Verticillium dahliae (V. dahliae) is a serious disease in cotton, resulting in massive yield losses and decline of fiber quality. Breeding resistant cotton cultivars is an efficient but elaborate method to improve the resistance of cotton against V. dahliae infection. However, the functional mechanism of several excellent VW resistant cotton cultivars is poorly understood at present. In our current study, we carried out RNA-seq to discover the differentially expressed genes (DEGs) in the roots of susceptible cotton Gossypium hirsutum cultivar Junmian 1 (J1) and resistant cotton G.hirsutum cultivar Liaomian 38 (L38) upon Vd991 inoculation at two time points compared with the mock inoculated control plants. The potential function of DEGs uniquely expressed in J1 and L38 was also analyzed by GO enrichment and KEGG pathway associations. Most DEGs were assigned to resistance-related functions. In addition, resistance gene analogues (RGAs) were identified and analyzed for their role in the heightened resistance of the L38 cultivar against the devastating Vd991. In summary, we analyzed the regulatory network of genes in the resistant cotton cultivar L38 during V. dahliae infection, providing a novel and comprehensive insight into VW resistance in cotton.Cotton is an important economic crop worldwide. Verticillium wilt (VW) caused by  R genes include the nucleotide-binding site (NBS), leucine-rich repeat (LRR), serine/threonine protein kinase domain (PK), drosophila toll domain, coiled-coil structure (CC), and transmembrane domain (TM) [Plants are under constant attack from various pests including bacteria, fungi, oomycetes, nematodes, and insects. As plant are unable to escape attacking pathogens by moving to a more favorable environment, they possess efficient defense mechanisms to protect themselves from disease, such as physical barriers and production of antimicrobial compounds ,2. Thus,ain (TM) . Moreoveain (TM) . SA-depeain (TM) .Gossypium spp.) is an important economic crop species cultivated worldwide. Verticillium wilt (VW) of cotton is a serious vascular disease that is detrimental to cotton yield and fiber quality, and is caused by the soil-born fungus Verticillium dahliae (V. dahliae). It is known that V. dahliae attacks host plants through their roots, generally takes around 2–4 days to reach the xylem vessels, and proliferates in the vascular to induce VW [V. dahliae is difficult to control owing to its ability to survive long-term as microsclerotia in the soil and adapt to a broad host range to survive and propagate [R genes [GbaNA1 [GhDSC1 [V. dahliae. Adenosine triphosphate (ATP)-binding cassette (ABC) genes have been identified that play a vital role in cotton against V. dahliae infection [GhOPR9 has been found to be a positive regulator under V. dahliae infection which can modulate the expression of genes related to the JA pathway [V. dahlia [GhWRKY70D13 negatively regulated cotton defense against V. dahliae through regulation of the genes involved in ET and JA biosynthesis and signaling pathways [V. dahlia [V. dahliae by interacting with the chitin sensory receptors [V. dahliae by activating JA biosynthesis and phenylpropanoid pathway [Cotton  and G. hirsutum susceptibility cultivar Junmian No. 1 (J1) response to V. dahliae infection, respectively. The aim of this study was to discover the differentially expressed genes after V. dahliae infection to identify the potential resistant network of L38. The significantly different expressed genes (DEGs) were classified and divided into six groups, including J1-early, J1-late, J1-sustained, L38-early, L38-late, and L38-sustained. The potential function of DEGs in these groups were analyzed and putative association with sectors associated with disease resistant pathways, such as phenylpropanoid biosynthesis, plant hormone signal transduction, Mitogen-activated protein kinase (MAPK) signaling pathway and plant-pathogen interaction were revealed from our study. In addition, the resistance gene analogues (RGAs) that displayed differential expression were further analyzed. Our findings suggest that the expression of several resistance-related genes was induced in the resistant cotton cultivar L38 in a manner different from the susceptible cultivar J1 during V. dahliae infection, thus providing an insight into the genomic networks at play that result in the VW resistance of the L38 cotton cultivar.RNA-seq has been widely applied to monitor the gene expression profiles and describe the response characteristics of cotton roots under nfection ,30,31,32G. hirsutum cultivar Junmian 1 (J1) and resistant cotton G.hirsutum cultivar Liaomian 38 (L38) were grown and maintained in a greenhouse at 28 °C under 16 h light/8 h dark photoperiod. Moreover, L38 and J1 were planted at a disease nursery in Liaoyang, located in northeast of China .The susceptible cotton V. dahliae strain Vd991 [6 conidia/mL which was then used for inoculating cotton seedlings [The highly virulent in Vd991  was culteedlings . Roots oThe disease index (DI) of Verticillium wilt was obtained according to the following formula:The index, comprising five grades , was used for assigning infected plants. Grade 0 indicates that the plant is healthy and has no disease symptoms, grades 1 to 4 represent the typical yellowing and wilting observed in 0–25%, 25–50%, 50–75%, and 75–100% leaves of investigated plants, respectively.Both J1 and L38 root samples were inoculated with Vd991 after 24 h and 72 h, in addition, the samples without inoculation and collected just prior to the treatments were designated as 0 h. For each experiment three technical replicates were performed with each replicate including 6 seedlings. Therefore, a total of 18 root samples were chosen for RNA extraction. Total RNA samples of cotton roots were extracted using Plant RNA Purification Kit . The RNA concentration and purity were measured by NanoDrop 2000 spectrophotometer . The RNA integrity was checked by agarose gel electrophoresis. Genomic DNA was removed by DNase treatment.Gossypium hirsutum (NCBI_GCF_000987745.1_ASM98774v1) by HISAT  [http://www.ncbi.nlm.nih.gov/sra/) (26 October 2021). The accession number is currently pending submission until acceptance of the manuscript.RNA-Seq library preparation and sequencing were performed using the standard Illumina protocols . Briefly, mRNAs were enriched from 1.5 μg total RNA by using magnetic beads with Oligo (dT) and used as templates to synthesize the first stranded cDNAs with random hexamers and produce double-stranded by using DNA Polymerase I. The products were enriched with PCR to create the final cDNA libraries after addition of A tail and ligation with sequencing adapters. Strand-specific sequencing was performed on an Illumina HiSeq X-Ten by BGI , which generated 150 bp paired-end reads. The raw reads were filtered by a series of steps, including removal of lower quality read, adapters, higher content base of N reads, to obtain clean reads. The clean reads were mapped onto reference genome of scripts) . The datp-value ≤ 0.05 [A total of six groups of cotton root sample  were used for DEGs analysis. Significantly, DEGs were identified from four comparisons, including J1-24 compared with J1-0 (J1-24/0), J1-72 compared with J1-0 (J1-72/0), L38-24 compared with L38-0 (L38-24/0) and L38-72 compared with L38-0 (L38-72/0). Fragments per kilobase per million mapped fragments (FPKM) were calculated by RSEM and used to estimate the effects of sequencing depth and gene length on the mapped read counts. Foldchange in gene expression value was calculated by FPKM J1-24/FPKM J1-0, FPKM J1-72/FPKM J1-0, FPKM L38-24/FPKM L38-0, and FPKM L38-72/FPKM L38-0. DEseq2 was used to analyze DEGs in cotton under the criteria of a corrected Foldchange ≥2 and adjusted e ≤ 0.05 . The heahttp://wego.genomics.org.cn/) (26 October 2021) in three terms, including cellular component, molecular functional and biological function, if the p-value < 0.05. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was then performed in KEGG Mapper (https://www.kegg.jp/kegg/mapper.html) (26 October 2021).Gene ontology (GO) term enrichment analysis of DEGs was performed using WEGO (http://www.bio-rad.com/) (26 October 2021). The relative quantification of RT-qPCR was measured by 2−ΔΔCt analysis method [18S gene (18S rRNA). Three biological replicates were performed for each experiment, with three technical replicates. The specific primers used are listed in RNA aliquots of 2.0 µg were used for cDNA synthesis by the TranScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit . Quantitative PCR (qPCR) was performed using SYBR Green PCR Master Mix  on a CFX96 Touch Real-time PCR Detection System . Duncan’s multiple range test was used for three samples (p < 0.05).Student’s V. dahliae, seedlings of both L38 and VW susceptible J1 cultivars at four weeks were inoculated with Vd991. L38 plants showed highly resistant phenotype to Vd991 compared to J1 at 14 days post-inoculation , and more than 60% of clean reads were uniquely mapped to the genome of G. hirsutum in each sample file to ensure the credibility of the RNA-seq data , J1-72h compared with J1-0h (J1-72/0), L38-24 compared with L38-0h (L38-24/0), and L38-72h compared with L38-0h (L38-72/0) respectively. Thousands of DEGs were detected in the J1-24/0, J1-72/0, L38-24/0, and L38-72/0 groups. Venn diagrams of DEGs illustrate both commonly expressed and specifically expressed genes between the J1 and L38 cultivars. For up-regulated DEGs, 490 were specifically expressed in J1-24/0, 1097 were specifically expressed in J1-72/0, while 85 genes had sustained elevated levels both in J1-24/0 and J1-72/0 timepoints. Similarly, 578 genes were specifically expressed in L38-24/0, 2556 were specifically expressed in L38-72/0 group, while 771 genes had sustained expression levels in both L38-24/0 and L38-72/0 treatment groups following the V. dahliae inoculation  were identified as culation B. For doculation C. Furtheculation D.To reveal potential functions of differentially expressed genes (DEGs), we identified the Gene Ontology (GO) terms of these genes. Therefore, the DEGs were divided into six groups for further analysis, including those specifically expressed in J1-24/0 (J1-Early), specifically expressed in J1-72/0 (J1-Late), expressed both in J1-24/0, J1-72/0 (J1-Sustained), expressed only in L38-24/0 (L38-Early), expressed only in L38-72/0 (L38-Late), and expressed both in L38-24/0 and L38-72/0 (L38-Sustained). Thus, 701, 899, 1422, 3524, 390, and 2078 DEGs matching to the above-mentioned groups were assigned GO terms. The regulation of macromolecule metabolic process and regulation of RNA metabolic process were highly enriched both in cultivar J1 and L38 A. Each gIn addition, KEGG enriched pathways from each group were analyzed, and more than 100 pathways were assigned in each group, including metabolic pathways, biosynthesis of secondary metabolites and so on . SeveralAmong these GO terms and KEGG pathways, several DEGs were classified in resistance function. Therefore, the typical resistance pathway was further identified, including plant-pathogen interaction, plant hormone signal transduction, MAPK signaling pathway.CDPK), flagellin-sensing 2 (FLS2), WRKY transcription factor, avrPphB-susceptible 1 (PBS1), heat shock protein 90 (HSP90), and so on , but only DEGs of L38 assigned in MAPK/ERK kinase kinase (MEKK1) and MAP kinase (MPK4)  and PBS1  A. In effand PBS1 A. Particand PBS1 . Moreove dahliae B.TIR1; in ethylene signal transduction, DEGs only expressed in L38 were assigned in ETR; in jasmonic acid signal transduction, DEGs uniquely expressed in L38 related to JAZ; in salicylic acid signal transduction, DEGs uniquely expressed in L38 regulated non-expression of pathogenesis-related gene 1 (NPR1) and pathogenesis-related gene 1 (PR1)  A. To fur dahliae B.Suppressor of mkk1 mkk2 2 (SUMM2), that is involved in reducing cell death defense response, was the only enriched DEG of J1 in the early stage which was different from other pathways , and PR1 were the only enriched DEGs of L38  genes have been shown to play an essential role in recognizing effectors from pathogens and in triggering downstream signaling during plant disease resistance [R genes, including nucleotide-binding sites (NBSs), leucine-rich repeat (LRR), Toll/Interleukin-1 receptor (TIR), coiled-coil (CC) and transmembrane (TM) [V. dahliae inoculation. A total of 486 RGAs were identified from six groups, including J1-early, J1-late, J1-sustained, L38-early, L38-late, and L38-sustained. 358 RGAs were identified in L38, significantly higher than in J1 which displayed only 128 such RGAs (LOC107902722 (NBS-LRR), which was down regulated in J1 while up regulated in L38  . Therefouch RGAs A. Moreovuch RGAs A. A totauch RGAs B. Intered in L38 B. For thd in L38 B. In addd in L38 C. RGAs od in L38 C. Howeved in L38 C. Furthed in L38 D. Thus, V. dahliae is becoming clearer, thus assisting in the breeding of resistant cotton cultivars. In fact, the gene regulation network underlying the molecular mechanisms leading to resistance seen in several cotton cultivars with excellent VW tolerance is relatively unexplored. For instance, the cultivar Liaomian bred from the Research of Economic Crops in Liaoning Province showed higher resistance during production, but the exact mechanism underlying its resistant function remained unclear. In this study, we first confirmed the resistance phenotype of L38 compared with VW susceptible cotton J1 . These results show that the expression pattern of genes in L38 and J1 respond differentially to V. dahliae infection , RPM1 interacting 4 (RIN4), avrPphB susceptible 1 (PBS1), and Resistance to Pseudomonas syringae 5 (RPS5) [FLS2 in plant-pathogen interaction pathway, but DEGs induced in L38 were assigned to RIN4 and PBS1 related pathways  . From oupathways A. In addresponse A. These  species A. These V. dahliae inoculation. Moreover, most of RGAs in the resistant L38 cultivar were different from those in the susceptible J1 cultivar. Interestingly, 14 such RGAs that were down-regulated in J1 were up-regulated in the resistant L38 cultivar  and transmembrane leucine rich repeat (TM-LRR). More RGAs were significantly expressed in L38 than in J1 following cultivar A,B. ThisV. dahliae. Many DEGs were found through subsequent GO and KEGG analysis that related to function of VW resistance in cotton. The expression level of candidate DEGs were reconfirmed to ensure the regulation of these genes related to defense response upon V. dahliae inoculation. Moreover, the cotton RGAs were also analyzed during V. dahliae inoculation revealing that RGAs in the resistant L38 cultivar were typically different from those in the susceptible J1 cultivar, implying that the regulation network of resistance-related genes in L38 was more novel and diverse than in the susceptible cotton cultivar. This study lays a solid foundation for further in-depth analysis of myriad of candidate resistance genes in the VW resistant L38 cotton cultivar. At present we are further exploring the functional mechanism of typical resistance-related genes revealed in our study presented here to elucidate the molecular basis of VW resistance observed in the resistant L38 cotton cultivar.In short, DEGs of cotton with a putative role in VW resistance were discovered by RNA-seq analysis of two cotton cultivars differing in their resistance to In conclusion, it is our understanding that the inter-connected regulatory network of these gene clusters in the resistant cotton cultivar L38 upon pathogen infection provides a novel and comprehensive insight into the mechanism of VW resistance in pest resistant cotton cultivars. The findings of this study can be harnessed to design increased tolerance to VW in cotton, which is currently under significant threat from this devastating disease worldwide.


TGF-β1- and IL-10-specific single-nucleotide polymorphism (SNP) combination on the risk for osteoporosis. We investigated and analyzed the relationships between three TGF-β1 SNPs , one IL-10 SNP (+1927A/C) and the level of bone mineral density (BMD), as well as the risk of osteoporosis in Taiwanese osteoporotic patients. A total of 217 subjects were recruited, including 88 osteoporotic patients and 129 healthy controls, for SNPs, BMD and clinical characteristics statistical analyses. Females with TGF-β1 SNP (−509 C/C) and IL-10 SNP (+1927 C/C) genotypes showed a great benefit for femoral neck T-scores. However, the combination of TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/A) genotypes in all subjects showed a significant decrease in total hip BMD T-scores. The TGF-β1 SNP (−509 C/T) genotype in all subjects and TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/C) genotypes in males showed positive effects on body height. The combination of the many SNPs in the anti-inflammatory TGF-β1 and IL-10 genes may be cooperatively involved in the development of osteoporosis. Our data suggested that the specific SNP combination of TGF-β1 (−509) and IL-10 (+1927) may act as a predictive factor for postmenopausal osteoporosis in Taiwanese women.Osteoporosis is a rising health threat in the increasingly aging world population. It is a common skeletal disease strongly linked to genetic predisposition. We aim to identify the effects of the anti-inflammatory  Osteoporosis is a common bone disease in humans and is rapidly increasing to become a major public health problem among elderly individuals worldwide . OsteopoTGF-β1), interleukin-10 (IL-10), vitamin D receptor (VDR), osteoprotegerin (OPG), collagen type I α 1 (COL1A1) and estrogen receptor (ER) genes, and may act as potential clinical markers for osteoporosis [Several candidate genes and their SNPs are strongly associated with osteoporosis pathogenesis, including transforming growth factor-β1  (−509 C/T) was significantly associated with its plasma concentration. Yamada et al. described that TGF-β1 SNPs + 29 T/C and −509 C/T alone [ process . TGF-β1  process ,12,13,14 process  observedtibility ,17. In atibility  found th/T alone  or in co/T alone  combinedIL-10 is an anti-inflammatory cytokine that is secreted by activated immune cells. It limits the secretion of proinflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-12, IL-2 and IFN-γ; modulates the differentiation and proliferation of immune cells, including macrophages, T cells and B cells; and attenuates monocyte recruitment or activation ,22.IL-10 knockout mice, the hallmarks of osteoporosis can be observed, including an increased level of RANKL and osteoprotegerin, promoted bone resorption, reduced bone formation and altered trabecular structure [IL-10 gene polymorphisms may affect its mRNA and protein productions and are relevant in osteoporotic development. The genotype of the IL-10 SNP (−627 C/C) correlated with a lower spinal BMD in postmenopausal osteoporotic patients [IL-10 also has potent inhibitory effects on osteoclastogenesis and acceleratory effects on osteoblastic differentiation . In IL-1tructure ,25. Studpatients .TGF-β1 and IL-10 genes and the incidence of low BMD in postmenopausal osteoporotic women.In this study, we aimed to elucidate the correlation of risk factors, including age, height and genetic factors, for postmenopausal osteoporosis in Taiwanese. This study focuses on the interaction between four polymorphisms in both the Osteoporotic patients were recruited after providing written informed agreement. The clinical trial protocol was approved by the Institutional Review Board of the Tri-Service General Hospital Medical Centre (TSGH-IRB #TC098-06). All subjects met the inclusion criteria based on BMD as an indicator of osteoporosis (T-score ≤ −2.5 standard deviations (SD)) or on a diagnosis of osteopenia with fracture (T-score < −1.0 and >−2.5 SD) using dual-energy X-ray absorptiometry (DXA) detection. Exclusion criteria for patients (in whom menses had stopped for more than 12 months) were as follows: (1) menolipsis at age < 40 years, or bilateral ovariectomies; (2) any medical history of illness, including primary hyperparathyroidism, overactive thyroid, diabetes, hepatic cirrhosis or renal failure; (3) drug treatment that affected bone metabolism, including bisphosphonates, calcitonin, glucocorticoids, thyroxin, or antiepileptics; or (4) hormone replacement therapy within the past 4 months ,28. Afte2), 0.25 U DNA polymerase , 0.4 μM each of forward primer and reverse primer  was used. Genomic DNA (100 ng) was used for genotyping by polymerase chain reaction—restriction fragment length polymorphism (PCR-RFLP). The following reaction buffers were used: 1 × reaction buffer  were used for incubation with PCR products of the TGF-β1 SNPs  and IL-10 SNP (+1927) at 37 °C overnight. Adequate numbers of informative bands were analyzed on a 3% high-resolution agarose gel  containing ethidium bromide. The resulting images were photographed for genotyping  was used in this study to assess the BMD values of the lumbar vertebra and bilateral hip joint of the whole body. Osteoporosis is defined as a disease in which the BMD (T-score) is lower than −2.5 SD below the average of a young healthy population, according to the WHO criteria .p value less than 0.05 was considered statistically significant. The distributional properties of the continuous variables were represented as the means ± SD and categorical variables were expressed relating to frequency and percentage. In the univariate analysis, the continuous variables of normal distributions were compared between the healthy and osteoporotic subjects with a two-sample t test. Continuous variables that did not fit normal distributions were examined by the Mann–Whitney U test (Wilcoxon rank-sum test). The chi-squared test or Fisher’s exact test was used to compare categorical variables between groups. In the multivariate analysis, the test was conducted by fitting linear regression models to the effects of risk factors on four continuous outcome variables .R2.15.1 software  was used for statistical analysis. A two-tailed p value > 0.05) one at a time until all regression coefficients were significantly different from 0. In addition, the coefficient of determination  was also examined to evaluate the GOF of the fitted regression model. A value of R2 near 1 indicates that most of the response variability is explained by the covariates used in the multiple linear regression model [The regression analysis procedures followed the recommended approach ,31. Brieon model ,32.TGF-β1 gene, including TGF-β1 (−509) at the promoter region, TGF-β1 (+29) and TGF-β1 (+869) at the exon 1 region and one SNP of IL-10 (+1927) at the exon 2 region, were analyzed in this study. Blood DNA obtained from patients with osteoporosis/osteopenia with fractures and from healthy subjects were individually amplified by four pairs of SNP PCR primers, as shown in Dde I for TGF-β1 SNP (−509 C/T), BsrB I for TGF-β1 SNP (+29 C/T), Pvu II for TGF-β1 SNP (+869 C/T) and Aci I for IL-10 SNP (+1927 A/C). The enzyme-digested fragment-length polymorphisms were predicted as shown in TGF-β1 SNPs  in each patient was found in the TGF-β1 SNPs: −509 C/C linked with the SNP (+29 T/T) and SNP (+869 C/C) types, −509 C/T linked with the SNP (+29 C/T) and SNP (+869 C/T) types, and −509 T/T linked with the SNP (+29 C/C) and SNP (+869 T/T) and types, respectively . The −509 T/C and +29 T/C SNPs showed a strong linkage disequilibrium with a D’ > 0.99. These SNPs also showed strong linkage disequilibrium in the studies of Langdahl et al. [TGF-β1 SNPs may therefore be caused by one of them and influence each other. Thus, we evaluated the patients in terms of the SNPs, TGF-β1 SNP (−509) and IL-10 SNP (+1927), which can also simultaneously represent the effects of the TGF-β1 SNPs (+29 and +869) on the risk of osteoporosis.Three SNPs of the ectively . We alsol et al.  and Tzakl et al. . The LD TGF-β1 SNP (−509) and IL-10 SNP (+1927) for patients with osteoporosis (n = 88) and control subjects (n = 129) are shown in p > 0.05) between the healthy controls and the patients with osteoporosis or osteopenia with fractures. The genotype frequency of the TGF-β1 SNP (−509) or IL-10 SNP (+1927) showed no significant difference between the healthy control subjects and the patients with osteoporosis . However, the bone fracture patients were significantly shorter (p < 0.001) and weighed less (p = 0.008) than the healthy control subjects in univariate analysis (p < 0.001). In addition, the DXA BMD values for spine, femoral neck, and total hip in patients with osteoporosis or osteopenia with fractures were significantly lower than that in the control subjects (p < 0.001), as shown in A total of 222 subjects were recruited in the beginning, but five subjects in the osteoporosis group were excluded for their use of glucocorticoid or thyroxin drugs. Thus, finally, 88 osteoporotic patients and 129 healthy control subjects were enrolled in this study. The means for gender, anthropometric parameters , BMD T-scores, vegetarian status, habitual dietary calcium (Ca) and analysis . The meaTo identify the predictors of the four continuous outcome variables, a multivariate analysis of the BMD T-scores was performed by fitting four multiple linear regression (MLR) models for lumbar spine (Y1), femoral neck (Y2), total hip (Y3) and height (Y4), as shown in TGF-β1 (−509 T/C) and IL-10 (+1927 A/C) are shown in p < 0.0001). Daily calcium (Ca) intake greater than 800 mg/day increased the spinal T-score by 0.3764 (p < 0.05). Younger age (33–55 years old) was also found to be a beneficial factor for osteoporosis, associated with an increase in spinal T-scores of 0.6541 (p < 0.001). After adjusting for the effects of the other covariates, the estimated mean value of the spinal BMD T-score, Y1, in the subjects with an age between 33 and 55 years would be 0.6541 higher than those younger than 33 years or older than 55 years. After adjustment for the covariates of height, age and Ca intake, the patients with both polymorphisms of the TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/A + A/C) genotypes and the female patients with both polymorphisms of the TGF-β1 SNP (−509 C/C) and IL-10 SNP (+1927 C/C) genotypes had spinal T-scores (Y1) of 0.4242 and 1.0417, respectively; however, these two interactions showed no significant difference . A higher level of spinal BMD T-scores could be found in younger age (33–55 years old), taller people and people with Ca intake > 800 mg/day, but there was no significant relation connected to the TGF-β1 SNP (−509 C/C) and IL-10 SNP (+1927 C/C) genotypes.The multiple-variable linear regression analysis results of the possible factors associated with the spinal BMD T-score (Y1) in those with osteoporosis or osteopenia with fractures and TGF-β1 (−509 T/C) and IL-10 (+1927 A/C) in patients are shown in p < 0.05), body height , and Ca intake > 855 mg/day  had beneficial effects on the mean value of the femoral neck T-score (Y2). The femoral neck T-scores in female patients and in middle-aged patients (≥55 years) were 0.5283 lower than those in the male patients of the same age (p < 0.01). Considering the interaction of gender and height, the femoral neck T-scores in female patients were 0.0049 higher than those in the male patients of the same height (p < 0.05).The multiple-variable linear regression analysis results of the correlations between the BMD T-score of femoral neck (Y2) and the polymorphisms of TGF-β1 SNP (−509 C/C) and IL-10 SNP (+1927 C/C) genotypes was 1.1666 higher than those in any other group (p < 0.05), which means these genotypes had a greater tendency to prevent osteoporosis. However, there was only a borderline significant increase in the female subjects with both polymorphisms of the TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 C/C + A/C) genotypes and the results showed a 0.4220 higher femoral neck T-score in these patients than in any other group (p = 0.0559). A higher level of femoral neck BMD T-scores could be found in the age range of 33–68 years old, in taller people, females, people with Ca intake > 855 mg/day, and the TGF-β1 SNP (−509 C/C) and IL-10 SNP (+1927 C/C) genotypes, but there was no significant relation connected to the TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 C/C + A/C) genotypes.The femoral neck T-score of female subjects with both polymorphisms of the TGF-β1 SNP (−509 T/C) and IL-10 SNP (+1927 A/C) in patients are shown in p < 0.05) and Ca intake > 875 mg daily  had beneficial effects on the mean value of the total hip BMD T-score (Y3). Considering the interaction of gender and age, the total hip T-score in female subjects was 0.0560 lower than those of male subjects of the same age (p < 0.0001). Considering the interaction of gender and height, the total hip T-scores in female patients were 0.0229 higher than that of male patients of the same height (p < 0.0001). The total hip T-score of subjects with both polymorphisms of the TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/A) genotypes was 1.4692 lower than that in any other group (p < 0.01). A higher level of total hip BMD T-scores could be found in taller people and people with Ca intake > 875 mg/day. Females who had the same height as males showed a higher T-score, but a lower T-score was found in females compared to males who were of the same age. A lower level of total hip BMD T-scores could be found for the TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/A) genotypes.The multiple-variable linear regression analysis of the correlations between the total hip BMD T-score (Y3) and the polymorphisms TGF-β1 SNP (−509 T/C) and IL-10 SNP (+1927 A/C) in patients with osteoporosis or osteopenia with fractures are shown in p < 0.0001) and patients who had these SNP genotypes had a one-year age difference associated with a 0.3503 cm shorter body height (p < 0.0001).The multiple-variable linear regression analysis of the possible correlations between body height (Y4) and the polymorphisms TGF-β1 SNP (−509 C/T) genotype were 1.8464 cm taller than those in any other group (p < 0.05). Females with both polymorphisms of the TGF-β1 SNP (−509 C/C + C/T) and IL-10 SNP (+1927 C/C) genotypes were 4.3845 cm shorter than the female subjects in any other group (p < 0.01). However, males who had both polymorphisms of the TGF-β1 (−509 T/T) and IL-10 (+1927 A/C) genotypes were 7.8140 cm taller than those in any other group (p < 0.01).Patients with the polymorphism Osteoporosis is a progressive disease with a multifactorial etiology that also represents an increasingly serious health problem around the world. Although the variation in BMD, a major index determining bone strength, is determined by environmental, behavioral, hormonal and nutritional factors, the most important component of the variation in BMD may be genetically controlled ,34. AlthTGF-β1 (−509 C/C) and IL-10 (+1927 C/C) genotypes showed the greatest tendency to prevent osteoporosis compared with those of all other groups  and IL-10 polymorphism  and osteoporosis/BMD can be found in the NHGRI-EBI Catalog of published genome-wide association studies . Langdahl et al. [TGF-β1 polymorphisms and osteoporosis, which showed that these polymorphisms were associated with BMD reduction or fracture risk increment in some populations. Accumulating evidence has shown that the SNP + 29 T→C transition in the signal sequence of the anti-inflammatory TGF-β1 gene is significantly associated with higher BMD at the spine and hip in Japanese and German women [TGF-β1 (+869 T/C) polymorphism was associated with increased risk of osteoporotic fracture at the lumbar spine and femoral neck [TGF-β1 (+869 T/C) and (+29 T/C) polymorphisms increased the susceptibility to osteoporosis in an Asian population. The combination of both the TGF-β1 (−509 C/T) and (+869 T/C) polymorphisms was associated with the risk of osteoporosis [TGF-β1 SNPs  in each patient (TGF-β1 (−509) and IL-10 (+1927), which can also represent the effects of TGF-β1 SNPs (+869 and +9) on the risk of osteoporosis at the same time.There are three SNPs in the l et al.  used thean women ,40, but an women . Althougral neck ,42. Sun ral neck  performeoporosis  and urinoporosis . In this patient . Thus, wIL-10 gene could affect protein expression and activity, therefore shifting the balance between bone formation and resorption and increasing the subsequent risk of osteoporosis. Animal studies have shown that a lack of IL-10 expression decreases femoral BMD, increases bone fragility and inhibits bone resorption, eventually leading to osteoporosis [IL-10 SNP (−592 C/C) and/or IL-10 ht2 (A-C-C-T) genotypes generally had relatively lower spinal BMD in Korean postmenopausal women. In addition, mounting studies have shown that the presence of the C allele at position—597 decreased the frequency of the G allele at position—1082, or a polymorphism of the C/C genotype at position—627 in the IL-10 promoter may constitute a risk for the development of osteoporosis [IL-10 (+1927) showed that the A/A genotype was related to a higher risk for hepatocellular carcinoma [IL-10 (+1927) polymorphism in osteoporosis has not been revealed until now.The polymorphic genotype of the anti-inflammatory oporosis . Park etoporosis  reportedoporosis ,46. The arcinoma ,48. HoweTGF-β1 or IL-10 gene polymorphisms in osteoporotic development have not yet been revealed. In this study, we identified body height as another predictor in the causal pathway between SNPs and spinal, femoral neck, and total hip T-scores. Furthermore, the TGF-β1 SNP (−509 C/T) genotype in all subjects and the TGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/C) genotypes in males showed positive associations with body height.Bone loss caused by the development of osteoporosis may be gradual and painless with no significant clinical symptoms. In this study, we found that bone loss also triggered a higher ratio of different types of fractures in osteoporosis or osteopenia patients . OsteopoTGF-β1 SNP (−509 T/T) and IL-10 SNP (+1927 A/A)—and adjusted age, habitual Ca intake, gender, BMI and height in Taiwanese subjects; it showed a significantly lower total hip BMD when compared with other SNP combinations. Furthermore, we also found that the combination of both polymorphisms of the TGF-β1 SNP (−509 C/C) and IL-10 SNP (+1927 C/C) genotypes showed a higher femoral neck BMD T-score in female subjects than in any other group, which means these genotypes had a potential to act as a predictive factor for postmenopausal osteoporosis in Taiwanese women. Therefore, early genetic testing of these predictive factors for postmenopausal osteoporosis in the Taiwanese female population can help to prescribe to patients with these factors the appropriate nutritional supplements or clinical treatments, eventually slowing down the bone mass loss caused by age and inheritance and preventing osteoporosis-related diseases. However, well-designed studies with larger sample sizes are required to further explicate these associations.This study is the first report to evaluate the risks of osteoporosis and the associations between the anti-inflammatory cytokine-specific SNPs—


Escherichia coli O157:H7 strains with different stx profiles, previously isolated from feedlot cattle , as well as five foodborne outbreak strains and six stx-negative strains from NCBI. Phylogenomic analysis demonstrated that the stx2c-carrying C1-010 and stx-negative C1-057 strains were grouped with the six NCBI stx-negative E. coli O157:H7 strains in Cluster 1, whereas the stx2a-carrying C1-067 and five foodborne outbreak strains were clustered together in Cluster 2. Based on different clusters, we selected the three newly sequenced strains, one stx2a-carrying strain, and the six NCBI stx-negative strains and identify their prophages at the stx insertion sites. All stx-carrying prophages contained both the three Red recombination genes  and their repressor cI. On the other hand, the majority of the stx-negative prophages carried only the three Red recombination genes, but their repressor cI was absent. In the absence of the repressor cI, the consistent expression of the Red recombination genes in prophages might result in more frequent gene exchanges, potentially increasing the probability of the acquisition of stx genes. We further investigated each of the 10 selected E. coli O157:H7 strains for their respective unique metabolic pathway genes. Seven unique metabolic pathway genes in the two stx2a-carrying strains and one in the single stx2c-carrying and seven stx-negative strains were found to be associated with an upstream insertion sequence 629 within a conserved region among these strains. The presence of more unique metabolic pathway genes in stx2a-carrying E. coli O157:H7 strains may potentially increase their competitiveness in complex environments, such as feedlot cattle. For the stx2c-carrying and stx-negative E. coli O157:H7 strains, the fact that they were grouped into the same phylogenomic cluster and had the same unique metabolic pathway genes suggested that they may also share closely related evolutionary pathways. As a consequence, gene exchange between them is more likely to occur. Results from this study could potentially serve as a basis to help develop strategies to reduce the prevalence of pathogenic E. coli O157:H7 in livestock and downstream food production environments.A comparative whole genome analysis was performed on three newly sequenced  Escherichia coli O157:H7 causes more than 96,000 cases of diarrheal illness and 3,200 hospitalizations annually in the United States (eae (encoding intimin) and stx (encoding Shiga toxin) harbored in foodborne pathogenic E. coli O157:H7 strains are central to the pathogenesis of HUS  and in feedlot environments by lysogenization of stx-carrying prophages into the stx insertion sites in bacterial genomes  and Sanger sequencing, have provided valuable information on the potential mechanisms of the acquisition or loss of the  genomes . Additio process .stx genes, studies have also been conducted to compare the antimicrobial resistance of stx-positive and stx-negative E. coli O157:H7 isolates. A recent study compared antibiotic resistance genes in 19 stx-positive and 8 stx-negative E. coli O157:H7 isolates from cattle , tet(B), sulI, sulII, sulIII, cmlA, catI, catII, catIII, and floR. None of the eight stx-negative E. coli O157:H7 strains carried any antibiotic resistance genes, whereas one of the stx-positive strains contained several antibiotic resistance genes including blaTEM, strA–strB, tet(A), and sulII, and another stx-positive strain harbored a single tet(A) gene  than the 17 stx-negative isolates (four of nine)  gene . Anothern method . The ninof nine) . These tstx-positive and stx-negative E. coli O157:H7 are limited by their capabilities to study only certain selected DNA sequences from the bacterial genome, such as individual genes and/or short DNA fragments. With the development of next generation sequencing technology and whole genome sequencing (WGS), researchers are now able to compare and analyze DNA sequences at the level of the entire bacterial genome. A whole genome comparison study of E. coli O157 isolates from patients and cattle indicated that stx-negative E. coli O157:H7/NM isolates shared 22 virulence genes, the locus of enterocyte effacement region, and plasmids with stx-positive E. coli O157:H7 isolates . In the present study, we conducted comparative genomic analysis of three isolates with different stx profiles to investigate their virulence genes, genes involved in the acquisition or loss of stx genes, and genes involved in metabolic pathways. Furthermore, the WGS data of these three strains were compared with those of five foodborne outbreak E. coli O157:H7 strains and six stx-negative E. coli O157:H7 strains from animal and food sources. Comparative whole genome analysis of E. coli O157:H7 strains with different stx gene profiles obtained from animal and food sources will expand our understanding of the mechanisms of exchange of stx genes and other genes associated with the exchange of stx genes between stx-positive and stx-negative E. coli O157:H7. Results from this study could potentially serve as a basis to help develop potential strategies to reduce the prevalence of pathogenic E. coli O157:H7 in livestock and downstream food production environments.The current study was conducted to better understand the detailed mechanisms involved in the acquisition or loss of ed study . The PCRE. coli O157:H7 strains with different stx profiles were selected for this study. These included strains C1-057 , C1-010 , and C1-067 , and all three strains were previously isolated from fecal samples collected from cattle in a commercial feedlot . Additionally, the two stx-positive E. coli O157:H7 strains, C1-010 and C1-067, were sequenced via the PacBio Sequel system. The two stx-positive E. coli O157:H7 strains were activated from frozen glycerol stocks (−80°C) by two transfers in tryptic soy broth  at 37°C for 24 h. The genomic DNA of each isolate was extracted using a QIAGEN Genomic DNA kit . For the PacBio platform, the extracted large genomic DNA needs to be fragmented into smaller size by EcoRI restriction enzyme prior to DNA sequencing. If EcoRI restriction recognition sites were methylated, the restriction enzyme would not be able to cleave the DNA. In our study, a small amount of extracted DNA sample was digested with EcoRI. Then, gel electrophoresis was used to prove that EcoRI restriction recognition sites were not methylated, and the genomic DNA could be fragmented into smaller size. The extracted DNA samples were sent out for sequencing service by Genewiz, Inc. .Three  feedlot . For thestx1−, stx2c+) and 497x for strain C1-067 . The lengths of raw reads were between 3 and 40 kb. The raw reads of strains C1-010 and C1-067 were subsequently de novo assembled into contigs separately by Canu (version 1.6). When fully assembled, the genomes of strains C1-010 and C1-067 consisted of 13 and 12 scaffolds, respectively. To identify plasmids, DNA sequences of pO157 (NCBI accession no. NC_002128) and pOsak1 (NCBI accession no. AB011548) of E. coli O157:H7 strain Sakai were used as a reference for Basic Local Alignment Search Tool (BLAST) analyses  (2) with the parameter set at 2,000 bootstraps  . The kSNotstraps .E. coli O157:H7 strains were selected for prophage identification. Phage Search Tool Enhanced Release (PHASTER) (available at3) is a web-based service for rapid identification and annotation of prophage sequences within bacterial genomes , C1-010 , and C1-067 , the reference strain Sakai , and the six aforementioned stx-negative strains  were extracted from the whole genome sequence of E. coli O157:H7 strain Sakai  identified by PHASTER.DNA sequences of six previously reported in Sakai . The stxg BLASTn . Each ofexo, bet, and gam) and Red operon repressor gene cI were extracted from the whole genome sequence of E. coli O157:H7 strain Sakai (stx insertion sites in the 10 selected strains using BLASTn (stx−), C1-010 , and C1-067  for these four genes.DNA sequences of three Red recombination genes  and strain Sakai were aligned by DNASTAR MegAlign Pro 14 to identify large specific sequence regions (SSRs) (> 10 kb) that were located in either strain C1-057 or Sakai. Genes that were harbored in SSRs and the sequence regions (10 kb) downstream or upstream of each SSR in either strain C1-057 or Sakai were mapped against the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using the Integrated Microbial Genomes and Microbiomes (accessible at4)  and C1-010 , and the six stx-negative strains from the NCBI database to identify if any of the metabolic pathway genes were present in any of these strains.The whole genome sequences of ble at4) . The uniE. coli O157:H7 strains previously isolated from feedlot cattle fecal samples. As already described, each of these three strains had different stx profiles. In the current study, the gapless chromosome of the stx-negative strain, C1-057 (5.45 Mb), was obtained. The chromosomes of the two stx-positive strains, C1-010  (5.57 Mb) and C1-067  (5.56 Mb), were composed of 12 and 11 scaffolds, respectively. In addition, the plasmid identification showed that strains C1-057, C1-010, and C1-067 each contained one pO157-like plasmid with sequence similarity of 97% (97.5 kb), 97% (91.9 kb), and 99% (92.7 kb) to the pO157 (92.7 kb) in strain Sakai, respectively. The DNA sequences of these three strains have been deposited to the NCBI  and C1-010 , together with the six stx-negative E. coli O157:H7 strains from the NCBI database, were grouped into Cluster 1, whereas C1-067  and the five foodborne outbreak strains were grouped into Cluster 2. More specifically, strains C1-057 and C1-010 were clustered in Cluster 1a with three stx-negative strains from the United States and one stx-negative strain from Malaysia. Strain C1-067 grouped into Cluster 2b with three of the foodborne outbreak strains  had the closest relationship with strain Sakai.Single nucleotide polymorphisms analysis was performed to evaluate the phylogenomic relationship among the 14  strains . The optE. coli O157:H7 strains. The 10 strains included all the strains that were grouped in Cluster 1  and strains C1-067  and Sakai from Cluster 2b since they had the closest phylogenomic relationship. The results of the identified prophages are shown in E. coli O157:H7 strains. These prophages contained genome sizes ranging from 6.3 to 124.9 kb. Strain Sakai is known to have one P4-like prophage Sp2 (12.9 kb) , strain C1-010 , and strain C1-067  harbored the identical Sp2 in their genomes. One stx-negative strain (NZRM3614) contained a Sp2-like prophage that shared 100% coverage and identity to Sp2 but with an insertion sequence of 1.3 kb.Prophage identification, using PHASTER, was conducted on 10 of the 14 12.9 kb) . The DNAstx1+, stx2a+) were grouped into Cluster 2b  and C1-010  from feedlot cattle were grouped into Cluster 1  and C1-067  belonging to Clusters 1 and 2, respectively, were also compared. Strains C1-057 and C1-067 shared three intact, one questionable, and two incomplete identical prophages. Strain C1-057 carried eight intact, one questionable, and one incomplete prophages that were not found in strain C1-067, whereas strain C1-067 contained eight intact, one questionable, and three incomplete unique prophages. In addition, in strain C1-067, two intact, one questionable, and five incomplete prophages shared similar but not identical sequences with the corresponding intact prophages in strain C1-057.Prophages in the newly sequenced strains C1-057  and seven stx-negative strains], but not in the two strains within Cluster 2b (Sakai and C1-067). PHASTER results indicated that this prophage was the most similar to a known phage P4  with 61% query coverage and 97.4% identity. It should be noted that when phage P4 infects E. coli cells, it has three known modes of propagation: (1) it can integrate within the host chromosome as a prophage; (2) it can replicate as a plasmid; or (3) it can replicate itself rapidly in its lytic cycle . All 10 strains carried all 6 stx insertion site genes. As shown in stx insertion sites of wrbA, yehV, and sbcB were occupied by prophages in 2, 10, and 3 strains, respectively, whereas the other 3 stx insertion sites of argW, yecE, and Z2577 remained accessible and unoccupied in all 10 strains.The chromosome of the 10 E. coli O157:H7 strains, strains Sakai and C1-067  belonging to Cluster 2b carried 2 identical stx-carrying prophages: 1 stx1-carrying prophage at the yehV stx insertion site and 1 stx2a-carrying prophage at the wrbA stx insertion site. All strains from Cluster 1a, except for strain C1-010 , harbored an identical stx-negative prophage at the yehV stx insertion site. The two strains from Cluster 1b and strain C1-010 carried an identical stx-negative prophage at the yehV stx insertion site, whereas strain C1-010 contained a stx2c-carrying prophage, and the two strains from Cluster 1b contained stx-negative prophages with varied DNA sequences at the sbcB stx insertion site.Among the 10 selected E. coli O157:H7 strains, one specific type of prophage with varied DNA sequences was observed at the yehV stx insertion site. The stx1-carrying prophage Sp15 (50.5 kb) was present in strains Sakai and C1-067 . An intact stx-negative prophage (47.6 kb), named Sp15-like, in all Cluster 1 strains had 72% query coverage and 95% identity to prophage Sp15. We observed a 10.2 kb DNA fragment without the stx genes of the prophage Sp15-like in the strains from Cluster 1, whereas a 12.8 kb DNA fragment containing the genes of stx1A and stx1B of the stx1-carrying prophage Sp15 was present in each strain of Sakai and C1-067  but with varied sizes and varied query coverages and identities were found in the stx2c-carrying strain C1-010 belonging to Cluster 1a and the two stx-negative strains from Cluster 1b. A stx2c-carrying prophage (61.7 kb), named 1717-like-A, in strain C1-010 had 98% query coverage and 99% identity to phage 1717. A stx-negative prophage , named 1717-like-B, and another stx-negative prophage , named 1717-like-C, were found in the stx-negative strains CV261 and NZRM3614, respectively. Sequence comparisons demonstrated that a 29.2 kb DNA fragment containing the stx2c gene was present in prophage 1717-like-A in strain C1-010 belonging to Cluster 1a, but this DNA fragment was absent in the stx-negative prophages 1717-like-B and 1717-like-C in strains CV261 and NZRM3614 from Cluster 1b, respectively.At the ion site , prophagstx insertion sites in the 10 selected strains were investigated for the presence/absence of Red homologous recombination genes  and/or their repressor gene cI  contained both the three Red recombination genes and their repressor cI. Depending on the strain, stx-negative prophages had three situations: (1) prophage 1717-like-C in one strain contained both the three Red recombination genes and their repressor cI; (2) prophage Sp15-like in eight strains harbored only the three Red recombination genes, but the repressor cI was absent; and (3) prophage 1717-like-B in one strain contained neither any of the Red recombination genes nor the repressor cI.The prophages located at the 3  gene cI . This anstx−), C1-010 , and C1-067  were also examined for any other prophages that were not located at the stx insertion sites but also harbored the Red recombination gene set. In addition to the prophages at the stx insertion sites, only one additional prophage  carrying exo and bet, but not gam and cI, was found in strains Sakai, C1-057, C1-010, and C1-067.The entire genomes of strains Sakai, C1-057  and C1-057 (stx−), respectively. The stx2a-carrying E. coli O157:H7 strains Sakai and C1-067 carried eight copies of unique metabolic pathway genes, including sodC, modD, fepC, fecD, fbpA, dhaM, dhaL, and dhaK. On the other hand, the stx2c-carrying E. coli O157:H7 strain C1-010 and seven stx-negative E. coli O157:H7 strains contained one copy of unique metabolic pathway gene, dhaR. Protein functions of the nine copies of unique metabolic pathway genes are listed in We found that there were nine SSRs and six SSRs uniquely present in strains Sakai  within a conserved region among these strains . These results suggested that the three E. coli O157:H7 strains from feedlot cattle may exhibit different levels of pathogenicity.In this study, we performed a whole genome analysis of three t cattle . Phylogestx-negative E. coli O157:H7 could potentially be converted to stx-positive E. coli O157:H7 in cattle, swine, and avian production systems. A previous evolutionary model suggested that stx-positive E. coli O157:H7 could have evolved stepwise from an ancestor E. coli O55:H7, following the steps of 1) acquiring stx2-carrying prophages, 2) losing the O55 rfb-gnd gene, acquiring the O157 rfb-gnd gene, and losing the ability to ferment sorbitol, and 3) acquiring stx1-carrying prophage  strains may potentially lead to an increase in their competitiveness in complex environments, such as feedlot cattle.The presence or absence of ce genes . In thismunities . Genes f ability . Genes dtabolism . The presodC gene encodes copper/zinc-superoxide dismutase (Cu–Zn SOD) that catalyzes the dismutation of the superoxide radical (O2–) into hydrogen peroxide (H2O2)  but was absent in strains C1-057 , C1-010 , and the six stx-negative strains from the NCBI database. The second copy of sodC may provide additional protection from the toxicity of superoxide radical. Thus, the stx-positive strains Sakai and C1-067 may be more resistant to exogenous superoxide radical than strains C1-057, C1-010, and the six stx-negative strains from the NCBI database.The e (H2O2) . The sodsurvival . We founfepC, fecD, and fbpA genes encode proteins that are involved in three separate iron uptake systems, respectively. The fepC, from the ferric enterobactin transport system FepABCDG, encodes an ATP-binding protein transport that provides energy to assist iron uptake from the environment  can potentially increase iron uptake ability compared with strains C1-057 , C1-010 , and the six stx-negative strains from the NCBI database.The ironment . The fecmembrane . The fbp protein . AlthougEscherichia coli can metabolize glycerol via two microaerobic pathways: the dihydroxyacetone kinase (DAK) pathway and the glycerol pathway  E. coli O157:H7 stx-positive strains Sakai and C1-067  harbored a dha operon containing dhaK, dhaL, and dhaM, but not dhaR; (3) E. coli O157:H7 stx-positive strain C1-010  harbored another dha operon comprising only dhaR but not dhaK, dhaL, or dhaM; and (4) seven E. coli O157:H7 stx-negative strains harbored the same disrupted dha operon as strain C1-010. Additionally, both two types of disruptions of dha systems as shown in strains C1-067 and C1-010 inactivated the metabolism of glycerol via the DAK pathway. dhaK led to a reduction in the efficacy of glycerol utilization by approximately 50%, resulting in a reduction in cell growth rate by 70%, in a minimal medium supplemented with glycerol as the sole carbon source. Another study also indicated that adding three levels of crude glycerin  in corn silage diets decreased the prevalence levels of E. coli O157 to 4.4, 3.2, and 1.8% in fecal samples from growing cattle, respectively (P < 0.01)  in feed decreased (P < 0.05) the DNA concentrations of fiber-fermenting bacteria Butyrivibrio fibrisolvens and Selenomonas ruminantium in rumen, potentially resulting in the reduction of fiber digestibility  E.  < 0.01) . Our restibility . Howeverosolvens . In addiglycerol . The addstx2a-carrying strains and one in the one stx2c-carrying and seven stx-negative strains were found downstream of an IS629 within a conserved region  can be found below: MJ conceived the presented idea and carried out the experiment and wrote the manuscript with the support from HY. HY helped supervise the project. All authors provided critical feedback and contributed to the final version of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The RAS oncogene is one of the most frequently mutated genes in human cancer, with K-RAS having a leading role in tumorigenesis. K-RAS undergoes alternative splicing, and as a result its transcript generates two gene products K-RAS4A and K-RAS4B, which are affected by the same oncogenic mutations, are highly homologous, and are expressed in a variety of human tissues at different levels. In addition, both isoforms localise to the plasma membrane by distinct targeting motifs. While some evidence suggests nonredundant functions for both splice variants, most work to date has focused on K-RAS4B, or even just K-RAS . This review aims to address the most relevant evidence published regarding K-RAS4A and to discuss if this “minor” isoform could also play a leading role in cancer, concluding that a significant body of evidence supports a leading role rather than a supporting (or secondary) role for K-RAS4A in cancer biology. The importance of gene alternative splicing has been well documented. This conserved biological process occurs when a single gene produces different mRNA transcripts, thus helping to contribute to the formation of a vast transcriptome and proteome . This prRAS mutations are frequently found in cancer   , where tcancers) . In factcancers) . Figure The discovery over 35 years ago  of the fPubmed search on “K-RAS” or “KRAS” yields 21,408 results, a search on “K-RAS4A” or “KRAS4A”or “K-Ras4A” or “KRas4A” or “K-Ras4a” or “KRas4a” yields 54 results and a search on “K-RAS4B” or “KRAS4B″or “K-Ras4B” or “KRas4B” or “K-Ras4B” or “K-Ras4b” or “KRas4b” yields 213 results . This finding suggests that most studies have not discriminated by K-RAS isoform. Nevertheless, the two splice variants exhibit differential tissue expression , which are common to both genes, thus rendering both oncogenic . The biopression . TherefoIt is well stablished that RAS isoforms exhibit distinct biological activities and subcellular localisations that depend mainly on the interaction between the C-terminal hypervariable region (HVR) and host membranes . The HVRRAS interaction with the plasma membrane is required for its function. K-RAS4A and K-RAS4B differ mainly in their C-terminal regions , which iStructural analysis using atom molecular dynamics simulations investigated K-RAS4A placement at membranes that contain anionic lipids (POPS or PIP2) . This stThe protein conformations of K-RAS4A and K-RAS4B have also been compared by all-atom molecular dynamics simulations to identify isoform-specific differences. The results suggested that the catalytic domain of GDP-bound K-RAS4A differs from that of K-RAS4B by presenting a more exposed nucleotide binding pocket, also showing distinct dynamic fluctuations in switch I and II regions, which could affect the interaction between the catalytic domain and downstream effectors .in vitro, K-RAS4A and K-RAS4B have been shown to differentially affect Raf-1  . For exaA quantitative RT-PCR assay has been developed to detect the splice junction region and thus measure variant expression in human cancer cell lines . Of the As previously mentioned, the K-RAS4A HVR sequence shares similarities with those of K-RAS4B and N-RAS. Nussinov and colleagues proposed that the N-RAS-like state of K-RAS4A  could influence its high expression in melanoma, and that the K-RAS4B-like state of K-RAS4A  could contribute to the high expression levels seen in colon cancer .Regarding benign tumour tissues, Shahrabi-Farahani and colleagues reported that during the proliferative phase of the menstrual cycle, K-RAS4A mRNA was upregulated (2.7-fold higher) in eutopic endometrium in patients with endometriosis compared to controls , whereasDifferent roles have been attributed to K-RAS4A. Studies of embryonic stem cells have suggested that K-RAS4A promotes apoptosis while K-RAS4B inhibits it, and that the K-RAS4A/4B isoform ratio regulates tumorigenesis by influencing stem cell differentiation and survival . In addiG12V in mice that lack K-RAS4B is sufficient to promote metastatic lung adenocarcinomas  present a reduction in leukemogenicity potential. Unlike the results seen with mutations at the palmitoylation site of N-RAS , palmitoylation-defective K-RAS4A could still induce leukaemia in mice (In human K-RAS-mutant leukaemia cell lines and in acute myeloid leukaemia (AML) cells, K-RAS4A is also expressed, and Zhao and colleagues showed that cells harbouring mutations at the palmitoylation site of oncogenic K-RAS4A (i.e., palmitoylation-defective mutant K-RAS4A in mice . The KIK in mice . These fG12C inhibitor able to reduce K-RASG12C tumours (G12C versus K-RAS4BG12C in different cancer types remains to be determined. It would be interesting to see the development of novel mutation- and splice variant-specific inhibitors in those cancers where both isoforms are simultaneously affected. Nevertheless, more analysis should be performed to better clarify if there are any significant differences between mutant K-RAS4A and mutant K-RAS4B in response to distinct therapies.The fact that both splice variants are identical in the region where most K-RAS oncogenic mutations occur suggests that previous reports of mutations in K-RAS may actually have uncovered mutations in both transcripts, not just in K-RAS4B. In addition, cancers harbouring K-RAS mutations may behave differently depending on which splice variant is predominantly affected, which could impact on therapy response. As K-RAS4A and K-RAS4B possess slightly different structures when in the GDP-bound state, with GDP-bound K-RAS4A presenting a more exposed nucleotide binding pocket than GDP-bound K-RAS4B , compoun tumours , is a maK-RAS4B research has historically overshadowed that of K-RAS4A, suggesting that K-RAS4A is a minor variant. Nevertheless, the fact that K-RAS4A is evolutionarily conserved and binds distinct effectors at different affinities compared to K-RAS4B, in addition to the fact that K-RAS4A expression varies across tissue types, argue for a more important role than previously thought. Additional work is needed to unravel the different roles that each splice variant plays in normal versus tumours tissues. Such knowledge may help inform understanding of therapy resistance and improve disease management of cancer types with differential splice variant expression. Personalised medicine has exploited K-RAS-mutation-specific tumour differences for the development of mutation-selective anti-RAS strategies; thus, it could be beneficial to place K-RAS4A in the spotlight and perhaps achieve more selective cancer treatment strategies.


Synechocystis sp. PCC 6803 is incapable of catabolism of L-arabinose as a source of carbon and energy. In this study, all the heterologous genes expressed in Synechocystis were derived from Escherichia coli K-12. Initially we constructed four Synechocystis strains that expressed AraBAD enzymes involved in L-arabinose catabolism, either in combination with or without one of the three arabinose transporters, AraE, AraFGH or AraJ. Among the recombinants, the strain possessing AraJ transporter was observed to be the most efficient in terms of dry biomass production and L-arabinose consumption. Later, an additional strain was generated by the expression of AraJ in the AraE-possessing strain. The resultant strain was shown to be advantageous over its parent. This study demonstrates that AraJ, a protein with hitherto unknown function plays a role in the uptake of L-arabinose to boost its catabolism in the transgenic Synechocystis strains. The work also contributes to the current knowledge regarding metabolic engineering of cyanobacteria for the utilization of pentose sugars.Lignocellulosic biomass can serve as an inexpensive and renewable source of carbon for the biosynthesis of commercially important compounds. L-arabinose is the second most abundant pentose sugar present in the plant materials. Model cyanobacterium The online version contains supplementary material available at 10.1186/s13568-021-01277-7. Synechocystis strains were engineered to transport and catabolize L-arabinose.L-arabinose transport took place even in the absence of heterologous transporters.araJ showed the most impressive results.Expression of putative transporter gene Biomass is an abundant and renewable raw material that can be converted into a number of useful products, transportation fuels and direct energy  is one of the most extensively studied primary phototrophs. It is able to grow under autotrophic, mixotrophic, and heterotrophic conditions, can integrate foreign DNA at a high frequency, and holds a proven record as a host strain for the synthesis of biofuels, health related compounds, and commodity chemicals  under consideration. AraE (proton symporter) and AraFGH are well characterized members of the Major Facilitator Superfamily (hereafter MFS) class and ATP Binding Cassette (hereafter ABC) class of transporter proteins, respectively  family  was used for cloning and plasmid construction steps. The strains were grown at 37 °C on solid LB medium or in liquid LB medium with shaking (220 rpm) in the presence of appropriate antibiotic, if necessary. Antibiotic concentrations used for selection were- ampicillin: 100 µg/mL, kanamycin: 25 µg/mL, erythromycin: 200 µg/mL, and spectinomycin: 50 µg/mL. The antibiotics were purchased from Thermo Fisher Scientific.Synechocystis wild-type strain (hereafter WT), kanamycin resistance cassette, upstream and downstream regions of a neutral site , a ~ 0.4 kb region encompassing the psbA2 promoter (denoted as psbA2 promoter) and a ~ 0.5 kb 5ST1T2 double terminator region were inserted into pBluescript II SK+ (Stratagene) as described  and 5′-ACTCCTGCAGTCAGACGCCGATATTTCTC-3′ (reverse primer) for araFGH;5′-ACTCGAATTCATGCACAAATTTACTAAAGCCCTG-3′ (forward primer) and 5′-ACTCCTGCAGTCAGACAGTGCGTTTCGCTTTTTG-3′ (reverse primer) for araJ.5′-ACTCGAATTCATGAAAAAAGTCATTTTATCTTTGGCTC-3′ (forward primer) and 5′-ACTCCTGCAGCTACCCCAGTGGTTTCGCCAG-3′ (reverse primer) for EcoRI-PstI sites of the plasmid to generate three individual plasmids. Note that, in the case of araFGH gene set, only the region from the start codon of araF to the end codon of araG was amplified; no other regulatory elements from the E. coli araFGH operon were included.The amplicons were inserted between Synechocystis strains possessing exogenous arabinose transporters and in the WT strain, erythromycin resistance cassette was amplified from plasmid pTSC   and 5′-ACTCGGATCCATCGATTCACAAAAAATAGGCACACG-3′ (reverse primer). The amplicon was inserted into pBluescript II SK+ plasmid at KpnI-XhoI and SpeI-SacII sites of the plasmid, respectively. The psbA2 promoter and 5ST1T2 double terminator were amplified and cloned into the plasmid as described  and 5′-ACTCCTCGACATGGGGATCAGCGCTAAATGC-3′ (reverse primer) for the upstream region, and 5′-ACTCACTAGTAAGCCCATTTACGTCGTGTTGGTC-3′ (forward primer) and 5′-ACTCCCGCGGCCAACGGTTCCAGGTGACTATC-3′ (reverse primer) for the downstream region. The upstream and downstream fragments were inserted between 5′-ACTCGAATTCATGGCGATTGCAATTGGCCTC-3′ (forward primer) andEcoRI-SbfI restriction sites present at its ends and inserted between the EcoRI-PstI sites of the plasmid. Similar to araFGH, in the case of araBAD gene set, only the region from the start codon of araB to the end codon of araD was amplified; no other regulatory elements from the E. coli araBAD operon were included.5′-ACTCCCTGCAGGTTACTGCCCGTAATATGCCTTC-3′ (reverse primer). The amplicon was digested at the araJ into the engineered strain possessing araE and araBAD, spectinomycin resistance cassette, upstream and downstream regions of a neutral site , psbA2 promoter and 5ST1T2 double terminator region were inserted into pBluescript II SK+ (Stratagene) as described . When OD730 reached ~ 0.7–0.8, the transformations were carried out as described (Porter d Porter . Upon coSynechocystis strains at OD730 ~ 0.7 as previously described (Mohamed and Jansson petA (sll1317) was used as the positive control as described  was used per manufacturer’s instructions. PCR products were analyzed on 0.8% agarose gel.Total RNA was isolated from cultures of Synechocystis strains was measured in terms DW as described , with the initial OD730 of 0.05. The cultures were provided with: for autotrophy, ~ 60 μE m-2 s-1 light; for mixotrophy, 20 mM L-arabinose (3.00 g/L) or 20 mM D-glucose (3.60 g/L) or 10 mM each of L-arabinose (1.50 g/L) and D-glucose (1.80 g/L) along with ~ 60 μE m-2 s-1 light. Biomass values were estimated at 6-h intervals for the first 3 days followed by 24-h intervals for the next 4 days.Growth exhibited by Enzymatic uptake assays were performed for the cultures grown only under mixotrophy. During the assays, 1 mL cultures were collected at the same time intervals specified for biomass measurements. The cultures were immediately filtered using 0.45 µm pore size 13 mm nylon membrane syringe filters (Fisher Scientific) to obtain cell-free media.340 values before and after the β-galactose dehydrogenase action.The amount of L-arabinose present in the filtered media was measured using L-Arabinose/D-Galactose Assay Kit (Megazyme) per manufacturer’s instructions. Sequential action of galactose mutarotase and β-galactose dehydrogenase generates NADH molecules. The amount of NADH is stoichiometric with that of L-arabinose which was calculated from the difference in OD510 values obtained for the samples, in reference to the OD510 value read for a glucose standard.The amount of D-glucose present in the filtered media was measured using D-Glucose Assay Kit-GOPOD Format (Megazyme) per manufacturer’s instructions. Action of glucose oxidase and peroxidase generates quinoneimine dye. The amount of quinoneimine formed is stoichiometric with that of D-glucose. The amount of D-glucose in the cell-free media was calculated from ODSynechocystis strains possessing arabinose transporter and catabolic genes were developed from the WT strain in two distinct rounds of transformation by homologous double recombination. In the first round, three recombinant strains possessing one of the three transporter genes, araE or araFGH or araJ of E. coli K-12 origin were generated. The strains were named A-Tr1, A-Tr2 and A-Tr3, respectively, and collectively referred to as A-Tr. In the subsequent round of transformation, four recombinant strains were generated by inserting the araBAD gene set of E. coli K-12 origin into the three strains generated in the first round of transformation and the WT strain. The recombinant strains were named A-Ut1, A-Ut2, A-Ut3 and A-Ut4, respectively, and collectively referred to as A-Ut  coding for apocytochrome f, a core subunit of cytochrome b6f complex was tested. As the negative control, DNase-treated RNA samples obtained from each strain were tested to ensure the absence of any leftover DNA in the RNA samples. In the presence of cDNA templates obtained from the respective strains, primers A1/A2 for araE, B1/B2 for araFGH, C1/C2 for araJ and E1/E2 for araBAD generated products of expected sizes. In negative control reactions, the aforementioned primer pairs failed to generate any product. A ~0.98-kb band representing petA was obtained for positive controls in all the transformants , biomass yields were observed to be similar for all the strains except for the AraJ-possessing A-Tr3 and A-Ut3 strains. A-Tr3 and A-Ut3 strains showed a degree of growth impairment in the presence of 20 mM glucose , the WT and the A-Tr strains showed biomass yields similar to those obtained under autotrophy, indicating the inability of the strains to utilize arabinose. However, the A-Ut strains showed greater dry weight (hereafter DW) yields under the mixotrophy-arabinose condition than under autotrophy. Biomass accumulation results demonstrate that all the A-Ut strains carrying the catabolic gene set were able to utilize arabinose. Under the mixotrophy-arabinose condition, at the 7th day of culture, the biomass reached by the A-Ut1, A-Ut2, A-Ut3 and A-Ut4 strains was ~ 31%, 43%, 112%, and ~ 50% greater than the average biomass for the four strains under autotrophy. In the presence of arabinose, AraJ-possessing A-Ut3 strain showed higher biomass values than the other A-Ut strains at each time point. At the 7th day of culture, A-Ut3 showed ~ 38% greater biomass than the A-Ut4 strain, which lacked any heterologous transporter . Under the mixotrophy-mixed sugar condition, at the 7th day of culture, the biomass reached by the arabinose-consuming strains, A-Ut1, A-Ut2, A-Ut3 and A-Ut4 was ~ 124%, 130%, 215%, and ~ 136% greater than the average biomass for the four strains accumulated under autotrophy. Similar to the mixotrophy-arabinose condition, the A-Ut3 strain showed higher biomass values than the other A-Ut strains at each time point tested. At the end of the last day of culture, A-Ut3 showed ~31% greater biomass than the A-Ut4 strain. Under mixotrophy-arabinose as well as mixotrophy-mixed sugar condition, the AraFGH-possessing A-Ut2 strain showed DW values similar to those shown by the A-Ut4 strain. On the other hand, the AraE-possessing A-Ut1 strain showed marginally lower DW values than those observed for the A-Ut4 strain at all time points. In the presence of arabinose and glucose, the A-Ut strains did not show two distinct growth phases  of the araBAD operon were able to function in Synechocystis. We are unable to comment about the RBSs of the araFGH gene set at this time. Based on the biomass accumulation and the arabinose uptake results, AraFGH-possessing A-Ut2 strain failed to show any advantage over the A-Ut4 strain which lacked any non-native transporter   family  A-Tr1, (b) A-Ut1, (c) A-Tr2, (d) A-Ut2, (e) A-Tr3, (f) A-Ut3, (g) WT and (h) A-Ut4 were grown as three biological replicates, and the data are presented as means ± standard deviations. DW and residual sugars were estimated every 6 hours for the first 3 days followed by every 24 hours for the next 4 days. Blue line with filled squares, red lines with filled diamonds, light green line with filled triangles and violet line with filled circles represent growth under autotrophy, mixotrophy (20 mM arabinose), mixotrophy (20 mM glucose) and mixotrophy (10 mM each of arabinose and glucose) respectively. Similarly, light blue line with empty squares, orange line with empty circles and sky-blue line with crosses represent arabinose uptake under mixotrophy (20 mM arabinose), arabinose uptake under mixotrophy (10 mM each of arabinose and glucose) and glucose uptake under mixotrophy (10 mM each of arabinose and glucose) respectively. BG-11 media with 20 mM arabinose, 20 mM glucose, 10 mM arabinose and 10 mM glucose contain 3.0026, 3.6032, 1.5013 and 1.8016 g/L of the sugars respectively. Figure S2. Growth of A-Ut3 under various arabinose concentrations. A-Ut3 was grown in the presence of 0 mM (autotrophy) to160 mM arabinose as three biological replicates, and the data are presented as means ± standard deviations. DW was estimated every 24 hours for 7 days. Lines with filled circles represent growth in the presence of various arabinose concentrations, as specified within the figure. Figure S3. Growth of Synechocystis strains under mixotrophy in the presence of 20 mM arabinose. Synechocystis strains were grown in the presence of 20 mM (3.0026 g/L) L-arabinose. DW values estimated at the end of the seventh day are presented in the figure. Data were collected from three biological replicates and presented as means ± standard deviations. Table S1. Additional Synechocystis strains generated by modification of ribosome binding sites (RBSs) within araBAD gene set.


Caenorhabditis elegans have been studied to better understand complex, integrative developmental decision-making. Although it is known that ascarosides play multiple roles in the development and behavior of nematode species other than C. elegans, these parallel pheromone systems have not been well-studied. Here, we show that ascarosides in the nematode Caenorhabditis briggsae are biosynthesized in the same manner as C. elegans and act to induce the alternative developmental pathway that generates the stress-resistant dauer lifestage. We show that ascr#2 is the primary component of crude dauer pheromone in C. briggsae; in contrast, C. elegans dauer pheromone relies on a combination of ascr#2, ascr#3, and several other components. We further demonstrate that Cbr-daf-22, like its C. elegans ortholog Cel-daf-22, is necessary to produce short-chain ascarosides. Moreover, Cbr-daf-22 and Cel-daf-22 mutants produce an ascaroside-independent metabolite that acts antagonistically to crude dauer pheromone and inhibits dauer formation.The biosynthetic pathways and functions of ascaroside signaling molecules in the nematode  Caenorhabditis elegans has long been used as model organism not only for understanding general biological phenomena but also for the specific questions of nematode development and behavior. However, as we further explore this species, it is important to expand those findings to show they are applicable to other nematodes, beyond C. elegans.The nematode Caenorhabditis briggsae has previously been used to further investigate findings in C. elegans   . Caenorhifecycle . The gro methods . In partfeasible .C. elegans and C. briggsae is triggered by the confluence of both physical and chemical signals indicating the abundance of food, the concentration of conspecific nematodes in the local area, and the local amount of various dauer-inducing pheromones , Cel-dhs-28 (Cbr-dhs-28), and Cel-daf-22 (Cbr-daf-22)   . CaenorhC. elegans, further modification to the 4′ position of an ascaroside to form increasingly complex signals occurs in the gut granules  at 20°C.The Indian strain AF16 was used as a wild type for Cbr-daf-22 mutants was done using the C. briggsae modifications of the universal STOP-IN cassette method as described in Cbr-daf-22(sy1524) was a successful insertion of the universal STOP-IN cassette, while Cbr-daf-22 (sy1525), contains a 34 bp insertion from the Cbr-dpy-10 locus; both shown below with the flanking sequences underlined.Construction of the 2 sy1524GTGAATAGTGCATTAGACGAGGGAAGTTTGTCCAGAGCAGAGGTGACTAAGTGATAAGCTAGCTTGTGGACTAAAATATGCCGsy1525AAAGAGGCTGTGAATAGTGCTAAATCCGATTTGAAGACCTGTGACACACCGGTAGCTAGCTTATCACTTGTGGACTAAAATATGCCGC. briggsae onto 10 cm NGM plates (each seeded with 800 µl of OP50 E. coli grown to stationary phase in Luria–Bertani broth) and incubated at 22°C. Once the food was consumed, each plate was then washed with 25 ml of S-complete medium into a 125 ml Erlenmeyer flask, and 1 ml of OP50 E. coli was added , shaking at 220 rpm and 22°C. After 70 h, cultures were centrifuged at 1,000g for 1 min. After discarding supernatant, 24 ml H2O was added along with 6 ml bleach, 900 µl 10 M NaOH, and the mixture was shaken for 3 min to prepare eggs. Eggs were centrifuged at 1,000g, the supernatant was removed, and the egg pellet was washed with 25 ml M9 buffer twice and then suspended in a final volume of 5 ml M9 buffer in a 50 ml centrifuge tube. Eggs were counted and placed on a rocker and allowed to hatch as L1 larvae for 24 h at 22°C. A total of 75,000 L1 larvae were seeded in 25 ml cultures of S-complete with 1 ml of OP50 and incubated at 220 rpm and 22°C in a 125 ml Erlenmeyer flask. After 72 h, nematodes were spun at 1,000g for 5 min and spent medium was separated from nematode body pellet. Separated medium and nematode pellet were flash frozen over liquid nitrogen and then lyophilized. Two biological replicates were grown for each strain. Mutants were grown with parallel wild-type controls, and biological replicates were started on different days.Culturing began by chunking Cbr-daf-22(sy1524), Cbr-glo-1(sy1382), N2, Cel-daf-22(ok693), and Cel-glo-1(zu391) were made using previously described methods  was grown until exhausted. The liquid culture supernatant was separated from the pellet, filtered, and then dried completely. The dried material was extracted using ethanol; the extract was then dried and redissolved in 1 ml of sterilized water.Crude pheromones of AF16,  methods . BrieflyE. coli grown in Luria–Bertani broth, spun down, and heated at 97°C for 5 min) in the center of the plate. Ten adult nematodes were placed on the plate and allowed to lay eggs for 3 h. The adult nematodes were removed, and the plates were seeded with an additional 10 µl of OP50 on top of the eggs. The plates were stored in an incubator at 25.5°C for 24 h, after which the plates were quickly checked to confirm normal hatching and L1 development. After a further 24 h in the 25.5°C in the incubator (48 h after the start of the experiment) the total number of nematodes and dauer-stage nematodes were counted for each plate. To continue to ensure that the pheromones did not affect development, plates were kept for a further 24 h after the end of the experiment to check for expected continued development .Dauer assays were run using the method described in g for 10 min at 4°C, and supernatants were transferred to 20 ml glass scintillation vials. Samples were then dried in a SpeedVac (Thermo Fisher Scientific) vacuum concentrator. Dried materials were resuspended in 1 ml methanol and vortexed for 1 min. Samples were pelleted at 10,000g for 5 min and 22°C, and supernatants were transferred to 2 ml HPLC vials and dried in a SpeedVac vacuum concentrator. Samples were resuspended in 100 μl of methanol, transferred into 1.7-ml Eppendorf tubes, and centrifuged at 18,000g for 20 min at 4°C. Clarified extracts were transferred to HPLC vials and stored at −20°C until analysis was performed.Lyophilized pellet and media samples were crushed and homogenized by shaking with 2.5 mm steel balls at 1,300 rpm for 3 min in 30 s pulses while chilled with liquid nitrogen (SPEX sample prep miniG 1600). Powdered media and pellet samples were extracted with 10 ml methanol in 50 ml centrifuge tubes, rocking overnight at 22°C. Extractions were pelleted at 5,000m/z range 150–1,000 m/z, AGC target 3e6. The instrument was calibrated with positive and negative ion calibration solutions (Thermo-Fisher) Pierce LTQ Velos ESI pos/neg calibration solutions. Peak areas were determined using Xcalibur 2.3 QualBrowser version 2.3.26 (Thermo Scientific) using a 5-ppm window around the m/z of interest.High-resolution LC–MS analysis was performed on a Thermo Fisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. One microliter of extract was injected and separated using a water-acetonitrile gradient on a Thermo Scientific Hypersil GOLD C18 column  and maintained at 40°C. Solvents were all purchased from Fisher Scientific as HPLC grade. Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 1% B for 3 min, then from 1% to 100% B over 20 min, 100% for 5 min, then down to 1% B for 3 min. Mass spectrometer parameters: 3.5 kV spray voltage, 380°C capillary temperature, 300°C probe heater temperature, 60 sheath flow rate, 20 auxiliary flow rate, 2.0 spare gas; S-lens RF level 50.0, resolution 240,000, Cbr-glo-1 loss-of-function mutant shares at least part of its phenotype with an orthologous Cel-glo-1 mutant: neither mutant is able to form gut granules  and modular (e.g. icas#2) short-chain ascarosides  and anthranilic acid glucosides (angl)   (Coburn s (angl) .Cbr-glo-1 and Cbr-daf-22 mutants would affect wild-type nematode dauer formation across a range of concentrations. Wild-type (AF16) crude pheromone exhibited an expected dose–response curve that showed a stable increase in the number of dauers as the amounts of pheromone increased (Cbr-daf-22 (Cbr-glo-1 mutants (Cbr-daf-22 crude dauer pheromone was unable to induce dauer formation at any concentration. Cbr-glo-1 crude pheromone appeared to induce dauer formation at fairly consistent but intermediate levels across a broad range of dosages. A Kolmogorov–Smirnov test showed that the dauer curve of Cbr-glo-1 pheromone is significantly different from that of AF16 (C. briggsae are simple (i.e. unmodified) short-chain ascarosides.We performed a series of dauer assays using wild-type (AF16) nematodes to determine how the various innate crude pheromone preparations from ncreased . At highr-daf-22  and Cbr- mutants . As hypo of AF16 . Even thC. elegans, testing the effects on wild-type nematodes of crude pheromone prepared from N2 wild type  and Cel-glo-1 (Cbr-glo-1) have been shown to be physiologically and metabolomically equivalent  and anthranilic acid glucosides (angl) at levels comparable to, or slightly lower than wild type. Functionally, the absence of short-chain ascarosides in Cel-daf-22 and Cbr-daf-22 crude pheromone causes a complete lack of dauer formation under the same conditions where increased amounts of C. briggsae or C. elegans wild-type crude pheromone, respectively, causes a linear increase in dauer formation.Cel-glo-1 and Cbr-glo-1 mutants have previously been shown to lack gut granules and the subsequent ability to form 4′-modified ascarosides (Cbr-glo-1 and Cel-glo-1 mutant crude pheromone to have unusual dose response curves where the amount of dauer formation appears to be independent of the amount of pheromone encountered. As phosphorylated ascarosides are upregulated in both Cel-glo-1 and Cbr-glo-1 (arosides . Functiobr-glo-1 , they arC. elegans and other nematode species, especially when their ascaroside profiles overlap.With these similarities of the main ascaroside-forming pathways, we can infer that other major components of the ascaroside formation pathways, such as the remainder of the beta-oxidation pathway, are likely to be similarly conserved. We also expect to see further conservation in the major components of ascaroside formation between Caenorhabditisbriggsae releases fewer types of known ascarosides than C. elegans and, subsequently, has seemingly fewer behaviors regulated by these ascarosides (arosides . The patC. briggsae. Thus, ascr#2 acts functionally the same in both C. briggsae and C. elegans. Combined with the conserved ascaroside formation pathways, this finding indicates that it may be an ascaroside originally used by the evolutionary ancestor of both species. However, the 2 species then diverge in other dauer-inducing ascaroside signals such as ascr#3, which is another major component of C. elegans dauer pheromone (C. briggsae metabolome.We confirmed that ascr#2 is the main component of dauer pheromone in heromone , but whiCaenorhabditiselegans and C. briggsae not only are closely related in the elegans group of the Caenorhabditis genus, but also have overlapping habitats (C. briggsae preferring warmer climates and seasons than C. elegans (habitats . The 2 s elegans . But thi elegans . FurtherC. briggsae (It has been reported that an indole-modified version ascr#2, icas#2, acts as a sex pheromone in briggsae . The unmCbr-daf-22 and Cel-daf-22 samples indicated something more than a simple absence of dauer ascarosides. Subsequently, we found that Cbr-daf-22 and Cel-daf-22 pheromone actively depresses dauer formation, even when in the presence of dauer-promoting crude pheromone from wild-type nematodes.Although the absence of short-chain ascarosides and their dauer-inducing signals staves off dauer-formation under favorable developmental conditions, the almost complete lack of dauer formation in Cbr-daf-22 and Cel-daf-22 metabolomes contain either a specific antidauer compound or a compound that acts as a receptor antagonist. Potential candidates for either process include the angl or iglu families of glycosides, as these small molecules are highly abundant in Cbr-daf-22 and Cel-daf-22 metabolomes. The iglu family of glycosides is especially interesting as a potential antidauer signal as previous studies have shown these small molecules represent detoxification products of indole derived from E. coli, the primary food source of lab-cultured nematodes (Cel-daf-22 and Cbr-daf-22 pheromones both are able to suppress dauer in both C. briggsae and C. elegans, the antidauer pheromone for both species is hypothesized to contain at least 1 identical compound.Our findings indicate that ematodes . It woulematodes . As the C. elegans and C. briggsae.In addition to food, population density, and known dauer pheromones, it is clear that the dauer-decision system is further adjusted through the use of additional pheromone signaling. Thus, the dauer decision is not simply made under unfavorable conditions opposite to the standard L3 reproductive stage—instead there is an active antidauer signal that adds to the complexity of the finely tuned dauer-decision process in Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.G3 online.jkac014_Supplementary_DataClick here for additional data file.