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During a terrestrial vertebrate survey of the Dabie Mountains in Anhui Province, eastern China, we collected four Asian shrew mole specimens (hereafter, shrew moles). Based on published literature and comparison with previously collected materials, the four specimens were similar to shrew moles from the mountains of Southwest China; however, no species in this group has been previously recorded from the Dabie Mountains. The genetic and morphological characteristics of the specimens were analyzed, based upon which a new species of shrew mole is described, named Uropsilus dabieshanensis sp. nov.

Protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) E3 ligase, was considered to be an inhibitor of STAT1 by inhibiting the DNA-binding activity of STAT1 and blocking STAT1-mediated gene transcription in response to cytokine stimulation. PIAS1 has been determined to be involved in modulating several biological processes such as cell proliferation, DNA damage responses, and inflammatory responses, both in vivo and in vitro. However, the role played by PIAS1 in regulating neurodegenerative diseases, including Alzheimer's disease (AD), has not been determined. In our study, significantly different expression levels of PIAS1 between normal controls and AD patients were detected in four regions of the human brain. Based on a functional analysis of Pias1 in undifferentiated mouse hippocampal neuronal HT-22 cells, we observed that the expression levels of several AD marker genes could be inhibited by Pias1 overexpression. Moreover, the proliferation ability of HT-22 cells could be promoted by the overexpression of Pias1. Furthermore, we performed RNA sequencing (RNA-seq) to evaluate and quantify the gene expression profiles in response to Pias1 overexpression in HT-22 cells. As a result, 285 significantly dysregulated genes, including 79 upregulated genes and 206 downregulated genes, were identified by the comparison of Pias1/+ cells with WT cells. Among these genes, five overlapping genes, including early growth response 1 (Egr1), early growth response 2 (Egr2), early growth response 3 (Egr3), FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1), were identified by comparison of the transcription factor binding site (TFBS) prediction results for STAT1, whose expression was evaluated by qPCR. Three cell cycle inhibitors, p53, p18 and p21, were significantly downregulated with the overexpression of Pias1. Analysis of functional enrichment and expression levels showed that basic region leucine zipper domain-containing transcription factors including zinc finger C2H2 (zf-C2H2), homeobox and basic/helix-loop-helix (bHLH) in several signaling pathways were significantly involved in PIAS1 regulation in HT-22 cells. A reconstructed regulatory network under PIAS1 overexpression demonstrated that there were 43 related proteins, notably Nr3c2, that directly interacted with PIAS1.

During a terrestrial vertebrate survey of the Dabie Mountains in Anhui Province, eastern China, we collected four Asian shrew mole specimens (hereafter, shrew moles). Based on published literature and comparison with previously collected materials, the four specimens were similar to shrew moles from the mountains of Southwest China; however, no species in this group has been previously recorded from the Dabie Mountains. The genetic and morphological characteristics of the specimens were analyzed, based upon which a new species of shrew mole is described, named Uropsilus dabieshanensis sp. nov.

In this study, we reassessed the taxonomic position of Typhlomys (Rodentia: Platacanthomyidae) from Huangshan, Anhui, China, based on morphological and molecular evidence. Results suggested that Typhlomys is comprised of up to six species, including four currently recognized species (Typhlomys cinereus, T. chapensis, T. daloushanensis, and T. nanus), one unconfirmed candidate species, and one new species (Typhlomys huangshanensis sp. nov. ). Morphological analyses further supported the designation of the Huangshan specimens found at mid-elevations in the southern Huangshan Mountains (600 m to 1 200 m a.s.l.) as a new species.

Here, we report the scalable fabrication of 2i-functionalized micro-pyramid-array (μPyA/+2i) inserts for use in commercial multi-well plates, as the alternative cultivation platform for maintaining long-term self-renewal and pluripotency of multiple mESCs and mouse induced pluripotent stem cells. Relevant evidence including cell morphology characterization increased alkaline phosphatase activity, high expression of mESC self-renewal markers, decreased levels of differentiation-associated markers, and high proportion of self-renewal marker cells are provided. Further studies demonstrated that μPyA/+2i could cause a higher cell density in mESC colony, and induce gene expression changes. Subsequent studies showed that μPyA/+2i can influence the cytoskeleton and promote cell adhesion through Cldn-7 upregulation. In summary, these μPyA/+2i inserts offer flexible and gelatin-free micro-envriomnets to maintain long-term self-renewal and pluripotency of mESCs. Enabled by the microstructured inserst, the facile stem cell manipulation and transfer among culture dishes will broaden stem cells both in routine and translational applications.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) is an efficient and precise gene-editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Dose- and time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.

Primordial germ cells (PGCs) are common ancestors of all germline cells. However, mechanistic understanding of how PGC specification occurs is limited. Here, we identified transcription factor CP2-like 1 (Tfcp2l1), an important pluripotency factor, as a pivotal factor for PGC-like cell (PGCLC) specification. High-throughput sequencing and quantitative real-time PCR analysis showed that Tfcp2l1 expression is gradually increased during mouse and human epiblast differentiation into PGCLCs in vivo and in vitro. Consequently, overexpression of Tfcp2l1 can enhance the specification efficiency even without inductive cytokines in mouse epiblast-like cells derived from embryonic stem cells, while knockdown of Tfcp2l1 significantly inhibits PGCLC generation. Mechanistic studies revealed that Tfcp2l1 exerts its function partially through the direct induction of PR domain zinc finger protein 14, a key PGC marker, as downregulation of the PR domain zinc finger protein 14 transcript can impair the ability of Tfcp2l1 to direct PGCLC commitment. Importantly, we finally demonstrated that the crucial role of the human homolog Tfcp2l1 in promoting PGCLC specification is conserved in human pluripotent stem cells. Together, our data uncover a novel function of Tfcp2l1 in PGCLC fate determination and facilitate a better understanding of germ cell development.

Stereoselective β-C(sp2)-H alkylation of enamides with redox-active N-hydroxyphthalimide esters via a photoredox-catalyzed decarboxylative cross-coupling reaction is demonstrated. This methodology features operational simplicity, broad substrate scopes, and excellent stereoselectivities and functional group tolerance, affording a diverse array of geometrically defined and synthetically valuable enamides bearing primary, secondary or tertiary alkyl groups in satisfactory yields.

CRISPR-Cas9 knockin of the HBB gene paves the way for a general therapeutic solution for treating hemoglobinopathies., Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate–mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)–encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

Ischemia reperfusion (I/R) injury, an inevitable event accompanying heart transplantation, is the primary factor leading to organ failure and graft rejection. In order to prevent I/R injury, we established murine heart transplantation model with I/R and cell culture system to determine whether β-catenin is a mediate factor in preventing I/R injury in heart transplantation. After successfully established heterotopic heart transplantation mice model, the I/R injury was induced, and two dynamic temporal were studied during different I/R phases. With the increase of ischemia and reperfusion time, heart damage was more severe. In the initial study, we observed that β-catenin was significantly decreased, while ROCK1 and PTEN increased during the perfusion phase from day 0 to day 1, and remain the same level until 3 days later. The similar pattern that β-catenin was down-regulated while ROCK1 and PTEN were up-regulated was also observed in the dynamic temporal ischemia study. To further investigate the role of β-catenin signaling in I/R injury in vitro, β-catenin over-expressing plasmid was transfected into HL-1 cells, a cardiac cell line. We noted that β-catenin over-expressing cardiomyocytes showed decreased ROCK1/PTEN expression both at mRNA and protein levels. In addition, cobalt dichloride (CoCl(2)) -induced oxidative stress model was further established to mimic cardiac I/R injury. We observed that CoCl(2)-induced activation of ROCK1/PTEN signaling pathway were attenuated by transient transfection of a β-catenin over-expressing plasmid. Taken together, our results suggest that cardiac transplant induced IR injury is closely associated with the down-regulation of β-catenin and up-regulation of ROCK1 and PTEN expression.

In this study, we detect depressive effect on leptin receptor (LEPR) by LEPR-specific short hairpin RNA (shRNA) expression plasmids in chicken preadipocytes, and effect on messenger RNA (mRNA) expression levels of genes related to signal transduction, including JAK2, STAT3, SOCS3 as well as CPT1, which is associated with fatty acid metabolism. shRNA expression vectors targeting LEPR were constructed and transfected into chicken preadipocytes. The transfection efficiency was evaluated by fluorescence microscopy. Real-time PCR was used to detect its effect on mRNA expression levels of JAK2, STAT3, SOCS3 and CPT1. Results showed that LEPR mRNA was knocked down by 99% (P

The mutation of somitogenesis protein Mesogenin 1 (Msgn1) has been widely used to study the direct link between somitogenesis and the development of an embryo. Several studies have used gene expression profiling of somitogenesis to identify the key genes in the process, but few have focused on the pathways involved and the coexpression patterns of associated pathways. Here we employed time-course microarray datasets of differentiating embryonic stem cells by overexpressing the transcription factor Msgn1 from the public database library of Gene Expression Omnibus (GEO). Then we applied gene set enrichment analysis (GSEA) to the datasets and performed candidate transcription factors selection. As a result, several significantly regulated pathways and transcription factors (TFs), as well as some of the specific signaling pathways, were identified during somitogenesis under Msgn1 overexpression, most of which had not been reported previously. Finally, significant core genes such as Hes1 and Notch1 as well as some of the TFs such as PPARs and FOXs were identified to construct coexpression networks of related pathways, the expression patterns of which had been validated by our following quantitative real-time PCR (qRT-PCR). The results of our study may help us better understand the molecular mechanisms of somitogenesis in mice at the genome-wide level.

In article number https://doi.org/10.1002/advs.201903432, Shih‐Hwa Chiou, Hsian‐Rong Tseng, Paul S. Weiss, and co‐workers introduce an in vivo CRISPR/Cas9‐mediated knockin approach using two supramolecular nanoparticle (SMNP) vectors. By intravitreally injecting the two SMNP vectors into the mouse eyes, the RS1/GFP gene is knocked into the Rosa26 site in mice retinas, offering a revolutionarily curative therapeutic solution for X‐linked juvenile retinoschisis.

Stromal microenvironment increases tumor cell survival, proliferation and migration, and promotes angiogenesis. In order to provide comprehensive information on the stromal heterogeneity of diverse tumors, here we employed the microarray datasets of human invasive breast and prostate cancer-associated stromals and applied Gene Set Enrichment Analysis (GSEA) to compare the gene expression profiles between them. As a result, 8 up-regulated pathways and 73 down-regulated pathways were identified in the breast tumor stroma, while 32 up-regulated pathways and 18 down-regulated pathways were identified in the prostate tumor stroma. Only 9 pathways such as tryptophan metabolism were commonly up or down regulated, but most of them (including ABC transporters) were specific for these two tumors. Several essential tumors stromal marker genes were also significantly identified. For example, CDH3 was significantly up-regulated in the stromals of both breast and prostate tumors, however EGFR was only significantly down-regulated in the stromal of breast tumor. Our study would be helpful for future therapeutic and predictive applications in breast and prostate cancers.

The present study makes an investigation into expression of genes related to cardiac development in chicken, quail and chicken-quail hybrids during the early stage of embryogenesis. Real-time PCR was used to detect mRNA expressions of Nkx2-5, GATA4 and TBX5 in the heart of chicken, quail and chicken-quail hybrids embryos during the 3rd to 7th days of incubation. Results showed that NKX2-5 mRNA displayed a similar expression trend in chicken, quail and chicken-quail hybrids. The initial and highest expression of Nkx2-5 was focused on the 3rd day of incubation, then it declined till 5th day of incubation, thereafter, it fluctuated. Expression of Nkx2-5 gene in quail was significantly higher than in chicken and chicken-quail hybrids, and no significant difference was observed between the two latter species. GATA4 mRNA showed a similar expression trend between chicken and quail, which displayed a steady increase from 3rd to 6th d, then, the expression level decreased. However, GATA4 mRNA expression in chicken-quail hybrids was significantly higher than that in chicken and quail from 3rd to 5th d (p

Cystic echinococcosis (CE), caused by infection with the larval stage of the cestode Echinococcus granulosus , is a chronic zoonosis, to which sheep are highly susceptible. Previously, we found that Kazakh sheep with different MHC haplotypes differed in CE infection. Sheep with haplotype MHCMva Ibc-Sac IIab-Hin 1Iab were resistant to CE infection, while their counterparts without this haplotype were not. MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at the post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. To identify microRNA controlling resistance to CE in the early stage of infection, microRNA profiling was conducted in the intestinal tissue of sheep with resistant and non-resistant MHC haplotypes after peroral infection with E. granulosus eggs. A total of 351 known and 186 novel miRNAs were detected in the resistant group, against 353 known and 129 novel miRNAs in the non-resistant group. Among these miRNAs, 83 known miRNAs were significantly differentially expressed, including 75 up-regulated and 8 down-regulated miRNAs. Among these known microRNAs, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, miR-26b, and miR-27a showed a significantly higher expression in CE-resistant sheep compared to the CE-non-resistant library, with the FC > 3. Functional analysis showed that they were NF-kB pathway-responsive miRNAs, which are involved in the inflammation process. The results suggest that these microRNAs may play important roles in the response of intestinal tissue to E. granulosus .

In order to study the potential gene function of ovine EST-SSR markers, nine original EST of Ovine Skin Derived polymorphic EST-SSR loci, which were developed in an early study by our lab, were ontology annotated and Electro localized. The results revealed that the original ESTs of the six loci had high homology with known genes and three of them probably played an important role in wool traits. Compared with its cDNA library, 8 loci were located on chromosomes of cattle. The homology of chromosomes between cattle and sheep was estimated based on the similarity coefficients calculated by positioning markers. Additionally, NJ clustering tree was establishedto serve for electro localization of ovine EST-SSR markers. Finally, 8 EST-SSR markers were successfully positioned on ovine chromosomes. The results from this study not only provide references for further studies on genetic mapping, in silico cloning of key genes for wool traits, but also are helpful to the researchs of chromosome evolution in animal.

In this study, we detect depressive effect on leptin receptor (LEPR) by LEPR-specific short hairpin RNA (shRNA) expression plasmids in chicken preadipocytes, and effect on messenger RNA (mRNA) expression levels of genes related to signal transduction, including JAK2, STAT3, SOCS3 as well as CPT1, which is associated with fatty acid metabolism. shRNA expression vectors targeting LEPR were constructed and transfected into chicken preadipocytes. The transfection efficiency was evaluated by fluorescence microscopy. Real-time PCR was used to detect its effect on mRNA expression levels of JAK2, STAT3, SOCS3 and CPT1. Results showed that LEPR mRNA was knocked down by 99% (P 0.01) after transfection for 72 h. In the knockdown preadipocytes, the mRNA levels of JAK2 and CPT1 were down-regulated by 47.56% (P 0.01) and 42.26% (P 0.05), respectively; while expression of STAT3 and SOCS3 increased 7.72-fold (P 0.01), 1.71-fold (P 0.01), respectively. It is concluded that knockdown of LEPR influences mRNA expression of its down-stream genes, suggesting that chicken LEPR play a certain role in regulating genes in the complicated gene network of preadipocytes.

Background Cystic Echinococcosis(CE), caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), is a chronic parasitic zoonosis, with highly susceptible infection in sheep. However, the comprehensive molecular mechanisms that underlie the process of E. granulosus infection in the early stage remain largely unknown. The objective of this present study was to gain a cluster of genes expression profiles in the intestine tissue of sheep infected with CE. Methods Nine healthy sheep were divided into infection group and healthy controls, with six infected perorally 5000 E. granulosus eggs suspended in 1000μl physiological saline and three controls perorally injected 1000μl physiological saline. All animals were sacrificed at 4 hours post-infection, respectively. The intestine tissue was removed and the RNA was extracted. In the infection group, the biology replicates were designed to make sure the accuracy of the data. The ovine microarrays were used to analyze changes of gene expression in the intestine tissue between CE infected sheep and healthy controls. Real-time PCR was used to assess reliability of the microarray data. Results By biology repeats, a total of 195 differentially expressed genes were identified between infected group and controls at 4 hours post-infection, with 105 genes related to immune responses, while 90 genes associated with functions including energy metabolism, fat soluble transport, etc. Among the 105 immunity genes, 72 genes showed up-regulated expression levels while 33 showed down-regulation levels. Function analysis showed that most of up-regulated genes were related to innate immune responses, such as mast cell, NK cell, cytokines, chemokines and complement. In addition, Real-time PCR analysis of a random selection of nine genes confirmed the reliability of the microarray data. Conclusion To our knowledge, this is the first report describing gene expression profiles in the intestine tissue of CE infection sheep. These results suggested that the innate immune system was activated to elicit immediate defense in the intestine tissue where E. granulosus invaded in at 4 hour-post infection. Furthermore, future interest will also focus on unraveling similar events, especially for the function of adaptive immunity, but at late stage infection.

The present study makes an investigation into expression of genes related to cardiac development in chicken, quail and chicken-quail hybrids during the early stage of embryogenesis. Real-time PCR was used to detect mRNA expressions of Nkx2-5, GATA4 and TBX5 in the heart of chicken, quail and chicken-quail hybrids embryos during the 3rd to 7th days of incubation. Results showed that NKX2-5 mRNA displayed a similar expression trend in chicken, quail and chicken-quail hybrids. The initial and highest expression of Nkx2-5 was focused on the 3rd day of incubation, then it declined till 5th day of incubation, thereafter, it fluctuated. Expression of Nkx2-5 gene in quail was significantly higher than in chicken and chicken-quail hybrids, and no significant difference was observed between the two latter species. GATA4 mRNA showed a similar expression trend between chicken and quail, which displayed a steady increase from 3rd to 6th d, then, the expression level decreased. However, GATA4 mRNA expression in chicken-quail hybrids was significantly higher than that in chicken and quail from 3rd to 5th d (p

Activation of leukemia inhibitor factor (LIF)–Stat3 or Wnt/β-catenin signaling promotes mouse embryonic stem cell (mESC) self-renewal. A myriad of downstream targets have been identified in the individual signal pathways, but their common targets remain largely elusive. In this study, we found that the LIF–Stat3 and Wnt/β-catenin signaling pathways converge on Sp5 to promote mESC self-renewal. Forced Sp5 expression can reproduce partial effects of Wnt/β-catenin signaling but mimics most features of LIF–Stat3 signaling to maintain undifferentiated mESCs. Moreover, Sp5 is able to convert mouse epiblast stem cells into a naïve pluripotent state. Thus, Sp5 is an important component of the regulatory network governing mESC naïve pluripotency., Summary: This study reveals a new function of Sp5 in mouse embryonic stem cell (ESC) self-renewal mediated by CHIR99021 and LIF, and reprogramming of EpiSCs into naїve ESCs.

In order to study the potential gene function of ovine EST-SSR markers, nine original EST of Ovine Skin Derived polymorphic EST-SSR loci, which were developed in an early study by our lab, were ontology annotated and Electro localized. The results revealed that the original ESTs of the six loci had high homology with known genes and three of them probably played an important role in wool traits. Compared with its cDNA library, 8 loci were located on chromosomes of cattle. The homology of chromosomes between cattle and sheep was estimated based on the similarity coefficients calculated by positioning markers. Additionally, NJ clustering tree was establishedto serve for electro localization of ovine EST-SSR markers. Finally, 8 EST-SSR markers were successfully positioned on ovine chromosomes. The results from this study not only provide references for further studies on genetic mapping, in silico cloning of key genes for wool traits, but also are helpful to the researchs of chromosome evolution in animal.

Mannose-binding lectin (MBL) is the archetypical pathogen recognition protein of the innate immune defence. In humans, three frequently occurring single nucleotide polymorphisms (SNPs) in the coding region of MBL gene are associated with the abnormal polymerization, decreased serum concentration and strongly impaired function of MBL protein. To understand whether or not SNPs in MBL gene are associated with serum concentration of MBL in sheep, we investigated 105 individuals of the Hu sheep by PCR single-strand conformation polymorphism (SSCP) analysis, DNA sequencing, and enzyme-linked immunosorbent assay. SSCP analyses of PCR amplicons from a 194-bp section of the exon-I region of the MBL gene revealed four patterns: A, B, C and D. In comparison with the sequences of the full-length MBL gene of sheep (GenBank accession numbers FJ977629 and AM933378; reference sequence hereafter), pattern A has a 3-bp deletion, a 6-bp deletion and 42 SNPs. Pattern B has 3 SNPs, pattern C has 2 SNPs, whereas pattern D is identical to the reference sequence. Twenty-four of the 47 SNPs of the four patters are synonymous whereas the other 23 SNPs are non-synonymous. The two deletions in the pattern A result in deletions of amino acids but there are no frame shifts in the putative MBL protein. The concentration of MBL protein in serum ranges from 1571 to 3657 μg/L in the Hu sheep. Our statistic analyses showed that patterns A and B are associated with reduced MBL protein level in serum, whereas pattern C is associated with increased MBL protein level in serum (P0.05) in the Hu sheep.