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End-stage renal disease is a worldwide health burden, but the pathogenesis of uremia-associated cognitive impairment (CI) is poorly recognized. We hypothesized that uremia brings about deficiency of thiamin and folic acid and causes CI by inducing oxidative stress. Therefore, 24 Sprague-Dawley rats were randomly divided into two groups: a 5/6 nephrectomy group (n = 12) and a sham-operated group (n = 12). The Morris water maze was used to assess the cognitive function eight weeks post-surgery, and serum levels of thiamin, folic acid and homocysteine were detected subsequently. Brain and kidney tissues were collected for pathological examination and 8-Hydroxy-2’-deoxyguanosine (8-OHdG) immunochemistry staining. Results showed that the escape latency on training days 1-2 was longer, and the time in quadrant IV on experimental day 6 was significantly shorter in 5/6 nephrectomy group. Meanwhile, the uremic rats showed decreased thiamin, folic acid and increased homocysteine. We also found the time in quadrant IV was positively correlated with thiamin and folic acid level, while negatively correlated with the blood urea nitrogen and 8-OHdG positive cell proportion. Furthermore, in 5/6 nephrectomy group, the hippocampal neuron count was significantly reduced, and a greater proportion of 8-OHdG positive cells were detected. Pretreating LPS-stimulated rat microglial cells with thiamin or folic acid in vitro alleviated the inflammatory impairment in terms of cell viability and oxidative stress. In summary, we applied a uremic rat model and proved that uremia causes serum thiamin and folic acid deficiency, homocysteine elevation, along with neuron reduction and severe oxidative stress in hippocampus, finally leading to CI.

Wool plays an irreplaceable role in the lives of livestock and the textile industry. The variety of hair quality and shape leads to the diversity of its functions and applications, and the finer wool has a higher economic value. In this study, 10 coarse and 10 fine ordos fine wool sheep skin samples were collected for RNA-seq, and coarse and fine skin/hair follicle RNA-seq datasets of other five animal breeds were obtained from NCBI. Weighted gene co-expression network analysis showed that the common genes were clustered into eight modules. Similar gene expression patterns in sheep and rabbits with the same wool types, different gene expression patterns in animal species with different hair types, and brown modules were significantly correlated with species and breeds. GO and KEGG enrichment analyses showed that, most genes in the brown module associated with hair follicle development. Hence, gene expression patterns in skin tissues may determine hair morphology in animal. The analysis of differentially expressed genes revealed that 32 highly expressed candidate genes associated with the wool fineness of Ordos fine wool sheep. Among them, KAZALD1 (grey module), MYOC (brown module), C1QTNF6 (brown module), FOS (tan module), ITGAM, MX2, MX1, and IFI6 genes have been reported to be involved in the regulation of the hair follicle cycle or hair loss. Additionally, 12 genes, including KAZALD1, MYOC, C1QTNF6, and FOS, are differentially expressed across various animal breeds and species. The above results suggest that different sheep breeds share a similar molecular regulatory basis of wool fineness. Finally, the study provides a theoretical reference for molecular breeding of sheep breeds as well as for the investigation of the origin and evolution of animal hair.

Spermatogenesis is a complex process intricately regulated by the hypothalamic-pituitary-testis (HPT) axis. However, research on the regulatory factors governing the HPT axis remains limited. This study addresses this gap by conducting a comprehensive analysis of transcriptomes from the pituitary and testis tissues across various developmental stages, encompassing embryonic day (E120), neonatal period (P0), pre-puberty (P90), and post-puberty day (P270). Utilizing edgeR and WGCNA, we identified stage-specific genes in both the pituitary and testis throughout the four developmental stages. Notably, 380, 242, 34, and 479 stage-specific genes were identified in the pituitary, while 886, 297, 201, and 3,678 genes were identified in the testis. Subsequent analyses unveiled associations between these stage-specific genes and crucial pathways such as the cAMP signaling pathway, GnRH secretion, and male gamete generation. Furthermore, leveraging single-cell data from the pituitary and testis, we identified some signaling pathways involving BMP, HGF, IGF, and TGF-β, highlighting mutual regulation between the pituitary and testis at different developmental stages. This study sheds light on the pivotal role of the pituitary-testis axis in the reproductive process of sheep across four distinct developmental stages. Additionally, it delves into the intricate regulatory networks governing reproduction, offering novel insights into the dynamics of the pituitary-testis axis within the reproductive system.

Abstract Background The adaptive evolution of plateau indigenous animals is a current research focus. However, phenotypic adaptation is complex and may involve the interactions between multiple genes or pathways, many of which remain unclear. As a kind of livestock with important economic value, cashmere goat has a high ability of plateau adaptation, which provides us with good materials for studying the molecular regulation mechanism of animal plateau adaptation. Results In this study, 32 Jiangnan (J) and 32 Tibetan (T) cashmere goats were sequenced at an average of 10. Phylogenetic, population structure, and linkage disequilibrium analyses showed that natural selection or domestication has resulted in obvious differences in genome structure between the two breeds. Subsequently, 553 J vs. T and 608 T vs. J potential selected genes (PSGs) were screened. These PSGs showed potential relationships with various phenotypes, including myocardial development and activity (LOC106502520, ATP2A2, LOC102181869, LOC106502520, MYL2, ISL1, and LOC102181869 genes), pigmentation (MITF and KITLG genes), hair follicles/hair growth (YAP1, POGLUT1, AAK1, HES1, WNT1, PRKAA1, TNKS, WNT5A, VAX2, RSPO4, CSNK1G1, PHLPP2, CHRM2, PDGFRB, PRKAA1, MAP2K1, IRS1, LPAR1, PTEN, PRLR, IBSP, CCNE2, CHAD, ITGB7, TEK, JAK2, and FGF21 genes), and carcinogenesis (UBE2R2, PIGU, DIABLO, NOL4L, STK3, MAP4, ADGRG1, CDC25A, DSG3, LEPR, PRKAA1, IKBKB, and ABCG2 genes). Phenotypic analysis showed that Tibetan cashmere goats has finer cashmere than Jiangnan cashmere goats, which may allow cashmere goats to better adapt to the cold environment in the Tibetan plateau. Meanwhile, KRTs and KAPs expression in Jiangnan cashmere goat skin was significantly lower than in Tibetan cashmere goat. Conclusions The mutations in these PSGs maybe closely related to the plateau adaptation ability of cashmere goats. In addition, the expression differences of KRTs and KAPs may directly determine phenotypic differences in cashmere fineness between the two breeds. In conclusion, this study provide a reference for further studying plateau adaptive mechanism in animals and goat breeding.

Abstract Background Merino sheep exhibit high wool production and excellent wool quality. The fleece of Merino sheep is predominantly composed of wool fibers grown from hair follicles (HFs). The HF is a complex biological system involved in a dynamic process governed by gene regulation, and gene expression is regulated by microRNAs (miRNAs). miRNA inhibits posttranscriptional gene expression by specifically binding to target messenger RNA (mRNA) and plays an important role in regulating gene expression, the cell cycle and biological development sequences. The purpose of this study was to examine mRNA and miRNA binding to identify key miRNAs and target genes related to HF development. This will provide new and important insights into fundamental mechanisms that regulate cellular activity and cell fate decisions within and outside of the skin. Results We analyzed miRNA data in skin tissues collected from 18 Merino sheep on four embryonic days (E65, E85, E105 and E135) and two postnatal days (D7 and D30) and identified 87 differentially expressed miRNAs (DE-miRNAs). These six stages were further divided into two longer developmental stages based on heatmap cluster analysis, and the results showed that DE-mRNAs in Stage A were closely related to HF morphogenesis. A coanalysis of Stage A DE-mRNAs and DE-miRNAs revealed that 9 DE-miRNAs and 17 DE-mRNAs presented targeting relationships in Stage A. We found that miR-23b and miR-133 could target and regulate ACVR1B and WNT10A. In dermal fibroblasts, the overexpression of miR-133 significantly reduced the mRNA and protein expression levels of ACVR1B. The overexpression of miR-23b significantly reduced the mRNA and protein expression levels of WNT10A. Conclusion This study provides a new reference for understanding the molecular basis of HF development and lays a foundation for further improving sheep HF breeding. miRNAs and target genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine-wool sheep.

Background: Hair follicle development is important for the fineness of wool. Our previous methylated DNA immunoprecipitation sequencing showed that IGFBP2 and IGFBP4 were differentially methylated at different development stages of the hair follicle of Super Merino sheep. This study selected these two genes as candidates to uncover the influence of their promoter methylation on hair follicle development. Results: The results showed that the expression levels of IGFBP2 and IGFBP4 in the skin of very fine wool Super Merino sheep were higher than that in the very thick wool group, and the difference for IGFBP4 was significant. The total methylation rate of IGFBP2 promoter was extremely low, and no significant difference was found in the skin tissue of the Super Merino sheep at different fineness groups. However, the total methylation rate of IGFBP4 promoter in the very thick group was significantly higher than that in the very fine group, and the total DNA methylation levels of the IGFBP4 were significantly negatively correlated with mRNA expression. Furthermore, the transcription factor prediction results showed 104 and 91 candidate binding sites for transcription factors in the promoter regions of IGFBP2 and IGFBP4, respectively. Among these, JUN, EP300, SP1, PAX6, ETS1, and MYOD1 may be related to hair follicle development. Conclusions: The total methylation level of IGFBP4 promoter was negatively correlated with its mRNA expression in Super Merino sheep skin tissues with different degrees of fineness, which could be used as an epigenetic marker for wool breeding in Super Merino sheep.How to cite: Tian Y, Du J, Yang X, et al. Sheep IGFBP2 and IGFBP4 promoter methylation regulates gene expression and hair follicle development. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.07.001.

Abstract Background Merino sheep are the most famous fine wool sheep in the world. They have high wool production and excellent wool quality and have attracted worldwide attention. The fleece of the Merino sheep is composed predominantly of wool fibers grown from secondary wool follicles. Therefore, it is necessary to study the development of hair follicles to understand the mechanism of wool production. The hair follicle is a complex biological system involved in a dynamic process governed by gene regulation. The hair follicle development process is very complex and poorly understood. The purpose of our research is to identify candidate genes related to hair follicle development, provide a theoretical molecular breeding basis for the cultivation of fine wool sheep, and provide a reference for the problems of hair loss and alopecia areata that affect human beings. Results We analyzed mRNAs data in skin tissues of 18 Merino sheep at four embryonic days (E65, E85, E105 and E135) and two postnatal days (P7 and P30). G1 to G6 represent hair follicles developmental at six stages (i.e. E65 to P30). We identified 7879 differentially expressed genes (DEGs) and 12623 novel DEGs, revealed different expression patterns of these DEGs at six stages of hair follicle development, and demonstrated their complex interactions. DEGs with stage-specific expression were significantly enriched in epidermal differentiation and development, hair follicle development and hair follicle morphogenesis and were enriched in many pathways related to hair follicle development. The key genes (LAMA5, WNT10A, KRT25, SOSTDC1, ZDHHC21, FZD1, BMP7, LRP4, TGFβ2, TMEM79, SOX10, ITGB4, KRT14, ITGA6, and GLI2) affecting hair follicle morphogenesis were identified by network analysis. Conclusion This study provides a new reference for the molecular basis of hair follicle development and lays a foundation for further improving sheep hair follicle breeding. Candidate genes related to hair follicular development were found, which provided a theoretical basis for molecular breeding for the culture of fine wool sheep. These results are a valuable resource for biological investigations of fleece evolution in animals.

Abstract Background Tibetan cashmere goats are served as a valuable model for high altitude adaptation and hypoxia complications related studies, while the cashmere produced by these goats is an important source of income for the herders. The aim of this study was to investigate the differences in protein abundance underlying the fine (average 12.20 ± 0.03 μm of mean fiber diameter) and coarse cashmere (average 14.67 ± 0.05 μm of mean fiber diameter) producing by Tibetan cashmere goats. We systematically investigated the genetic determinants of fiber diameter by integrated analysis with proteomic and transcriptomic datasets from skin tissues of Tibetan cashmere goats. Results We identified 1980 proteins using a label-free proteomics approach. They were annotated to three different databases, while 1730 proteins were mapped to the original protein coding genes (PCGs) of the transcriptomic study. Comparative analyses of cashmere with extremely fine vs. coarse phenotypes yielded 29 differentially expressed proteins (DEPs), for instance, APOH, GANAB, AEBP1, CP, CPB2, GPR142, VTN, IMPA1, CTSZ, GLB1, and HMCN1. Functional enrichment analysis of these DEPs revealed their involvement in oxidation-reduction process, cell redox homeostasis, metabolic, PI3K-Akt, MAPK, and Wnt signaling pathways. Transcription factors enrichment analysis revealed the proteins mainly belong to NF-YB family, HMG family, CSD family. We further validated the protein abundance of four DEPs (GC, VTN, AEBP1, and GPR142) through western blot, and considered they were the most potential candidate genes for cashmere traits in Tibetan cashmere goats. Conclusions These analyses indicated that the major biological variations underlying the difference of cashmere fiber diameter in Tibetan cashmere goats were attributed to the inherent adaptations related to metabolic, hypoxic, and stress response differences. This study provided novel insights into the breeding strategies for cashmere traits and enhance the understanding of the biological and genetic mechanisms of cashmere traits in Tibetan cashmere goats.

Summary: Comparing transcriptome can help us reveal the genetic and evolutionary architecture underlying complex phenotypes within and between species. Here, by analyzing 386 publicly available RNA sequencing samples using a uniform bioinformatics pipeline, we systematically compared expression profiles of 10 immune-relevant tissues across humans, mice, pigs, cattle, sheep, and chickens. In general, we demonstrated that gene expression of orthologous genes was conserved within tissues across species. By integrating our findings with results of genome-wide association studies (GWAS) from 17 health-relevant traits in humans and 16,539 health-relevant quantitative trait loci (QTLs) in animals, we found that transcriptionally conserved genes were significantly enriched for more heritability of complex traits, compared to species-specific genes. In conclusion, our results advanced the knowledge of the transcriptome evolution of immune tissues and demonstrated that multi-species transcriptome comparison is highly informative for understanding the genetics of complex traits/disease.

Abstract Background Characterization of the molecular mechanisms underlying hair follicle development is of paramount importance in the genetic improvement of wool-related traits in sheep and skin-related traits in humans. The Merino is the most important breed of fine-wooled sheep in the world. In this study, we systematically investigated the complexity of sheep hair follicle development by integrating transcriptome and methylome datasets from Merino sheep skin. Results We analysed 72 sequence datasets, including DNA methylome and the whole transcriptome of four gene types, i.e. protein-coding genes (PCGs), lncRNAs, circRNAs, and miRNAs, across four embryonic days (E65, E85, E105, and E135) and two postnatal days (P7 and P30) from the skin tissue of 18 Merino sheep. We revealed distinct expression profiles of these four gene types across six hair follicle developmental stages, and demonstrated their complex interactions with DNA methylation. PCGs with stage-specific expression or regulated by stage-specific lncRNAs, circRNAs, and miRNAs were significantly enriched in epithelial differentiation and hair follicle morphogenesis. Regulatory network and gene co-expression analyses identified key transcripts controlling hair follicle development. We further predicted transcriptional factors (e.g. KLF4, LEF1, HOXC13, RBPJ, VDR, RARA, and STAT3) with stage-specific involvement in hair follicle morphogenesis. Through integrating these stage-specific genomic features with results from genome-wide association studies (GWAS) of five wool-related traits in 7135 Merino sheep, we detected developmental stages and genes that were relevant with wool-related traits in sheep. For instance, genes that were specifically upregulated at E105 were significantly associated with most of wool-related traits. A phenome-wide association study (PheWAS) demonstrated that candidate genes of wool-related traits (e.g. SPHK1, GHR, PPP1R27, CSRP2, EEF1A2, and PTPN1) in sheep were also significantly associated with dermatological, metabolic, and immune traits in humans. Conclusions Our study provides novel insights into the molecular basis of hair follicle morphogenesis and will serve as a foundation to improve breeding for wool traits in sheep. It also indicates the importance of studying gene expression in the normal development of organs in understanding the genetic architecture of economically important traits in livestock. The datasets generated here are useful resources for functionally annotating the sheep genome, and for elucidating early skin development in mammals, including humans.

Pituitary gonadotropins perform essential functions in mammalian reproduction by stimulating gametogenesis and steroidogenesis in the ovaries and testicles. EZH2 is a histone methyltransferase that inhibits proliferation and aggravates apoptosis in stem cells subjected to pathological stimuli. However, the expression and molecular mechanisms of EZH2 in pituitary cells in vitro have not been extensively studied. In this study, the relative abundances of EZH2 mRNA (p < 0.01) and protein (p < 0.05) expression were larger in the pituitary cells of Hu sheep with relatively greater fecundity (GF) compared to those with lesser fecundity (LF). Loss-of-function examinations demonstrated that EZH2 gene knockdown led to an earlier induction of apoptosis in sheep pituitary cells (PCs). The relative abundance of CASP3, CASP9, and BAX was increased (p < 0.01), while BCL2’s abundance was less decreased (p < 0.01) in PCs where there was EZH2 gene knockdown. Additionally, cell proliferation (p < 0.01) and viability (p < 0.01) were decreased in EZH2-knockdown sheep PCs, and the cell cycle was blocked compared to a negative control (NC). Notably, EZH2 gene knockdown led to reduced abundances of gonadotropin subunit gene transcripts (FSHβ, p < 0.05) and reduced FSH release (p < 0.01) from PCs. EZH2 gene knockdown led to reduced phosphorylation of AKT, ERK, and mTOR (p < 0.01). The results suggest that EZH2 regulates pituitary cell proliferation, apoptosis, and FSH secretion through modulation of the AKT/ERK signaling pathway, providing a foundation for further study of pituitary cell functions.

Abstract Background Genetic improvement of wool and growth traits is a major goal in the sheep industry, but their underlying genetic architecture remains elusive. To improve our understanding of these mechanisms, we conducted a weighted single-step genome-wide association study (WssGWAS) and then integrated the results with large-scale transcriptome data for five wool traits and one growth trait in Merino sheep: mean fibre diameter (MFD), coefficient of variation of the fibre diameter (CVFD), crimp number (CN), mean staple length (MSL), greasy fleece weight (GFW), and live weight (LW). Results Our dataset comprised 7135 individuals with phenotype data, among which 1217 had high-density (HD) genotype data (n = 372,534). The genotypes of 707 of these animals were imputed from the Illumina Ovine single nucleotide polymorphism (SNP) 54 BeadChip to the HD Array. The heritability of these traits ranged from 0.05 (CVFD) to 0.36 (MFD), and between-trait genetic correlations ranged from − 0.44 (CN vs. LW) to 0.77 (GFW vs. LW). By integrating the GWAS signals with RNA-seq data from 500 samples (representing 87 tissue types from 16 animals), we detected tissues that were relevant to each of the six traits, e.g. liver, muscle and the gastrointestinal (GI) tract were the most relevant tissues for LW, and leukocytes and macrophages were the most relevant cells for CN. For the six traits, 54 quantitative trait loci (QTL) were identified covering 81 candidate genes on 21 ovine autosomes. Multiple candidate genes showed strong tissue-specific expression, e.g. BNC1 (associated with MFD) and CHRNB1 (LW) were specifically expressed in skin and muscle, respectively. By conducting phenome-wide association studies (PheWAS) in humans, we found that orthologues of several of these candidate genes were significantly (FDR

Abstract Diagnostic testing of biological macromolecules is of great significance for early warning of disease and cancer. Nevertheless, restricted by limited surface area and large steric hindrance, sensitive detection of macromolecules with interface‐based sensing method remains challenging. Here, a “biphasic replacement” electrochemical aptamer‐based (BRE‐AB) sensing strategy which placed capture reaction of the biomacromolecule in a homogeneous solution phase and replaced with a small diameter of single‐stranded DNA to attach to the interface is introduced. Using the BRE‐AB sensor, the ultrasensitive detection of luteinizing hormone (LH) with the detection limit of 10 × 10−12 m is demonstrated. Molecular Dynamics simulations are utilized to explore the binding mechanism of aptamer and target LH. Moreover, it is confirmed that the BRE‐AB sensor has excellent sensing performance in whole blood and undiluted plasma. Using the BRE‐AB sensor, the LH concentrations in 40 clinical samples are successfully quantified and it is found that LH is higher expressed in breast cancer patients. Furthermore, the sensor enables simple, low‐cost, and easy to regenerate and reuse, indicating potentially applicable for point‐of‐care biological macromolecules diagnostics.

Tibetan cashmere goats are famous for producing the finest, softest and lightest cashmere fiber in China. The growth and development of skin are closely related to fineness and are the key factors affecting the quality of cashmere. To investigate the specific role of long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) in regulating cashmere fineness of Tibetan Cashmere goats in the anagen phase, we conducted high-throughput RNA sequencing of fine-type and coarse-type skin tissues. We identified 2,059 lncRNA candidates (1,589 lncRNAs annotated, 470 lncRNAs novel), and 80 differentially expressed (DE) lncRNAs and their potential targets were predicted. We also identified 384 DE messenger RNAs (mRNAs) out of 29,119 mRNAs. Several key genes in KRT26, KRT28, KRT39, IFT88, JAK3, NOTCH2 and NOTCH3 and a series of lncRNAs, including ENSCHIT00000009853, MSTRG.16794.17, MSTRG.17532.2, were shown to be potentially important for regulating cashmere fineness. GO and KEGG enrichment analyses of DE mRNAs and DE lncRNAs targets significantly enriched in positive regulation of the canonical Wnt signaling pathway, regulation of protein processing and metabolism processes. The mRNA-mRNA and lncRNA-mRNA regulatory networks further revealed potential transcripts involved in cashmere fineness. We further validated the expression patterns of DE mRNAs and DE lncRNAs by quantitative real-time PCR (qRT-PCR), and the results were consistent with the sequencing data. This study will shed new light on selective cashmere goat breeding, and these lncRNAs and mRNAs that were found to be enriched in Capra hircus RNA database.

Genomic evaluations are a method for improving the accuracy of breeding value estimation. This study aimed to compare estimates of genetic parameters and the accuracy of breeding values for wool traits in Merino sheep between pedigree-based best linear unbiased prediction (PBLUP) and single-step genomic best linear unbiased prediction (ssGBLUP) using Bayesian inference. Data were collected from 28,391 yearlings of Chinese Merino sheep (classified in 1992–2018) at the Xinjiang Gonaisi Fine Wool Sheep-Breeding Farm, China. Subjectively-assessed wool traits, namely, spinning count (SC), crimp definition (CRIM), oil (OIL), and body size (BS), and objectively-measured traits, namely, fleece length (FL), greasy fleece weight (GFW), mean fiber diameter (MFD), crimp number (CN), and body weight pre-shearing (BWPS), were analyzed. The estimates of heritability for wool traits were low to moderate. The largest h2 values were observed for FL (0.277) and MFD (0.290) with ssGBLUP. The heritabilities estimated for wool traits with ssGBLUP were slightly higher than those obtained with PBLUP. The accuracies of breeding values were low to moderate, ranging from 0.362 to 0.573 for the whole population and from 0.318 to 0.676 for the genotyped subpopulation. The correlation between the estimated breeding values (EBVs) and genomic EBVs (GEBVs) ranged from 0.717 to 0.862 for the whole population, and the relative increase in accuracy when comparing EBVs with GEBVs ranged from 0.372% to 7.486% for these traits. However, in the genotyped population, the rank correlation between the estimates obtained with PBLUP and ssGBLUP was reduced to 0.525 to 0.769, with increases in average accuracy of 3.016% to 11.736% for the GEBVs in relation to the EBVs. Thus, genomic information could allow us to more accurately estimate the relationships between animals and improve estimates of heritability and the accuracy of breeding values by ssGBLUP.

Immune and targeted therapy are becoming the first-line treatment for renal cell carcinoma (RCC). However, therapeutic outcomes are limited due to the low efficiency and side effect. Here, it is found that helicenes are able to exhibit an anticancer capability through changing the molecular structure from planar to nonplanar. Furthermore, the cytotoxicity in vitro and cancer inhibition ability of nonplanar helicenes increase with its aromatic rings’ number. It is further demonstrated that benzo[4]helicenium shows the specific killing efficiency against the RCC cancer as compared to normal kidney cells. This is majorly originated from a more selective damage of benzo[4] helicenium for mitochondria and DNA in RCC cancer cells, not the normal kidney. The selective killing ability of benzo[4]helicenium makes it have potential to be used as a targeted drug for the precise treatment of RCC. [ABSTRACT FROM AUTHOR]