Active fractions from Jingfang Baidu Powder alleviate Klebsiella-induced Pneumonia by inhibiting TLR4/Myd88-ERK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jingfang Baidu Powder (JFBDP) is a classic traditional Chinese medicine prescription. Although Jingfang Baidu powder obtained a general consensus on clinical efficacy in treating pneumonia, there were many Chinese herbal drugs in formula, complex components, and large oral dosage, which brings certain obstacles to clinical application. AIM OF THE STUDY: Therefore, screening of the active fraction that exerts anti-pneumonia helps improve the pharmaceutical preparation, improve the treatment compliance of patients, and further contribute to the clinical application, and the screening of the new active ingredients with anti-pneumonia. The histopathological observation, real-time quantitative PCR, western blotting, and immunofluorescence were applied to evaluate the anti-pneumonia efficacy of active fractions from JFBDP. RESULTS: Three fractions from JFBDP inhibit the gene expression of IL-1ß, IL-10, CCL3, CCL5, and CCL22 in lung tissue infected by Klebsiella at various degrees, and presented a good dose-response relationship. JF50 showed stronger anti-inflammatory effects among three fractions including JF30, JF50, and JF75. Besides, JF50 significantly reduced the protein expression of TLR4 and Myd88 in lung tissue infected with Klebsiella, and it also significantly inhibited p-ERK and p-NF-κB p65. JF50 significantly inhibits the protein expression of Caspase 3, Caspase 8, and Caspase 9 in lung tissue infected with Klebsiella at the dose of 25 mg/kg and 50 mg/kg. CONCLUSION: JF50 improves lung pathological damage in Klebsiella pneumonia mice by inhibiting the TLR4/Myd88/NF-κB-ERK signaling pathway, and inhibiting apoptosis of lung tissue cells. These findings provide a reference for further exploring the active substance basis of Jingfang Baidu Powder in treating bacterial pneumonia.

Endogenous retrovirus HERVH-derived lncRNA UCA1 controls human trophoblast development.

Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.

Targeting DNA Damage Repair and Immune Checkpoint Proteins for Optimizing the Treatment of Endometrial Cancer.

The dependence of cancer cells on the DNA damage response (DDR) pathway for the repair of endogenous- or exogenous-factor-induced DNA damage has been extensively studied in various cancer types, including endometrial cancer (EC). Targeting one or more DNA damage repair protein with small molecules has shown encouraging treatment efficacy in preclinical and clinical models. However, the genes coding for DDR factors are rarely mutated in EC, limiting the utility of DDR inhibitors in this disease. In the current review, we recapitulate the functional role of the DNA repair system in the development and progression of cancer. Importantly, we discuss strategies that target DDR proteins, including PARP, CHK1 and WEE1, as monotherapies or in combination with cytotoxic agents in the treatment of EC and highlight the compounds currently being evaluated for their efficacy in EC in clinic. Recent studies indicate that the application of DNA damage agents in cancer cells leads to the activation of innate and adaptive immune responses; targeting immune checkpoint proteins could overcome the immune suppressive environment in tumors. We further summarize recently revolutionized immunotherapies that have been completed or are now being evaluated for their efficacy in advanced EC and propose future directions for the development of DDR-based cancer therapeutics in the treatment of EC.

Integrative analysis of single-cell embryo data reveals transcriptome signatures for the human pre-implantation inner cell mass.

As the source of embryonic stem cells (ESCs), inner cell mass (ICM) can form all tissues of the embryo proper, however, its role in early human lineage specification remains controversial. Although a stepwise differentiation model has been proposed suggesting the existence of ICM as a distinct developmental stage, the underlying molecular mechanism remains unclear. In the present study, we perform an integrated analysis on the public human preimplantation embryonic single-cell transcriptomic data and apply a trajectory inference algorithm to measure the cell plasticity. In our results, ICM population can be clearly discriminated on the dimension-reduced graph and confirmed by compelling evidences, thus validating the two-step hypothesis of lineage commitment. According to the branch probabilities and differentiation potential, we determine the precise time points for two lineage segregations. Further analysis on gene expression dynamics and regulatory network indicates that transcription factors including GSC, PRDM1, and SPIC may underlie the decisions of ICM fate. In addition, new human ICM marker genes, such as EPHA4 and CCR8 are discovered and validated by immunofluorescence. Given the potential clinical applications of ESCs, our analysis provides a further understanding of human ICM cells and facilitates the exploration of more unique characteristics in early human development.

PARP12 regulates mouse oocyte meiotic maturation.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification of proteins that involves the transfer of ADP-ribose moieties, and plays important roles in many biological processes including DNA repair, gene expression, RNA processing, ribosome biogenesis, and protein translation. Though it is accepted that PARylation is crucial for oocyte maturation, little is known about how Mono(ADP-ribosyl)ation (MARylation) regulates this process. Here, we report that Parp12, a mon(ADP-ribosyl) transferase of poly(ADP-ribosyl) polymerase (PARP) family, was highly expressed at all stages of oocytes during meiotic maturation. At germinal vesicle (GV) stage, PARP12 was mainly distributed in cytoplasm. Interestingly, PARP12 formed granular aggregation near to spindle poles during metaphase I (MI) and metaphase II (MII). PARP12 depletion results in abnormal spindle organization and chromosome misalignment in mouse oocytes. Chromosome aneuploidy frequency in PARP12 knockdown oocytes was significantly increased. Importantly, PARP12 knockdown triggers activation of spindle assembly checkpoint as shown by active BUBR1 in PARP12-KD MI oocytes. Besides, F actin was significantly attenuated in PARP12-KD MI oocytes which may affect the asymmetric division process. Transcriptomic analysis demonstrated that PARP12 depletion disrupts transcriptome homeostasis. Collectively, our results showed that the maternally expressed mono(ADPribosyl) transferases PARP12 was essential for oocyte meiotic maturation in mouse.

Generation of NANOS3-mCherry reporter human embryonic stem cell line SYSUe-009-A using CRISPR/Cas9.

NANOS3 is a zinc-finger containing RNA-binding protein that has been demonstrated to be highly expressed in human primordial germ cell(hPGC), thus NANOS3 can serve as a marker for hPGC development. Due to the ethical and technical restrictions, it is difficult to get primary human germline cells. Human primordial germ cell-like cells (hPGCLCs) generated from pluripotent stem cells is an excellent alternatives in human germ cell-related studies. This hESC line with an mCherry knock-in at the site before NANOS3's stop codon serves as a useful tool to learn human PGC specification.

Dynamic reprogramming of H3K9me3 at hominoid-specific retrotransposons during human preimplantation development.

Reprogramming of H3K9me3-dependent heterochromatin is required for early development. How H3K9me3 is involved in early human development remains, however, largely unclear. Here, we resolve the temporal landscape of H3K9me3 during human preimplantation development and its regulation for diverse hominoid-specific retrotransposons. At the 8-cell stage, H3K9me3 reprogramming at hominoid-specific retrotransposons termed SINE-VNTR-Alu (SVA) facilitates interaction between certain promoters and SVA-derived enhancers, promoting the zygotic genome activation. In trophectoderm, de novo H3K9me3 domains prevent pluripotent transcription factors from binding to hominoid-specific retrotransposons-derived regulatory elements for inner cell mass (ICM)-specific genes. H3K9me3 re-establishment at SVA elements in the ICM is associated with higher transcription of DNA repair genes, when compared with naive human pluripotent stem cells. Our data demonstrate that species-specific reorganization of H3K9me3-dependent heterochromatin at hominoid-specific retrotransposons plays important roles during early human development, shedding light on how the epigenetic regulation for early development has evolved in mammals.

Recapitulating early human development with 8C-like cells.

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.

Genomic and Transcriptomic Analyses Reveal Pathways and Genes Associated With Brittle Stalk Phenotype in Maize.

The mechanical strength of the stalk affects the lodging resistance and digestibility of the stalk in maize. The molecular mechanisms regulating the brittleness of stalks in maize remain undefined. In this study, we constructed the maize brittle stalk mutant (bk5) by crossing the W22:Mu line with the Zheng 58 line. The brittle phenotype of the mutant bk5 existed in all of the plant organs after the five-leaf stage. Compared to wild-type (WT) plants, the sclerenchyma cells of bk5 stalks had a looser cell arrangement and thinner cell wall. Determination of cell wall composition showed that obvious differences in cellulose content, lignin content, starch content, and total soluble sugar were found between bk5 and WT stalks. Furthermore, we identified 226 differentially expressed genes (DEGs), with 164 genes significantly upregulated and 62 genes significantly downregulated in RNA-seq analysis. Some pathways related to cellulose and lignin synthesis, such as endocytosis and glycosylphosphatidylinositol (GPI)-anchored biosynthesis, were identified by the Kyoto Encyclopedia of Gene and Genomes (KEGG) and gene ontology (GO) analysis. In bulked-segregant sequence analysis (BSA-seq), we detected 2,931,692 high-quality Single Nucleotide Polymorphisms (SNPs) and identified five overlapped regions (11.2 Mb) containing 17 candidate genes with missense mutations or premature termination codons using the SNP-index methods. Some genes were involved in the cellulose synthesis-related genes such as ENTH/ANTH/VHS superfamily protein gene (endocytosis-related gene) and the lignin synthesis-related genes such as the cytochrome p450 gene. Some of these candidate genes identified from BSA-seq also existed with differential expression in RNA-seq analysis. These findings increase our understanding of the molecular mechanisms regulating the brittle stalk phenotype in maize.

Chemical-induced chromatin remodeling reprograms mouse ESCs to totipotent-like stem cells.

Totipotent cells have more robust developmental potency than any other cell types, giving rise to both embryonic and extraembryonic tissues. Stable totipotent cell cultures and deciphering the principles of totipotency regulation would be invaluable to understand cell plasticity and lineage segregation in early development. Our approach of remodeling the pericentromeric heterochromatin and re-establishing the totipotency-specific broad H3K4me3 domains promotes the pluri-to-totipotency transition. Our protocol establishes a closer match of mouse 2-cell (2C) embryos than any other 2C-like cells. These totipotent-like stem cells (TLSCs) are stable in culture and possess unique molecular features of the mouse 2C embryo. Functionally, TLSCs are competent for germline transmission and give rise to both embryonic and extraembryonic lineages at high frequency. Therefore, TLSCs represent a highly valuable cell type for studies of totipotency and embryology.

In-depth analysis reveals complex molecular aetiology in a cohort of idiopathic cerebral palsy.

Cerebral palsy is the most prevalent physical disability in children; however, its inherent molecular mechanisms remain unclear. In the present study, we performed in-depth clinical and molecular analysis on 120 idiopathic cerebral palsy families, and identified underlying detrimental genetic variants in 45% of these patients. In addition to germline variants, we found disease-related postzygotic mutations in ∼6.7% of cerebral palsy patients. We found that patients with more severe motor impairments or a comorbidity of intellectual disability had a significantly higher chance of harbouring disease-related variants. By a compilation of 114 known cerebral-palsy-related genes, we identified characteristic features in terms of inheritance and function, from which we proposed a dichotomous classification system according to the expression patterns of these genes and associated cognitive impairments. In two patients with both cerebral palsy and intellectual disability, we revealed that the defective TYW1, a tRNA hypermodification enzyme, caused primary microcephaly and problems in motion and cognition by hindering neuronal proliferation and migration. Furthermore, we developed an algorithm and demonstrated in mouse brains that this malfunctioning hypermodification specifically perturbed the translation of a subset of proteins involved in cell cycling. This finding provided a novel and interesting mechanism for congenital microcephaly. In another cerebral palsy patient with normal intelligence, we identified a mitochondrial enzyme GPAM, the hypomorphic form of which led to hypomyelination of the corticospinal tract in both human and mouse models. In addition, we confirmed that the aberrant Gpam in mice perturbed the lipid metabolism in astrocytes, resulting in suppressed astrocytic proliferation and a shortage of lipid contents supplied for oligodendrocytic myelination. Taken together, our findings elucidate novel aspects of the aetiology of cerebral palsy and provide insights for future therapeutic strategies.

Hypomorphic and hypermorphic mouse models of Fsip2 indicate its dosage-dependent roles in sperm tail and acrosome formation.

Loss-of-function mutations in multiple morphological abnormalities of the sperm flagella (MMAF)-associated genes lead to decreased sperm motility and impaired male fertility. As an MMAF gene, the function of fibrous sheath-interacting protein 2 (FSIP2) remains largely unknown. In this work, we identified a homozygous truncating mutation of FSIP2 in an infertile patient. Accordingly, we constructed a knock-in (KI) mouse model with this mutation. In parallel, we established an Fsip2 overexpression (OE) mouse model. Remarkably, KI mice presented with the typical MMAF phenotype, whereas OE mice showed no gross anomaly except for sperm tails with increased length. Single-cell RNA sequencing of the testes uncovered altered expression of genes related to sperm flagellum, acrosomal vesicle and spermatid development. We confirmed the expression of Fsip2 at the acrosome and the physical interaction of this gene with Acrv1, an acrosomal marker. Proteomic analysis of the testes revealed changes in proteins sited at the fibrous sheath, mitochondrial sheath and acrosomal vesicle. We also pinpointed the crucial motifs of Fsip2 that are evolutionarily conserved in species with internal fertilization. Thus, this work reveals the dosage-dependent roles of Fsip2 in sperm tail and acrosome formation.

Complete chloroplast genome of Alternanthera philoxeroides by de novo sequencing.

Alternanthera philoxeroides (Mart.) Griseb. (Alternanthera philoxeroides) is an important herbage species, which could provide high-quality feed for livestock and poultry breeding. This paper is the first to report the A. philoxeroides's chloroplast genomes, which were detected by de novo sequencing. The results showed that the length of A. philoxeroides' chloroplast genome sequence was 152,255 bp, including a large single-copy (LSC) region (84,670 bp), a small single-copy (SSC) region (17,343 bp), and two inverted repeat (IR) regions (25,121 bp). Alternanthera philoxeroides' chloroplast genome encoded 132 genes including 8 rRNA, 38 tRNA, and 86 protein-coding genes. After phylogenetic and cluster analysis, A. philoxeroides was closest to Amaranthaceae, and the relationship between Amaranthus and Achyranthes was closest.

Arhgef2 regulates neural differentiation in the cerebral cortex through mRNA m6A-methylation of Npdc1 and Cend1.

N 6-methyladenosine (m6A) is emerging as a vital factor regulating neural differentiation. Here, we report that deficiency of Arhgef2, a novel cause of a neurodevelopmental disorder we identified recently, impairs neurogenesis, neurite outgrowth, and synaptic formation by regulating m6A methylation. Arhgef2 knockout decreases expression of Mettl14 and total m6A level significantly in the cerebral cortex. m6A sequencing reveals that loss of Arhgef2 reduces m6A methylation of 1,622 mRNAs, including Npdc1 and Cend1, which are both strongly associated with cell cycle exit and terminal neural differentiation. Arhgef2 deficiency decreases m6A methylations of the Npdc1 and Cend1 mRNAs via down-regulation of Mettl14, and thereby inhibits the translation of Npdc1 and nuclear export of Cend1 mRNAs. Overexpression of Mettl14, Npdc1, and Cend1 rescue the abnormal phenotypes in Arhgef2 knockout mice, respectively. Our study provides a critical insight into a mechanism by which defective Arhgef2 mediates m6A-tagged target mRNAs to impair neural differentiation.

Generation of GPAM knockout human embryonic stem cell line SYSUe-008-A using CRISPR/Cas9.

GPAM (glycerol-3-phosphateacyltransferase1) is a mitochondrial enzyme that catalyze an essential step in glycerolphospholipids and triacylglycerol biosynthesis process. Loss-of-function mutation of GPAM has been shown to lead to hypomyelination of corticospinal tract in cerebral palsy patient. To model this rare disease with human brain organoid, we generated a GPAM knockout human embryonic stem cell line SYSUe-008-A by CRISPR/cas9. The GPAM knockout cell line maintains a normal karyotype and shows comparable level of pluripotent stem cell marker expression and differentiation potential as wild-type human embryonic stem cells.

A Chromosome-Level Genome Assembly of Dendrobium Huoshanense Using Long Reads and Hi-C Data.

Dendrobium huoshanense is used to treat various diseases in traditional Chinese medicine. Recent studies have identified active components. However, the lack of genomic data limits research on the biosynthesis and application of these therapeutic ingredients. To address this issue, we generated the first chromosome-level genome assembly and annotation of D. huoshanense. We integrated PacBio sequencing data, Illumina paired-end sequencing data, and Hi-C sequencing data to assemble a 1.285 Gb genome, with contig and scaffold N50 lengths of 598 kb and 71.79 Mb, respectively. We annotated 21,070 protein-coding genes and 0.96 Gb transposable elements, constituting 74.92% of the whole assembly. In addition, we identified 252 genes responsible for polysaccharide biosynthesis by Kyoto Encyclopedia of Genes and Genomes functional annotation. Our data provide a basis for further functional studies, particularly those focused on genes related to glycan biosynthesis and metabolism, and have implications for both conservation and medicine.

Generation of an induced pluripotent stem cell line SYSUi-004-A from a child of microcephaly with TYW1 mutations.

We generated an induced human pluripotent stem cell line from a child with microcephaly carrying compound heterozygous mutations of TYW1 inherited from healthy parents. This iPS cell line showed typical embryonic stem cell-like morphology, expressed pluripotent markers that comparable to human embryonic stem cells. Moreover, these cells have the ability to differentiate into three germ layers and maintain a normal karyotype.

[Analysis of the chloroplast genome characteristics of Rhus chinensis by de novo sequencing].

Rhus chinensis is an important economic species, which could provide raw materials for pharmaceutical and industrial dyes. Rhus chinensis is famous for its resistance to drought, cold, and salt. It grows in temperate, warm temperate, and subtropical regions. We report here Rhus chinensis chloroplast genomes by de novo sequencing. The results show that the length of Rhus chinensis was 159 082 bp, exhibiting a typical four-part structure with two single-copy regions (long single copy [LSC] and short single copy [SSC] sections) separated by a pair of inverted repeats (IRs). The length of LSC and SSC was 85 394 bp and 18 663 bp, respectively. The genomes contained 126 genes, including 88 protein encoding genes, 8 rRNA and 30 tRNA genes. In the chloroplast genome, 61.97% of the sequence were gene coding region. In the sequence of gene encoding region, the vast majority of sequences were protein encoding region, accounting for 86.65%, followed by rRNA (10 620 bp, 10.77%) and tRNA (2 540 bp, 2.58%). In Rhus chinensis chloroplast genome, only 8 genes contain introns, all containing 1 intron except ycf3 gene (2 introns). The Rhus chinensis chloroplast genome contains 755 SSR locies. SSR mainly consists of dinucleotide and mononucleotide, accounting for 60% (453) and 28.74% (217) respectively. The clustering results show that Anacardiaceae were closest to Rhus chinensis, followed by Aceraceae and Sapindaceae. This study provides a molecular basis for the classification of Rhus chinensis.

Generation of an induced pluripotent stem cell line SYSUi-003-A from a child with epilepsy carrying GRIN2A mutation.

We generated iPSCs from peripheral blood mononuclear cells of a child with epilepsy carrying heterozygous missense mutation in GRIN2A, using integration free episomal vectors. These iPSCs express pluripotent markers, represent a normal karyotype and have the ability to differentiate into three germ layers.

Correction: Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration.

[This corrects the article DOI: 10.1371/journal.pone.0075344.].