Induced pluripotent stem cells (iPSCs) derived from somatic cells have enormous potential for clinical applications. Notably, it was recently reported that reprogramming from somatic cells to iPSCs can induce genomic copy number variation (CNV), which is one of the major genetic causes of human diseases. However it was unclear if this genome instability is dependent on reprogramming methods and/or the genetic background of donor cells. Furthermore, genome-wide CNV analysis is technically challenging and CNV data need to be interpreted with care.

Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest β-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained P(luxS) and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of P(luxS) containing a putative promoter. This putative promoter was a hybrid promoter and its β-Gal activity was higher than P(luxS). Reconstructing the hybrid promoter from pShuttle-09 to P(lapS) further improved the β-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters.

Ruminant animals possess a characteristic four-compartment stomach (rumen, reticulum, omasum, and abomasum) that is specialized for pre-intestinal digestion of plant materials. Of these four compartments, the rumen is the largest. The rumen's diverse microbial community has been well studied. However, the current understanding of microbial profiles in the reticulum, omasum and abomasum are lacking. In the present study, fluid samples from the reticulum, omasum, and abomasum of goats at 3, 7, 14, 21, 28, 42, and 56 days after birth, as well as the negative controls (NC) used for microbial DNA extraction, were subjected to 16S rRNA sequencing. By filtering operational taxonomic units (OTUs) in NC, distinct temporal distributions of microbes were observed in the different compartments, we showed that the OTUs in control samples had a large effect to the samples with low microbial density. In addition, Proteobacteria gradually decreased with age from days 3 to 56 in all three compartments, and the relative abundance of Bacteroidetes increased from 24.15% (Day 3) to 52.03% (Day 56) in abomasum. Network analysis revealed that Prevotellaceae_UGG-03 and Rikenellaceae_RC9 were positively correlated with Prevotella_1, lending support to the well understood fact that cellulose is well digested in compound stomachs prior to the rumen. Pathway analysis revealed that gene expression in abomasum at Day 3 were primarily related to Glycolysis/Gluconeogenesis and Pyruvate metabolism, suggesting that colostrum digestion is the dominant function of the abomasum at an early age. These findings combined with other recent rumen microbiota data show that the microbiome landscape represents three distinct stages in ruminant stomachs. The first stage is to gain access to external microorganisms at Day 0-14, the secondary stage is for microbial transition at Day 14-28, and the third stage is for exogenous and endogenous microbial colonization beyond Day 28 of age. Our results provide insight into microbiota dynamics in ruminant stomachs, and will facilitate efforts for the maintenance of gastrointestinal balance and intervention with starter diets in juvenile ruminants during early development.

Microvesicles (MVs) were involved in the pathogenesis of many diseases, such as cardiovascular diseases and diabetes. Oxidative stress played a key role in the development and progression of diabetic nephropathy (DN). Our aim of the present study was to investigate whether high glucose (HG) could provoke MVs generation from podocytes and its potential mechanism. Mouse podocyte clone 5 (MPC-5) was stimulated by HG. The intracellular reactive oxygen species (ROS) of podocytes were measured by fluorescence microscopy with the probe of CM-H2DCFDA and MitoSOX™. Antioxidants N-Acetyl-l-cysteine (NAC) and α lipoic acid (α-LA) were used to treat podocytes after HG stimulation. The rate of podocyte apoptosis was evaluated with Annexin V-FITC by flow cytometry. NOX4 expression was examined and siRNA were performed to explore the mechanism of MVs generation. The quantities of MVs from MPC-5 cells was significantly increased (P<0.05) by 4.6-times after 30 mM glucose stimulation, accompanied with double increased apoptosis. Cellular ROS generation was increased by HG at the peak of 48 h stimulation. HG-induced MVs were significantly decreased by 52.9% after pretreatment by antioxidant NAC. Nevertheless, mitochondrial ROS in podocytes reached a peak at 4 h stimulation, but specific antioxidant α-LA had no effect on the production of MVs (P>0.05). Levels of NOX4 mRNA and protein expression were significantly up-regulated by HG (P<0.05). Podocyte-derived MVs by HG were eliminated by NOX4 siRNA. HG can provoke MVs generation from glomerular podocytes through ROS/NOX4 pathway, not from mitochondrial pathway.

Recent studies have demonstrated that dermal papilla cell-derived exosomes (DPC-Exos) promote the anagen stage of hair follicle (HF) growth and delay the catagen stage. However, the roles of DPC-Exos in regulating hair follicle stem cell (HFSC) quiescence and activation remain unknown. Here, we found that HFSC differentiation was induced by co-culture with DPCs, and that DPC-Exos attached to the surface of HFSCs. Using micro RNA (miRNA) high-throughput sequencing, we identified 111 miRNAs that were significantly differentially expressed between DPC-Exos and DPCs, and the predicted target genes of the top 34 differentially expressed miRNAs indicated that DPC-Exos regulate HFSCs proliferation and differentiation via genes involved in cellular signal transduction, fatty acid expression regulation, and cellular communication. The overexpression of miR-22-5p indicated that it negatively regulates HFSC proliferation and LEF1 was revealed as the direct target gene of miR-22-5p. We therefore propose the miR-22-5p-LEF1 axis as a novel pathway regulating HFSC proliferation.

In China, the combination of herb Salvia miltiorrhiza Bge. (Danshen) and Carthamus tinctorius L. (Honghua) is an effective treatment for stroke. A previous study showed that the combination of four herbal components: danshensu (DSS), hydroxysafflor yellow A (HSYA), salvianolic acid A (SAA), and salvianolic acid B (SAB) was effective for treatment of cerebral ischemia-reperfusion (I/R) injury in rats. However, the pharmacokinetic characteristics of this formula require further investigation. The present study investigated the pharmacokinetic differences between each component of in two formulas in cerebral I/R injury rats. The influencing factors may affect the compatibility of components were analyzed.

Jet-lag symptoms arise from temporal misalignment between the internal circadian clock and external solar time when traveling across multiple time zones. Light is known as a strong timing cue of the circadian clock. We here examined the effect of daily light on the process of jet lag by detecting c-Fos expression in the master clock neurons in the suprachiasmatic nucleus (SCN) under 8-hr phase-advanced jet lag condition. In WT mice, c-Fos-immunoreactivity was found at 1-2 hours on the first day after light/dark (LD) phase-advance. This induction was also observed on the second and third days, although their levels were diminished day by day. In contrast, c-Fos induction in the SCN of V1a-/-V1b-/- mice, which show virtually no jet lag symptoms even after 8-hr phase-advance, was only detected on the first day. These results indicate that external light has affected SCN neuronal activity for 3 days after LD phase-advance in WT mice suggesting the continuous progress of activity change of SCN neurons under jet lag conditions. Noteworthy, limited c-Fos induction in V1a-/-V1b-/- SCN is also consistent with the rapid reentrainment of the SCN clock in mutant mice after 8-hr LD phase-advance.

An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a crucial prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. Besides, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine, and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification.

Goat domestication was critical for agriculture and civilization, but its underlying genetic changes and selection regimes remain unclear. Here, we analyze the genomes of worldwide domestic goats, wild caprid species, and historical remains, providing evidence of an ancient introgression event from a West Caucasian tur-like species to the ancestor of domestic goats. One introgressed locus with a strong signature of selection harbors the MUC6 gene, which encodes a gastrointestinally secreted mucin. Experiments revealed that the nearly fixed introgressed haplotype confers enhanced immune resistance to gastrointestinal pathogens. Another locus with a strong signal of selection may be related to behavior. The selected alleles at these two loci emerged in domestic goats at least 7200 and 8100 years ago, respectively, and increased to high frequencies concurrent with the expansion of the ubiquitous modern mitochondrial haplogroup A. Tracking these archaeologically cryptic evolutionary transformations provides new insights into the mechanisms of animal domestication.

Temperature type is one of the key traits determining the cultivation regime of Lentinula edodes. However, the molecular and metabolic basis underling temperature type remain unclear. Here, we investigated the phenotypic, transcriptomic, and metabolic features of L. edodes with different temperature types under both control (25 °C) and high (37 °C) temperature conditions. We found that under the control condition, the high- and low-temperature types of L. edodes harbored distinct transcriptional and metabolic profiles. The high-temperature (H-)-type strain had a higher expression level of genes involved in the toxin processes and carbohydrate binding, while the low-temperature (L-)-type strain had a high expression level of oxidoreductase activity. Heat stress significantly inhibited the growth of both H- and L-type strains, while the latter had a higher growth inhibition rate. Upon exposure to heat, the H-type strain significantly up-regulated genes associated with the components of the cellular membrane, whereas the L-type strain markedly up-regulated genes involved in the extracellular region and carbohydrate binding. Metabolome data showed that thermostress altered purine and pyrimidine metabolism in the H-type strain, whereas it altered cysteine, methionine, and glycerophospholipid metabolism in the L-type strain. Transcriptome and metabolome integrative analysis was able to identify three independent thermotolerance-related gene-metabolite regulatory networks. Our results deepen the current understanding of the molecular and metabolic basis underlying temperature type and suggest, for the first time, that thermotolerance mechanisms can be temperature-type-dependent for L. edodes.

Currently, endoscopic submucosal dissection (ESD) has gradually become the diagnosis and treatment of choice for initial esophageal cancer. However, the formation of esophageal stricture after ESD is one of its important complications. In this paper, we intend to identify the risk factors of esophageal stricture to develop a nomogram model to predict the risk of esophageal stricture and validate this model.A total, 159 patients were included in this study, including 21 patients with esophageal stenosis. Multivariate analysis showed that age greater than 60 years, high neutrophil-to-lymphocyte ratio, the extent of esophageal mucosal defect greater than 1/2, and postoperative pathological type of early esophageal squamous cell carcinoma were independent risk factors for predicting esophageal stricture. We constructed a nomogram model to predict esophageal stenosis by these 4 independent predictors.The prediction performance of the model was verified by the area under the receiver operating characteristic curve, the area under the receiver operating characteristic curve of the model was 0.889, and the sensitivity and specificity were 80.00% and 91.28%, respectively, indicating that the prediction performance of the model was good; The calibration curve constructed by internal cross-validation suggested that the predicted results of the nomogram agreed well with the actual observed values.The nomogram model has a high accuracy for predicting esophageal stricture after esophageal ESD and is extremely important to reduce or avoid the occurrence of esophageal stricture. But it needs more external and prospective validation.

Extracellular vesicles (EVs), which exist in the follicular fluid of ruminant ovaries, are considered as cargo carriers for the transfer of biomolecules to recipient cells. However, the functions and changes in EVs in antral follicles remain ambiguous. In the present study, we isolated and characterized EVs from goat follicular fluid by means of differential ultracentrifugation and Western blotting of marker proteins. Bioinformatics tools were used to detect miRNA expression levels in EVs. Different miRNA expression patterns of EVs exist in small to large follicles. Thirteen differentially expressed miRNAs (seven upregulated and six downregulated) were identified and used for analysis. A total of 1948 predicted target genes of 13 miRNAs were mapped to signaling pathways, and three significantly enriched pathways (FoxO, MAPK, and PI3K-AKT signaling pathways) were involved in follicular development, as revealed by KEGG enrichment analysis. Our findings suggest that EVs in follicular fluid play biofunctional roles during follicular development in goats.

Fibroblasts and myofibroblasts are major mesenchymal cells in the lamina propria of colon mucosa and in colon cancer tissues. Detailed insight into the highly specific populations of fibroblasts and myofibroblasts is required to understand the integrity and homeostasis of human colon mucosa and colon cancer. Based on gene expression profiles of single cells, we identified fibroblast populations that produce extracellular matrix components, Wnt ligand- and BMP-secreting fibroblasts, chemokine- and chemokine ligand-generating fibroblasts, highly activated fibroblasts, immune-modulating fibroblasts, epithelial cell-modulating myofibroblasts, stimuli-responsive myofibroblasts, proliferating myofibroblasts, fibroblast-like myofibroblasts, matrix producing myofibroblasts, and contractile myofibroblasts in human colon mucosa. In colon cancer tissue, the compositions of fibroblasts and myofibroblasts were highly altered, as were the expressing patterns of genes including BMPs, Wnt ligands, chemokines, chemokine ligands, growth factors and extracellular matrix components in fibroblasts and myofibroblasts. Our work expands the working atlas of fibroblasts and myofibroblasts and provides a framework for interrogating the complexity of stromal cells in human healthy colon mucosa and colon cancer tissues.

Wnt/β-catenin signalling is aberrantly activated in most colorectal cancer (CRC) and is one key driver involved in the initiation and progression of CRC. However, mutations of APC gene in CRC patients retain certain activity of APC protein with decreased β-catenin signalling and DKK4 expression significantly upregulates and represses Wnt/β-catenin signalling in human CRC tissues, suggesting that a precisely modulated activation of the Wnt/β-catenin pathway is essential for CRC formation and progression. The underlying reasons why a specifically reduced degree, not a fully activating degree, of β-catenin signalling in CRC are unclear. Here, we showed that a soluble extracellular inhibitor of Wnt/β-catenin signalling, DKK4, is an independent factor for poor outcomes in CRC patients. DKK4 secreted from CRC cells inactivates β-catenin in fibroblasts to induce the formation of stress fibre-containing fibroblasts and myofibroblasts in culture conditions and in mouse CRC xenograft tissues, resulting in restricted expansion in tumour masses at primary sites and enhanced CRC metastasis in mouse models. Reduced β-catenin activity by a chemical inhibitor MSAB promoted the CRC metastasis. Our findings demonstrate why reduced β-catenin activity is needed for CRC progression and provide a mechanism by which interactions between CRC cells and stromal cells affect disease promotion.

Analysis of the proteome of myostatin (MSTN) knockout (KO) mouse C2C12 cells has proven valuable to studies investigating the molecular mechanisms by which MSTN regulates skeletal muscle development. To identify new protein/pathway alterations and candidate biomarkers for skeletal muscle development, we compared proteomic profiles of MSTN KO C2C12 cells (KO) with corresponding wild-type cells (NC) using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. A total of 2637 proteins were identified and quantified in KO cells. Among these proteins, 77 proteins were significantly differentially expressed, 38 upregulated, and 39 downregulated, in MSTN KO C2C12 cells. These significantly altered proteins are involved in metabolic processes, developmental processes, immune system processes, and the regulation of other biological processes. Enrichment analysis was utilized to link these alterations to biological pathways, which are predominantly related to oxidative phosphorylation, protein digestion and absorption, mitochondrion localisation, antigen processing and presentation, the MAPK signaling pathway, the PPAR signaling pathway, the PI3K-Akt signaling pathway, and the JAK-STAT signaling pathway. Upregulation of several proteins, including epoxide hydrolase, tropomyosin 1, Cyb5a, HTRA1, Cox6a1, CD109, Synap29, and Ugt1a6, likely enhanced skeletal muscle development, the immune system, and energy metabolism. Collectively, our results present a comprehensive proteomics analysis of MSTN KO C2C12 myoblast cells; we hypothesize that MSTN KO could activate p38MAPK signaling pathway by CDC42, and we further deciphered the function of MSTN in the regulation of skeletal muscle development, immune processes, and mitochondrial energy metabolism.

Dermal papilla cells (DPCs), the "signaling center" of hair follicle (HF), delicately master continual growth of hair in mammals including cashmere, the fine fiber annually produced by secondary HF embedded in cashmere goat skins. Such unparalleled capacity bases on their exquisite character in instructing the cellular activity of hair-forming keratinocytes via secreting numerous molecular signals. Past studies suggested microRNA (miRNAs) and long non-coding RNAs (lncRNAs) play essential roles in a wide variety of biological process, including HF cycling. However, their roles and related molecular mechanisms in modulating DPCs secretory activities are still poorly understood.

This present research work reports the comparative analysis of the entire nucleotide sequence of mitochondrial genomes of Serranochromis robustus and Buccochromis nototaenia and phylogenetic analyses of their protein-coding genes in order to establish their phylogenetic relationship within Cichlids. The mitochondrial genomes of S. robustus and B. nototaenia are 16,583 and 16,580 base pairs long, respectively, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and one control region (D-loop) which is 888 and 887 base pairs long, respectively, showing the same gene order and identical number of gene or regions with other well-elucidated mitogenomes of Cichlids. However, with exception of cytochrome-c oxidase subunit-1 (COX-1) gene, all the identified PCGs were initiated by ATG-codons. Structurally, 11 tRNA genes in B. nototaenia species and 9 tRNA genes in S. robustus species, folded into typical clover-leaf secondary structure created by the regions of self-complementarity within tRNA. All the 22 tRNA genes in both species lack variable loop. Moreover, 28 genes which include 12-protein-coding genes are encoded on the H-strand and the remaining 9 genes including one protein-coding gene are encoded on the L-strand. Thirteen sequences of concatenated mitochondrial protein-coding genes were aligned using MUSCLE, and the phylogenetic analyses performed using maximum likelihood and Bayesian inference showed that S. robustus and B. nototaenia had a broad phylogenetic relationship. These results may be a useful tool in resolving higher-level relationships in organisms and a useful dataset for studying the evolution of the Cichlidae mitochondrial genome, since Cichlids are well-known model species in the study of evolutionary biology, because of their extreme morphological, biogeographical, parental care behavior for eggs and larvae and phylogenetic diversities.

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM(-/-)) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM(-/-) HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM(-/-) mice. Instead, ATM and Gadd45a double knockout (ATM(-/-) Gadd45a(-/-)) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM(-/-) HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM(-/-) Gadd45a(-/-) HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM(-/-) Gadd45a(-/-) mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM(-/-) mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM(-/-) HSCs.

Unintended off-target mutations induced by CRISPR/Cas9 nucleases may result in unwanted consequences, which will impede the efficient applicability of this technology for genetic improvement. We have recently edited the goat genome through CRISPR/Cas9 by targeting MSTN and FGF5, which increased muscle fiber diameter and hair fiber length, respectively. Using family trio-based sequencing that allow better discrimination of variant origins, we herein generated offspring from edited goats, and sequenced the members of four family trios (gene-edited goats and their offspring) to an average of ∼36.8× coverage. This data was to systematically examined for mutation profiles using a stringent pipeline that comprehensively analyzed the sequence data for de novo single nucleotide variants, indels, and structural variants from the genome. Our results revealed that the incidence of de novo mutations in the offspring was equivalent to normal populations. We further conducted RNA sequencing using muscle and skin tissues from the offspring and control animals, the differentially expressed genes (DEGs) were related to muscle fiber development in muscles, skin development, and immune responses in skin tissues. Furthermore, in contrast to recently reports of Cas9 triggered p53 expression alterations in cultured cells, we provide primary evidence to show that Cas9-mediated genetic modification does not induce apparent p53 expression changes in animal tissues. This work provides adequate molecular evidence to support the reliability of conducting Cas9-mediated genome editing in large animal models for biomedicine and agriculture.

The ability of rumen microorganisms to use fibrous plant matter plays an important role in ruminant animals; however, little information about rumen colonization by microbial populations after weaning has been reported. In this study, high-throughput sequencing was used to investigate the establishment of this microbial population in 80 to 110-day-old goats. Illumina sequencing of goat rumen samples yielded 101,356,610 nucleotides that were assembled into 256,868 reads with an average read length of 394 nucleotides. Taxonomic analysis of metagenomic reads indicated that the predominant phyla were distinct at different growth stages. The phyla Firmicutes and Synergistetes were predominant in samples taken from 80 to 100-day-old goats, but Bacteroidetes and Firmicutes became the most abundant phyla in samples from 110-day-old animals. There was a remarkable variation in the microbial populations with age; Firmicutes and Synergistetes decreased after weaning, but Bacteroidetes and Proteobacteria increased from 80 to 110 day of age. These findings suggested that colonization of the rumen by microorganisms is related to their function in the rumen digestive system. These results give a better understanding of the role of rumen microbes and the establishment of the microbial population, which help to maintain the host's health and improve animal performance.