varying levels of dairy products with nutritional adequacy, dietary cost and GHGE, acidification and eutrophication potentials.https://www.data-archive.ac.uk) [n = 710 and females: n = 945), unless otherwise stated. The mean quantity of individual foods consumed per day was aggregated into food sub-groups  according to the NDNS classification. Non-consumers of each food group were included in the analysis. The dairy food group included milk, cheese, yogurt, fromage frais, ice cream, other dairy, cream and dairy desserts, but not butter as this is classified as part of the fats and oils food group in NDNS. Milk alternatives such as almond, rice and soya milks were removed from the food groups and analyzed as a separate food group. The nutritional contribution of each food group to average estimated requirement (EAR) for energy, dietary reference values (DRV) for macronutrients and reference nutrient intakes (RNI) for vitamins and minerals was also calculated. All micronutrients reported in the NDNS were used in the analysis. The NDNS was conducted according to the guidelines laid down in the Declaration of Helsinki, and ethical approval for all procedures was granted by Local Research Ethics Committees covering all areas in the survey. All participants  gave informed consent.Data files from years 1 to 4 (2008/2009–2011/2012) of the NDNS were obtained from the UK Data Archive (e.ac.uk) . OverallwEI) and repeated BMR measurements (CVwBMR) was 23% and 8.2%, respectively [tP) was 15% [Energy misreporting was assessed using Goldberg’s cut-off 2 criterion , 17, whi was 15% , 17. SubEstimates of GHGEs, eutrophication potential and acidification potential were used to calculate the environmental impact of diets containing varying levels of dairy products. Briefly, data for GHGEs, eutrophication potential and acidification potential associated with each of the 136 sub-food groups from the NDNS were collected during the period of June–September 2014, from relevant literature from the UK and other European countries and were cross-referenced with at least one other source to ensure representativeness. Data were collected from primary productionto retail using life-cycle assessment (LCA). Within each NDNS sub-food group, environmental data on the most commonly consumed foods were averaged to produce a single value for each sub-food group. For some composite dishes or processed foods, where a single value was not available, the environmental impact was estimated using component ingredients. This was particularly the case for eutrophication and acidification potentials where data were only available for main food items –26. A den = 3420) reported in years 1–4 of the NDNS. Briefly, the retail price of food items was collected online from Asda and Waitrose supermarkets during the period of June–September 2014, and was updated in July 2015. The collection of food item costs was standardized, and whenever possible, the minimum weight of a food or product as sold was used. Furthermore, any offers or multi-buys were avoided to ensure that the true cost of a food item was captured. The majority of costs was based on own brand products/house brands for Asda and Waitrose supermarkets. The retail price of each of the food items was calculated as an average cost between Asda and Waitrose, and was aggregated into food groups as described above. In the case of food groups with a large number of individual foods, we took the average cost of the most consumed foods by weight within that food group. A table showing a summary of the food prices used for each sub-food group can be found in Supplemental Table 2.The monetary cost of the diets was estimated by collecting UK retail prices of all food items  of diets per unit of nutrient were calculated using the following equations:A cut-off was also applied when 100% of the RNI was met for a particular nutrient. For example, if the unit  consumed per day of a nutrient exceeded the RNI for that particular nutrient, then the RNI was used in the calculation instead of the unit nutrient per day.Anthropometric , blood pressure and blood biomarker data , low-density lipoprotein cholesterol (LDL-C), triacylglycerol (TAG), C-reactive protein, glycated hemoglobin and glucose) were obtained for participants in the NDNS. Details of how blood samples were taken, stored and analyzed have been described in detail elsewhere .A ‘baseline’ diet was created which was based on the average UK adult (19–64 years) male and female diets from the NDNS. The lower and higher dairy diets were created by splitting the male and female ‘baseline’ diet into quartiles of total grams of dairy, with the lowest quartile representing the lower dairy diet (Q1) and the highest quartile representing the higher dairy diet (Q4). The Alternative Healthy Eating Index (AHEI) score was calculated for each of the diets using the methods previously described  with minV) test or partial Eta2 (η2) for non-categorical variables. We regarded the P value of  0.05 as statistically significant. All statistical analyses were performed in SPSS version 21 (SPSS Inc.). In this population, it was estimated that 13% (n = 194) of subjects were classed as under-reporters of energy intake. However, underreporting was not considered further in statistical analysis. LRNI was used in this study as it represents a diet that will be insufficient for 97.5% of the population and is also used in NDNS as a marker of diet quality.The primary outcome of this study was to determine the associations between UK diets containing varying levels of dairy products with nutritional adequacy, dietary cost and three elements of environmental impact . The secondary outcome was to calculate GHGE and financial cost per unit nutrient to estimate the quality costs of each diet. Analysis of covariance (ANCOVA) was used to detect statistically significant differences between diets based on dairy quartiles and nutrient intakes, environmental impact, dietary cost, food groups and GHGEs and financial cost per nutrient controlling for age, sex and total energy intake (kJ). We controlled for total energy intake in our analysis of the environmental impact because GHGEs have been shown to be positively associated with total energy intake . BonferrP = 0.005, partial η2 = 0.030), qualifications , socio-economic classification  ethnic group  and smoking status .The sociodemographic and health characteristics of adults by dairy quartile are presented in Table P < 0.0001), bread (P = 0.001), vegetables and potatoes (P = 0.014), biscuits (P = 0.02), eggs and dishes (P = 0.039) and preserves and sweet spreads (P = 0.004) across quartiles of dairy intake. There were no significant differences in red meat (P = 0.18) or processed meat (P = 0.20) across increasing quartiles of dairy intake. Bonferroni post hoc analysis comparing the highest (Q4) with the lowest (Q1) dairy quartiles showed that subjects in Q4 had significantly higher intakes of high-fiber breakfast cereals, other breakfast cereals, milk, other milk and cream, cheese, ice cream, yogurt and fromage frais, fruit, tea, coffee and water , bread (P = 0.001), vegetables and potatoes (P = 0.007) and preserves and sweet spreads (P = 0.002) and significantly lower intakes of milk alternatives (P < 0.0001), eggs and egg dishes (P = 0.031), chips (P = 0.001), soft drinks  (P < 0.0001) and alcoholic beverages (P < 0.0001) compared to subjects in Q1.Diets for the total population and for different quartiles of dairy intake are shown in Table P < 0.0001), with the diets of adults in Q2 (P = 0.022), Q3 (P < 0.0001) and Q4 (P < 0.0001) having significantly higher AHEI scores compared with the diets of adults in Q1  and iron (P = 0.014) across increasing quartiles of dairy intake. For total energy intake, carbohydrate, protein, saturated fat, cis-MUFA, PUFA, calcium, magnesium, potassium, iodine, zinc, thiamine, riboflavin, vitamin B12, folate and iron, there was a significantly higher intake by adults in Q4 compared with Q1 . In addition, for the nutrients that were significantly different across dairy quartiles , the percentage of subjects below the LRNI was less in Q4 compared with Q1  with the diets containing the highest amount of dairy (Q4) having significantly higher eutrophication potential (29%)  compared with the diets containing the lowest amount of dairy , there was a significant difference in eutrophication potential across increasing quartile of dairy intake  with the diets containing the highest amount of dairy (Q4) having significantly higher GHGE and acidification potential (both P < 0.0001) compared with the diets containing the lowest amount of dairy (Q1). However, the significance was lost when the analysis was adjusted for energy intake, age and gender ; however, there was no significant difference in dietary cost between Q4 and Q1 , with diets in Q4 being on average 19% (mean SEM: £1.1/day 0.09) cheaper than Q1.There was a significant difference between dietary costs (£/day) across increasing quartile of dairy intake in the non-adjusted model P = 0.05; howeveP = 0.039) in the non-adjusted model (Table P = 0.019) and DBP (P = 0.037) across quartiles of total dairy intake, with individuals in Q4 having significantly lower SBP (P = 0.028) compared with individuals in Q1. There were no other significant differences between quartiles of total dairy intake and biomarkers of health.There was a significant difference across increasing quartiles of total dairy intake for height (el Table . When adP < 0.0001), calcium (P < 0.0001), magnesium (P = 0.013), potassium (P < 0.0001), zinc (P = 0.018) and iodine (P < 0.0001) (Table P = 0.0001), calcium , magnesium , potassium  and iodine  and significantly higher for zinc  in diets in Q4 compared to diets in Q1.The GHG emissions per unit nutrient with a cut-off of 100% RNI for each nutrient were significantly different across quartiles of dairy intake for riboflavin (1) Table . Bonferr12, folate, iron, calcium, magnesium, potassium, zinc and iodine  less in Q4 for energy intake (9%), protein (1%), SFA (20%), riboflavin (17%), folate (4%), iron (4%), calcium (19%), magnesium (14%), potassium (19%), zinc (13%) and iodine (25%) and significantly higher for thiamine (2%) and vitamin B12 (3%) compared with Q1.The financial cost per unit nutrient with a cut-off of 100% RNI for each nutrient was significantly different across dairy quartiles for energy intake, protein, SFA, thiamine, riboflavin, vitamin BThe association between the average UK diet and diets containing varying quantities of dairy intake with GHGEs was further explored in general linear models that examined the contribution of 15 individual food groups to GHGEs, adjusted for age, sex and energy intake . The food groups contributing most to GHGEs in the total population were meat and meat products, vegetables and potatoes, dairy products, cereals and cereal products and alcohol contributing 24, 16, 15, 14 and 11%, respectively, of total GHGEs.P-trend < 0.0001; fruit = 0.002) contributing to GHGEs in Q4 compared with Q1 . In addition, three food groups  were associated with significantly lower  contribution to GHGEs in Q4 compared with Q1 . Among the 15 food groups, the food group contributing most to GHGEs was dairy products, in which diets containing the highest amount of dairy (Q4) had 0.88 kg CO2 eqv/day (376%) greater GHGEs than diets containing the least amount of dairy (Q1).Food groups were differentially associated with GHGEs across dairy quartiles, with two food groups  significantly (dairy products P < 0.0001) across quartiles of total dairy intake is shown in Supplemental Tables 5–17. Food groups were differentially associated with nutrient intakes across increasing quartiles of dairy intake, with cereal and cereal products significantly contributing to intakes of energy (P-trend = 0.001), carbohydrate (P-trend = 0.0001), zinc (P-trend = 0.001), magnesium (P-trend = 0.0001), thiamine (P-trend = 0.0001) and folate (P-trend = 0.0001) in the higher diary diets (Q4) compared with the lower dairy diets (Q1). Milk and milk products contributed significantly more to intakes of SFA, PUFA, protein, calcium, potassium, iodine, riboflavin and vitamin B12 intakes in Q4 compared with Q1 .The relationship between nutrients and food groups that were significantly different  have minimal effects on blood lipid concentrations . Howevern = 994) in the health analysis, which means that bias may have incurred. This study is representative of actual dietary intakes in the UK, but may not be representative of the diets of other countries. Despite these limitations, we believe that this study is an important step forward in investigating the environmental impact of typical UK diets using multiple measures of diet-related environmental impact.This study faced a number of limitations, including relying on approximate environmental data collected from a number of different sources. There were potential methodological differences, the limited availability of environmental data for every NDNS food group  and data were not available for environmental impacts associated with the consumption phase, such as food preparation and waste. Similar considerations apply to the collection of financial data, which were obtained using retail food prices, and therefore only reflect costs at one point in time and only for the foods reported in the NDNS. In addition, the lack of measures of uncertainty in the prices and environmental impacts is another important limitation of this analysis. The cross-sectional design of the NDNS, with no prospective follow-up, is also a limitation. There were quite a few missing samples Below is the link to the electronic supplementary material.


This study provides new evidence on the physiological changes of catecholamines in follicular fluid during the follicular growth in the mare. Both dopamine and epinephrine increase in the follicular fluid with the advance of follicular development, although norepinephrine decreases. These changes could be related to the existence of systemic, autocrine and/or paracrine mechanisms of synthesis, metabolism and interconversion of catecholamines for the regulation of follicular growth and development.n = 20), medium  and large . Follicular fluid (FF) samples were extracted from each follicle. Intrafollicular DA, NA and AD concentrations were significantly higher than the systemic concentrations (p < 0.05). Intrafollicular DA concentrations were higher in medium than small and large follicles (p < 0.05). Intrafollicular NA concentrations were higher in small than medium and large follicles (p < 0.05). Intrafollicular AD concentrations were higher in large than small and medium follicles (p < 0.05). Follicle diameter was significantly and negatively correlated with NA and AD (p < 0.05). A significant correlation of the same hormone concentration in FF and in systemic fluid was observed (p < 0.05). In summary, the FF can serve as an intraovarian catecholamine-storing compartment, with the ability to release neurotransmitters in a regulated way. These results provide novel insights into the neuronal nature of the follicle, suggesting the involvement of catecholamines in normal ovarian functions in mares.In some species, catecholamines in follicular fluid (FF) are related to local physiological events responsible for the regulation of ovarian functions and oocyte maturation. The aim of the present study was to determine and compare intrafollicular and systemic concentrations of dopamine (DA), noradrenaline (NA) and adrenaline (AD) in cycling mares. Sixty ovaries were collected during breeding season from 30 mares raised for slaughterhouse meat production, with clinically normal reproductive tracts, were evaluated. Blood samples were collected prior to slaughter. Follicles were classified into three categories in relation to size: small (20–30 mm;  In mares, the involvement of dopamine (DA), noradrenaline (NA) and adrenaline (AD) on reproductive physiology is documented, since the catecholamines regulate gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL) secretion ,2,3. FurIn pig’s follicular fluid (FF), NA concentrations increase significantly during the follicular phase, with physiological implications in preovulatory events, as during luteinization . FernándAlthough DA was detected in the FF of mare follicles of all sizes, NA has not been identified in medium or large follicles . It was From the clinical point of view, the study of FF has an immense value for better understanding the regulatory mechanisms of fertility in female reproduction. Considering that FF is a compartment with the ability to store and release catecholamines and that it could serve as a reserve to maintain a higher availability of these molecules, the hypothesis of this study was to verify the existence of catecholamines in FF for their possible contribution by systemic sources in mares. On this basis, the objective of the present study was to determine DA, NA and AD concentrations in FF and blood samples, by taking into account the correlations between molecules and the possible contribution of systemic catecholamine concentrations to FF contents.All procedures involved in this study were approached by the CEU-Cardenal Herrera University Committee of Ethics in Animal Research and were carried out by considering the RD 37/2014 that regulates the protection of animals at the time of slaughter and the EU directive 2010/63/EU.The study was conducted in the northern hemisphere during months of the breeding season (April and May 2018). During this period, the ambient temperature ranged from 27–31 °C, with a relative humidity of 40–60%. A total of 30 clinically healthy mares  aged 6.6 ± 1.3 years were studied. According to system described by Henneke et al. , the ani®; Belgium) and were transferred into a polypropylene tube containing EDTA (1 mg/mL of blood). Samples were centrifuged at 1200× g for 10 min, and plasma was collected and stored at 4 °C in portable coolers for later transport to the laboratory. After the slaughter, only the ovaries of the normal reproductive tracts were collected. The period between the slaughter and collection of ovaries in no case exceeded 2 h, as proposed by Hinrichs et al. [From each animal blood samples (20 mL) were collected from the jugular vein 1 h before slaughtering (gunshot), while mares were in lairage, a procedure that took just a few seconds for each horse. Blood samples were collected using evacuated tubes , medium  or large . FF was aspirated using a different sterile syringe and 22 G needle for each follicle. Following collection, the FF samples were centrifuged for 10 min at 1200× g to eliminate the cumulus oocyte complexes. Only the supernatant (pure FF) was removed and stored in 0.5 mL aliquots at −20 °C until subsequent analysis.Ovaries were washed three times with sterile saline and the follicles were direct measured with a digital Vernier caliper and categorized according to diameter as small , NA (ng/mL) and AD (ng/mL) concentrations were determined by competition EIA-Technical 3-CAt EIA  specifically validated for the equine species . The perp < 0.05.Descriptive statistics mean ± standard deviation (SD) for DA, NA and AD concentrations in FF of small, medium and large follicles and in blood plasma were calculated. Normality was verified in all the data groups, using the Kolmogorov–Smirnov test. To determine the magnitude of variation in the concentrations of these constituents of FF and plasma in follicles of different diameters, data were subjected to one-way ANOVA analysis. Post-hoc comparisons were performed using Tukey’s test. The relationship between FF and systemic DA, NA and AD concentrations was examined by linear regression analysis, and the correlation was expressed by Pearson’s correlation coefficient. Differences were considered statistically significant when p < 0.05), and are presented as mean ± SD.In all the samples, intrafollicular concentrations of DA, NA and AD were significantly higher than the systemic ones (p < 0.05). Medium follicles showed higher DA concentrations than small and large follicles  concentrations were higher in small (306.2 ± 113.8 ng/mL), medium (707.9 ± 360.6 ng/mL) and large (351.2 ± 132.5 ng/mL) follicles than systemic fluid . Large follicles showed higher NA concentrations than small and medium follicles  concentrations were higher in small (472.2 ± 227.7 ng/mL), medium (244.7 ± 93.9 ng/mL) and large (210.1 ± 81.6 ng/mL) follicles than systemic fluid . Large follicles showed higher AD concentrations than small and medium follicles  concentrations were higher in small (48.9–83.2 ng/mL), medium (53.1–71.8 ng/mL) and large (47.7–93.3 ng/mL) follicles than systemic fluid  and was significantly and negatively correlated with NA and AD (p < 0.05).Follicular diameter increased significantly in medium and large respect to small follicle sizes (p < 0.05; A significant correlation of the same hormone concentration in FF and in systemic fluid was observed (The intrafollicular concentration of catecholamines in this study completely differed from those previously reported in the same species , despiteSubstantial intrafollicular catecholaminergic activity in mares suggests an extra- or intra-ovarian production or synthesis. In women, the FF of large follicles accumulates NA in concentrations higher than systemic ones and on similar form to mare, significant correlations between both fluids were obtained . AlthougAnother point is that DA antagonist treatment of acyclic mares provides controversial results. Hence, some studies reported that treatment with sulpiride, domperidone or perphenazine stimulated ovarian recrudescence, advancing the first ovulation of the year in mares maintained outside ,5,7; othFurthermore, NA targets β-adrenergic receptors of granulosa cells and elevates cAMP, allowing them to serve as catecholamine-storing cells within the ovarian follicle and ensuring that DA and/or NA are present in the granulosa cell compartment . In addiMetanephrine, a metabolite of DA and NA in FF and granulosa cells, also provides clear evidence of the follicular metabolism of catecholamines . In addiIn summary, the presence of DA, NA and AD in the FF of the mare indicates the existence of interspecific differences regarding the content of catecholamines with respect to other species. The great amount of DA, NA and AD in FF could indicate the existence of systemic and autocrine and/or paracrine mechanisms related to catecholamine synthesis, metabolism and subsequent use, guaranteeing the successful growth and development of ovarian follicles. This physiological response could have pivotal applications in the reproductive clinic for the diagnosis of pathological processes that occur with infertility in the mare. However, future investigations would be required to clarify these complex mechanisms of interconversion and sources of catecholamines in the FF of mares.


Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease, affecting approximately 3 in 10 obese children worldwide.PNPLA3 rs738409, a strong genetic contributor to NAFLD.We aimed to investigate the potential relationship between gut microbiota and NAFLD in obese youth, while considering the role of PNPLA3 rs738409 genotyping. Fecal samples were collected to analyze the V4 region of the 16S rRNA gene for intestinal bacteria characterization.In this cross-sectional study, participants completed an abdominal magnetic resonance imaging to measure hepatic fat fraction (HFF), oral glucose tolerance test, and Yale Pediatric Obesity Clinic.Obese youth (body mass index >95th percentile) with NAFLD  and without NAFLD .PNPLA3 genotype.Shannon-Wiener diversity index values and proportional bacterial abundance by NAFLD status and P = 0.013). Subjects with NAFLD showed a higher Firmicutes to Bacteroidetes (F/B) ratio (P = 0.019) and lower abundance of Bacteroidetes (P = 0.010), Prevotella (P = 0.019), Gemmiger (P = 0.003), and Oscillospira (P = 0.036). F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were associated with HFF when controlling for group variations. We also observed an additive effect on HFF by PNPLA3 rs738409 and Gemmiger, and PNPLA3 rs738409 and Oscillospira.Subjects with NAFLD had decreased bacterial alpha-diversity compared with those without NAFLD (PNPLA3 rs738409.Obese youth with NAFLD have a different gut microbiota composition than those without NAFLD. These differences were still statistically significant when controlling for factors associated with NAFLD, including  Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in the pediatric population, affecting approximately 3 in 10 obese children worldwide , 3. NotaPNPLA3), a gene encoding for a protein called adiponutrin, which has been found to confer susceptibility to increased hepatic fat levels and hepatic inflammation  were recruited from the Yale Pediatric Obesity Clinic. All participants completed an oral glucose tolerance test (OGTT) to assess glucose metabolism and a fast-magnetic resonance imaging (MRI) abdominal scan to assess body fat partitioning and intrahepatic fat content. In addition, fasting blood samples were collected at the OGTT visit to obtain liver function tests, a lipid panel, and a genetic test. Metabolic studies were conducted at the Yale Center for Clinical Investigation at 8:00 AM after a 12-hour overnight fast. A stool sample from each subject was also collected. Exclusion criteria included known hepatic diseases (except for NAFLD), alcohol consumption, smoking, and medication use. Written parental informed consent and written child assent were obtained from all participants. Yale University Human Investigation Committee approved the study.Measurement of liver fat content was performed by MRI using the 2-point Dixon method, and hepatic fat fraction (HFF) was calculated from the mean pixel signal intensity data. MRI studies were performed on a GE or Siemens Sonata 1.5 Tesla system.Genomic DNA was extracted from peripheral blood leukocytes from 8 cc of whole blood using the guanidine HCl DNA extraction protocol. To genotype the rs738409 single-nucleotide polymorphism, the following pair of primers was used: forward = 5′-GCCCTGCTCACTTGGAGAAA-3′ and reverse = 5′-TGAAAGGCAGTGAGGCATGG-3′. Polymerase chain reaction (PCR) was performed using an annealing temperature of 65.0°C and PCR products were analyzed by automatic sequencing at the Yale W.M. Keck Facility.Samples were processed in 2 separate batches; the larger subset (60 samples) in 2016 and the smaller subset (13 samples) in 2019. DNA was extracted using the MoBio PowerMag Soil 96-well kit  for the larger subset of samples. The smaller subset samples were extracted using the DNeasy PowerSoil Kit , as the MoBio kit was no longer available. Based on available information, the 2 kits only differ in the method of purification. The lysis step in both methods is identical, including the size of beads for mechanical lysis and the composition of the lysis buffer.Using either the MoBio PowerMag Soil 96 well kit or the Qiagen DNeasy PowerSoil kit, DNA was extracted from 0.25 g of fecal sample. DNA extracts were quantified using the Quant-iT PicoGreen kit  for the larger subset of samples and the Qubit High Sensitivity kit  for the smaller subset. The V4 region of the 16S rRNA gene was amplified using primers described in Caporaso et al  with duaThe 16S rRNA V4 gene sequence data was demultiplexed through BaseSpace . Paired-end data was then imported into QIIME 2 2019.4  and readData was imported into RStudio 3.5.2 using the qiime2R plugin  and analt tests, Mann-Whitney U tests, and chi-square tests were utilized to evaluate differences in the characteristics of the 2 groups. The following characteristics were compared using Student’s t tests (parametric) between the 2 HFF groups: age, height, weight, BMI, BMI z-score, 2-hour glucose, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), HFF, and visceral fat. Mann-Whitney U tests (nonparametric) were utilized for the following characteristics: fasting glucose, fasting insulin, whole-body insulin sensitivity index (WBISI), triglycerides, alanine aminotransferase (ALT), and subcutaneous fat. Chi-square tests were utilized to evaluate differences between the 2 groups in sex, race, and PNPLA3 genotype. When appropriate, data are presented as mean (standard deviation).The distribution of continuous variables was evaluated for skewness and kurtosis, and by using a histogram with the normal curve imposed. When appropriate, nonparametric tests were utilized. When needed, data were transformed into ranks to reduce the influence of outliers. Subjects were divided into 2 groups, HFF <5.5% (n = 29) and HFF ≥5.5% (n = 44). Student’s t test, to account for unequal variances between the 2 groups. The abundance of each bacterial phyla and genera was compared between the 2 HFF groups using Student’s t test or Mann-Whitney U test. Phyla where a phylum did not reach 1% abundance in at least one sample were excluded from the analysis. Therefore, 8 out of 13 phyla were included in the analysis. Genera in which a genus did not reach 1% abundance in at least one sample were not included in the analysis. Thus, 51 out of 125 phyla were included in the analysis. The abundance of each phylum and genus was compared between the 2 groups using Mann-Whitney U test for all phyla and genera, except for Firmicutes and Faecalibacterium, in which Student’s t tests were used.Shannon-Wiener diversity index values, an alpha diversity measure that accounts for the number of species and evenness of their abundance, were calculated for all stool samples using the R package phyloseq. The values were compared between the 2 HFF groups using Welch’s PNPLA3 genotype, race, and BMI z-score, multiple linear regression was conducted for each phylum and genus that was significantly different in abundance between the 2 HFF groups to determine if the respective phylum/genus was found to be predictive of HFF. For a phylum or genus that was not normally distributed, abundance values were ranked and the ranked abundance values were used in the regression model. Additionally, each multiple linear regression model was checked for normality using a Predicted Probability plot and for absence of multicollinearity using variance inflation factor values.Simple linear regression, with HFF as the outcome, was conducted for each phylum and genus that was significantly different in abundance between the 2 HFF groups. While controlling for P = 0.616), sex (P = 0.316), height (P = 0.166), and BMI (P = 0.061). The groups significantly differed in race (P = 0.008), with a greater proportion of African American participants in the group without NAFLD. Despite both groups having BMI greater than 95th percentile, the group with NAFLD was found to have greater weight (P = 0.047) and BMI z-score (P = 0.008), compared with the group without NAFLD. In regards to glucose metabolism, the groups had comparable fasting glucose (P = 0.450) and 2-hour glucose (P = 0.442) values, but the group with NAFLD had increased fasting insulin (P < 0.001) and decreased WBISI (P < 0.001). The lipid profile between the 2 groups was similar in total cholesterol (P = 0.468) and LDL (P = 0.948); however, the group with NAFLD had lower HDL (P = 0.014) and higher triglycerides (P < 0.001). Participants with NAFLD displayed significantly higher HFF (P < 0.001) and ALT (P < 0.001). Comparing body fat distribution between the 2 groups, they did not differ in subcutaneous fat composition (P = 0.253), but visceral fat composition was higher in the group with NAFLD (P < 0.001). The groups differed in the PNPLA3 genotype (P < 0.001), with the GG genotype  present only in the group with NAFLD.We compared characteristics between the groups with and without NAFLD . There wThe distribution of phyla and genera per NAFLD status for each participant is shown in Supplementary Figure 1 , 43. In P = 0.013) bacterial diversity compared with those without NAFLD (P = 0.019) in participants with NAFLD (P = 0.010) in the group with NAFLD. Among the 51 examined genera, Prevotella (P = 0.019), Gemmiger (P = 0.003), and Oscillospira (P = 0.036) were lower in proportional abundance in the group with NAFLD, compared with the group without NAFLD.Comparing the Shannon-Wiener diversity index values between the 2 HFF groups revealed that subjects with NAFLD have decreased  ratio as independent variable was significant and explained 15.3% of the variation in HFF . The mule in HFF . This moPNPLA3 genotype, race, and BMI z-score, found that as Bacteroidetes abundance increased, HFF decreased   . In thisOscillospira as independent variable was significant and explained 10.3% of the variation in HFF   . In thisPNPLA3 variant on HFF, we categorized the bacterial abundances in tertiles, from smallest to largest  and third tertile (highest abundance) of Gemmiger had a mean HFF value of 7.98% (14.5%), whereas participants with PNPLA3 genotype GG  and first tertile (lowest abundance) of Gemmiger had a mean HFF value of 22.1% (11.5%). Participants with PNPLA3 genotype CC and third tertile (highest abundance) of Oscillospira had a mean HFF value of 5.10% (10.0%), whereas participants with PNPLA3 genotype GG and first tertile (lowest abundance) of Oscillospira had a mean HFF value of 20.03% (11.3%).To illustrate the effect of the bacteria and the  largest . ParticiPrevotella, Gemmiger, and Oscillospira in the group with NAFLD, compared with the group without NAFLD. When controlling for PNPLA3 genotype, race, and BMI z-score, regression analyses demonstrated that F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were all individually associated with HFF. These regression models with PNPLA3 genotype, race, BMI z-score, and F/B ratio, Bacteroidetes, Gemmiger, or Oscillospira explained over 20% of the variation in HFF. We also discovered an additive effect on HFF from PNPLA3 and Gemmiger, and PNPLA3 and Oscillospira.Our data substantiate recent findings that NAFLD is associated with changes of the intestinal microbiota. Subjects with NAFLD were found to have decreased bacterial diversity and richness compared with those without NAFLD. Additionally, we observed a significantly higher Firmicutes to Bacteroidetes ratio and lower abundance of Bacteroidetes, P < 0.05). Since the gut microbiota protects the host from colonization and invasion of other pathogens, having a greater bacterial diversity may better protect against injurious colonization of other pathogens and prevent bacteria and toxins from gaining access to the portal circulation and releasing inflammatory cytokines in the liver. For over a decade, studies of lean and obese mice have proposed that gut microbiota dysbiosis may increase energy harvest from the diet and alter how this energy is utilized and stored (Our results on fecal microbiome alpha diversity agree with those of Del Chierico et al  and Schwd stored , 36. In d stored . Therefod stored , 19.Oscillospira in the group with NAFLD, compared with the group without NAFLD, which is consistent with the findings from the Michail et al (Oscillospira decrease coupled to up-regulation of 2-butanone was identified as signature of NAFLD onset, although this was based on comparisons with lean controls. While the abundance of Oscillospira was higher in obese children without NAFLD relative to those with NAFLD, the study does not disclose if this difference was significant. Our multiple linear regression model, which controlled for PNPLA3 genotype, race, and BMI z-score, showed that both Oscillospira and PNPLA3 genotype were predictive of HFF. Thus, there is an additive effect between Oscillospira and PNPLA3 rs738409 variant in conveying susceptibility to NAFLD, in which both factors independently affect HFF.We observed a significantly lower abundance of il et al  study. IUnlike the studies by Del Chierico et al  and MichPrevotella copri was associated with more severe fibrosis, while our study noticed lower abundance of Prevotella in the group with NAFLD, compared with the group without NAFLD. When we accounted for PNPLA3 genotype, Prevotella was no longer predictive of NAFLD status. The discrepancy between our findings and that of Schwimmer et al (While Schwimmer et al  also fouer et al  could poer et al . In conter et al  revealeder et al  study alPNPLA3 rs738409. Additional information, such as liver biopsies and shotgun metagenome data for more patients, would strengthen the current study and include limitations of our study; this will be pursued in the future. Another limitation of this study is its descriptive nature; thus, future directions include testing the causal relationship of intestinal bacteria on NAFLD by modifying bacterial abundance and composition with the appropriate probiotics as an adjunct to weight loss and/or dietary changes while measuring HFF at baseline and after intervention. While descriptive, two important strengths of our study are the thorough phenotype that allows consideration of metabolic and genetic factors and the young age of the studied population.This study provides important insights regarding the interaction of the microbiome and NAFLD. A diverse gut microbiome correlates with reduced prevalence of NAFLD, providing further support for microbial community richness improving host health. Our results show that the F/B ratio and certain bacterial genera have statistically significant associations with HFF and NAFLD status, even when controlling for factors associated with NAFLD, including PNPLA3 rs738409 genotype. Additionally, we identified specific bacterial candidates for testing causal relationships and that may be beneficial as probiotics in the treatment of NAFLD. Overall, our data support previous experimental observations while also providing the foundation for testing novel hypotheses in future studies.Our findings agree with the notion that gut microbiota composition and diversity could potentially be used to detect susceptibility to NAFLD, which has been mentioned in previous studies , 17, 20.


Dicraeus orientalis feeds on the seeds of Poaceae. The complete mitochondrial genome of D. orientalis was sequenced and annotated as the first representative of the family Chloropidae. The full length of mitogenome was 16,188 bp, consisting of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rRNAs). The nucleotide composition was highly A + T biased, accounting for 79.1% of the whole mitogenome. All PCGs start with ATN codons except COI, which end with TAN or incomplete stop codon. ML analysis revealed that Carnoidea was closely related to Ephydroidea and the phylogenetic relationship within Acalyptratae was Tephritoidea + ((Carnoidea + Ephydroidea) + Opomyzoidea). The Chloropidae, also known as the grass fly, is a large family in the superfamily Carnoidea  with about 3000 described species worldwide Nartshuk . They arDicraeus orientalis Becker used for this study were collected from Banzhai village , Maolan, Libo, Guizhou by Ding Yang on 13 October 2013 and identified by Xiaoyan Liu. Specimens are deposited in Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory in Huazhong Agricultural University, Wuhan, China (Accession number: HZAU0010025). The genomic DNA was extracted from the adult’s whole body (except head) using the DNeasy DNA Extraction kit (TIANGEN) and stored at −20 °C until needed. DNA samples were pooled for next-generation sequencing library construction following Gillett et al.  was 16,188 bp in length, which consisted of 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs, and control region. The structure of this mitochondrial genome was similar to other Dipteran flies reported previously  and NADH-ubiquinone oxidoreductase chain 1 gene (ND1) use TCG and ATA, respectively. The termination codon of these protein-coding genes had four types .The specimens of t et al. . All quat et al. . The resD. orientalis could provide valuable information for future studies of Carnoidea phylogeny.Based on the nucleotide sequences of 13 PCGs from 14 Diptera species, we conducted a phylogenetic analysis with maximum likelihood (ML) method using RAxML 7.0.3 (Stamatakis


KLK) expression in recurrent and non-recurrent prostate tumors and adjacent healthy prostate tissues.To assess kallikrein (KLK genes in 34 recurrent and 36 non-recurrent prostate cancer samples and 19 adjacent healthy prostate tissue samples was assessed with quantitative reverse-transcription polymerase chain reaction. The samples were obtained from Baylor College of Medicine, Houston, TX, USA between 2013 and 2016 .The expression levels of 15 KLK1, KLK4, KLK9, and KLK14. Recurrent samples were negative for KLK1, KLK2, and KLK14 but demonstrated higher levels of KLK3, KLK4, and KLK9 than controls. Other KLKs were not significantly expressed.Compared with controls, prostate cancer samples showed a strong decrease in KLK gene family in recurrent prostate cancer. KLKs could be used as recurrence markers for prostate cancer.This study for the first time showed a difference in the expression levels of the  Prostate cancer is the most common visceral malignancy and the second deadliest cancer in men. In most cases, the cancer grows gradually, is initially limited to the prostate gland, and does not cause significant morbidity. However, some prostate cancers are aggressive and rapidly spreading .Prostate specific antigen (PSA) or kallikrein (KLK) 3 is an enzyme released into the seminal fluid. It is secreted from the cells surrounding and forming the inner part of the prostate acini and luminal epithelial cells . ElevateKLK genes are localized on the 19q13 chromosome and encode 15 serine proteases, the largest protease family in the human genome. These proteases play a role in different physiological processes, such as semen liquefaction and skin shedding. Various amounts of KLK genes are expressed in a broad range of tissues, indicating a various degree of their functional involvement in physiological processes  have a reduced expression in the prostate, breast, and some other cancers. NES1 has been shown to be a potent angiogenesis inhibitor of a new breast cancer tumor suppressor protein and PSA  spectrophotometer device with absorbance values of 260 nm and 280 nm wavelengths.Complementary DNAs (cDNAs) were synthesized with Transcriptor High Fidelity Reverse Transcription kit  using the same number of samples from the isolated RNAs. Reverse transcriptase PCR protocol with Oligo (dt) primers for single chain cDNA synthesis was used as per the manufacturer’s protocol. qPCR analysis was performed with SYBR Green Master Mix . All primer sequences are shown in KLKs expression between the groups was assessed with the t test. Spearman’s rho coefficient was used to assess the correlation between KLK genes, serum PSA levels, and Gleason scores. For normally distributed variables, the significance of differences among groups was assessed with one way ANOVA with post-hoc Tamhane T2 for the groups in which the variances were not equal. If the variances were homogeneous, the Fisher LSD test was performed. For not normally distributed variables (KLK9), the Kruskal-Wallis H test was used. All significance values were adjusted by the Bonferroni correction. Recurrent and non-recurrent patients were compared according to race with the χ2 test. The level of statistical significance was set at P ≤ 0.05. Statistical analyses were performed with SPSS v. 16.0 , and graphs were created with the GraphPad Prism Trial Version .Normality testing was performed with the Kolmogorov-Smirnov test. Data are expressed as mean ± standard deviation. Significance of differences in KLK1 expression was significantly decreased in recurrent (P = 0.048), non-recurrent (P = 0.03), and both tumor tissues combined (R+NR) compared with healthy tissues (P = 0.007). KLK2 expression was significantly decreased in recurrent tumor tissues compared with non-recurrent tissues (P = 0.021), and in non-recurrent tissues compared with healthy tissues (P = 0.026). There was no significant difference between healthy tissues and R + NR tissues. The expression of both KLK3 and KLK4 was increased in recurrent tissues  and R + NR tissues compared with healthy tissues . KLK8 expression did not significantly differ between recurrent tissues and healthy tissues and between R + NR tissues and healthy tissues, and it was increased in non-recurrent tissues compared with recurrent tissues (P = 0.045). KLK9 was significantly increased in recurrent tissues (P < 0.001) and R + NR tissues compared with healthy tissues (P < 0.001). KLK14 was the only KLK significantly decreased in R + NR tissues compared with healthy tissues (P = 0.004). KLK14 was significantly decreased in recurrent tissues compared with healthy tissues (P < 0.001) and significantly increased in recurrent tissues compared with non-recurrent tissues (P = 0.001)  . We did P = 0.019, KLKs expression, except for KLK1 and KLK14, in the recurrent group. We only found a significant negative correlation between Gleason score and KLK14 in the non-recurrent group  plays a vital role in both normal biology and tumor growth and progression  were elevated in ovarian cancer tissue samples and cell lines compared with the healthy ovary  are underexpressed in prostate cancer tissues, while KLK3, KLK4, KLK8, and KLK9 are overexpressed in both recurrent and non-recurrent tissues. The limitations of this study include a relatively small size, retrospective study design, and a single-center experience.Our results point to the fact that KLK1, KLK2, KLK3 KLK4, KLK8, KLK9, and KLK14, which are differentially expressed in prostate cancer, could be used as promising biomarkers of prostate cancer progression. In our opinion, KLKs play a role in promoting or inhibiting tumor growth and metastasis by regulated gene expression. However, our findings need to be validated in studies with a much larger number of participants. In addition, more novel markers should be assessed.In conclusion, we propose that


Camellia brevistyla is an economic species for its seeds with high oil content and ornamental value, which is cultivated widespreadly across southern China. In this study, the complete chloroplast (cp) genome sequence of C. brevistyla was assembled and annotated in order to future genetic research. The whole cp genome of C. brevistyla is 159,281 bp in size, composed of a small single copy (SSC) region of 15,662 bp and a large single copy (LSC) region of 86,251 bp separated by a pair of inverted repeats . The overall GC content of C. brevistyla cp genome is 37.19%, with the base content A (31.03%), T (31.78%), C (18.94%), and G (18.25%). Phylogenetic analysis of 20 species based on 74 protein-coding genes shows that C. brevistyla is evolutionarily closest to Camellia danzaiensis. Camellia brevistyla, one of more than 250 species of the genus Camellia, grows mainly in thickets or pine forests on hillsides at an elevation of about 600 m and is found commonly in southeast China. Camellia brevistyla is listed as an economic tree species for oil production and horticulture  and discuss the phylogenetic relationship among its populations, which will provide important reference for its future biological research.C. brevistyla were collected from Jinhua International Camellia Species Park . Voucher specimen (CD_01) was deposited in State Key Laboratory of Tree Genetics and Breeding, Research Institute of Subtropical Forestry, Chinese Academy of Forestry. C. brevistyla was sequenced using Illumina Hiseq 2500 sequencing systems  at Genesky Biotechnologies . The 24,696,066 raw reads were under quality control by Trimmomatic . The longest genes of protein-coding genes, tRNAs and rRNAs is ycf2_2 with 6867 bp, rrn23 with 2809 bp and tRNA-Ile3 with 239 bp, respectively.The complete cp genome sequence of Camellia species. The evolutionary distances were computed using the JTT matrix-based method  and also clustered closely to Camellia pitardii (NC_022462.1).A Minimum-Evolution tree based on 74 protein-coding genes that were screened by the Perl script (Wang et al.


Background. Acute kidney injury (AKI) requiring dialysis during pregnancy is uncommon. We present a case of a young female diagnosed with antiglomerular basement membrane (anti-GBM) disease during pregnancy. Case Presentation. A 23-year-old woman approximately 15 weeks pregnant experienced weakness, nausea, vomiting, and anorexia for one week and anuria for 48 hours. No known drug allergies and no significant social or family history for kidney or genitourinary disease were reported. Laboratory analysis revealed anemia, life-threatening hyperkalemia, AKI, and elevated antiglomerular basement membrane (GBM) antibodies. Renal biopsy revealed 100% cellular crescents, confirming the diagnosis. The patient was treated using plasmapheresis and methylprednisolone followed by oral steroids, azathioprine, and tacrolimus. At 24 weeks and 4 days of gestation, the patient had hypoxic respiratory failure as well as preterm premature rupture of membranes. Due to the development of infection and lack of renal recovery, immunosuppression was discontinued. At 28 weeks and 0 days of gestation, the patient developed uncontrollable hypertension requiring emergent delivery. Postpartum, her hypertension improved without signs of preeclampsia though still requires dialysis. Discussion. Pregnancy presents a unique challenge for providers treating patients with anti-GBM disease. Fetal safety should be considered and risks thoroughly discussed with the patient when choosing an immunosuppressive regimen for this condition. Antiglomerular basement membrane (anti-GBM) disease is a rare, life-threatening, small vessel vasculitis usually presenting as rapidly progressive glomerulonephritis characterized by glomerular cellular crescents and linear deposits of IgG along the GBM . Anti-GBHere, we present a young woman diagnosed with anti-GBM disease early in her pregnancy and later giving birth prematurely with pre- and postpartum maternal, fetal, and neonatal course of events.2, vaginal delivery of a healthy female infant at 41 weeks of gestation 10 months prior to the current presentation, and a first-trimester miscarriage six months prior to presentation. Her medications included prenatal vitamins, folic acid, vitamin C, and ondansetron for nausea. She denied alcohol intake, tobacco use, or substance abuse. She had no known drug allergies. There was no previous history of renal or pulmonary disease. No family disease was known and she had no other relevant findings in her medical history.A 23-year-old previously healthy woman at 15 weeks and 3 days gestation of her third pregnancy presented to the emergency department with complaints of persistent watery diarrhea, nausea, and vomiting for one week, bilateral lower extremity numbness, tingling, and weakness for five days, and anuria for 48 hours. Her past medical history was significant for obesity with a body mass index of 37.2 kg/mThe patient was afebrile on presentation with a blood pressure of 114/53 mmHg, a heart rate of 68 beats/minute, a respiratory rate of 31 breaths/minute, and an oxygen saturation of 98% on room air. The remainder of her systemic examination findings, including those for her chest, were within normal limits.3 μL, mean corpuscular volume (MCV) of 72 fL, low serum albumin of 3.1 g/dL, hemoglobin level of 7.8 g/dL, and hematocrit of 24.1 % and elevated red cell distribution width (RDW). Electrolyte and metabolite analysis revealed low sodium level (129 mmol/L) and elevated potassium, creatinine, and blood urea nitrogen (BUN) levels of 7.1 mmol/L, 19.8 mg/dL, and 113 mg/dL, respectively, with a decreased estimated glomerular filtration rate (eGFR) of 2.2 mL/min compared to normal values from a metabolic profile checked six months prior and an elevated hemoglobin level of 12.3 g/dL one month prior to presentation. Transaminases were within normal limits. Urinalysis showed hematuria with >100 red blood cells per field and proteinuria with 100 mg/dL protein. Furthermore, the patient was found to have an elevated erythrocyte sedimentation rate (ESR) of 117 mm/hr and mildly elevated haptoglobin of 298 mg/dL. Hematological analysis and workup for evidence of disseminated intravascular coagulation revealed a normal (INR) of 1.0, activated partial thromboplastin time (APTT) of 36.0 seconds, and normal platelet response yet elevated levels of fibrinogen (798 mg/dL) and D-dimer . Iron panel detected a normal serum iron level of 46 μg/dL and low total iron-binding capacity (TIBC) of 225 μg/dL with a corresponding transferrin saturation percentage of 20%, reticulocyte count of 25.5 × 102 μL, and reticulocyte percentage of 0.78.Initial laboratory workup included a complete blood count and comprehensive metabolic panel which showed anemia and electrolyte imbalance. Blood cell analysis revealed a normal total protein level, white blood cell count of 10.1 × 10Further laboratory workup for autoimmune conditions, infectious disease, or history of drug use revealed elevated levels of serum anti-GBM antibodies at 7.6 units (U)/mL and no evidence for underlying microbial infection or drug-induced toxicity. Serum samples were negative for cryoglobulins, antiphospholipid, antinuclear, and cytoplasmic antibodies as well as extractable nuclear antigen and antibodies to red blood cells or ADAMS TS 13. Complement levels (C3 and C4) were within normal limits.A renal ultrasound was performed and measured the right kidney at 14.6 × 7.7 × 7.8 cm and 13.7 × 7.2 × 7.2 cm for the left kidney and increased resistance pattern in the renal vasculature with no evidence of hydronephrosis, renal calculi, or focal lesions. The bladder appeared normal. No cause of acute renal failure was identified by diagnostic imaging. Obstetrical extended ultrasound revealed a single, living intrauterine gestation at 15 weeks and 3 days based on menstrual dates and fetal biometric measurements. Fetal growth was found to be appropriate for gestational age. Anatomic survey was limited due to early gestational age but overall appeared to be unremarkable. The fetus was found to be active in normal amniotic fluid volume. No CT chest was obtained on the initial presentation due to the absence of pulmonary symptoms and in an effort to avoid radiation exposure.The patient received hemodialysis on day one via the right internal jugular temporary hemodialysis catheter placed emergently in ICU. The urgent need for pathological diagnosis and its associated risk to the mother and the fetus was thoroughly discussed with the patient and obstetrics/gynecology with the decision made to proceed with renal biopsy with continuous fetal monitoring and administration of desmopressin for bleeding risk associated with acute kidney injury and uremic platelets. Renal biopsy revealed findings consistent with rapidly progressing glomerulonephritis with 100% cellular crescents and immunofluorescence exhibiting diffuse linear staining along the glomerular basement membrane with IgG (3+), C3 (3+), kappa (3+), and lambda (2+) stains Figures . ElectroGiven concerns for shortness of breath and chest tightness, a chest X-ray and chest computed tomography (CT) scan  were perLater, the patient developed severe hypertension which was not manageable with antihypertensive drugs, and the decision was made to deliver the fetus. Emergent cesarean section was performed at 28–0/7 weeks, and a male neonate, 1.1 kg with Apgar scores of 1/6/7, was delivered. The patient's postpartum course was relatively uncomplicated with no development of postpartum preeclampsia. The patient remained on hemodialysis until the end of her postpartum period, following which she transitioned to peritoneal dialysis and is currently awaiting a transplant.Anti-GBM antibody disease during pregnancy is a very rare occurrence. There are a few case reports in the literature with biopsy-proven anti-GBM disease and varied presentations. The outcomes of infants from mothers with anti-GBM disease during pregnancy are generally poor, often resulting in stillbirth and both natural and artificial abortion –8. MoreoDeubner et al.  describeThe present case is unique in terms of diagnosis, flare-up, and treatment for anti-GBM disease during the course of pregnancy followed by a successful preterm delivery. In our case, the patient presented with anti-GBM disease during the early phase of her third pregnancy with no evidence of kidney dysfunction during her previous pregnancies. The anti-GBM antibody titers were very high at the time of diagnosis and unlike the cases reported by Deubner et al.  and YankMaternal, pregnancy, and fetal outcomes of anti-GBM disease in pregnancy are poor and are associated with uncontrolled vasculitis –8. The tAnti-GBM disease should be considered in pregnant patients presenting with oligoanuric renal failure. Plasmapheresis and immunosuppression remain the treatments of choice. When choosing an immunosuppressive regimen in pregnancy, fetal safety should be considered and risks thoroughly discussed prior to treatment.


CAR and DSG2 genes are highly co-expressed in early stage LUAD and associated with significantly poorer survival (p = 0.0046). TMA also showed that CAR/DSG2 expressions were altered in lung cancer tissue. CAR in the TMA was correlated with proliferation, apoptosis, and epithelial–mesenchymal transition (EMT), while DSG2 was associated with proliferation only. The Kaplan–Meier survival analysis revealed that CAR, DSG2, or a co-expression of CAR/DSG2 was associated with poorer overall survival. Conclusions: The co-expression of CAR/DSG2 predicted a worse overall survival in LUAD. CAR combined with DSG2 expression can predict prognosis.Introduction: Coxsackievirus/adenovirus receptors (CARs) and desmoglein-2 (DSG2) are similar molecules to adenovirus-based vectors in the cell membrane. They have been found to be associated with lung epithelial cell tumorigenesis and can be useful markers in predicting survival outcome in lung adenocarcinoma (LUAD). Methods: A gene ontology enrichment analysis disclosed that DSG2 was highly correlated with CAR. Survival analysis was then performed on 262 samples from the Cancer Genome Atlas, forming “Stage 1A” or “Stage 1B”. We therefore analyzed a tissue microarray (TMA) comprised of 108 lung samples and an immunohistochemical assay. Computer counting software was used to calculate the H-score of the immune intensity. Cox regression and Kaplan–Meier analyses were used to determine the prognostic value. Results:  For decades, lung cancer has been recognized as the leading cause of cancer-related deaths worldwide. In general, around 80% of all lung cancers are non-small-cell lung cancers (NSCLCs), which comprise lung adenocarcinoma (LUAD), squamous cell carcinoma (LSCC), and large-cell carcinoma. Small-cell lung cancers account for only 10% to 15% of all lung cancers but are typically more aggressive . The 5-yThe coxsackievirus and adenovirus receptor (CAR) is a tight-junction protein localized to the cell membrane as part of the immunoglobulin (Ig) superfamily. It is associated with cell–cell adhesions as part of the apical junctional complex . PreviouThere are four desmoglein (DSG) proteins—DSG1, DSG2, DSG3, and DSG4—that are members of the cadherin family, which are transmembrane proteins that bind with other cadherins. DSG2 participates in cell growth, proliferation, apoptosis, and tumorigenic migration, as well as in invasion ,20. A prCAR and DSG2 are similar molecules and can be recognized by adenovirus-based vectors in the cell membrane . FurtherCAR gene sequential changes from NL: atypical adenomatous hyperplasia (AAH) to invasive LUAD. One group was the same expressed trend genes as CAR gene. Another group was the contrary expressed trend as CAR gene. The genes of at least three datasets that had the same or contrary expressed trends as CAR gene were selected in each group. Then, these two groups of genes were screened to only include cancer-related genes. The ClusterProfiler R package was used to perform gene function analysis for the two groups of cancer-related genes [www.string-db.org).To investigate gene expression in lung cancer, four public domain datasets were collected from the gene expression omnibus (GEO) from the National Center for Biotechnology Information. The accession numbers were GSE102511, GSE32863, GSE52248, and GSE68571 . Dataseted genes . Gene inhttp://cancergenome.nih.gov). “FPKM-UQ” was set as the measurement unit of the gene expression level. According to the clinical information from metadata of 594 samples, 262 samples that were “Stage 1A” or “Stage 1B” were included for further analysis. The expression of CAR was divided by the average expression level of the housekeeping genes from each sample. Two group of genes expressed either the same or contrary trends as CAR gene, which were also used to identify additional potential biomarkers. DSG2 gene was found to be highly correlated with CAR gene. In the 262 samples, the expression levels of CAR and DSG2 gene were defined as high if their expression was >90% of the samples. Survival analysis of the 262 samples was performed by the R package “survminer”.To assess a survival analysis associated with the two groups of gene expression, 594 LUAD RNA-seq data were obtained from the the Cancer Genome Atlas . The TMA  was comprised of 108 lung cancer formalin-fixed samples, paraffin-embedded samples, and two normal adjacent non-tumor tissues, which were purchased from Tech Smart Hong Kong, Ltd., Hong Kong. All human samples met the requirements of the Human Material Transfer Agreement (MTA). TMA patients consisted of 63 males and 45 females, with a median age of 67.1 years . All patients had follow-up records for >10 years. The survival time was calculated from the date the specimen was obtained to the follow-up deadline or mortality. Post-mortem specimens and surgically obtained tissues were collected under ethical conditions, and both the donor and their relatives (where appropriate) provided informed consent. All tissue samples underwent standard medical care to protect the donor’s privacy. Informed consent was recorded and retained by the tissue banks of the certified hospitals that obtained the tissue. All enrolled patients were de-linked anonymously for protection. This study protocol was approved by the institutional review board of the institution.Statistical analysis was performed using SPSS version 20.0 . Correlations between two categorical variables were examined using Pearson’s chi-square test. Progression-free survival (PFS) and overall survival (OS) were calculated at 12 and 36 months after the initial diagnosis, respectively. The Kaplan–Meier method was used to analyze the distributions of PFS and OS, and log-rank tests were performed to compare the differences between the two categories. Univariate analysis and multivariate survival analysis were conducted using the Cox proportional hazards regression model to obtain hazard ratios (HRs) with 95% confidence intervals (CIs), as well as to identify independent prognostic factors for PFS and OS. Statistical significance was set at α = 0.05.IHC assays were completed according to the standard method. Array slides were shipped at room temperature. All tissue array slides were covered with an extra layer of paraffin and were stored at 4 °C. The slides were heated at 60 °C for at least 30 min before de-paraffinization via xylene. The tissue blocks were cut into sections for IHC stain. The IHC stain was performed using a Ventana BenchMark XT automated stainer . Briefly, 4 μm-thick sections were cut consecutively from formalin-fixed tissue and paraffin-embedded tissue. Sections would be mounted on salinized slides and allowed to dry overnight at 37 °C. After deparaffinization and rehydration, slides would be incubated with 3% hydrogen peroxide solution for 5 min. After a washing procedure with the supplied buffer, tissue sections were repaired for 40 min with ethylenediamine tetraacetic acid . The slides were incubated with the primary antibody for 60 min at 37 °C and then overnight at 4 °C. The primary antibodies, CAR  and DSG2 , were used. The IHC stain results are shown in In order to evaluate the immunostaining results objectively, we performed whole-slide scanning with computer counting. The slides were scanned with a slide scanner  in 200× magnification. Computer counting software of CellQuant and PatternQuant were used . Briefly, PatternQuant was trained to recognize regions of interest following the CellQuant to evaluate the H-Score. The H-score was defined by immuno-intensity, which multiplied staining percentage (range from 0 to 300). Immuno-intensity was recorded as 0 for no staining, 1 for faint staining, 2 for moderate staining, and 3 for intense staining C,D. The The IHC results for the potential correlation between clinicopathological characteristics and survival outcomes were illustrated by Cox regression analyses, multiple testing (Bonferroni) correction, and the Kaplan–Meier method. Co-expressed genes surveilled by the bi-variant correlation were measured in proliferation, apoptosis, angiogenesis, and epithelial–mesenchymal transition , which were the calculated using Pearson correlation analysis.CAR gene expression for NL tissue advancing to AAH and to invasive LUAD presented elevated levels in concordance with various genes, as shown by gene ontology   . The genCAR and DSG2 gene co-expression was higher in early stage lung adenocarcinoma and was shown to have a significantly poorer survival outcome, when compared with lower CAR and DSG2 gene expression (p = 0.0046)  (p = 0.051) ( 0.0046) . Based o= 0.005) . Interes= 0.051) .p < 0.001), apoptosis (p < 0.001), and EMT (p = 0.009) (p = 0.001) , correlations among these markers were evaluated. Our results demonstrated a strong correlation between the TMA CAR expression levels, proliferation (= 0.009) A. DSG2 w= 0.001) B. The co= 0.001) C.p = 0.045);  .To evaluate the potential association between prognosis and the expression of CAR, DSG2, and the co-expression of CAR/DSG2, a Kaplan–Meier survival analysis was performed. The expression of CAR and DSG2 was divided into high and low expression levels according to the cutoff value. As shown in = 0.001) A,B. Of n= 0.001) , but was= 0.001) . FurtherCAR and DSG2 gene in LUAD. Initially, GEO datasets, ClusterProfiler, STRING, TCGA gene expression data, and survminer were used to examine the survival prediction value of elevated CAR gene expression from NL tissue that transformed into early LUAD. There were 372 expressed genes that had the same or different expression profiles as CAR gene, which were illustrated by a heatmap. The concordance of the upregulated genes that expressed CAR were determined, and 16 highly associated genes, as determined by gene ontology, were screened for further analysis. This revealed that CAR and DSG2 genes were expressed at >90% in 262 of the TCGA samples. This was the most important feature for survival outcome prediction. Later, we validated this finding using TMA with pathologists and a comprehensive computer counting software analysis, which assessed survival. The Pearson analysis was utilized to depict the association between co-expressed markers commonly seen in tumorigenesis, such as proliferation, angiogenesis, apoptosis, and EMT. The findings of co-expressed high CAR and DSG2 gene expression with poor survival from the TCGA database were consistent with the IHC staining prediction model in the 108 TMA LUAD patients.In this study, we elucidated the innovative value of CAR is associated with various physiological and pathophysiological functions. CAR participates in cancer biology as a critical regulator of survival and growth. Multitumor array analysis has shown altered CAR expression in different cancer types . CAR wasSeveral previous studies have also demonstrated that DSG2 is highly expressed in several tumors, such as gastric cancer, skin squamous cell carcinomas, and melanoma ,24,32. DDSG2 is a cell adhesion molecule similar to CAR and is a receptor for adenovirus-based vectors. It had been widely reported that the adenovirus can be used as an oncolytic virus vector for gene therapy cancer treatment . HoweverCAR and DSG2 gene in combination as a model independent prognostic predictor for LUAD. Firstly, surveillance from GEO enrichment analysis and gene ontology listed the dominant genes categorized from molecular functions and cellular components, which were selected for mapping with the TCGA database. This generated 15 genes predominantly related to CAR gene . We found that DSG2 gene was the most important. When expressed with CAR gene at >90%, the survival prediction was statistically significant.Taken together, increased levels of CAR and DSG2 could help target oncolytic adenovirus vectors for use as a therapeutic tool in lung cancer ,21; howep = 0.002). The co-existing marker also indicated a strong association with cell proliferation, as opposed to apoptosis, angiogenesis, or EMT. Our findings suggest that CAR and DSG2 participate in cell–cell junctions, which is the linkage between cell membranes that regulates tissue homeostasis in certain mechanisms, such as tissue barrier integrity, cell proliferation, and migration.Later, we examined CAR and DSG2 expression from a paraffin-embedded TMA. When compared with the NL tissue, CAR and DSG2 both increased in intensity and percentage of tissue stained among the specimens. Subsequent analysis determined the H-score was defined as immuno-intensity multiplied by staining percentage (range from 0 to 300). The cutoff H scores for CAR and DSG2 were 50.5 and 120, respectively. CAR and DSG2 had a positive correlation in the bi-variant correlation analysis (p = 0.008). When the two markers were taken into consideration and divided into high or low expression levels according to cutoff values, high CAR/DSG2 co-expression could predict worsening outcomes both in the long term (250 months) and the short term (60 months). Other than early stage disease (stage I and IIA), CAR/DSG2 co-expression could hamper survival outcomes when disease staging was relatively advanced. We proposed that a possible mechanism might be associated with enhanced cell proliferation or EMT. Thus, our findings suggest that CAR/DSG2 expression is a comprehensive predictor of overall survival in LUAD. This could be beneficial in clinical practice and could play an important role as a potential therapeutic target for factors linked with LUAD survival.In order to evaluate the association between CAR, DSG2, co-expression of CAR/DSG,2 and survival prognosis, a Kaplan–Meier survival analysis was conducted. Increased levels of CAR or DSG2 were associated with poorer overall survival in all stages up to 250 months, although high DSG2 expression played a much more significant role, as determined by the Cox regression analysis  -dependent host molecule interaction, virus tropism, antibody–capsid chimera engineering, modification of antigen-specific targeting peptides, OAd transduction-sensitive or refractory, would help optimize the corresponding vectors.The current study showed that CAR, DSG2, or a co-expression of CAR and DSG2 in LUAD is a predictor of overall survival. High CAR expression was positively associated with cell proliferation, apoptosis, and EMT; while DSG2 was only positively correlated with proliferation. No confounding factors were influenced by age, gender, or histologic type. LUAD patients with high CAR or DSG2, and especially CAR and DSG2, had significantly reduced overall survival rates. The usage of CAR/DSG2 combined with IHC staining had a potential clinical value as a predictor of LUAD patient survival. Further in vitro and/or in vivo studies are warranted to confirm the clinical usage concept of tumor gene delivery therapy. Combination therapy using OAd for treating cancer is vital for virus uptake and gene transfer. In summary, CAR combined with DSG2 expression could be a robust biomarker for predicting prognosis and evaluating the efficiency of gene therapy. Co-expression and enhancement could promote cellular sensitivity to adenovirus infection. Therefore, additional studies are needed to elucidate the potential mechanism of CAR/DSG2 as an optimal target for viral vectors as part of gene therapy.


Schistosoma mansoni tegument surface in comparison to tissue sections of whole worms or couples. We found that phosphatidylcholines (PC) and specific phosphatidylethanolamines (PE) are significantly more abundant inside the worm body compared to the tegument. On the other hand, the latter was found to be enriched in sphingomyelins (SM), phosphatidylserines (PS), lysophosphatidylcholines (LPC), and specific PE species. We further investigated lipid classes concerning number of carbon atoms in fatty acyl chains as well as the degree of unsaturation and found pronounced differences between the tegument and whole-worm body. Furthermore, differences between male and female teguments were found. The lipid composition of S. mansoni tissues has been investigated in an untargeted, spatially resolved manner for the first time.Schistosomes are human pathogens causing the neglected tropical disease schistosomiasis, which occurs worldwide in (sub-)tropical regions. This infectious disease is often associated with poverty, and more than 700 million people are at risk of infection. Exploitation of novel habitats and limited therapeutic options brought schistosomes into research focus. Schistosomes are the only trematodes that have evolved separate sexes. They are covered by their metabolically active tegument, a surface area representing the interface between male and female in their permanent mating contact but also between parasite and host. The tegument comprises, besides others, numerous specific lipid compounds. Limited information is available on the exact lipid composition and its spatial distribution. We used atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to characterize the  Schistosoma mansoni can be supported by investigations of the specific molecular communication in male/female and in worm/host interactions. Improving the knowledge about S. mansoni is mandatory, since there is justified fear of the possibility of resistance development against the only available drug Praziquantel. We used mass spectrometry imaging as a powerful tool to provide topographic and tissue-specific information on the parasite. We investigated single male and female worms, as well as mating couples, regarding both, their inner tissue, and their intact surfaces, the tegument. We found highly specific lipid species and visualized their local distributions and abundances in high-resolution molecular images. Our findings may help to improve knowledge of the complex life cycles and of molecular communication mechanisms of schistosomes and may help to develop new drugs and strategies for treatment of the infectious disease.WHO-defined Neglected Tropical Diseases, including schistosomiasis, are a burden for a significant part of the human world population. The fight against the diecious trematode  Schistosoma . The World Health Organization (WHO) has classified schistosomiasis as one of the neglected tropical diseases due to global spreading and its poverty-related endemicity.+). The inlet capillary was heated to 250°C, and a potential of 3 kV was applied between sample holder and inlet capillary. This setup enabled molecular topographic analyses of complex surfaces with high accuracy in mass and space at scan rates of about 1 pixel/s. Image sizes are listed in For MS imaging, an autofocusing “AP-SMALData was converted into mzXML or MS2 file formats using Proteowizard (v3.0.11https://metaspace2020.eu/project/Kadesch-2019-Smansoni). Annotations were further processed manually for all measurements removing non-reproducible signals found in two or less samples . One RoI per MSI dataset was defined, to yield signal intensities for three biological replicates per class , enabling statistical analysis. For statistical analyses, the data matrix was imported to Perseus+ (Δm = 3.1 ppm) at m/z 780.5537 ± 5 ppm. B–Signal intensity  vs mass deviation in ppm. A double peak can be observed shifted by approximately 1.5 ppm and 2.8 ppm. C–MS ion signal at m/z 780.55254 ± 0.2 ppm showing an increased signal intensity on the worm surface assigned to protonated PE (39:5). D–MS ion at m/z 780.55151 ± 0.2 ppm assigned to PE (37:2) as sodium adduct. By hierarchical clustering, the signal at m/z 780.5537 was determined to be more abundant in the worm body compared to tegumental surface (see A–MS ion image of nearly isobaric PE-adduct species [PE (39:5) + H]face see . The sig(TIF)Click here for additional data file.S9 Fig+. B–distribution of m/z 804.5514 assigned to [PE (39:4) + Na]+. This difference in distribution could be explained by different concentrations of salt in tegument and inner tissue or by isobaric interferences that were not contained in the LC-MS/MS-database.A—Distribution of m/z 782.5694 assigned to [PE (39:4) + H](TIF)Click here for additional data file.


The impact of postoperative complications (POCs) on the long-term prognosis of patients with colorectal carcinoma was analysed with respect to their severity according to the Clavien-Dindo classification (CDC).The prospectively collected data of 2158 patients who underwent curative resection of a colorectal carcinoma  without distant metastases from 1995 to 2014 were analysed. The POCs were documented in a standardized form and graded with the CDC. Patients who died postoperatively  were excluded.p = 0.005). The 5-year distant metastasis rate was 15.9% in CDC 0 patients and 19.5% in CDC I–IV patients. In multivariate analysis, distant metastasis was highest in CDC III patients . The 5-year overall survival rate was 83.5% in patients without POCs and 73.5% in patients with POCs. It was worst in CDC IV patients (63.1%), which was confirmed by multivariate analysis .In total, 467 patients (21.6%) had POCs: CDC I, 141 (6.5%); CDC II, 162 (7.5%); CDC III, 112 (5.2%); and CDC IV, 52 (2.4%). More POCs and higher CDC grades were found in men, ASA III-IV patients, rectal carcinoma patients, and patients who underwent abdominoperineal excisions or multivisceral resections. The 5-year locoregional recurrence rate was 5.3% in patients without POCs and 6.6% in patients with POCs. It was highest in CDC III patients (12.9%), which was confirmed in multivariate analysis (HR 2.2; Patients with POCs after colorectal surgery have a poor long-term prognosis. As the CDC grade increases, survival deteriorates. Quality management in colorectal carcinoma is usually divided into short- and long-term results. How closely these two are linked was first demonstrated with anastomotic leaks. In 1991 Akyol et al.  showed tIn 1992, Clavien et al.  classifiThe Erlangen Registry for Colorectal Carcinoma (ERCRC) was established in 1978. Quality management and prognostic factor analysis are the main objectives of this prospective cancer registry. The aim of the current study was to analyse the impact of postoperative complications on long-term prognosis and to assess the severity of complications graded by the CDC and their influence on locoregional recurrence, distant metastasis, disease-free survival, and overall survival.The study refers to 2215 consecutive patients who underwent colorectal carcinoma surgery at the Department of Surgery of the University Hospital Erlangen, Germany, between 1995 and 2014. We evaluated the prospectively collected database of the Erlangen Registry for Colorectal Carcinomas (ERCRC) according to the following inclusion criteria: solitary invasive colorectal carcinoma (at least into the submucosa); no appendiceal carcinoma; no other previous or synchronous malignancies; carcinomas not related with familial adenomatous polyposis, ulcerative colitis or Crohn’s disease; no distant metastases; radical elective surgery; and residual tumour classification R0 . All patients underwent resection with regional lymph node dissection according to the standards of total or partial mesorectal excision   or complCarcinomas with a distal margin ≤ 16 cm to the anal verge were classified as rectal carcinomas and those with a distal margin > 16 cm were classified as colon carcinomas .n = 250) received chemoradiation according to the protocol presented by the German CAO/ARO/AIO-94 study [2 per day during the first and fifth week. A total of 91 patients received oral capecitabine instead of an infusion of 5-FU. In addition, 235 patients received oxaliplatin, 17 patients received irinotecan, and 8 patients received additional cetuximab. During the last few years, hyperthermia was provided to some patients within clinical trials. Six to eight weeks after the completion of preoperative chemoradiation, the patients underwent open TME surgery [After preoperative staging, long-term preoperative chemoradiation was administered for locally advanced rectal carcinomas  and for selected lower-third carcinomas (< 6 cm) to enable sphincter preservation. Of the 545 patients 46.7%) with neoadjuvant therapy, the majority of the patients  classification system .Postoperative complications (POCs) were documented in a standardized form. To assess the severity of these POCs, the Clavien-Dindo classification (CDC) , 12 was Anastomotic leaks were classified according to Rahbari et al. . SevereThe American Society of Anaesthesiologists (ASA) classifies the comorbidities of the patients into 6 levels . The levThe patients were followed up for at least 5 years, every 3 months for the first 2 years and every 6 months thereafter; the follow-up included a physical examination, carcinoembryonic antigen (CEA) analysis, abdominal ultrasonography, chest X-ray, computed tomography (CT) of the pelvis and rectoscopy/colonoscopy, depending on localization (rectum or colon) and stage of the primary tumour. In 2004, the follow-up examinations were changed to semi-annually for the first 2 years and then yearly for a total of 5 years according to the first edition of the German S3-Guidelines for Colorectal Carcinoma . A locor2 test and Fisher’s exact test were used to compare frequencies, and the Mann-Whitney U test was used to analyse continuous data. The Kaplan-Meier method was used to calculate the 5-year rates of locoregional recurrence, distant metastasis and survival. Survival curves were compared using a log-rank test. Disease-free survival was defined as the time to the first occurrence of locoregional recurrence, distant metastasis or death by any cause. The endpoint of overall survival was death from any cause. Factors that were found to be significant in the univariate analysis were included in a multivariate Cox regression model. A p value less than 0.05 was considered significant. Statistical analysis was carried out using the statistics software package SPSS® version 21 .The χn = 141 (6.5%); grade II, n = 162 (7.5%); grade III, n = 112 (5.1%); and grade IV, n = 52 (2.4%). Table n = 28) and urinary bladder dysfunction (n = 40); for CDC grade II complications, urinary tract infection (n = 40) and central venous catheter infection (n = 23); for CDC grade III, anastomotic leak (n = 27), abdominal wall dehiscence (n = 14) and postoperative bleeding (n = 11); and for CDC grade IV, anastomotic leak (n = 23) and pulmonary complication (n = 12). Compared to other patients, male patients, patients with an ASA level > II, tumour localization in the rectum, abdominoperineal excision and multivisceral resection had significantly more postoperative complications and higher CDC grades.The characteristics of all 2158 patients and their tumours are shown in Table The median number of regional lymph nodes examined in all specimens was 25 (1–145). In patients with rectal carcinoma, the median number of regional lymph nodes examined was 22 (2–76). In patients with primary surgery, it was 27 (4–76), and in those with neoadjuvant treatment, it was 18 (2–62). In patients with colon carcinoma, the median number of regional lymph nodes examined was 29 (1–145). In those who underwent standard colon resections, it was 27 (4–76), and in extended resections, it was 37 (12–145).The pathological circumferential resection margin (pCRM) was positive (≤ 1 mm) in 20 (2.0%) of the 997 rectal cancer specimens who underwent respective examinations.The quality of TME/PME  was evalAnastomotic leaks of grade B and C were observed in 45 (4.8%) of 942 patients after rectal resection and in 23 (2.3%) of 985 patients after colon resection.p < 0.001).Six hundred fifty-eight patients (30.5%) received adjuvant treatment. The higher the CDC grade, the less frequently adjuvant therapy was administered . The locoregional recurrence rate was significantly higher in men than in women and increased with stage. The rate was higher in patients with POCs  and was highest in patients with CDC grade III POCs . In the multivariate Cox regression analysis, sex (p = 0.050), tumour site (p < 0.001), stage III tumours (p = 0.005) and CDC grade III POCs were found to significantly influence the locoregional recurrence rate . The rate was 8.5% for rectal carcinomas compared to 2.2% for colon carcinomas p < 0.00. The locp = 0.009). The univariate analysis also found that ASA classification (p = 0.028), tumour site (p < 0.001) and tumour stage (p < 0.001) were significant factors. The 5-year distant metastasis rate increased as the CDC grade advanced, but this association did not reach significance (p = 0.066). In the multivariate analysis, ASA classification (p = 0.045), tumour site (p < 0.001) and stage III tumours (p = 0.020) were found to be independent prognostic factors. In addition, CDC grade III POCs were again significantly associated with the highest risk for distant metastases . The rate differed significantly between patients with and without POCs . In the univariate analysis, significant differences were found for age, sex, ASA classification, tumour site, tumour stage and CDC grade. In the multivariate analysis adjusted for age, all of these factors were found to have a significant influence on disease-free survival. Patients with CDC grade IV POCs were found to have the worst disease-free survival compared to patients without POCs . The patients with POCs had a significantly worse disease-free survival (65.2%) than patients without POCs . The univariate analysis was significant for age (p < 0.001), sex (p = 0.001), tumour stage (p < 0.001) and CDC grade (p < 0.001). In the multivariate analysis, all of these factors were found to be significant. Patients with CDC grade IV POCs had the worst overall survival . The patients with POCs had a strikingly decreased overall survival (73.5%) compared to patients without POCs (83.5% n = 1097) and 2005–2014 (n = 1061), are compared, we found fewer POCs in the second period , in particular a decrease in POCs of CDC grade IV was observed . With regard to long-term outcome, the 5-year locoregional recurrence rate decreased significantly from 7.3% (95%CI 5.7–8.9) to 3.8% , while improvements in distant metastasis , disease-free survival  and overall survival  did not reach significance.If the study time is divided and the two periods, 1995–2004  of the patients with CDC grade III POCs had a surgical complication. These patients required endoscopic, radiological or surgical interventions. Anastomotic leak is a typical example of a CDC grade III complication frequently associated with increased rates of locoregional recurrences and even poor overall survival  patients. This caCDC grade IV complications are life-threatening and require the patient to be admitted to an intermediate or intensive care unit. The results of the present study show that these patients had the worst disease-free and overall survival rates compared to other patients. It should be noted that these severe complications are not necessarily surgical complications. In the present study, 40% of CDC IV patients had non-surgical complications. Preoperative comorbidities, such as chronic heart failure, chronic obstructive pulmonary disease (COPD) and renal failure, represent a particular risk for non-surgical complications. These comorbidities can lead to single- or multi-organ dysfunction after colorectal surgery, which affects survival outcomes regardless of the original oncological prognosis.Artinyan et al.  showed tThe ASA classification, which assesses the preoperative status of the patient, is an independent factor for morbidity and mortality. An older age is frequently associated with more comorbidities and thus with an increased ASA classification. The present study showed that patients with more serious comorbidities (ASA III or IV classification) were associated with more frequent complications and a higher CDC grade. This is reflected in the worse long-term prognosis with shorter disease-free and overall survival when patients with comorbidities do not recover properly from the complications of surgery. Patients who are more ill are more likely to have a higher complication rate and thus a lower survival rate. As a complication-free course is important for the overall prognosis, good preoperative preparation is essential, particularly for older and high-risk patients. In addition to ensuring appropriately treated comorbidities, such as good control of diabetes and hypertension, optimal preoperative and postoperative nutrition and physical activity play an important role. Malietzis et al. found an association between muscle mass and postoperative morbidity and mortality. They identified myopenia as an independent risk factor for disease-free and overall survival in patients with colorectal cancer . It has Patients with complications have an increased risk of not receiving adequate adjuvant therapy. The lack of receiving adjuvant therapy postoperatively is known to increase metastasis rate and to decrease survival. In addition, if adjuvant treatment is indicated after surgery, it should start on time; otherwise, the prognosis will deteriorate . AccordiNevertheless, the anatomical extent of the tumour (TNM classification) and presence of residual tumour (R classification) are still the most important prognostic factors for the oncologic outcome. High-quality surgery, as part of a multidisciplinary care strategy, is essential for a complication-free postoperative course and a favourable long-term outcome. The increasing specialization in colorectal surgery contributes to this conclusion .Our study has some limitations, particularly the single-centre design and its retrospective nature. During the long study time of 20 years, treatment protocols have changed, and multimodal treatment has gained importance. For example, the administration of adjuvant chemotherapy for stage III colon cancer nearly doubled from 44% in 1995–1999 to 79% in 2010–2014.In conclusion, this study identified postoperative complications as an inverse prognostic factor in colorectal cancer with a strong relationship to the Clavien-Dindo classification. It emphasizes the importance of a complication-free postoperative course, not only in view of the immediate postoperative problems and costs but also for the long-term course. This relevance should be known to the entire treating and caring team during the preoperative preparation, the actual surgery and in the postoperative course. It also should be discussed with the patient, so that he/she will definitely attend all follow-up examinations.