Approximately 10% of patients with gastric cancer (GC) have a genetic predisposition toward the disease. However, there is scant knowledge regarding germline mutations in predisposing genes in the Chinese GC population. This study aimed to determine the spectrum and distribution of predisposing gene mutations among Chinese GC patients known to have hereditary high-risk factors for cancer.A total of 40 GC patients from 40 families were recruited from seven medical institutions in China. Next-generation sequencing was performed on 171 genes associated with cancer predisposition. For probands carrying pathogenic/likely pathogenic germline variants, Sanger sequencing was applied to validate the variants in the probands as well as their relatives.CDH1 (n = 1), MLH1 (n = 1), MSH2 (n = 1), CHEK2 (n = 1), BLM (n = 1), EXT2 (n = 1), PALB2 (n = 1), ERCC2 (n = 1), and SPINK1 (n = 2). In addition, 129 variants of uncertain significance were identified in 27 patients.According to sequencing results, 25.0% (10/40) of the patients carried a combined total of 10 pathogenic or likely pathogenic germline variants involving nine different genes: This study indicates that approximately one in every four Chinese GC patients with hereditary high risk factors may harbor pathogenic/likely pathogenic germline alterations in cancer-susceptibility genes. The results further indicate a unique genetic background for GC among Chinese patients. MUTYH-associated adenomatous polyposis (MAP) [CDH1 in 1998 [CDH1, MLH1, MSH2, MSH6, PMS2, EPCAM, TP53, STK11, BRCA1, BRCA2, MUTYH, APC, SMAD4, BMPR1A, and PTEN [CDH1-negative families that meet HDGC criteria.Gastric cancer (GC) is the third most common cause of cancer-related mortality worldwide . Approxiis (MAP) , familiais (MAP) , juvenilis (MAP) , 19, andis (MAP) . Since t in 1998 , >20 GC-and PTEN . NeverthIn general, the marked differences in ethnicity, diet, and living habits between Chinese and Caucasian populations suggest that Western genetic-screening guidelines may not be suitable for Eastern populations. To date, research on hereditary GC in the Chinese population is scarce. Nonetheless, it is important to identify patients with genetic aberrations because such mutations may influence clinical management, yet the underlying genetic factors conferring susceptibility to GC remain largely unknown. Accordingly, this study aimed to explore the frequency and spectrum of predisposing germline gene variants among Chinese GC patients with hereditary high risk factors for cancer. For probands harboring pathogenic or likely pathogenic germline variants, Sanger sequencing was applied to validate the variants in both the probands and their family members.Between January 2017 and August 2018, gastric-adenocarcinoma patients with hereditary high risk factors were recruited from seven hospitals throughout six provinces in China . PatientThis study was approved by the Ethics Committee of Peking University Cancer Hospital and the relevant ethics committees of each of the participating centers. All procedures were performed in accordance with the ethical standards of the respective committees on human experimentation  and with the Helsinki Declaration of 1964 and its later versions. All patients and their family members provided written informed consent to participate.The solution-phase panel was designed to cover all exons (including parts of the introns) of 171 cancer-predisposing genes selected following a thorough literature review and a review of unpublished data .The processes of genomic DNA extraction, NGS, bioinformatic analysis, and variant filtering and annotation were supported by BGI Genomics  and performed as a previous study has described .2) test. P < 0.05 was considered statistically significant.Statistical analyses were performed with SPSS Statistics 21.0 software . For continuous variables (age at diagnosis), data with non-normal distributions are presented as medians with interquartile ranges; rank tests were used for all other analyses. Categorical variables are presented as ratios. Differences in mutation rates between groups were compared and analysed with the chi-square  associated with LS. Pathogenic or likely pathogenic variants of homologous recombination repair genes  were detected in three probands. One proband harbored a CDH1 variant associated with HDGC that was, therefore, likely pathogenic. Another two probands carried likely pathogenic variants of other genes known to be associated with a genetic predisposition toward cancer . Two patients were identified with SPINK1 mutations, which have not been reported previously  were found to carry pathogenic or likely pathogenic variants. Two probands carried MMR pathogenic variants  carried the same mutation; his mother was diagnosed with breast cancer at 45 years old. The patient from the BZ13 family with early-onset GC harbored an EXT2 likely pathogenic variant inherited from his mother. The patient had a second-degree relative with GC. The patient from the BZ16 family who carried the CHEK2 likely pathogenic mutation was diagnosed with GC at the age of 25 years and did not have a family history of cancer. His mother and twin brother had the same variant, as verified by Sanger sequencing, whereas his father and aunt did not carry this variant. The XJ1 family proband harbored a PALB2 mutation and was diagnosed with GC at 30 years, but there was no family history. The Sanger-sequencing results for his parents revealed that the variant was inherited from his father. The probands of the BZ2 family and BZ10 family each harbored a pathogenic mutation in SPINK1 and were diagnosed with two primary cancers. The former was diagnosed with renal clear cell carcinoma at the age of 62 and subsequently diagnosed with breast cancer at 63. He had a family history of cancer, but his younger brother and younger sister did not carry the same variant, as verified by Sanger sequencing. The patient from the BZ10 family was diagnosed with thyroid cancer and GC at the age of 31 and 37 years, respectively.The proband of the LZ2 family, who harbored an P = 0.011; No significant differences in age of onset, family history, or clinical stage were found between patients carrying pathogenic or likely pathogenic variants and those with variants of uncertain significance (VUSs). However, patients with pathogenic or likely pathogenic mutations were more likely to have multiple-onset primary malignancies than those without these mutations , MSH2 (n = 1), CDH1 (n = 1), BLM (n = 1), PALB2 (n = 1), EXT2 (n = 1), CHEK2 (n = 1), ERCC2 (n = 1), and SPINK1 (n = 2).Although research on hereditary GC in China is limited, investigations on other neoplasms, such as breast cancer, ovarian cancer, and prostate cancer, have suggested that the genetic spectrum of Chinese patients with hereditary tumors may differ from that of Caucasians. In general, the heterogeneous clinical features of hereditary tumor syndromes and the atypical presentations of cancer family histories hamper attempts to summarize and cluster genotypes and phenotypes using a traditional single-gene resolution approach. In contrast, this study enrolled Chinese GC patients with hereditary high risk factors for cancer, and clinical information including age at diagnosis, special histologic types, family history of malignant tumors, and microsatellite status was used to explore the frequency and spectrum of germline variants of cancer-predisposing genes. To the best of our knowledge, this is the first multicenter research study in China aiming to reveal GC-related germline variants in CDH1, MSH2, and PALB2 have been identified [The spectrum of germline variants in Chinese GC patients in this study revealed a pattern distinct from that in previous studies. Although genes such as entified , there wMLH1 splice-site variant c.790 + 1G>A, which results in the loss of amino acids 227 to 295 of the MLH1 protein and has been shown to functionally render MLH1 defective in MMR activity [MSH2 nonsense mutation c.610G>T (p.G204*). Sheng et al. [We identified two pathogenic variants in MMR genes associated with LS. First, the proband of the LZ2 family carried the activity , 31, andg et al.  detectedg et al. . Both prCDH1 is a cancer-predisposing gene that is mutated in families meeting the criteria for clinically defined HDGC, with ∼40.0% of HDGC families harboring germline mutations in CDH1. For example, Hansford et al. [CDH1 mutations were found in 31 cases. The CDH1 germline mutation rate correlates negatively with the morbidity of GC worldwide. In countries with low GC morbidity, such as Canada, the USA, the UK, and the Netherlands, the CDH1 mutation rate can be as high as 51.6% in patients meeting HDGC clinical criteria [CDH1 mutation rate is 15.4% in Japan, which has the highest GC morbidity [CDH1 gene germline mutation . Thus, the CDH1 mutation rate in our study for this subset of patients was only 6.7%.Clinically defined HDGC is characterized by early-onset, multigenerational diffuse GC, and lobular breast cancer. Clinical criteria for HDGC have been established by the International Gastric Cancer Linkage Consortium (IGCLC) . CDH1 isd et al.  identificriteria . Conversorbidity . In our ERCC2 missense mutation c.1532G>A (p.R511Q) was not found in any of the databases queried. Validation of ERCC2 c.1532G>A at the functional level is, therefore, warranted. The ERCC2 p. R511Q variant is located in the region mediating interaction with MMS19, which fits the PM1 criteria. Furthermore, the frequency of this variant in GnomAD is 0, fitting the PM2 criteria; however, missense mutations of ERCC2 result in a high rate of functional impact, which meets the PP2 criteria. On the basis of these findings, as well as the in silico predictions providing PP3-level evidence, we categorized this variant as likely pathogenic (PM1+PM2+PP2+PP3).The BLM is the causative gene of Bloom syndrome (BS). BS is an autosomal recessive disorder characterized by proportionate pre- and post-natal growth deficiency; sun sensitivity; telangiectatic, hypo-, and hyperpigmented skin; predisposition toward malignancy; and chromosomal instability [tability . This vaEXT2 gene is causative of hereditary multiple exostoses (HME). HME is an autosomal dominant disorder characterized by multiple exostoses most commonly arising from the juxta-epiphyseal region of the long bones [EXT2 frameshift variant harbored was classified as likely pathogenic.The ng bones . The BZ1CHEK2 variant c.1553_1554insG (p.S518Rfs*7) is a well-described, lower-penetrance mutation that is mainly associated with breast cancer as well as colorectal cancer and prostate cancer. Previous studies have shown that CHEK2 mutations predispose individuals toward GC, particularly in cases of early onset; regardless, the association between CHEK2 and GC and the underlying molecular mechanisms require extensive investigation [The tigation . The fratigation . This vaPALB2 is a breast-cancer-susceptibility gene [PALB2 mutations have also been identified in four recent studies on GC, supporting their association with GC [PALB2 splice-site mutation c.1684 + 1G>A results in abnormal mRNA splicing, which affects the function of the protein. This variant was detected in one patient with high-risk neuroblastoma and classified as likely pathogenic, in accordance with a previous report [PALB2 colocalizes with BRCA2 in the nuclear foci, promoting its localization and stability in nuclear structures and facilitating its recombinational repair and checkpoint functions. A previous study reported that ity gene . PALB2 m with GC , 42, 43.s report .SPINK1 splice-site mutation c.194 + 2T>C was identified in the probands of the BZ2 and BZ10 families. This mutation affects a donor splice site in intron 4 of the gene: it is predicted to affect mRNA splicing, resulting in a significantly altered protein due to exon skipping, shortening, or the inclusion of intronic material. Experimental studies have shown that this splice-site variant completely abolishes SPINK1 mRNA and protein expression in cell culture. Furthermore, this variant is recurrent in individuals of Asian descent with chronic pancreatitis [The reatitis , 46. Mulin silico predicted to be high-risk variants. The pathogenic classification of two of these VUSs is controversial, as described below. We employed Sanger sequencing to validate these VUSs in the two patients. MUTYH VUS and CHEK2 VUS.We found a total of 129 VUSs in 27 of the patients in this study. Most of these VUSs were missense mutations; 27 VUSs were MUTYH splice-site mutation c.934 – 2A>G, which alters a conserved intronic nucleotide and causes aberrant splicing based on in vitro studies [MAP is an autosomal recessive disease that typically presents with multiple colorectal adenomas and an increased risk for colorectal cancer. GC among these patients is uncommon and is reported in only 2.0% of cases . The pro studies . However studies . MultiplCHEK2 missense mutation c.1111C>T (p.H371Y). Liu et al. [in vitro functional studies, this missense mutation causes a decrease in phosphorylation and enzymatic activity compared with the wild-type CHEK2 protein. Nevertheless, the decreased activity caused by this variant is not as pronounced as the effect caused by a known kinase-disrupting variant [In silico analyses support that this variant does not alter protein structure or function. Despite some indication that this variant may cause disease, the evidence is insufficient at present. Therefore, we classified this mutation as a VUS.The proband of the BZ14 family carried the u et al.  reportedu et al. , 54. Acc variant . In siliThere are many differences in ethnicity, diet, and living habits between Chinese and Western populations, and clinicians in China should not simply adopt Western screening criteria for assessing hereditary GC. Thus, one of the purposes of this study was to establish screening criteria relevant to hereditary GC in China. To enroll patients, the present study referenced the clinical criteria for HDGC and LS for screening, and only patients with hereditary high risk factors were included. We found that Chinese patients with multiple-onset primary malignancies were almost 7-fold more likely to carry pathogenic germline mutations than those without such malignancies .in vitro and in vivo should be performed to correctly classify these variants.Our study had several limitations. As the size of the cohort recruited was limited, we were unable to unequivocally define disease-causing variants and, therefore, GC-predisposing genes. A large number of VUSs were detected with the application of NGS, and several candidate VUSs were considered to be potentially pathogenic based on certain ACMG criteria and bioinformatic prediction tools. Further functional studies CHEK2, BLM, EXT2, ERCC2, PALB2, and SPINK1) identified in our study confer susceptibility to GC needs to be deciphered in future work. In addition to refining the enrollment criteria to improve patient selection, future studies should focus on assessing the functional impact of all of these variants through in vitro testing, tumor analysis, and segregation data.This prospective multicenter study enrolled 40 GC patients with hereditary high risk factors for cancer to explore the prevalence of germline genetic alterations in cancer-susceptibility genes by NGS. By using multiplexed panel sequencing, we found that 25.0% of patients carried pathogenic/likely pathogenic germline mutations in 9 of 171 genes tested. Several of these variants are located in GC-associated susceptibility genes or genes associated with other, clinically heterogeneous cancer-predisposition syndromes. Whether novel variants (CDH1 gene mutation rate was 6.7% in the 15 families meeting the HDGC clinical criteria in our study, which is significantly lower than that in Western countries. This finding indicates the unique genetic background of GC in Chinese patients. Because patients with pathogenic or likely pathogenic germline variants have a dismal clinical outcome and a high rate of multicancer occurrence, it is strongly recommended to provide genetic counseling, genetic screening, and family surveillance and management for patients with hereditary high risk factors. By screening populations with hereditary high risk factors, multiple-gene sequencing can be effectively applied to discover novel disease-causing genes of hereditary disease.The Gastroenterology Report online.L.S. and X.C.W. directed, conceived of, and designed the project. Y.J.Z., Y.Y., T.X., Q.W., and S.Y.T. collected the data. Y.J.Z. drafted the manuscript. X.T.Z. and J.G. revised the manuscript. B.R.L., J.D.Z., X.B.C., Z.J.W., M.Q., X.W., and L.S. were the principal investigators from the seven institutions involved in the study. L.S. and X.C.W. supervised the study and revised the manuscript. All authors read and approved the final manuscript.None.goab020_Supplementary_DataClick here for additional data file.


Dietary-based primary prevention guidelines for chronic kidney disease (CKD) treatment are lacking due to limited evidence. Single nutrient intake studies do not account for complex dietary interactions. We assessed associations between dietary patterns and renal function in the Northern Ireland Cohort for the Longitudinal Study of Ageing (NICOLA).a posteriori dietary patterns. Renal function was characterised by estimated glomerular filtration rate (eGFR) using serum creatinine and cystatin-C. Associations were assessed according to quintiles of dietary pattern adherence and multivariable regression analysis examined associations with eGFR.A cross-sectional observational study used NICOLA baseline dietary data collected between February 2014 and March 2016 via a food frequency questionnaire for 2590 participants aged ≥ 50 years. Principal component analysis identified 2  lower than the most adherent. Those with lowest adherence to the ‘unhealthy’ dietary pattern 2 had a mean eGFR 1.9 ml/min/1.73m2  higher than those with highest adherence. Participants with lowest adherence to dietary pattern 3, characterised by a high consumption of alcohol and coffee, had a mean eGFR 1.8 ml/min/1.73m2  lower than those with greatest adherence.Variation in three dietary patterns was significantly associated with eGFR. After adjustment for potential confounders, participants with least adherence to the ‘healthy’ dietary pattern 1 had a mean eGFR 3.4 ml/min/1.73mOur findings identify independent associations between dietary patterns and eGFR. These findings can inform the development of diet-related primary prevention advice for CKD.The online version contains supplementary material available at 10.1007/s00394-021-02579-z. Dietary contributions to the aetiology of chronic kidney disease (CKD) are not well characterised and, as a result, guidelines for primary prevention specific to CKD are lacking due to limited published evidence. Existing dietary guidelines target the primary prevention of important risk factors for CKD, including hypertension , 2 and tDiets in free-living populations are variable and complex and comprise a myriad of foods, eaten separately or together as meals. Given this complexity and the inherently multidimensional impact of foods , the association with health outcomes may depend on overall dietary pattern rather than single macro- or micronutrient intake . Single a-priori derived dietary patterns, such as the Mediterranean diet, can necessitate changes in food choice and food preparation methods that may present a barrier to adherence.Conceptually healthier interventions based on dietary patterns such as the Dietary Approaches to Stop Hypertension (DASH) diet have been shown to be associated with reduced CKD risk –11 and pStudies to identify a posteriori dietary patterns in local populations and to examine the associations with renal health have been conducted in diverse populations including cohorts in the USA , 22, 23,In a Taiwanese population, a dietary pattern characterised primarily by a greater consumption of meat, offal, processed food, rice and flour products,, and low consumption of dark-coloured fruit and vegetables, was associated with a small, increased risk of poorer renal function . In a TaThese studies highlight the benefits of evaluating the effects of conceptually healthy and naturally existing dietary patterns in a region-specific manner. However, there is little evidence in British and Irish populations, which likely differ markedly in their dietary habits and the type and quality of available foods compared to other populations. In the Irish Nun Eye Study (INES ), greateData from Wave 1 of NICOLA were used to test associations between renal function and dietary patterns specific to the Northern Ireland population. This study collected health and lifestyle data from 8452 free-living individuals in Northern Ireland, aged 50 years and over with planned 10-year follow-up assessments for consenting participants. Baseline data were collected between December 2013 and March 2016 (NCT number: NCT01938898 ). The stData collection included a health assessment, a self-completion questionnaire and a computer-assisted personal interview. Blood samples were collected as part of the health assessment. Renal function was assessed as the estimated glomerular filtration rate (eGFR) calculated using the combined CKD-EPI equation for serum creatinine and cystatin-C from a blood sample collected at a single time point . DietarySystolic blood pressure (SBP) and diastolic blood pressure (DBP) measurements were the average of two independent readings. Diabetic status was defined using a combination of participant percentage haemoglobin A1c (HbA1c > 6.5%), diabetic medication use or self-reported diabetes. Self-reported medication use was defined according to the Anatomical Therapeutic Chemical (ATC) Classification System based on the active ingredients of drugs according to the organ or system on which they act and their therapeutic, pharmacological and chemical properties. Educational attainment was classified as completing primary, secondary or tertiary level education. Smoking status was characterised as current, past or non-smoker. Alcohol consumption was categorised into three groups: non-drinker, drinker and ex drinker.Statistical analyses were conducted using SPSS v24 . Continuous variables were summarised using means and standard deviations. Categorical variables were summarised using frequencies and percentages. The dietary pattern analysis has been described previously . In briepost hoc test. Differences in categorical variables between quintiles of adherence were assessed using the Kruskal–Wallis test with post hoc pairwise Mann–Whitney U tests to determine the location of differences. Multivariable linear regression analysis was used to assess associations between eGFR and dietary pattern adherence. Adjusted models controlled for age, sex, diabetes status, SBP, smoking status, alcohol consumption, waist circumference, high-density lipoprotein (HDL), low-density lipoprotein (LDL), lipid-modifying agent use, antihypertensive and diabetes drug use, multiple-deprivation score, and education level. In a sensitivity analysis, we tested for associations of interactions between eGFR and dietary pattern adherence by BMI category and for those 65 < age ≤ 65 years. For all analyses, p < 0.05 was considered statistically significant. Study datasets are available from the corresponding author following reasonable request.Differences in mean eGFR between quintiles of dietary pattern adherence were assessed using one-way analysis of variance (ANOVA). Adjustment for multiple comparison was carried out using Student–Neuman–Keuls (SNK) 2 with 589 (23%) having a BMI < 25 kg/m2, 1139 (44%) BMI ≥ 25 kg/m2 but < 30 kg/m2 and 862 (23%) had a BMI ≥ 30 kg/m2. Mean HDL cholesterol was 1.61 mmol/L (SD = 0.44) and mean LDL cholesterol was 3.34 mmol/L (SD = 1.10). Nine percent of participants had diabetes mellitus and 40% were hypertensive (defined as a SBP ≥ 140 mm Hg or DBP ≥ 90 mm Hg or if use of antihypertensive medication was self-reported). The mean eGFR was 73.5 ml/min/1.73m2 (SD = 16.3). The majority (85%) of participants had completed at least secondary level education, 54% had never smoked, and 69% currently consumed alcohol. A lipid-modifying agent was being used by 34% of participants; 6% used medication to treat their diabetes, and 2% used antihypertensive medication.NICOLA baseline characteristics for the 2590 participants with both dietary pattern and renal function data are shown in Table Three dietary patterns met the retention criteria of Eigenvalues greater than 1 and above the scree plot breakpoint. The food components and factor loadings of each dietary pattern are described in Table 2  compared to the highest adherence eGFR 76.0 ml/min/1.73m2 . These differences remained significant in the adjusted model where the least adherent quintile had an eGFR mean difference of − 3.4 ml/min/1.73m2  compared to the most adherent reference category. The association between adherence to dietary pattern 1 and eGFR was significant in both the unadjusted and adjusted models (p for trend < 0.001). In a sensitivity analysis dichotomised by 65 < age ≥ 65 years, the effect size was diminished in those aged ≥ 65 years but was no longer significant in the smaller sample size, although the trend remained the same .The difference in mean renal function for participants, divided by quintiles of adherence to dietary patterns is shown in Table 2  compared to the most adherent eGFR 73.8 ml/min/1.73m2 . These differences remained significant in the adjusted model where the least adherent quintile had an eGFR mean difference 1.9 ml/min/1.73m2  compared to the most adherent reference category. The association between adherence to dietary pattern 2 and eGFR was significant for both the unadjusted (p = 0.04) and adjusted models (p for trend = 0.02) and this effect was largely driven by those aged ≥ 65 years .For the unhealthy dietary pattern 2, those in the lowest quintile of adherence had significantly better renal function than those in the reference category, i.e. the highest intake of foods associated with dietary pattern 2. Least adherent quintile: eGFR 76.2 ml/min/1.73m2  compared to the most adherent quintile: eGFR 78.1 ml/min/1.73m2 . These differences remained significant in the adjusted model where the least adherent quintile: eGFR − 1.8 ml/min/1.73m2  compared to the most adherent reference category. The association between adherence to dietary pattern 3 and eGFR was significant for both the unadjusted (p for trend < 0.001) and adjusted models (p for trend = 0.001). In a sensitivity analyses stratified by those aged < 65 years remained significant (P = 0.002), although the association with those aged ≥ 65 years was just above the significance threshold for .For dietary pattern 3, characterised by a high consumption of alcohol and coffee, those with lower adherence had significantly poorer renal function than those with the highest intake of foods associated with this dietary pattern .In this free-living, population-based cohort, comprising adults  ≥ 50 years old in Northern Ireland, three a posteriori dietary patterns were identified. Low adherence to a dietary pattern characterised by consumption of processed meats, white bread, confectionary, fried potatoes, red meat, added sugar, etc. (dietary pattern 2), was associated with higher eGFR and this effect was stronger in those aged < 65 years. In contrast, poor adherence to the two other dietary patterns were associated with lower eGFR. These were dietary pattern 1, characterised by consumption of vegetables, wholegrains, fruits, oily fish, dairy, white fish etc., and dietary pattern 3, characterised by high intakes of drinks such as coffee, wine, beer and other alcohol, but low intakes of butter, margarine, confectionary, higher fibre breads, cereals, natural potatoes and tea. These associations were independent of diabetes status, blood pressure and other measured potential confounding factors.2 increase in eGFR compared to a 6 ml/min/1.73m2 increase in renal function for those in the least adherent group for the unhealthy dietary pattern in the INES. The reductions in eGFR associated with low adherence to dietary patterns 1 and 3 were closer in magnitude at 3.4 ml/min/1.73m2 and 1.75 ml/min/1.73m2. The magnitude of these associations is equivalent to a year or more of annual age-related loss of renal function as reported in the Cardiovascular Health Study [2 in those aged ≥ 65 years versus 81 ml/min/1.73m2 in those aged < 65 years. Given the possibility of over adjustment for age  thereby reducing statistical power, and given its role in estimating glomerular filtration rate, the findings from the sensitivity analyses should be interpreted with caution.There was considerable overlap between the characteristics of the dietary patterns observed and the components of other conceptually healthy and unhealthy dietary patterns. The healthy dietary pattern 1 was characterised by consumption of foods similar to those found in the Mediterranean and DASH diets, while the unhealthy dietary pattern 2 was principally comprised of foods similar to those found in “Western diets” which have been associated with conditions such as diabetes and cardiovascular disease. Likewise, both of these dietary patterns were similar to those reported previously in the geographically overlapping INES , whereinth Study . AlthougDietary pattern 3 from the NICOLA study did not resemble any of the dietary patterns from the INES, despite the similar geographic location. This dietary pattern was characterised by a high intake of coffee, alcohol, crisps and pizza and a low intake of high-fibre breads, spreads and tea. The non-existence of this dietary pattern in the INES population may be unsurprising among those practicing a cloistered and religious lifestyle. Although conceptually unhealthy, low adherence to this dietary pattern was associated with poorer renal function in the current study. However, this finding may yet be related to confounding influences which were not controlled for, or, may result from the high intake of coffee  and alcoSeveral other active food constituents in the dietary patterns observed have been previously associated with renal function. Reduced endogenous antioxidant production has been reported in individuals with end-stage renal disease (ESRD) requiring haemodialysis , and antOily fish are rich in long-chain, n-3 polyunsaturated fatty acids which act on the cyclooxygenase pathway and arachidonic acid pathway to reduce the production of a variety of inflammatory mediators . MoreoveThe findings of this study add further support to previous studies reporting associations between conceptually healthy diets and CKD risk and provide novel evidence for the association between dietary patterns and eGFR in British and Irish populations, for which there is limited data, independent of the effect of diet on diabetes risk, blood pressure and other potential confounding factors.These analyses had several strengths. A large, population-based cohort enabled the generalisation of the findings, and unlike a number of previous studies which were limited in their ability to adjust for important confounding factors, the current study included a wide range of biological and lifestyle covariates. Dietary patterns and CKD prevalence vary by region , 52, andThe study design has several limitations. NICOLA has an inherent sample bias resulting from the recruitment of individuals aged 50 years and older. In addition, the sample excluded institutionalised individuals and those with dementia. Therefore, the generalisability of the study results may not extend to younger individuals, who may commonly adhere to other dietary patterns not identified in the current study, or to individuals with dementia in whom dietary habits may be profoundly affected. Longitudinal and experimental study designs could provide evidence of cause and effect but, at the time of this analysis, only cross-sectional data were available and so firm causal inferences cannot be drawn. Furthermore, dietary data were only captured at one time-point which may not reflect changes in dietary intakes. Direct measurement of glomerular filtration rate would provide an assessment of renal function with smaller inherent error than eGFR measures, however, eGFR was estimated using the clinically relevant CKD-EPI estimating equation and made use of the more accurate SCys and SCr-based measures which also better predicts complications associated with CKD , 56. MorIn summary, the findings of this study provide novel evidence for associations between culturally existing eating patterns and renal function, relevant to British and Irish populations, independent of the effect of diet on diabetes risk, blood pressure and other potential confounding factors. The results of this study will support diet-related primary prevention advice for CKD and inform future longitudinal analyses within the NICOLA.Supplementary file1 (DOCX 23 kb)Below is the link to the electronic supplementary material.


HYDIN) gene and g.61190482A > G  in the ubiquitin-specific protease 34 (USP34) gene. After Sanger sequencing, we found that rs777591“AA” under different genetic models except for the dominant model , could significantly reduce COPD risk, but rs12449210T > A was not related to COPD. In stratified analysis of smoking status, rs777591“AA” reduced COPD risk significantly among the nonsmoker group. Protein and mRNA expression of USP34 in cigarette smoke extract-treated BEAS-2b cells increased significantly compared with those in the control group. Our findings associate the USP34 rs777591“AA” genotype as a protector factor in COPD.Genetic factors are important factors in chronic obstructive pulmonary disease (COPD) onset. Plenty of risk and new causative genes for COPD have been identified in patients of the Chinese Han population. In contrast, we know considerably little concerning the genetics in the Kashi COPD population (Uyghur). This study aims at clarifying the genetic maps regarding COPD susceptibility in Kashi (China). Whole-exome sequencing (WES) was used to analyze three Uyghur families with COPD in Kashi . Sanger sequencing was also used to verify the WES results in 541 unrelated Uyghur COPD patients and 534 Uyghur healthy controls. WES showed 72 single nucleotide variants (SNVs), two deletions, and small insertions (InDels), 26 copy number variants (CNVs), and 34 structural variants (SVs), including g.71230620T > A  in the HYDIN axonemal central pair apparatus protein ( Chronic obstructive pulmonary disease (COPD) is a common and complicated disease of the lungs. It is characterized by continuous and irreversible airflow destruction due to chronic inflammation . As of 2Our epidemiological investigation shows the COPD prevalence in Kashi  to be 17.01% , which iKashi city is located in the northwestern China, and more than 90% of the total local population in Kashi are Uyghurs . The UygHYDIN) gene is located on human chromosome 16 with a length of 423 kb (HYDIN mutations can cause primary ciliary dyskinesia (PCD)  . PCD is ia (PCD) . Respiraia (PCD) .USP34) gene is a member of the ubiquitin-specific protease family. USPs can regulate cell growth  were determined to be studied because they were found for the first time in the Kashi COPD population. Furthermore, the association between the two SNVs and COPD risk have not been previously described. Based on the WES data sets, we continued to evaluate their relationship with clinical characteristics in a case-control study of 1075 people. Then, we hypothesized that the presence of SNVs combined with environmental factors  might regulate gene expression, thereby increasing the risk of COPD. Also, protein and mRNA expression of them were determined in cigarette smoke extract (CSE)-treated bronchial epithelial beas-2b (BEAS-2b) cells.In the present study, eight people with COPD and one healthy person from three Uyghur families with COPD in Kashi were subjected to whole-exome sequencing (WES) to screen for the susceptibility genes and polymorphisms related to COPD. The two single nucleotide variants (SNVs)  were selected for WES. All nine people were permanent residents of Kashi and aged >40 years. These Uyghur families were derived from a previous epidemiological investigation of COPD in Kashi. Families had ≥2 generations with COPD in three generations. The inclusion and exclusion criteria for the subjects were described by 2 Vacutainer tubes for DNA extraction. Apart from blood samples, the basic information of patients and HC were also collected: age, sex, body mass index (BMI), pulmonary function, smoking history, and other data. All pulmonary function tests were undertaken using a spirometer .Peripheral blood samples (5 ml) were obtained from each participant and transferred to EDTA-K1)/forced vital capacity (FVC) < 0.70 after bronchodilator inhalation denotes airflow limitation. The exclusion criteria are identical to COPD family patients. For the HC group, the exclusion criteria were the same as HC of COPD family.Recruited individuals were aged >40 years and from Kashi during 2018–2019. The study cohort was 541 Uyghur unrelated COPD patients and 534 Uyghur HCs from the First People’s Hospital of Kashi. For the COPD group, the inclusion criteria were people whose forced expiratory volume in the first second  were employed for statistical analyses. Quantitative data are the mean ± SD or median (interquartile range). The independent t-test was used to compare the difference between age and BMI. We used the chi-square test to ascertain the influence of sex, smoking status, coal consumption, wood consumption, cigarettes per day, and cumulative quantity of active smoking. The difference in quitting smoking years was assessed by Fisher’s exact test. For parameters that did not have a normal distribution , we used the Mann–Whitney U-test to evaluate differences. We tested for the Hardy–Weinberg equilibrium on each SNV among samples in the case-control study. Akaike’s information criteria (AIC) were used to calculate the genetic model for rs12449210 T > A and rs777591A > G . We used odds ratios (ORs) and corresponding 95% confidence intervals (CIs) by logistic regression analysis after correcting for sex, age, BMI, and smoking status to assess the relationship between SNVs and case–control groups. R function genpwr in package devtools was used to perform post hoc power estimation. The homogeneity test of stratified analyses was evaluated by the chi-square test. In the enrichment analyses, verification criteria of multiple SNVs were evaluated using the false discovery rate (FDR) with multiple corrections. The experimental data of Western blotting and real-time RT-qPCR were the mean ± SEM, and differences between groups were evaluated by one-way ANOVA. The replicates for the experiments were at least three times. p < .05 was considered significant.SPSS 18  and PLINK v1.07 , 2915 copy number variants (CNVs), and 29,016 structural variants (SVs). To facilitate observation of all variants in family members with COPD, we used Circos to display all variants .After WES, we obtained 100,193 SNVs. We undertook statistical analyses of all SNVs . We founThen, 3096 SNVs were obtained after calculating the corresponding scores, SIFT and PolyPhen-2 . FinallyThe top three regions with the largest number of InDels were the intron (71.90%), exon (8.28%), and noncoding RNA (8.28%) . FinallyAfter drawing a heat map for all 2915 CNVs, we detected the gain and loss of each chromosome . FinallyAfter analyzing the distribution of SVs in different regions of the genome, we found that the top three regions with the largest number of SVs were the intron (55.99%), exon (18.65%), and 3′ UTR (8.24%) . FinallyIn conclusion, we obtained 72 SNVs, two CNVs, 26 InDels, and 34 SVs by the aforementioned filtering strategy.q-value GO terms, pathways, and diseases to draw a bubble chart  but not for annual household income, coal consumption, wood consumption, cigarettes per day, cumulative quantity of active smoking, and quitting smoking years   .p > .05) (Rs12449210T > A and rs777591A > G both met the criteria for the Hardy–Weinberg equilibrium (p > .05) . Therefode novo mutations  and 99.44% .We undertook genetic model analysis  on the SNVs of rs12449210T > A and rs777591A > G . Becauseutations  in the pUSP34, after correcting for sex, age, smoking status, and BMI, this site significantly decreased the risk of COPD with the “AA” genotype based on the genotype model  and recessive model . In the allele model, the “A” genotype was related to a reduced risk of COPD . For the additive model, a reduced tendency of COPD risk was also present .For rs777591A > G located on HYDIN before correction “TA” or “AA + TA” increased the COPD risk under the genotype and dominant models . After correction, a significant effect was not present in the five models (p > .05).For rs12449210T > A located on 1%.We undertook stratified analysis for the relationship between rs12449210T > A and rs777591A > G and COPD risk based on smoking status and FEVThe beneficial effects of “AA” in rs777591A > G was more evident in nonsmokers in the genotype model , recessive model , allele model , and additive model . However, in the smoker group, the protective effects were not significant .For rs12449210T > A , no diff1%  at the genetic level, and found 72 SNVs of 55 genes that might be associated with COPD. Scholars have found four genes that might be related to COPD using WES in 49 families with COPD: mericans . Also, tpothesis  and mostpothesis . We did pothesis . ConsequWe evaluated only one HC as a reference; the selected 72 SNVs might contain the SNVs of healthy people. Hence, it was necessary to verify the relationship between these 72 SNVs and COPD in a case-control study.via NCBI database: https://www.ncbi.nlm.nih.gov/). Therefore, our data determined “AA” in rs777591A > G (USP34) to be a protective factor.In the case-control study of two SNVs (rs12449210T > A and rs777591A > G), they were analyzed through genetic models, tobacco smoking, and pulmonary function. Because the PLINK software defaulted a higher mutation frequency to a wild type, in Asia, the “G” mutant frequency of rs777591A > G was higher than “A”  signal transduction by deubiquitinating and stabilizing the NF-κB inhibitor Iκbα (USP34 might have a key role in the occurrence and development of COPD. rs777591A > G is located in the intron region at HaploReg v4.1 (ANNOVAR annotated it in 3′UTR of USP34). Although rs777591A > G is located in the noncoding regions, genome-wide association studies (GWASs) indicate that only 7% single nucleotide polymorphisms (SNPs) related to complex diseases were located in the coding regions, whereas 93% were in the noncoding regions  are also required.USP34 was a protective factor against COPD, especially in the nonsmoking population. USP34 mRNA and protein expression levels were increased in CSE-treated BEAS-2b cells. Therefore, our data provide new clues for the relationship between USP34 polymorphisms and COPD susceptibility in a Chinese Uyghur population.In the present study, WES revealed 72 SNVs, two CNVs, 26 InDels, and 34 SVs in three families with COPD. rs777591 “AA” of


Myasthenia gravis (MG) is an autoimmune disorder that frequently affects females at reproductive age. Herein, we aimed to assess the associations of clinical factors with pregnancy-related outcome in MG.We searched PubMed and EMBASE for case–control and cohort studies that reported the MG status during or after pregnancy and relevant clinical variables. The data was extracted in proportions and odds ratios (ORs) with 95% confidence intervals (CIs) in subsequent meta-analysis.Fifteen eligible articles reporting on 734 pregnancies with 193 worsening and 51 improved episodes were included out of 1765 records. The estimated worsening proportions in total, antepartum and postpartum periods were 0.36 (95% CI 0.25–0.40), 0.23 (95% CI 0.14–0.34) and 0.11 (95% CI 0.04–0.22) respectively. The proportion of pregnancy-related improvement in enrolled patients was 0.28 (95% CI 0.17–0.40), with 0.07 (95% CI 0.00–0.28) during pregnancy and 0.14 (95% CI 0.02–0.34) after pregnancy. No significant associations were disclosed between the clinical factors and MG worsening. Thymectomy before delivery is a strong predictor for MG improvement in postpartum period .The total proportion of pregnancy-related MG worsening and improvement in MG was 0.36 (95% CI 0.25–0.40) and 0.28 (95% CI 0.17–0.40), respectively. Thymectomy before the delivery may aid in clinical improvements associated with pregnancy. Future prospective cohort studies are required to determine more relevant factors.The online version contains supplementary material available at 10.1186/s13023-022-02205-z. Myasthenia gravis (MG) is an autoimmune disorder in neuromuscular junction with a prevalence ranged from 15 to 179 per million person . MG has The overall proportion for clinical worsening ranged from 10 to 90% in MG patients with pregnancy, with varied distributions in 1st, 2nd, 3rd trimester or puerperium . A shortTo date, there is no comprehensive meta-analysis on risk or protective factors for the course of MG during/after pregnancy. Given the current inconclusive results, our review mainly aims to assess the clinical status in female MG patients during or after pregnancy, and explore the associations between the clinical factors with MG exacerbation or improvement. Herein, we conducted a systemic review and meta-analysis to estimate the overall proportion for MG worsening related with pregnancy and to identify risk factors that predict pregnancy-related clinical outcome.The systematic review was reported according to the PRISMA  statement . EthicalTo identify all cases who had an episode of MG worsening and improvement during/after pregnancy, we searched PubMed and Embase with a last update on 12 August, 2021 with search terms including “myasthenia”, “myasthenia gravis”, “pregnancy”, “pregnant”, “labor”, “labour”, “maternal” and “gestation”. Reference lists of articles were also examined, and full-text papers were accessed and analyzed by all authors.Studies eligible for the systemic review and meta-analysis met the pre-defined criteria: (1) case–control or cohort studies; (2) MG was diagnosed before or during pregnancy with supportive evidence from clinical, neurophysiological or serological testing; (3) Full description of MG status before/during/after pregnancy can be clearly identified; (4) a publication written in English to permit easy access to the source information of all included articles.Articles were excluded based on the following exclusion criteria: (1) less than 5 cases included; (2) publication out of scope such as single case reports, case series, reviews, conference abstracts, commentary editorials or letters that reported no new data.Included studies were scrutinized for potential clinical data of myasthenia outcomes related to pregnancy. For the initial screening, the studies were independently evaluated by two authors (MS and XL) for inclusion. The conflicts if any were resolved after mutual discussions with the third author (SL). When studies included the overlapping data from the same patient sources, data from the largest cohort will be retained for further analysis. The following data were also extracted in a standard form from each study: name of the first author, year of publication, country, study design, number of patients, number of pregnancies, number of miscarriages, times of MG worsening and improvement, definition of worsening and improvement  improvement of clinical myasthenic manifestations or a significant reduction in MG medications than those they received before pregnancy; (2) clearly described as “improved” or “better” in original articles , otherwise the random effects analysis was used. Statistical significance was set at p < 0.05 with a 95% CI. Statistical tests of Funnel plots were conducted to visualize the publication bias (N > 10) and Egger’s test was used to assess funnel plot asymmetry. Trim and fill methods were then used to detect symmetry. To evaluate the stability of results and explain the possible source of heterogeneity (N > 2), sensitivity analysis for potential risk factors was performed using the leave-one-out analysis by omitting one study at a time. A quantitative synthesis of the collective findings was performed through R language (v. 4.1.1) (http://www.r-project.org) with the meta package [Proportions and ORs were extracted and calculated based on raw data from primary studies. Freeman-Turkey double arcsine transformation method was used to calculate the combined proportion of worsening in different and the 95% confidence interval (CI). Q test and I package .Various database searches resulted in 1765 records, of which 386 had to be excluded on the basis of titles or abstracts and 1379 were identified for full-text review. Finally, we identified 15 cohort studies that fulfilled the inclusion criteria and included in qualitive synthesis   (N = 8) , the trim and fill method was applied to analyze the publication bias following original funnel plots  and 0.28 (95% CI 0.17–0.40) respectively. From the nine studies entered the final analysis for preferential trimester, a slightly elevated worsening proportion during the third trimester was observed .RUNX3 [During and after pregnancy, distinct hormones with different concentrations may influence the immune network for MG pathogenesis in bidirectional modulation effect. In comparison to healthy controls, MG patients with anti- AChR/MuSK antibodies had low levels of galactosylated IgG2 . In contRUNX3 . RegulatRUNX3 , 36. HowRUNX3 . ProgestRUNX3 , 39. OthRUNX3 .As one of the mainstays in the treatments of myasthenia gravis, thymectomy has been accepted as a beneficial and effective treatment that is recommended for all patients concurrence with thymoma or refractory generalized MG with positive AChR antibodies –43. For There are several limitations that exist in our review. As noted already, the publication bias existed in our analysis, as there was a lack of high-qualified investigations in this field such as randomized trials and longitudinal prospective cohort studies. As the objectives and methods were heterogeneous in enrolled studies, the meta-analysis was performed across different designs, various definitions for MG worsening. There was a limitation of results as we could only provide overestimation of results as many concerned factors could not be analyzed. Future prospective cohort studies are anticipated to explore the optimal managements in controlling myasthenia gravis during or after pregnancies.The MG worsening in relation to pregnancy was still unpredictable, which could occur in every period during/after pregnancy. Thymectomy before delivery did not significantly decreased the risk of worsening but was found to improve the prognosis. For the better management of MG around pregnancy, thymectomy can be recommended to pursue a better short-term prognosis.Additional file 1. Characteristics of individuals from included studies except case reports.Additional file 2. Newcastle–Ottawa scale for assessing the quality of the included studies in meta-analysis.Additional file 3. Myasthenia gravis status scale and definitions.Additional file 4. Details of risk factors for worsening not included in the meta-analysis .Additional file 5. Details of clinical factors for improvement mot included in the meta-analysis .Additional file 6. Funnel plots of the meta-analysis before and after applying the trim-and-fill method.


Asthma is a chronic inflammatory disorder of the airways involving many different factors. This study aimed to screen for the critical genes using DNA methylation/CpGs and miRNAs involved in childhood atopic asthma.DNA methylation and gene expression data (Access Numbers GSE40732 and GSE40576) were downloaded from the Gene Expression Omnibus database. Each set contains 194 peripheral blood mononuclear cell (PBMC) samples of 97 children with atopic asthma and 97 control children. Differentially expressed genes (DEGs) with DNA methylation changes were identified. Pearson correlation analysis was used to select genes with an opposite direction of expression and differences in methylation levels, and then Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Protein–protein interaction network and miRNA–target gene regulatory networks were then constructed. Finally, important genes related to asthma were screened.CCL2, directly related to asthma, was involved. This gene is targeted by eight asthma related miRNAs . The genes IL2RG and CCl4 were also involved in this pathway.A total of 130 critical DEGs with DNA methylation changes were screened from children with atopic asthma and compared with control samples from healthy children. GO and KEGG pathway enrichment analysis found that critical genes were primarily related to 24 GO terms and 10 KEGG pathways. In the miRNA–target gene regulatory networks, 9 KEGG pathways were identified. Analysis of the miRNA–target gene network noted an overlapping KEGG signaling pathway, hsa04060: cytokine-cytokine receptor interaction, in which the gene The present study provides a novel insight into the underlying molecular mechanism of childhood atopic asthma. Asthma is a respiratory disease caused by the interaction of genetic and environmental factors, known to be mediated by epigenetics . ApproxiCTLA4) and interleukin-10 (IL-10) which are involved in immune system regulation and inflammation [Genetic variants drive the onset and development of asthma, including cytotoxic T-lymphocyte-associated protein 4  [The DNA methylation dataset, superserie GSE40736, was downloaded from the National Center of Biotechnology Information Gene Expression Omnibus (GEO) database(ih.gov/) , which chttps://bioconductor.org/packages/release/bioc/html/limma.html) [After downloading the original microarray data, the limma package in R software   was usedhttps://cran.r-project.org/package=pheatmap) [Moreover, the pheatmap package   in R sofheatmap) , 18. Thehttps://stat.ethz.ch/R-manual/R-devel/library/stats/html/cor.test.html) was used to calculate the Pearson correlation coefficient. The expression and methylation level differences in opposite directions were reserved for further analysis. Subsequently, the Database for Annotation, Visualization and Integrated Discovery tool, version 6.8 [https://david.ncifcrf.gov/) was used to perform GO function and KEGG pathway enrichment analysis for the mRNAs with an opposite direction of difference in expression and methylation levels. The threshold value was considered to be P < 0.05.Initially, we compared the collection of DEGs and DMGs, kept the intersection of the two data sets, and analyzed the overall correlation between the degree of difference in methylation and expression levels. The cor.test function , was used to search for the interaction between gene product proteins for genes with opposite expression and methylation levels, and an interactive network was built. The interactive network was visualized by Cytoscape version 3.7.2 software [http://www.cytoscape.org/). The GO biological process and KEGG signal pathway analysis based on DAVID were then performed on the gene nodes that constituted the interaction network. The threshold value was considered to be P < 0.05.String, version 10.5  (https:/software  (http://http://www.cuilab.cn/hmdd) to search for miRNAs directly associated with asthma. The target genes of asthma miRNAs were then screened using the starBase version 2.0 database [http://starbase.sysu.edu.cn/). The starBase database provides the comprehensive target gene prediction information from five databases: TargetScan, picTar, RNA22, PITA, and miRanda. We selected regulatory relationships included in at least one of the databases as miRNA-target gene relationship pairs to construct a miRNA -mRNA regulation relationship. Cytoscape 3.7.2 was used to display the networks. Finally, KEGG pathway analysis for target genes was performed using DAVID software.We used the Human MicroRNA Disease Database  , using “asthmatic” as a keyword, we searched for KEGG pathways and genes directly related to asthma, and compared them with pathways in which the genes in the constructed interaction network were significantly involved in the relevant pathways. We selected disease pathways with direct involvement of the asthma genes, constructed this part of the network separately, screened genes directly related to the disease, and conducted mechanism research through the important pathways of gene participation.In the Comparative Toxicogenomics Database, 2019 update   DEGs and 751 (412 hypomethylated and 339 hypermethylated) DMGs were identified between the asthmatic and healthy control groups. Volcano plots for the DEGs and differentially methylated sites were shown in Fig. We screened a total of 284 intersection genes that were differentially expressed in the DNA methylation and gene expression data set, and analyzed the relationship between gene expression and DNA methylation changes by calculating the correlation coefficient Fig. . The GO In the protein–protein interaction network, a total of 119 nodes were identified. This included 33 hypermethylated, downregulated genes and 86 hypomethylated, upregulated genes with 426 pairs of co-expression interactions , which is annotated to HADH, PCCA, and ACAA1; pyrimidine metabolism (P = 9.54e− 3), which is annotated to ENTPD5, POLR3A, and ENTPD1; adhesion molecules (P = 1.64e− 2), which is annotated to NCAM1, CDH1, and SPN; neuroactive ligand-receptor interaction (P = 1.65e− 2), which is annotated to in S1PR2, S1PR3, S1PR1, and S1PR5; primary immunodeficiency (P = 1.76e− 2), which is annotated to IL2RG, and RFXANK; fatty acid metabolism (P = 1.99e− 2), which is annotated to HADH and ACAA1; purine metabolism (P = 2.05e− 2), which is annotated to ENTPD5, POLR3A, and ENTPD1; cytosolic DNA-sensing pathway (P = 2.63e− 2), which is annotated to POLR3A and CCL4, and cytokine − cytokine receptor interaction (P = 4.27e− 2), which is annotated to CCL2, IL2RG, and CCL4.As noted in Table CCL2) gene directly related to asthma is involved. This gene is targeted by eight asthma related miRNAs . As noted in Fig. IL2RG and CCl4, are involved in this pathway.We screened 119 KEGG pathways and 116 genes that were directly associated with asthma by searching the CTD database. After comparison with the genes in the constructed regulatory network and the pathways in which genes participate significantly, an overlapping KEGG signaling pathway, hsa04060: cytokine-cytokine receptor interaction, was obtained in which the C−C motif chemokine ligand 2 (CCL2 gene directly related to asthma is involved, and the gene is targeted by 8 asthma related miRNAs. Two other genes, IL2RG and CCL4, are known to be involved in this pathway.Asthma is a complex multifactorial disease caused by the interaction of genetic and environmental factors. In our study, the hub genes were explored via an analysis of multiple data sets that included samples from asthmatics and healthy controls. A total of 130 critical DEGs that were differentially expressed in DNA methylome were detected. In the miRNA−target gene regulatory network directly related to asthma, an overlapping KEGG pathway, hsa04060: cytokine−cytokine receptor interaction, was noted, in which the CCL2, also known as MCP-1, is one of several cytokine genes clustered on the q-arm of chromosome 17. Chemokines are a superfamily of secreted proteins involved in immunoregulatory and inflammatory processes. CCL2 is a member of the CC subfamily which is characterized by two adjacent cysteine residues. It binds to chemokine receptors CCR2 and CCR4. The results of our study also show that CCL2 and CCl4 are involved in the cyclokine receptor interaction signaling pathway. CCL2 is closely involved in the inflammatory response in children with asthma [h asthma . Multiplh asthma . Downregh asthma . Chen eth asthma . These fIL2RG, is an important signaling component of many interleukin receptors, including IL-2, IL-4, IL-7, and IL-21, and is thus referred to as the common gamma chain [IL2RG was involved in primary immunodeficiency and cytokine-cytokine receptor interaction. We speculate that IL2RG and CCL4 might be important genes related to childhood atopic asthma, and together with CCL2 participate in the cytokine receptor interaction signaling pathway that plays a role in childhood atopic asthma.Interleukin 2 receptor subunit gamma, the protein encoded by ma chain . Mutatioma chain . The patThere are some limitations in this study. The key genes obtained in this study have not been further verified. In addition, the samples in these two data sets are all PBMCs, which would be more convincing if they were airway epithelial cells. But our research provides new biological insights into the development of asthma.CCL2 gene, directly related to asthma, is involved, and this gene is targeted by eight asthma related miRNAs. We speculate that IL2RG and CCL4, which are also involved in this pathway, might be critical genes related to childhood specific asthma, and together with CCL2 play a role in this disease. The bioinformatics analysis in this study may provide a valuable and reliable basis for determining biomarkers for the development of childhood atopic asthma.In conclusion, our study identified a total of 130 DEGs with significant DNA methylation changes. In a regulatory network directly related to asthma, the KEGG signaling pathway hsa04060:cyclokine-cyclokine receptor interaction was found, in which the


Biston thoracicaria (Lepidoptera: Geometridae) is 15,538 bp in length, containing 13 PCGs, 22 tRNAs, two rRNAs, and an A + T-rich region. All PCGs initiate with typical start codon of ATN and share the complete stop codon of TAA, whereas cox1 starts with CGA. The ML analysis was performed using a dataset matrix containing 13 PCGs concatenated from the mitogenomes of Geometridae species. Our study presented the phylogenetic relationship of (Larentiinae + ((Sterrhinae + (Ennominae + Geometrinae))). Within the genera Biston, B. thoracicaria grouped with other species as the sister group.The complete mitochondrial genome (mitogenome) of  Biston betularia) was a typical example for evolutionary study in 20th century, allowing biologists to solve questions about gene flow, rates of selection, and the neutral theory  under the voucher number 20200540. Total genomic DNA was extracted from a single female with the QIAamp DNA Mini Kit  following the manufacturer’s instruction. The sample was sequenced by Illumina HiSeq 4000 in Novogene , with a read length of 150 bp and the size of raw data 7.1 Gb (GenBank accession No. SRR13958261). The mitogenome was assembled using GetOrganelle v1.6.4 program   in July 2020, and the species was identified by Wu Yupeng from Taiyuan University of Science and Technology. A specimen was deposited at Herbarium of Institute of Plant Protection, Shanxi Academy of Agricultural Sciences (B. thoracicaria (GenBank accession No. MN956510) is a circular molecule of 15,538 bp in length. It contains the typical set of 37 genes as in most insect mitogenomes (Cameron cox1 starts with CGA. The rrnL is located between trnL1 and trnV, with the length of 1399 bp. The rrnS is located between trnV and the A + T-rich region, with the length of 778 bp. The control region is 446 bp in length, and is located between rrnS and trnM.The complete mitogenome of  Cameron , includi Cameron . The J-sBiston, B. thoracicaria grouped with other species as the sister group.Phylogenetic analysis was performed on concatenated nucleotide sequences of 13 PCGs derived from 18 Geometridae species, with three Larentiinae species as the outgroups. Phylogenetic inference was conducted using IQ-TREE 1.6.12 (Nguyen et al.


Oil spills in the marine environment have a devastating effect on marine life and biogeochemical cycles through bioaccumulation of toxic hydrocarbons and oxygen depletion by hydrocarbon-degrading bacteria. Oil-degrading bacteria occur naturally in the ocean, especially where they are supported by chronic inputs of oil or other organic carbon sources, and have a significant role in degradation of oil spills. Colwellia and Cycloclasticus. We show that metagenome-assembled genomes (MAGs) belonged to these naphthalene degraders by matching their 16S-rRNA gene with experimental stable isotope probing data. Surprisingly, we did not find a full PAH degradation pathway in those genomes, even when combining genes from the entire microbial community, leading us to hypothesize that promiscuous dehydrogenases replace canonical naphthalene degradation enzymes in this site. We compared metabolic pathways identified in 29 genomes whose abundance increased in the presence of naphthalene to generate genomic-based recommendations for future optimization of PAH bioremediation at the POLA, e.g., ammonium as opposed to urea, heme or hemoproteins as an iron source, and polar amino acids.Bacterial biodegradation is a significant contributor to remineralization of polycyclic aromatic hydrocarbons (PAHs)—toxic and recalcitrant components of crude oil as well as by-products of partial combustion chronically introduced into seawater via atmospheric deposition. The Deepwater Horizon oil spill demonstrated the speed at which a seed PAH-degrading community maintained by chronic inputs responds to acute pollution. We investigated the diversity and functional potential of a similar seed community in the chronically polluted Port of Los Angeles (POLA), using stable isotope probing with naphthalene, deep-sequenced metagenomes, and carbon incorporation rate measurements at the port and in two sites in the San Pedro Channel. We demonstrate the ability of the community of degraders at the POLA to incorporate carbon from naphthalene, leading to a quick shift in microbial community composition to be dominated by the normally rare IMPORTANCE Oil spills in the marine environment have a devastating effect on marine life and biogeochemical cycles through bioaccumulation of toxic hydrocarbons and oxygen depletion by hydrocarbon-degrading bacteria. Oil-degrading bacteria occur naturally in the ocean, especially where they are supported by chronic inputs of oil or other organic carbon sources, and have a significant role in degradation of oil spills. Polycyclic aromatic hydrocarbons are the most persistent and toxic component of crude oil. Therefore, the bacteria that can break those molecules down are of particular importance. We identified such bacteria at the Port of Los Angeles (POLA), one of the busiest ports worldwide, and characterized their metabolic capabilities. We propose chemical targets based on those analyses to stimulate the activity of these bacteria in case of an oil spill in the Port POLA. Cycloclasticus, Colwellia, Pseudomonas, Alteromonas, and others (12–12–Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant, mutagenic, and carcinogenic components of fossil fuel as well as by-products of incomplete combustion but are also part of cosmic dust, hydrothermal vent plumes, and algae , 2. Micr6–d others , 12–14. thers 12–, 12–18. 13C-labeled PAHs are added to samples and incorporated into the DNA of PAH-utilizers, causing its density to increase. Heavier DNA can then be physically separated in a density gradient. A large-scale SIP study was performed on DWH surface and deep-plume water, revealing local strains of PAH-degrading bacteria that responded to the input of hydrocarbons (13C-labeled) fractions under the assumption that the most heavily labeled organisms, and thus the main targets, will be found there. However, this strategy may lead to overlooking degraders with low-GC  genomes, whose DNA may not appear in the heaviest fractions even if they include moderate amounts of 13C (Stable isotope probing (SIP) is a well-established method for the identification of environmental bacteria utilizing targeted substrates, in this case PAHs , 20. 13Cocarbons . Some geocarbons . The stus of 13C , 23. Tags of 13C , 25.One of the main motivations to study PAH-degrading organisms is to characterize their metabolic requirements. An understanding of the suite of nutrients and cofactors those organisms require could potentially be applied toward bioremediation and biostimulation . The com27–13C-from PAHs. Naphthalene was used as a model substrate to select for communities involved in catabolism of PAHs  would be major incorporators of  of PAHs . We thenOur main hypothesis was that at the Port of Los Angeles (POLA) there would be a community of PAH-degrading bacteria likely sustained by chronic inputs and eutrophication. One indication of the existence of such a community would be measurable uptake of naphthalene-derived carbon upon amendment with PAHs, in this case naphthalene. We also wanted to test whether this degradation potential extended out into the San Pedro Channel at the San Pedro Ocean Time-series (SPOT) and Two Harbors (CAT) . Isotopi13C-naphthalene-enrichment of POLA seawater led to significant incorporation of labeled carbon (>0.005 g · ml−1 buoyant density increase corresponding to 9 atom percent excess) by 34 out of 180 operational taxonomic units (OTUs) (Colwellia spp. and Cycloclasticus spp. (Gammaproteobacteria) made up 40% of the planktonic (0.2 to 1 μ) microbial community. These OTUs were enriched at 53 (Colwellia) and 47 (Cycloclasticus) atom percent excess (Colwellia or Cycloclasticus) to nondetectable prior to enrichment (t0) at all sites on all dates. While both taxa were represented by multiple OTUs, there was always a dominant OTU which matched the 16S-rRNA genes from the metagenome-assembled genomes . The most abundant OTU accounted for 95% and 99% of the Colwellia and Cycloclasticus amplicons, respectively. Additional significantly enriched OTUs included Gammaproteobacteria , Flavobacteriales , Sphingobacteriales, Deferribacterales (Marine group A), and Rhodospirillales (The s (OTUs) . After 8t excess  , taxonomy determined using GTDB-Tk, Anvi’o, GToTree, and 16S-rRNA and mean coverage in metagenomes (second and third quartiles—Anvi’o setting Q2Q3). Download Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 0) metagenomes to those MAGs in order to pinpoint potential degraders, under the assumption that potential degraders would be more abundant in amended metagenomes than in t0 metagenomes  and less than 10% redundant  from naphthalene-amended POLA water. We then mapped reads from naphthalene-amended and unamended  only in naphthalene-amended POLA water from May 2015 and October 2014 , which is the first step in naphthalene degradation, but only parts of the remainder of the pathway known from pure cultures    and S5.  pathway . This ory nirBD; .10.1128/mSystems.00245-21.2FIG S2Colwellia and Cycloclasticus MAGs are labeled with a star. Download FIG S2, PDF file, 0.5 MB.Basic metabolic pathways in MAGs represented as fractions of the enzymes present. Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00245-21.4FIG S4Colwellia and Cycloclasticus MAGs. The MAGs are labeled in purple. The tree is based on 117 single-copy genes of Gammaproteobacteria and was built in GToTree. Complete reference genomes were downloaded from RefSeq on 20 August 2019. Download FIG S4, PDF file, 0.2 MB.Phylogenomic tree showing the placement of our Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00245-21.8DATA SET S3Data Set S3, XLSX file, 0.1 MB.Results of METABOLIC analysis of the 29 MAGs whose abundance increased in the presence of naphthalene. Each column represents a MAG, and each row represent a KEGG module. Download Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00245-21.9DATA SET S4Colwellia MAG and Cycloclasticus MAG. Download Data Set S4, XLSX file, 0.5 MB.Results of DRAM hits to open reading frames of the Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Cycloclasticus MAG  was detected in POLA naphthalene-amended metagenomes from May 2015, July 2014, and October 2014 (Cycloclasticus zancles 78-ME (Cycloclasticus MAG assembled from the Deepwater Horizon oil spill (nahD), trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (nahE), and catechol 2,3-dioxygenase (xylE) , which may also point to a potential antibacterial activity.Both MAGs contained secondary metabolite clusters of homoserine lactone (hserlactone) and lasso protein, which imply quorum sensing and a potential antimicrobial activity, respectively , 36. TheColwellia and Cycloclasticus, 27 MAGs were significantly more abundant in naphthalene-amended samples . One of these MAGs, Bin_47_1 (Porticoccus) contained a 16S-rRNA gene that matched at 100% identity to an OTU that was significantly enriched with naphthalene-derived 13C.In addition to l-rhamnosidase, beta-xylosidase, and pullulanase  degradation pathways defined by pure culture studies, but 12 of them had the KEGG pathway for degradation of the aromatic hydrocarbon cymene . Howeverlulanase .Colwellia and Cycloclasticus, nine of these MAGs also contained secondary metabolite clusters of homoserine lactone , terpene , and bacteriocin (Rhodobacteraceae).Similar to nahA, nahD, nahE, and salicylate hydroxylase, but not nahB, nahC, or salicylaldehyde dehydrogenase.Since several studies suggested that full degradation of PAHs may require bacterial consortia , 37, we 13C metagenome against all nucleotide sequences of naphthalene dehydrogenase ferredoxin subunit  from NCBI GenBank . We chose this gene because it was not represented in the MAGs but is necessary for the function of naphthalene-1,2-dioxygenase. We found 248 reads  that hit with a relaxed cutoff of E value 10−3 . Using a conservative estimate that this gene makes up 0.1% of the metagenome (similar to 16S-rRNA), and assuming one copy per cell and that each read maps to one copy of the gene, this would imply that only 8 out of 10,000 cells carry the naphthalene dioxygenase nahAc gene. This fraction would decrease even further if we dropped the single-copy and one read per gene assumptions.Moreover, to confirm that we did not miss additional naphthalene degradation genes because they did not assemble into contigs, we searched all forward reads from the POLA May 2015 In order to identify potential targets for biostimulation experiments, i.e., potentially limiting substances that might aid oil degradation if added after a spill, we performed a cross-comparison of the 29 MAGs that were significantly more abundant in naphthalene-amended water and searched for amino acid biosynthetic pathways, vitamin synthesis, potential nitrogen sources, and transporters.l-amino acids.While most MAGs had the ability to synthesize hydrophobic amino acids  and some polar amino acids , as well as charged amino acid lysine and amphipathic amino acid tryptophan, only one had the biosynthetic pathway for methionine, and none had biosynthetic pathways for arginine, phenylalanine, or tyrosine. However, 15 MAGs had a putative transport system for polar amino acids or branched amino acids, and 9 had a transport system for Colwellia and Cycloclasticus, eight MAGs can degrade urea to ammonia via urease, and four MAGs have genes coding for nirBD nitrite reductase. Five MAGs contain a phosphonate transport system whereas twenty, including Colwellia and Cycloclasticus, can transfer phosphate.Only five MAGs contained a urea transport system, and while 34% of the naphthalene-enriched genomes had a urease gene, so did 43% of the genomes that did not respond to naphthalene amendment. In addition to Colwellia and Cycloclasticus, 4 MAGs can synthesize biotin and 12 MAGs can synthesize pantothenate (vitamin B5). Colwellia and four additional MAGs can synthesize riboflavin (vitamin B2)  .Rhodobacteraceae and two Halioglobus), and 18 encode proteorhodopsins  and Rhodobacteraceae. Both Pseudohongiella and OM182 MAGs were matched by 16S-rRNA genes to OTUs that did not incorporate carbon from naphthalene, indicating that their increased abundance in the presence of naphthalene is not due to them being primary degraders. We speculate that their increased abundance in amended water is due to tolerance to naphthalene rather than utilization of naphthalene as a carbon source. We also note a difference in response time between the primary degraders. In three out of four 24-h incubations, Cycloclasticus abundance was already comparable to its abundance after 88 h, whereas Colwellia was only abundant in one 24-h incubation, and its abundance was orders of magnitude lower compared to 88 h. This may indicate a difference in growth rates and/or utilization of substrates and nutrients, as both taxa were undetectable in unamended water. Gutierez et al. . Incubation in the dark (88 h) would promote heterotrophy, while incubation in the light should still allow cyanobacteria and photosynthetic picoeukaryotes to compete over nutrients with potential naphthalene degraders. Sixteen of the MAGs that were enriched in amended 24-h incubations contained genes coding for proteorhodopsin. Proteorhodopsin can enhance ATP synthesis –45 and s–45–13C-labeled. Only one of these MAGs, representing Porticoccus, a bacterium previously associated with PAH degradation  but were still significantly enriched (9 to 15%). While two of them  may be involved directly in degradation of naphthalene but either grow more slowly or were simply outcompeted by Colwellia and Cycloclasticus   .Cycloclasticus has been shown to completely degrade PAHs in culture  oil spill , 21. WitColwellia and Cycloclasticus were nondetectable in both 16S-rRNA amplicons and MAG coverage before enrichment. However, after 88 h of incubation, they were the most dominant taxa in the mesocosms and exhibited very significant enrichment, indicating incorporation of naphthalene-derived carbon into their DNA over a substantial number of replication cycles.Both Colwellia MAG was the only one assembled here that contained two of the four annotated subunits of naphthalene 1,2-dioxygenase . As this is the first step of the pathway, requiring investment of reducing power , which could control transcription of hydrocarbon degradation genes. Dioxygenases require iron and an iron-binding domain, such as ferredoxin, that can be shared by multiple enzymes  pathway and nitrite/nitrate transporters , as well as a urea transporter and the ammonium-assimilating glutamate synthase pathway. Nitrate, nitrite, and ammonium are always detectable at POLA in surface seawater  or Fe(III), as 27/29 enriched MAGs contain heme transporters. Marine bacterioplankton have been shown to be able to incorporate iron from heme groups , 68. FinIn the current climate of excessive use of fossil fuels, chronic deposition of toxic and recalcitrant polycyclic aromatic hydrocarbons into the coastal ocean is inevitable. PAH-degrading bacteria may provide some control over the remineralization of these inputs and could serve as targets for bioremediation technologies. Identification of naturally occurring biodegraders is a crucial first step, but optimization of the degradation process requires knowledge of the metabolic requirements of local organisms . Moreovehttps://etc-cte.ec.gc.ca/databases/OilProperties/pdf/WEB_Marine_Diesel_Fuel_Oil.pdf], 22 November 2017), and it is surrounded by the second-largest metropolitan area in the United States, which is likely a source of consistent atmospheric PAH deposition. The LA-Long Beach port is the busiest port in the United States and the 10th-busiest in the world according to the International Association of Ports and Harbors . A report published in 2010 revealed detectable levels of many PAHs at various stations within the port and as far as outside the port entrance, corresponding to our sampling site (While the Port of Los Angeles (POLA) is not routinely monitored for PAHs, it is located in an area with natural oil seeps, it houses a ship-refueling station of high-aromatic-content marine diesel  naphthalene or fully labeled 13C-10-naphthalene  as crystals at a maximum final concentration of 400 nM was added to each 10-liter container of water collected without replication. This concentration is roughly 3 orders of magnitude lower than the solubility of naphthalene in water (30 mg/liter = 234 μM). Ammonium-chloride at a final concentration of 1 μM was also added to each container to prevent nitrogen limitation of PAH degraders. Naphthalene-amended seawater from all three sites from July and October 2014 was incubated in 10% ambient light, which is comparable to the light at 5-m depth at SPOT, at surface water temperature (17°C) for 24 h. POLA water from May 2015 was incubated in the dark at surface water temperature (20°C) for 88 h. Incubation of POLA water in the dark was performed to simulate more closely the conditions in that site, as light attenuation at POLA is much steeper than that of the other sites  profiles at the Port of Los Angeles (POLA), the San Pedro Ocean Time-series (SPOT), and Santa Catalina Island (CAT) in spring (April), winter (January), and fall (October). Download Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 13C labeled naphthalene. Ammonium-chloride at a final concentration of 1 μM was also added to each bottle, similar to the previously described incubations. A single bottle from each site was filtered immediately after amendment to establish a t0 atom percent 13C of the particulate carbon as a baseline for incorporation rate measurement. The remaining bottles (3 replicates per site) were incubated in a temperature-controlled room (see above). Incubations were carried out for ∼24 h and were terminated by filtration of microbial biomass onto precombusted (∼5 h at 400°C) 47-mm GF/F filters . The filters were then dried at 60°C and kept in the dark until analysis. Isotopic enrichment was measured on an IsoPrime continuous-flow isotope ratio mass spectrometer (CF-IRMS). IRMS data were corrected for both size effect and drift before being calculated as previously described  from all three sites was incubated in quadruplicate 2-liter polycarbonate bottles with 400 nM escribed .Total DNA was extracted from the Sterivex filters using a modified DNeasy plant kit protocol . The Sterivex filters containing STE buffer were thawed; 100 μl 0.1 mm glass beads was added into the filter casing and put through two 10-min cycles of bead beating in a TissueLyser device . The flowthrough, pushed out using a syringe, was incubated for 30 min at 37°C with 2 mg/ml lysozyme followed by another 30-min incubation at 55°C with 1 mg/ml proteinase K and 1% SDS. The resulting lysate was loaded onto the DNeasy columns, followed by the protocol described in the kit instructions. Only samples from the POLA May 2015 sample yielded enough DNA for ultracentrifugation of both the labeled and unlabeled DNA. However, metagenomes were sequenced from all dates and sites except SPOT October 2014, as the amount of extracted DNA was insufficient for library preparation.Isopycnic ultracentrifugation and gradient fractionation were performed as described in previous work , 72. Bric-13.593)  . DNA in YCAGCMGCCGCGGTAA), 1 μl (10 pg) indexed 926R reverse primer (5′‐CCGYCAATTYMTTTRAGTTT), 1 ng DNA, and 10 μl molecular-grade water.PCR was performed on each fraction with detectable DNA. Each reaction tube contained 12 μl 5 Prime HotMaster mix , 1 μl (10 pg) barcoded 515F-Y forward primer , quantified with PicoGreen, and diluted to 1 ng/μl. A pool of 1 ng of each uniquely barcoded product was cleaned and concentrated again with 0.8× Agencourt AMPure XP beads.The pooled amplicons were sequenced on an Illumina MiSeq instrument  for 600 cycles. Each pool was also spiked with 1 ng of an even and a staggered mock community in order to assess the sequencing run quality . All exp10.1128/mSystems.00245-21.6DATA SET S1P values of Kolmogorov-Smirnov normality test of 12C- and 13C-naphthalene-enriched OTU distribution as a function of density, density shift, corresponding atom percent excess, and SILVA-assigned taxonomy of 180 OTUs analyzed from POLA 5/15. Download Data Set S1, XLSX file, 0.02 MB.Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The raw reads were quality-trimmed using Trimmomatic  version https://www.r-project.org/) for the top 200 most abundant OTUs in each sample. The abundance of each OTU was normalized to a sum of 1 across all fractions.Plots of normalized abundance as a function of density were generated in R  with 9 amplification cycles. The libraries were bead purified as described above and sequenced on an Illumina MiSeq device for 600 cycles  or on an Illumina HiSeq rapid run for 500 cycles . See sourceforge.net/projects/bbmap/) requiring 95% identity. Binning was performed using two approaches: (i) binning the POLA 5/15 13C metagenome with CONCOCT  CONCOCT , 85, and CONCOCT , CONCOCT CONCOCT , and Met CONCOCT . These b CONCOCT . The res CONCOCT , and onl10.1128/mSystems.00245-21.5TABLE S1Table S1, DOCX file, 0.01 MB.Sample details—number of metagenomics (MG) reads and 16S-rRNA amplicons. *, Numbers of amplicons in enriched mesocosms are combined across all density fractions. Download Copyright © 2021 Sieradzki et al.2021Sieradzki et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Initial taxonomic assignment of MAGs was performed using GTDB-Tk . We thenReads from additional unenriched seawater metagenomes from all three sites were also mapped to the dereplicated MAG set to detect MAGs whose abundance increased in the presence of naphthalene. 16S-rRNA genes found in MAGs were searched against the amplicon OTUs.Open reading frames (ORFs) in the final set of metagenome-assembled genomes (MAGs) were predicted using Prodigal  and annoSecondary metabolite clusters were identified using the antiSMASH bacterial version 5 online platform  with theRead recruitment from different samples to the MAGs and viral contigs was analyzed with Anvi’o  using thPRJEB26952 and samples ERS2512855 to ERS2512864. The metagenomic library blank is under sample ERS2507713. Amplicon reads can be found under sample accession numbers ERS2507470 to ERS2507679, and PCR blanks and mock communities, under samples ERS2507702 to ERS2507712. Metagenomic t0 raw reads can be found under project PRJEB12234 and samples ERS2512914 to ERS2512919.Metagenomic and amplicon raw reads from enrichment mesocosms can be found on EMBL-ENA under project


P < 0.05). In addition, Trt dogs had lower recovery BT at weeks 2 and 5 than Ctl dogs (P < 0.05). Treatment dogs had greater fecal short-chain fatty acid concentrations than Ctl (P < 0.05). Diet had no effect on RR or body composition (P > 0.10), but exercise resulted in an overall 7% increase in lean and 3.5% decrease in fat mass (P < 0.05). These data suggest that increasing dietary soluble fiber may positively influence BT and gut health; however, it has no effect on RR or body composition. Soluble fiber did not negatively impact any measures of overall health and performance and should be considered for use in performance dogs.Dietary fiber affects canine physiology in many ways, such as increasing colonic absorption of water and improving gut health, both of which may positively impact exercise performance. The objectives of this study were to investigate the effects of increased dietary soluble fiber and incremental training on respiratory rate (RR), internal body temperature (BT), body composition, and fecal metabolites in mid-distance training sled dogs. Fourteen dogs (12 Siberian and 2 Alaskan Huskies) were blocked by age, sex, and body weight (BW) and then randomly allocated into one of two diet groups. Seven dogs were fed a dry extruded control diet (Ctl) with an insoluble:soluble fiber ratio of 4:1 (0.74% soluble fiber on a dry-matter basis), and seven dogs were fed a dry extruded treatment diet (Trt) with an insoluble:soluble fiber ratio of 3:1 (2.12% soluble fiber on a dry-matter basis). Fecal samples were taken once a week. All dogs underwent 9 weeks of incremental exercise conditioning where the running distance was designed to increase each week. Every 3 weeks, external telemetry equipment was used to non-invasively measure and record RR and internal BT at resting, working, and post-exercise recovery states. Body composition was measured on weeks −1 and 9 using quantitative magnetic resonance. Body composition, RR, BT, and fecal metabolites were analyzed using a mixed model with dog as a random effect and week and diet group as fixed effects. Dogs on Trt had lower working and post-exercise BT than Ctl ( Exercise has the capacity to affect whole-body physiology; however, high-intensity endurance training, such as that experienced by sled dogs, can lead to gastrointestinal (GI) disturbances, heat stress, and possible dehydration , 2. NutrDietary fiber can be divided into two categories based on its solubility in water . SolSoluble fiber may also impact body composition of actively training sled dogs. For example, sedentary humans who increased their soluble fiber intake had lower body weights (BWs), body mass index, and fat mass (FM) than those ingesting maltodextrin as a control . FM and Therefore, the objective of this study was to investigate the effects of an increased soluble fiber diet and an incremental training regimen on the outcomes of RR, BT, body composition, and fecal metabolites in mid-distance training huskies. We hypothesized that increased soluble fiber supplementation would decrease exercise-induced internal BT and FM, increase LBM and fecal SCFAs, and have no effect on RR in actively training sled dogs.2) containing anywhere from 2 to 10 dogs each.The study was approved by the University of Guelph's Animal Care Committee . Twelve client-owned domestic Siberian Huskies  and 2 Alaskan Huskies  with an average age of 3.75 ± 2.7 years (mean ± SD) and BW of 21.54 ± 2.83 kg were used in the study. Dogs resided and trained at an off-site facility  that had been previously visited and approved by the University of Guelph's Animal Care Services. During the study, dogs were group-housed in free-run, outdoor kennels  or treatment . For 2 weeks prior to the study period, all dogs were acclimated to a dry extruded Ctl diet  using a Siemens ADVIA 2120 hematology analyzer . Separate samples (4 ml) were centrifuged at 2,000 × g for 20 min at 4°C using a Beckman J6-MI centrifuge . Serum aliquots were collected and analyzed for serum biochemical components using a Roche Cobas 6000 c501 biochemistry analyzer .Blood samples were collected and analyzed as described by Templeman et al. . In brieBody composition  were measured using quantitative magnetic resonance (QMR) technology  on weeks −1 and 9. With the use of QMR technology, dogs were able to be imaged while avoiding anesthetization .During fecal collection, whole samples were collected within 15 min of being voided, and all visible contaminants  and portions of samples that were in contact with the ground were removed. Samples were transferred into a Whirl-pak bag  to be homogenized. Homogenized samples were then stored in 50-ml sterile centrifuge tubes , frozen, and kept at −20°C until further analysis. Samples were analyzed for SCFAs , branched-chain fatty acids , lactic acid, formic acid, and monosaccharides using an Agilent HP1000 series high-performance liquid chromatography .HPLC analysis was based on a previous study from our laboratory by Templeman et al. . In brieP ≤ 0.05 and trends at 0.05 < P ≤ 0.10.Statistical analyses were performed with Statistical Analysis System (SAS) . RR and BT data were analyzed for outliers by removing the data points that exceeded an RR <200 breaths/min and lower than 5 breaths/min. For BT, data points higher than 42°C and lower than 36°C were also considered outliers. Also, the periods during the exercise challenges when a four-dog team was stopped were removed, as these interruptions reduced the dogs' ability to maintain a wRR. These stops included instances of defecation and urination. Following data cleanup, a TRANSREG procedure was used to optimally transform the RR data. BT was calculated using the means of each 1-min interval from the start of each exercise challenge until 30 min post-exercise. Variances in RR, BT, fecal metabolites, fasted blood analytes (CBC and serum biochemistry), feed intake, BW, and body composition data were analyzed using PROC GLIMMIX of SAS . Dog was treated as a random effect; and activity level , week, and diet group  were treated as fixed effects. Activity level was analyzed against diet group and week, as well as week against diet group. RR and fecal metabolite means were compared using the Tukey honestly significant difference (HSD), whereas BT means were compared using a Fishers least significant difference (LSD). Statistical significance was declared at During data analysis, 18% (27/153) of the RR observations and 31%  of BT observations were removed due to either software malfunctions, which caused missing data, or movement of rectal probes during the challenge run. Two dogs were removed from the trial  due to exercise-related injuries; all data collected from these dogs until their removal were included in the results. Due to receiver limitations, only eight dogs were monitored for BT throughout the study .P > 0.10), but all variables differed by week ; however, BW did not differ from week −1 to weeks 0–1 (P > 0.10) and weeks 4–8  at weeks −1 and 9 did not differ between diet groups (P > 0.10); however, when data were pooled to evaluate the effects of exercise, all variables differed by week . Working BT tended to be lower at week 8 compared with week −1 (P < 0.10). Post-exercise BT was greater at week 5 compared with weeks 2 and 8 (P < 0.05) but was not different than week −1 (P > 0.10).No differences in rBT were observed between dietary treatments  and butyric acid , but no other weeks differed  prior to a race performed better than dogs with a greater level of these bacteria  in exercising dogs. Overall, these results can be used to improve training regimens and diets that may influence exercise physiology, health, and performance of sled dogs.The findings in the current study suggest that the addition of 1.38% soluble fiber to achieve an insoluble:soluble fiber ratio of 3:1 may have contributed to a reduction in internal BT at both working and recovery post-exercise states and resulted in greater fecal SCFA concentrations. Supplemental soluble fiber had no effect on RR or body composition; however, a 9-week training regimen resulted in increased LBM and decreased FM. Future research is warranted to investigate how a diet with a soluble fiber inclusion of 2.12% influences hydration status, and various cardiorespiratory measurements (i.e., heart rate and VOThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by University of Guelph's Animal Care Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.ET, JT, KS, and AS designed the experiment. ET, ER, and JT conducted the research. ET and LB analyzed the data. AS had primary responsibility for the final content. All authors read and approved the final manuscript. All authors contributed to the writing of the manuscript.MB was employed by the company emka Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Champion Petfoods Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.