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The mechanisms of modulating milk production traits remain largely unknown. Based on our previous RNA-seq, DDIT3 was presumed as a novel, promising candidate gene for regulating milk protein and fat traits in dairy cattle. To further detect the genetic effect of DDIT3 and its potential molecular mechanisms in regulating milk production traits in dairy cattle, here, we performed a genotype-phenotype association study. Two SNPs, g.-1194 C>T and g.-128 C>T, were significantly associated with MY (p = 0.0063), FY (p = 0.0001) and PY (p = 0.0216), respectively. A luciferase assay demonstrated that the allele T of g.-128 C>T increased the promoter activity by binding the HSF2, while allele C did not. To further reveal the molecular regulatory mechanisms, the DDIT3-knockdown MAC-T cells were established. It was observed that DDIT3 silencing could induce apoptosis and increase the number of PI-positive cells. Meanwhile, DDIT3 silencing led to increased expression of inflammatory markers, such as IL-6, IL6R, IL1B, IL7R, IL1RL2, IL1A, STAT1-5, MYC, IGFBP4, and IGFBP5, and especially for IL-6 (log2FC = 4.22; p = 3.49 × 10−112). Additionally, compared with the control group, increased lipid accumulation was found in the DDIT3-knockdown MAC-T cells. Thus, our results proved that lower expression of DDIT3 could result in increased lipid accumulation and apoptosis via up-regulating the expression of IL-6. These findings provided clues about the regulatory mechanisms of milk production traits in dairy cattle.

Circular RNA (circRNA), as a novel endogenous biomolecule, has been emergingly demonstrated to play crucial roles in mammalian lipid metabolism and obesity. However, little is known about their genome-wide identification, expression profile, and function in chicken adipogenesis. In present study, the adipogenic differentiation of chicken abdominal preadipocyte was successfully induced, and the regulatory functional circRNAs in chicken adipogenesis were identified from abdominal adipocytes at different differentiation stages using Ribo-Zero RNA-seq. A total of 1,068 circRNA candidates were identified and mostly derived from exons. Of these, 111 differentially expressed circRNAs (DE-circRNAs) were detected, characterized by stage-specific expression, and enriched in several lipid-related pathways, such as Hippo signaling pathway, mTOR signaling pathway. Through weighted gene co-expression network analyses (WGCNA) and K-means clustering analyses, two DE-circRNAs, Z:35565770|35568133 and Z:54674624|54755962, were identified as candidate regulatory circRNAs in chicken adipogenic differentiation. Z:35565770|35568133 might compete splicing with its parental gene, ABHD17B, owing to its strictly negative co-expression. We also constructed competing endogenous RNA (ceRNA) network based on DE-circRNA, DE-miRNA, DE-mRNAs, revealing that Z:54674624|54755962 might function as a ceRNA to regulate chicken adipogenic differentiation through the gga-miR-1635-AHR2/IRF1/MGAT3/ABCA1/AADAC and/or the novel_miR_232-STAT5A axis. Translation activity analysis showed that Z:35565770|35568133 and Z:54674624|54755962 have no protein-coding potential. These findings provide valuable evidence for a better understanding of the specific functions and molecular mechanisms of circRNAs underlying avian adipogenesis.

Breast cancer is the most frequently diagnosed malignant tumor in women. Foxtail millet is an important cereal food and exhibits a wide range of nutrition. We previously extracted total polyphenols composed of 12 compounds from foxtail millet bran (named BPIS). In this study, we find that BPIS inhibits the proliferation of breast cancer cells by restraining the cellular membrane synthesis via suppressing the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA). Mechanically, BPIS decreases the glucose regulated protein 78 (GRP78) level, which further inhibits the expression of SREBP-1 and downstream-target SCD1. Combining molecular docking, MTT screening and in vivo model, we ultimately identify that vitexin and syringic acid are the main active ingredients. Our data showed that the combination of vitexin and syringic acid suppress the proliferation of breast cancer cells, which support further study of BPIS as a new dietary assistant strategy to improve breast cancer.

Abstract Background Peutz–Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disease resulting in multiple gastrointestinal hamartomatous polyps, mucocutaneous pigmentation, and an increased risk of various types of cancer, and is caused by variations in the serine/threonine protein kinase STK11 (LKB1). Methods STK11 gene variations were identified by analyzing STK11 cDNA and genomic DNA. Minigenes carrying the wild‐type and mutant sequences were subjected to in vitro splicing assay to dissect the features of these mutations. The different distribution of wild‐type and mutant protein in cells were tested by Immunofluorescence assays and the functional analysis of the variation were performed using Western blot. Results A novel heterozygous splice‐acceptor site variation (c.921‐2 A>C) in intron 7 of the STK11 gene which is co‐segregates with the PJS phenotypes in the proband and all the affected family members and three previously reported variations (c.180C>G, c.580G>A, c.787_790del) were identified in the four families. The c.921‐2 A>C substitution resulted in the inactivation of a splice site and the utilization of a cryptic splice acceptor site surrounding exon 8, generating three different aberrant RNA transcripts, leading to frameshift translation and protein truncation. The results of minigenes indicated that the spliceosome can use a variety of 3’ acceptor site sequences to pair with a given 5’ donor site. The immunofluorescent visualization showed that the distribution of mutant STK11 was different from that of wild‐type STK11, suggesting the mutation may be the causative effect on the dysfunction of the mutant protein. The rescue experiments indicated that the failure of suppressing mTOR phosphorylation by shRNA STK11 could be eliminated by supply of wild‐type STK11 rather than mutant STK11. Conclusion We identified a novel heterozygous mutation (c.921‐2 A>C) in the STK11 in a Chinese PJS family. Haploinsufficiency of STK11 might contribute to the pathogenesis of the disease.

Discrepancies in human hepatitis delta virus (HDV) genotypes impact the virus’ biological behavior, clinical manifestation, and treatment response. Herein, this report aims to explore the role of recombination in the worldwide genotypic distribution and genetic diversity of HDV. Three-hundred-forty-eight human HDV full-length genomic sequences of ~1678 nt in length, isolated in twenty-eight countries worldwide between 1986 and 2018, were analysed. Similarity analysis and recombination mapping were performed, and forty-eight recombination events were identified, twenty-nine of which were isolated from Kyrgyzstan and determined to be involved in the diversity and extension of HDV sub-genotypes. HDV recombination occurred only between the genetically close genotypes (genotype 5 and genotype 2) or mainly within genotype 1, suggesting the complex replicative molecular mechanisms of HDV-RNA. The global distribution and classification of HDV genotypes have been updated, indicating that HDV recombination is one of the driving forces behind the biodiversity and the evolution of human HDV genomes. The outcome analysis suggests that the expansion of HDV sub-genotypes and the complex recombination networks might be related to the genomic character of Kyrgyzstan circulating strains and extensive mobility within countries and across borders. These findings will be of great importance in formulating more effective public health HDV surveillance strategies and guiding future molecular and epidemiological research to achieve better clinical outcomes.

WDR45 is an autophagy-related protein that involves in the formation of autophagosome. Mutations in WDR45 lead to the impairment of autophagy which is associated with the human β-propeller protein-associated neurodegeneration (BPAN). However, the relationship between autophagy and brain iron accumulation in patients with BPAN remains unclear. Here, we demonstrated that transferrin receptor (TfRC) which is critical for the iron import of cells was degraded via autophagy. TfRC was accumulated after the inhibition of autophagy by treatment with autophagic inhibitor chloroquine or knockdown of ATG2A. The intracellular iron content was increased in cells overexpressing TfRC or mutant WDR45, however, ferritin H (FTH) chain was decreased. Increased TfRC and simultaneously decreased FTH consequently resulted in an elevated level of ferrous iron (Fe2+) which further promoted cell ferroptosis, demonstrated by the increased lipid peroxidation and reactive oxygen species (ROS) and the decreased glutathione peroxidase 4 (GPX4) and cell viability. Taken together, these findings provide a piece of important evidence that WDR45 deficiency impairs autophagic degradation of TfRC, therefore leading to iron accumulation, and the elevated iron promotes ferroptosis which may contribute to the progression of BPAN.

Tuberculosis (TB), caused by Mycobacterium tuberculosis attacking the lungs and other organs, is one of the most common infectious disease worldwide. According to the WHO's 2020 report, a quarter of the world's population were infected with M. tuberculosis, and ~1.4 million people died of TB. Therefore, TB is a significant public health concern, which requires cost-effective strategies for prevention and treatment. The microbiota has been considered as a “forgotten organ” and a complex dynamic ecosystem, which plays a significant role in many physiological processes, and its dysbiosis is closely associated with infectious disease. Recently, a few studies have indicated associations between TB and microbiota. This review summarizes studies concerning the alterations of the gut and respiratory microbiota in TB, and their relationship with host susceptibility to M. tuberculosis infection, indicating that microbiota signatures in different stages in TB progression could be considered as biomarkers for TB diagnosis and control. In addition, the potential role of probiotics and postbiotics in TB treatment was discussed.

Objective: Wolcott–Rallison syndrome is a rare autosomal recessive inheritance disorder caused by the defectiveness of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3), which encodes the PKR-like endoplasmic reticulum kinase (PERK). Defect in EIF2AK3 results in a permanent diabetes in early infancy or newborn period, a tendency to develop skeletal fractures and other associated disorders such as severe liver and renal dysfunction, and central hypothyroidism. Two patients with Wolcott–Rallison syndrome-like manifestations in a Chinese family and family members were genetically analyzed to identify if any variations that occurred in EIF2AK3, which may cause Wolcott–Rallison syndrome.Methods: Whole-exome sequencing (WES) was performed to identify genetic variations, and Sanger sequencing was conducted to verify the identified variations in the family members with Wolcott–Rallison syndrome (WRS) clinical manifestations. Several bioinformatics tools were employed to predict the effect of EIF2AK3 variations on the protein function. The impact on PERK protein was analyzed by sequential analysis and evolution conservation study.Results: Two novel EIF2AK3 heterozygous single base variations (c.2818C>T and c.2980G>C) were detected in the proband. PERK has two functional domains: one is regulatory domain (aa 1–576), and the other is catalytic domain (aa 577–1,115). Both variations are missense mutations and locate in catalytic domain of PERK; c.2818C>T resulted in a residue substitution of proline for serine at amino acid site 940 (p.Pro940Ser), and variation c.2980G>C caused an amino acid change at position 994 from glutamic acid to glutamine (p.Glu994Gln). These novel missense variations may affect the physiological functions of PERK protein.Conclusions: Two novel compound heterozygous EIF2AK3 variations (c.2818C>T, p.Pro940Ser and c.2980G>C, p.Glu994Gln) were found in a Chinese family. The identification of the variations and verification of their pathogenicity extended the variation spectrum of EIF2AK3 variations causing Wolcott–Rallison syndrome and enriched valuable information for precise medical intervention for Wolcott–Rallison syndrome in China.

DIP2 protein contains three members: DIP2A, DIP2B and DIP2C, and are broadly expressed in the nervous system from Drosophila to human during embryonic development. Dip2c gene-associated mutations have been reported in tumors and neuronal diseases. However, the role of Dip2c in the context of mouse embryonic stem (mES) cells has not been explored. To investigate the biological function of Dip2c during early embryo development, we generated Dip2c-/- mES line using a CRISPR/Cas9 system. This cell line has contributed to further investigation of molecular mechanism of Dip2c during cell differentiation, as well as a cell model for screening for neurogenic drug and cancer clinical cure.

Mitochondria are known to generate approximately 90% of cellular reactive oxygen species (ROS). The imbalance between mitochondrial reactive oxygen species (mtROS) production and removal due to overproduction of ROS and/or decreased antioxidants defense activity results in oxidative stress (OS), which leads to oxidative damage that affects several cellular components such as lipids, DNA, and proteins. Since the kidney is a highly energetic organ, it is more vulnerable to damage caused by OS and thus its contribution to the development and progression of chronic kidney disease (CKD). This article aims to review the contribution of mtROS and OS to CKD progression and kidney function deterioration.

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

The feather rate phenotype in chicks, including early-feathering and late-feathering phenotypes, are widely used as a sexing system in the poultry industry. The objective of this study was to obtain candidate genes associated with the feather rate in Shouguang chickens. In the present study, we collected 56 blood samples and 12 hair follicle samples of flight feathers from female Shouguang chickens. Then we identified the chromosome region associated with the feather rate by genome-wide association analysis (GWAS). We also performed RNA sequencing and analyzed differentially expressed genes between the early-feathering and late-feathering phenotypes using HISAT2, StringTie, and DESeq2. We identified a genomic region of 10.0–13.0 Mb of chromosome Z, which is statistically associated with the feather rate of Shouguang chickens at one-day old. After RNA sequencing analysis, 342 differentially expressed known genes between the early-feathering (EF) and late-feathering (LF) phenotypes were screened out, which were involved in epithelial cell differentiation, intermediate filament organization, protein serine kinase activity, peptidyl-serine phosphorylation, retinoic acid binding, and so on. The sperm flagellar 2 gene (SPEF2) and prolactin receptor (PRLR) gene were the only two overlapping genes between the results of GWAS and differential expression analysis, which implies that SPEF2 and PRLR are possible candidate genes for the formation of the chicken feathering phenotype in the present study. Our findings help to elucidate the molecular mechanism of the feather rate in chicks.

Long non-coding RNAs (lncRNAs) are implicated in mammalian adipogenesis and obesity. However, their genome-wide distribution, expression profiles, and regulatory mechanisms during chicken adipogenesis remain rarely understood. In the present study, lncRNAs associated with adipogenesis were identified from chicken abdominal adipocytes at multiple differentiation stages using Ribo-Zero RNA-seq. A total of 15,179 lncRNAs were identified and characterized by stage-specific expression patterns. Of these, 840 differentially expressed lncRNAs were detected, and their cis- and trans-target genes were significantly enriched in multiple lipid-related pathways. Through weighted gene co-expression network analysis (WGCNA) and time-series expression profile clustering analysis, 14 key lncRNAs were identified as candidate regulatory lncRNAs in chicken adipogenic differentiation. The cis- and trans-regulatory interactions of key lncRNAs were constructed based on their differentially expressed cis- and trans-target genes, respectively. We also constructed a competing endogenous RNA (ceRNA) network based on the key lncRNAs, differentially expressed miRNAs, and differentially expressed mRNAs. MSTRG.25116.1 was identified as a potential regulator of chicken abdominal preadipocyte adipogenic differentiation by acting as a transcriptional trans-regulator of fatty acid amide hydrolase (FAAH) gene expression and/or a ceRNA that post-transcriptionally mediates FAAH gene expression by sponging gga-miR-1635.

Ovarian follicle selection largely depends on the transition of granulosa cells from an undifferentiated to a fully differentiated state, which is accompanied by morphological and functional changes in follicles. The processes and transcriptional regulation of follicles during follicle selection are unclear; we thus used follicles from the prehierarchal to the hierarchal stage to investigate histology, reproductive endocrinology, and transcription. The morphology of follicles changed markedly during follicle selection. The numbers of large white, small yellow, and large yellow follicles (LWF, SYF, and LYF, respectively) were 11.83 ± 2.79, 6.83 ± 2.23, and 1.00, respectively, per ovary. LYF showed thicker granulosa cell layers than those of other prehierarchal follicles. Progesterone concentrations were significantly higher in LYF than that in LWF and SYF. In total, 16,823 genes were positively expressed in LWF, SYF, and LYF. Among follicle types, 1290 differentially expressed genes were enriched regarding cell differentiation, blood vessel morphogenesis, and response to steroid hormones. Candidate genes associated with follicle selection participated in the Wnt signaling pathway, steroid hormone biosynthesis, and the TGF-β signaling pathway. We produced insights into crucial morphological characteristics of transcriptional regulation in follicle development. Our results provide an important basis for revealing the mechanism of follicle selection and potential impact on the poultry industry.

The ADP ribosylation factor (ARF) GTPase activation protein ASAP1 possesses multiple biological functions, including regulation of cytoskeletal dynamics, small GTP-binding protein receptor recycling, and intracellular vesicle trafficking. Recently, ASAP1 polymorphisms have been reported to be associated with human susceptibility to tuberculosis (TB) according to a large-scale genome-wide association study (GWAS); ASAP1 expression affects dendritic cell migration, which may be involved in TB predisposition. However, it remains unclear whether ASAP1 affects TB in vivo. To address this issue, we used zebrafish as a model system to examine the effects of Asap1 against Mycobacterium marinum, an organism closely related to Mycobacterium tuberculosis. Two zebrafish asap1 homologs (asap1a and asap1b) were identified and characterized. By morpholino knockdown of asap1a and asap1b as a whole, we found that the asap1 morphants showed a higher mycobacterial load than the controls, which was almost rescued by injecting asap1 mRNA that confers resistance to mycobacterial infection. These Asap1-depleted zebrafish also exhibited decreased macrophage migration in response to tail injury or upon infection with M. marinum in the hindbrain ventricle, which was also proved in THP1-derived macrophages of knockdown ASAP1. Together, these findings represent a new perspective on the role of Asap1 in resistance to mycobacterial infection.

DIP2A mutation is associated with abnormal brain development and diseases including dyslexia, autism and Alzheimer's disease. However, the role and the involved mechanisms remain unknown. To study the biological function of DIP2A during mESCs neural differentiation in early neural development, we generated a Dip2a homozygous knockout 46C ESC cell line using CRISPR/Cas9 genome editing technology. The eighth exon of Dip2a gene was replaced with PGK-Puro-P2A-mCherry. This 46C-Dip2a KO cell line offers a useful resource to investigate the molecular mechanisms of DIP2A in the process of cell fate determination, as well as a potential source of building disease mouse model.

Extremely low nucleotide diversity of modern horse Y-chromosome has been reported, and only poor phylogenetic resolution could be resulted from limited Y-chromosome markers. In this study, three types of horse Y-chromosome markers, including Single-nucleotide polymorphisms (SNPs), copy number variants (CNVs), and allele-specific CNVs, were developed by screening more than 300 male horses from 23 indigenous Chinese horse populations and 4 imported horse breeds. Fourteen segregating sites including a novel SNP in the AMELY gene were found in approximately 53 kb of male-specific Y-chromosome sequences. CNVs were detected at 11 of 14 sites, while allele-specific CNVs at 6 polymorphic sites in repeated fragments were also determined. The phylogenetic analyses with the SNPs identified in this study and previously published 51 SNPs obtained mainly from European horses showed that indigenous Chinese horses exhibit much deeper divergence than European and Middle Eastern horses, while individuals of Chinese horses with the C allele of the AMELY gene constituted the most ancient group. Via SNPs, CNVs, and allele-specific CNVs, much higher diversity of paternal lines can be detected than those identified with merely SNPs. Our results indicated that there are ancient paternal horse lines preserved in southwestern China, which sheds new light on the domestication and immigration of horses, and suggest that the priorities of the conservation should be given to the ancient and rare paternal lines. These three marker types provided finer phylogenetic resolution of horse patrilineal lines, and the strategies used in the present study also provide valuable reference for the genetic studies of other mammalian patrilineages.

The nutritional value of cow milk mainly depends on its fatty acid content and protein composition. The identification of genes controlling milk production traits and their regulatory mechanisms is particularly important for accelerating genetic progress in the breeding of dairy cows. On the basis of mammary gland transcriptome analyses, in this study we identified an miRNA, bta-miR-125a, that could control bovine milk-fat production by targeting the 3′ untranslated region (UTR) of the serum amyloid A-1 (SAA1) mRNA. The presence of synthetic bta-miR-125a (i.e., an miR-125a mimic) significantly down-regulated the expression of luciferase from mRNAs containing the binding sequence for bta-miR-125a in the 3′-UTR in a dual-luciferase reporter assay. Furthermore, the presence of the miR-125a mimic decreased the steady-state level of the SAA1 protein, but increased the accumulation of triglycerides and cholesterol content in bovine mammary epithelial cells (MAC-Ts). Blocking the function of bta-miR-125a using a specific inhibitor decreased the level of triglycerides and cholesterol content in the cells. These results indicate that bta-miR-125a can serve as a positive regulator of lipid synthesis in mammary epithelial cells, which acts by targeting SAA1 gene expression.

Currently, assays for rapid therapeutic drug monitoring (TDM) of β-lactam antibiotics in blood, which might be of benefit in optimizing doses for treatment of critically ill patients, remain challenging. Previously, we developed an assay for determining the penicillin-class antibiotics in blood using a thermometric penicillinase biosensor. The assay eliminates sample pretreatment, which makes it possible to perform semicontinuous penicillin determinations in blood. However, penicillinase has a narrow substrate specificity, which makes it unsuitable for detecting other classes of β-lactam antibiotics, such as cephalosporins and carbapenems. In order to assay these classes of clinically useful antibiotics, a novel biosensor was developed using New Delhi metallo-β-lactamase-1 (NDM-1) as the biological recognition layer. NDM-1 has a broad specificity range and is capable of hydrolyzing all classes of β-lactam antibiotics in high efficacy with the exception of monobactams. In this study, we demonstrated that the NDM-1 biosensor was able to quantify multiple classes of β-lactam antibiotics in blood plasma at concentrations ranging from 6.25 mg/L or 12.5 mg/L to 200 mg/L, which covered the therapeutic concentration windows of the tested antibiotics used to treat critically ill patients. The detection of ceftazidime and meropenem was not affected by the presence of the β-lactamase inhibitors avibactam and vaborbactam, respectively. Furthermore, both free and protein-bound β-lactams present in the antibiotic-spiked plasma samples were detected by the NDM-1 biosensor. These results indicated that the NDM-1 biosensor is a promising technique for rapid TDM of total β-lactam antibiotics present in the blood of critically ill patients.

Abstract Background Fabry disease (FD), a rare X‐linked α‐galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide in a variety of cell types. More and more disease‐causing mutations in GLA have been identified in FD due to the advancement of molecular diagnostic tools. We found a novel mutation in a Chinese family with predominant Fabry's disease nephropathy. Methods All coding regions and exon–intron splice junctions of the GLA gene were sequenced to find sequence variations. We evaluated the impact on the GLA protein by analysis of the GLA mRNA, by sequential analysis and homology modeling, and by site‐directed mutagenesis and in vitro expression studies. Results We identified a novel heterozygous missense mutation c.280T>C in our patient with variable phenotypic presentations of renal involvement. The novel GLA variant results in low expression of GLA mRNAs, impaired or loss of the disulfate bridge structure of wild‐type GLA, reduced GLA activity and defected nuclear shape in the GFP‐GLA‐MT transfected HEK293T cells. Conclusion A novel GLA missense mutation, c.280T>C (Cys94Arg), was found in a Chinese family with predominant renal manifestations of FD. Our study reveals the pathogenesis of c.280T>C mutation to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

Abstract Background Beta‐propeller protein‐associated neurodegeneration (BPAN, OMIM 300894) is an X‐linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3‐year‐old Chinese girl with BPAN. Methods The protein structure was constructed using SWISS‐MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag‐tagged wild‐type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry‐LC3 by confocal microscopy. Results The mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3‐II and p62 were increased in cells overexpression of wild‐type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3‐positive puncta were increased in cells expressing both wild‐type and mutant WDR45 while the number of LC3‐positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1. Conclusion Our study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.

Artificial selection is often associated with numerous changes in seemingly unrelated phenotypic traits. The genetic mechanisms of correlated phenotypes probably involve pleiotropy or linkage of genes related to such phenotypes. Dongxiang blue-shelled chicken, an indigenous chicken breed of China, has segregated significantly for the dermal hyperpigmentation phenotype. Two lines of the chicken have been divergently selected with respect to comb color for over 20 generations. The red comb line chicken produces significantly higher number of eggs than the dark comb line chicken. The objective of this study was to explore potential mechanisms involved in the relationship between comb color and egg production among chickens. Based on the genome-wide association study results, we identified a genomic region on chromosome 20 involving EDN3 and BMP7, which is associated with hyperpigmentation of chicken comb. Further analyses by selection signatures in the two divergent lines revealed that several candidate genes, including EDN3, BMP7, BPIFB3, and PCK1, closely located on chromosome 20 are involved in the development of neural crest cell and reproductive system. The two genes EDN3 and BMP7 have known roles in regulating both ovarian function and melanogenesis, indicating the pleiotropic effect on hyperpigmentation and egg production in blue-shelled chickens. Association analysis for egg production confirmed the pleiotropic effect of selected loci identified by selection signatures. The study provides insights into phenotypic evolution due to genetic variation across the genome. The information might be useful in the current breeding efforts to develop improved breeds for egg production.

Fabry disease (FD) is a rare X-linked α-galactosidase A (GLA) deficiency, resulting in progressive lysosomal accumulation of globotriaosylceramide (Gb3) in a variety of cell types. Here, we report a novel splicing mutation (c.801 + 1G > A) that results in alternative splicing in GLA of a FD patient with variable phenotypic presentations of renal involvement. Sequencing of the RT-PCR products from the patient’s blood sample reveals a 36-nucleotide (nt) insertion exists at the junction between exons 5 and 6 of the GLA cDNA. Splicing assay indicates that the mutated minigene produces an alternatively spliced transcript which causes a frameshift resulting in an early termination of protein expression. Immunofluorescence shows puncta in cytoplasm for mutated GLA whereas uniform staining small dots evenly distributed inside cytoplasm for wild type GLA in transfected HeLa cells. The increased senescence and decreased GLA enzyme activity suggest that the abnormalities might be due to the altered localization which further might result from the lack of the C-terminal end of GLA. Our study reveals the pathogenesis of splicing mutation c.801 + 1G > A to FD and provides scientific foundation for accurate diagnosis and precise medical intervention for FD.

The eggshell blueness is an interesting object for chicken genetic studies and blue-shelled chicken industry, especially after the discovery of the causative mutation of chicken blue eggshell. In the present study, genome wide association study (GWAS) was conducted in Chinese Dongxiang blue-shelled chicken underlying four traits of blue eggshell pigments: quantity of biliverdin (QB), quantity of protoporphyrin (QP), quantity of total pigment (QT), and color density trait (CD). A total of 139 individuals were randomly collected for GWAS. We detected two SNPs in genome-wise significance and 35 in suggestive significance, 24 out of the 37 SNP were located either within intron/exon or near 15 genes in a range of ~1.17 Mb on GGA21. For further confirmation of the identified SNP loci by GWAS, the follow-up replication studies were performed in two populations. A total of 146 individuals of the second generation derived from the former GWAS population, as well as 280 individuals from an alternative independent population were employed for genotyping by MALDI-TOF MS in a genotype-phenotype association study. Eighteen SNPs evenly distributed on the GGA21 significant region were successfully genotyped in the two populations, of which 4 and 6 SNP loci were shown significantly associated with QB, QT and QP in the two repeat populations, respectively. Further, the SNPs were narrowed down to a region of ~ 653.819 Kb on GGA21 that harbors five candidate genes: AJAP1, TNFRSF9, C1ORF174, CAMTA1, and CEP104. Shell gland of chickens laying dark and light blue eggshell was chosen for detection of mRNA expression of the five candidate genes. The results showed differential expression levels of these genes in the two groups. The specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are needed to explore the new functional role in chicken eggshell blueness.

Depression has been correlated with metabolic disorders, and the gut microbiota and its metabolites have been reported to be key factors affecting metabolic disorders. Several metabolites generated by the gut microbiota have been reported to exert antidepressant-like effects, including the short chain fatty acid (SCFA) butyrate. However, recent work has suggested that the abundance of butyrate is not significantly changed in neither human nor experimental animals with depression, and butyrate has been reported to decrease upon the administration of prebiotics with antidepressant-like effects. Supplementation of endogenous metabolites that are unchanged in depression may induce additional metabolic disorders and may lead to poorer clinical outcomes. However, the endogenous metabolites that are imbalanced in depression may include several antidepressant candidates that could circumvent these problems. In this study, we used GC-MS spectrometry to study the fecal metabolome of rats under Chronic Unpredictable Mild Stress (CUMS). We carried out static and dynamic metabolomics analyses to identify the differential metabolites between the CUMS rats and control rats. We identified propionic acid, rather than butyric acid, as a differential metabolite of the CUMS rats. Consistent with this, a 1-week intrarectal administration of sodium propionate (NaP, the salt form of propionic acid) induced antidepressant-like effects and partially rebalanced the plasma metabolome. The antidepressant-like effects of NaP were correlated with differential rescue of neurotransmitters in the prefrontal cortex, which may be achieved through the reduction of catabolism of noradrenaline, tryptophan and dopamine, rather than serotonin. These findings support NaP as a potential candidate in fighting depression by administering an endogenous metabolite.

Objectives There are large populations of indigenous horse (Equus caballus) in China and some other parts of East Asia. However, their matrilineal genetic diversity and origin remained poorly understood. Using a combination of mitochondrial DNA (mtDNA) and hypervariable region (HVR-1) sequences, we aim to investigate the origin of matrilineal inheritance in these domestic horses. Methods To investigate patterns of matrilineal inheritance in domestic horses, we conducted a phylogenetic study using 31 de novo mtDNA genomes together with 317 others from the GenBank. In terms of the updated phylogeny, a total of 5,180 horse mitochondrial HVR-1 sequences were analyzed. Results Eightteen haplogroups (Aw-Rw) were uncovered from the analysis of the whole mitochondrial genomes. Most of which have a divergence time before the earliest domestication of wild horses (about 5,800 years ago) and during the Upper Paleolithic (35–10 KYA). The distribution of some haplogroups shows geographic patterns. The Lw haplogroup contained a significantly higher proportion of European horses than the horses from other regions, while haplogroups Jw, Rw, and some maternal lineages of Cw, have a higher frequency in the horses from East Asia. The 5,180 sequences of horse mitochondrial HVR-1 form nine major haplogroups (A-I). We revealed a corresponding relationship between the haplotypes of HVR-1 and those of whole mitochondrial DNA sequences. The data of the HVR-1 sequences also suggests that Jw, Rw, and some haplotypes of Cw may have originated in East Asia while Lw probably formed in Europe. Conclusions Our study supports the hypothesis of the multiple origins of the maternal lineage of domestic horses and some maternal lineages of domestic horses may have originated from East Asia.

Outer membrane protein 25 (OMP25), a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK) signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308) and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10) were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK), and Jun-N-terminal kinase (JNK) from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

[This corrects the article DOI: 10.1371/journal.pone.0141138.].

Autophagy and the ubiquitin proteasome system (UPS) are the two major cellular degradation pathways, which are critical for the maintenance of cell homeostasis. The two pathways differ in their mechanisms and clients. The evolutionary conserved ATG16 plays a key role in autophagy and appears to link autophagy with the UPS. Here, we review the role of ATG16 in different species. We summarize the current knowledge of its functions in autophagosome membrane expansion and autophagosome formation, in Crohn’s disease, and in bacterial sequestration. In addition, we provide information on its autophagy-independent functions and its role in the crosstalk between autophagy and the UPS.

Transcriptome profiles established using high-throughput sequencing can be effectively used for screening genome-wide differentially expressed genes (DEGs). RNA sequences (from RNA-seq) and microRNA sequences (from miRNA-seq) from the tissues of longissimus dorsi muscle of two indigenous Chinese pig breeds (Diannan Small-ear pig [DSP] and Tibetan pig [TP]) and two introduced pig breeds (Landrace [LL] and Yorkshire [YY]) were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to muscle growth and lipid deposition. We obtained 27.18 G clean data through the RNA-seq and detected that 18,208 genes were positively expressed and 14,633 of them were co-expressed in the muscle tissues of the four samples. In all, 315 DEGs were found between the Chinese pig group and the introduced pig group, 240 of which were enriched with functional annotations from the David database and significantly enriched in 27 Gene Ontology (GO) terms that were mainly associated with muscle fiber contraction, cadmium ion binding, response to organic substance and contractile fiber part. Based on functional annotation, we identified 85 DEGs related to growth traits that were mainly involved in muscle tissue development, muscle system process, regulation of cell development, and growth factor binding, and 27 DEGs related to lipid deposition that were mainly involved in lipid metabolic process and fatty acid biosynthetic process. With miRNA-seq, we obtained 23.78 M reads and 320 positively expressed miRNAs from muscle tissues, including 271 known pig miRNAs and 49 novel miRNAs. In those 271 known miRNAs, 20 were higher and 10 lower expressed in DSP-TP than in LL-YY. The target genes of the 30 miRNAs were mainly participated in MAPK, GnRH, insulin and Calcium signaling pathway and others involved cell development, growth and proliferation, etc. Combining the DEGs and the differentially expressed (DE) miRNAs, we drafted a network of 46 genes and 18 miRNAs for regulating muscle growth and a network of 15 genes and 16 miRNAs for regulating lipid deposition. We identified that CAV2, MYOZ2, FRZB, miR-29b, miR-122, miR-145-5p and miR-let-7c, etc, were key genes or miRNAs regulating muscle growth, and FASN, SCD, ADORA1, miR-4332, miR-182, miR-92b-3p, miR-let-7a and miR-let-7e, etc, were key genes or miRNAs regulating lipid deposition. The quantitative expressions of eight DEGs and seven DE miRNAs measured with real-time PCR certified that the results of differential expression genes or miRNAs were reliable. Thus, 18,208 genes and 320 miRNAs were positively expressed in porcine longissimus dorsi muscle. We obtained 85 genes and 18 miRNAs related to muscle growth and 27 genes and 16 miRNAs related to lipid deposition, which provided new insights into molecular mechanism of the economical traits in pig.

The lungs undergo changes that are adaptive for high elevation in certain animal species. In chickens, animals bred at high elevations (e.g., Tibet chickens) are better able to hatch and survive under high-altitude conditions. In addition, lowland chicken breeds undergo physiological effects and suffer greater mortality when they are exposed to hypoxic conditions during embryonic development. Although these physiological effects have been noted, the mechanisms that are responsible for hypoxia-induced changes in lung development and function are not known. Here we have examined the role of a particular microRNA (miRNA) in the regulation of lung development under hypoxic conditions. When chicks were incubated in low oxygen (hypoxia), miR-15a was significantly increased in embryonic lung tissue. The expression level of miR-15a in hypoxic Tibet chicken embryos increased and remained relatively high at embryonic day (E)16-20, whereas in normal chickens, expression increased and peaked at E19-20, at which time the cross-current gas exchange system (CCGS) is developing. Bcl-2 was a translationally repressed target of miR-15a in these chickens. miR-16, a cluster and family member of miR-15a, was detected but did not participate in the posttranscriptional regulation of bcl-2. Around E19, the hypoxia-induced decrease in Bcl-2 protein resulted in apoptosis in the mesenchyme around the migrating tubes, which led to an expansion and migration of the tubes that would become the air capillary network and the CCGS. Thus, interfering with miR-15a expression in lung tissue may be a novel therapeutic strategy for hypoxia insults and altitude adaptation.

Basic situation of poultry genetic resources and poultry production in China was briefly introduced. The strategy on conservation and utilization were to breed new breeds or lines, to use heterosis and to develop new poultry products. Methods of selection, breeding procedure of quality meat yellow chickens, functional eggs with green shell, breeding systems of pure and cross breeds were also introduced.

Capons are male chickens whose testes have been surgically incised. Capons show a significant increase in fat accumulation compared to intact male chickens. However, while caponization leads to a significant reduction in androgen levels in roosters, little is known about the molecular mechanisms through which androgen status affects lipogenesis in avian species. Therefore, investigation of the influence of androgens on fat accumulation in the chicken will provide insights into this process. In this study, Affymetrix microarray technology was used to analyze the gene expression profiles of livers from capons and intact male chickens because the liver is the major site of lipogenesis in avian species. Through gene ontology, we found that genes involved in hepatic lipogenic biosynthesis were the most highly enriched. Interestingly, among the upregulated genes, the cytosolic form of the phosphoenolpyruvate carboxykinase (PCK1) gene showed the greatest fold change. Additionally, in conjunction with quantitative real-time PCR data, our results suggested that androgen status negatively regulated the PCK1 gene in male chickens.

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F(2) chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5' flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.

Thymoma is one of the thymic epithelial tumors arising from the thymic epithelial cells of a variety of animal species including cat, cattle, dog, goat, horse, and pig. The cancer cells of thymoma are always localized within thymus. Feline thymoma was usually identified in the cranial mediastinum of elder cats. In this report, we present the full diagnostic characteristics of a young cat with a cranial mediastinal mass diagnosed as a cystic type B1 thymoma. A 3-yearold male neutered domestic short-haired cat was referred for further diagnosis due to the shortness of breath and pleural accumulation. One single cystic extra-pericardial mass was seen and the boundary between the mass and the surrounding tissue was clearly demarcated in three-view thoracic radiographs. Ultrasound showed pleural effusion and intra-thoracic mass. Pleural fluid biochemistry showed high level of triglycerides and complete blood count revealed the elevated number of basophils and high level of feline serum amyloid A (fSAA). Pleural effusion sediment cytology showed a large number of round cells, mainly small lymphocytes, as well as a moderate number of neutrophils and a small number of large lymphocytes. The high level of triglycerides in pleural fluid instead of serum and high level of fSAA seem to provide informative clues to the diagnosis of thymoma and are worthy of further investigation. [ABSTRACT FROM AUTHOR]

Excessive alcohol consumption has led to the prevalence of gastrointestinal ailments. Alleviating gastric disorders attributed to alcohol-induced thinning of the mucus layer has centered on enhancing mucin secretion as a pivotal approach. In this study, foxtail millet bran polyphenol BPIS was divided into two components with MW < 200 D and MW > 200 D by molecular interception technology. Combined with MTT, cell morphology observation, and trypan blue staining, isoferulic acid (IFA) within the MW < 200 D fraction was determined as the effective constituent to mitigate ethanol-induced damage of gastric epithelial cells. Furthermore, a Wistar rat model with similar clinical features to alcohol-induced gastric mucosal injury was established. Then, gastric morphological observation, H&E staining, and assessments of changes in gastric hexosamine content and gastric wall binding mucus levels were carried out, and the results revealed that IFA (10 mg/Kg) significantly ameliorated alcohol-induced gastric mucosal damage. Finally, we applied techniques including Co-IP, molecular docking, and fluorescence spectroscopy and found that IFA inhibited the alcohol-induced downregulation of N-acetylgalactosamintransferase 2 (GALNT2) activity related to mucus synthesis through direct interaction with GALNT2 in gastric epithelial cells, thus promoting mucin synthesis. Our study lays a foundation for whole grain dietary intervention tailored to individuals suffering from alcoholic gastric mucosal injury [ABSTRACT FROM AUTHOR]