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WNT signaling represents an attractive target for cancer therapy due to its widespread oncogenic role. However, the molecular players involved in WNT signaling and the impact of their perturbation remain unknown for numerous recalcitrant cancers. Here, we characterize WNT pathway activity in small cell lung cancer (SCLC) and determine the functional role of WNT signaling using genetically engineered mouse models. β-Catenin, a master mediator of canonical WNT signaling, was dispensable for SCLC development, and its transcriptional program was largely silenced during tumor development. Conversely, WNT5A, a ligand for β-catenin–independent noncanonical WNT pathways, promoted neoplastic transformation and SCLC cell proliferation, whereas WNT5A deficiency inhibited SCLC development. Loss of p130 in SCLC cells induced expression of WNT5A, which selectively increased Rhoa transcription and activated RHOA protein to drive SCLC. Rhoa knockout suppressed SCLC development in vivo, and chemical perturbation of RHOA selectively inhibited SCLC cell proliferation. These findings suggest a novel requirement for the WNT5A–RHOA axis in SCLC, providing critical insights for the development of novel therapeutic strategies for this recalcitrant cancer. This study also sheds light on the heterogeneity of WNT signaling in cancer and the molecular determinants of its cell-type specificity. Significance: The p130–WNT5A–RHOA pathway drives SCLC progression and is a potential target for the development of therapeutic interventions and biomarkers to improve patient treatment.

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

Context The giant panda (Ailuropoda melanoleuca) is a rare and endangered species to be preserved in China. The giant panda has a low reproductive capacity, and due to the scarcity of samples, studies on testes from giant panda are very limited, with little knowledge about the process of spermatogenesis in this species. Aims To establish the gene expression profiles in cells from the testis of a giant panda. Methods The 10× Genomics single-cell RNA-sequencing platform was applied to cells from the testis of an adult giant panda. Key results We identified eight testicular cell types including six somatic and two germ cell types from our single-cell RNA-sequencing datasets. We also identified the differentially expressed genes (DEGs) in each cell type, and performed functional enrichment analysis for the identified testicular cell types. Furthermore, by immunohistochemistry we explored the protein localisation patterns of several marker genes in testes from giant panda. Conclusions Our study has for the first time established the gene expression profiles in cells from the testis of a giant panda. Implications Our data provide a reference catalogue for spermatogenesis and testicular cells in the giant panda, laying the foundation for future breeding and preservation of this endangered species.

Aims This study aims to investigate the effect of hydroxy-selenomethionine supplementation on the in vitro rumen fermentation characteristics and microorganisms of Holstein cows. Methods and Results Five fermentation substrates, including control (without selenium supplementation, CON), sodium selenite supplementation (0.3 mg kg−1 DM, SS03), and hydroxy-selenomethionine supplementation (0.3, 0.6 and 0.9 mg kg−1 DM, SM03, SM06 and SM09, respectively) were incubated with rumen fluid in vitro. The results showed that in vitro dry matter disappearance and gas production at 48 h was significantly higher in SM06 than SM03, SS03 and CON; propionate and total volatile fatty acid (VFA) production was higher in SM06 than CON. Moreover, higher species richness of rumen fluid was found in SM06 than others. Higher relative abundance of Prevotella and Prevotellaceae-UCG-003 and lower relative abundance of Ruminococcus-1 were detected in SM06 than CON. Besides, higher relative abundance of Ruminococcaceae_UCG-005 was found in CON than other treatments. Conclusions It is observed that 0.6 mg kg−1 DM hydroxy-selenomethionine supplementation could increase cumulative gas production, propionate, and total VFAs production by altering the relative abundance of Prevotella, Prevotellaceae-UCG-003, Ruminococcaceae_UCG-005 and Ruminococcus-1, so that it can be used as a rumen fermentation regulator in Holstein cows. Significance and Impact of the Study This study provides an optimal addition ratio of hydroxy-selenomethionine on rumen fermentation and bacterial composition via an in vitro test.

Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates mast cells (MCs) activation, which is a key target for the treatment of allergic diseases. However, there are few drugs targeting MRGPRX2. Leukocyte mono-immunoglobulin-like receptor 3 (CD300f) is a negative regulator of FcεRΙ-mediated MC activation. However, the regulatory effect of CD300f on MRGPRX2 remains unclear. Dehydroandrographolide (DA) is a main contributor of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) have been shown to inhibit type I hypersensitivity. The aim of this study was to determine whether DA negatively regulated MRGPRX2-mediated MC activation via CD300f and showed therapeutic effect on pseudo-allergic reactions. Mouse allergic models and MC degranulation were detected in vivo and in vitro, and inflammatory mediators were detected. siRNA interference and Biacore were used to verify the target. DA inhibited pseudo-allergic reactions by reducing vasodilation and serum cytokine levels in mice and inhibited MRGPRX2-mediated MC activation. The regulatory effect of DA was significantly decreased after the knockdown of CD300f expression. Moreover, DA upregulated the phosphorylation level of Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2, which are key kinases in the negative regulatory signaling pathways associated with CD300f. In conclusion, DA negatively regulates MRGPRX2-mediated MC activation via CD300f to inhibit pseudo-allergic reactions.

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I–MAVS complex to attenuate the RIG-I–mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

Intestinal epithelial barrier destruction occurs earlier than mucosal immune dysfunction in the acute stage of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. At present, however, the cause of compromised gastrointestinal integrity in early SIV infection remains unknown. In the current study, we investigated the effects of SIV infection on epithelial barrier integrity and explored oxidative stress-mediated DNA damage and apoptosis in epithelial cells from early acute SIVmac239-infected Chinese rhesus macaques (Macaca mulatta). Results showed that the sensitive molecular marker of small intestinal barrier dysfunction, i.e., intestinal fatty acid-binding protein (IFABP), was significantly increased in plasma at 14 days post-SIV infection. SIV infection induced a profound decrease in the expression of tight junction proteins, including claudin-1, claudin-3, and zonula occludens (ZO)-1, as well as a significant increase in the active form of caspase-3 level in epithelial cells. RNA sequencing (RNA-seq) analysis suggested that differentially expressed genes between pre- and post-SIV-infected jejuna were enriched in pathways involved in cell redox homeostasis, oxidoreductase activity, and mitochondria. Indeed, a SIV-mediated increase in reactive oxygen species (ROS) in the epithelium and macrophages, as well as an increase in hydrogen peroxide (H2O2) and decrease in glutathione (GSH)/glutathione disulfide (GSSG) antioxidant defense, were observed in SIV-infected jejuna. In addition, the accumulation of mitochondrial dysfunction and DNA oxidative damage led to an increase in senescence-associated β-galactosidase (SA-β-gal) and early apoptosis in intestinal epithelial cells. Furthermore, HIV-1 Tat protein-induced epithelial monolayer disruption in HT-29 cells was rescued by antioxidant N-acetylcysteine (NAC). These results indicate that mitochondrial dysfunction and oxidative stress in jejunal epithelial cells are primary contributors to gut epithelial barrier disruption in early SIV-infected rhesus macaques.

Axon regeneration after spinal cord injury (SCI) is limited by both a decreased intrinsic ability of neurons to grow axons and the growth-hindering effects of extrinsic inhibitory molecules expressed around the lesion. Deletion ofphosphatase and tensin homolog(Pten) augments mechanistic target of rapamycin (mTOR) signaling and enhances the intrinsic regenerative response of injured corticospinal neurons after SCI. Because of the variety of growth-restrictive extrinsic molecules, it remains unclear how inhibition of conserved inhibitory signaling elements would affect axon regeneration and rewiring after SCI. Moreover, it remains unknown how a combinatorial approach to modulate both extrinsic and intrinsic mechanisms can enhance regeneration and rewiring after SCI. In the present study, we deletedRhoAandRhoC, which encode small GTPases that mediate growth inhibition signals of a variety of extrinsic molecules, to remove global extrinsic pathways.RhoA/RhoCdouble deletion in mice suppressed retraction or dieback of corticospinal axons after SCI. In contrast,Ptendeletion increased regrowth of corticospinal axons into the lesion core. Although deletion of bothRhoAandPtendid not promote axon regrowth across the lesion or motor recovery, it additively promoted rewiring of corticospinal circuits connecting the cerebral cortex, spinal cord, and hindlimb muscles. Our genetic findings, therefore, reveal that a combinatorial approach to modulate both intrinsic and extrinsic factors can additively promote neural circuit rewiring after SCI.SIGNIFICANCE STATEMENTSCI often causes severe motor deficits because of damage to the corticospinal tract (CST), the major neural pathway for voluntary movements. Regeneration of CST axons is required to reconstruct motor circuits and restore functions; however, a lower intrinsic ability to grow axons and extrinsic inhibitory molecules severely limit axon regeneration in the CNS. Here, we investigated whether suppression of extrinsic inhibitory cues by genetic deletion ofRhoas well as enhancement of the intrinsic pathway by deletion ofPtencould enable axon regrowth and rewiring of the CST after SCI. We show that simultaneous elimination of extrinsic and intrinsic signaling pathways can additively promote axon sprouting and rewiring of the corticospinal circuits. Our data demonstrate a potential molecular approach to reconstruct motor pathways after SCI.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3β and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3β-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression.

In nature, the folding and conformation of proteins can control the cell or organelle membrane permeability and regulate the life activities. Here we report the first example of synthetic polypeptide vesicles that regulate their permeability via ordered transition of secondary conformations, in a manner similar to biological systems. The polymersomes undergo a β-sheet to α-helix transition in response to reactive oxygen species (ROS), leading to wall thinning without loss of vesicular integrity. The change of membrane structure increases the vesicular permeability and enables specific transport of payloads with different molecular weights. As a proof-of-concept, the polymersomes encapsulating enzymes could serve as nanoreactors and carries for glucose-stimulated insulin secretion in vivo inspired by human glucokinase, resulting in safe and effective treatment of type 1 diabetes mellitus in mouse models. This study will help understand the biology of biomembranes and facilitate the engineering of nanoplatforms for biomimicry, biosensing, and controlled delivery applications.

Larval metamorphosis in bivalves is a key event for the larva-to-juvenile transformation. Previously we have identified a thyroid hormone receptor (TR) gene that is crucial for larvae to acquire “competence” for the metamorphic transition in the mussel Mytilus courscus (Mc). The mechanisms of thyroid signaling in bivalves are still largely unknown. In the present study, we molecularly characterized the full-length of two iodothyronine deiodinase genes (McDx and McDy). Phylogenetic analysis revealed that deiodinases of molluscs (McDy, CgDx and CgDy) and vertebrates (D2 and D3) shared a node representing an immediate common ancestor, which resembled vertebrates D1 and might suggest that McDy acquired specialized function from vertebrates D1. Anti-thyroid compounds, methimazole (MMI) and propylthiouracil (PTU), were used to investigate their effects on larval metamorphosis and juvenile development in M. coruscus. Both MMI and PTU significantly reduced larval metamorphosis in response to the metamorphosis inducer epinephrine. MMI led to shell growth retardation in a concentration-dependent manner in juveniles of M. coruscus after 4 weeks of exposure, whereas PTU had no effect on juvenile growth. It is hypothesized that exposure to MMI and PTU reduced the ability of pediveliger larvae for the metamorphic transition to respond to the inducer. The effect of MMI and PTU on larval metamorphosis and development is most likely through a hormonal signal in the mussel M. coruscus, with the implications for exploring the origins and evolution of metamorphosis.

BackgroundCancer immunotherapy has taken center stage in cancer treatment. However, the current immunotherapies only benefit a small proportion of patients with cancer, necessitating better understanding of the mechanisms of tumor immune evasion and improved cancer immunotherapy strategies. Regulatory T (Treg) cells play an important role in maintaining immune tolerance through inhibiting effector T-cell function. In the tumor microenvironment, Treg cells are used by tumor cells to counteract effector T cell-mediated tumor suppression. Targeting Treg cells may thus unleash the antitumor activity of effector T cells. While systemic depletion of Treg cells can cause excessive effector T-cell responses and subsequent autoimmune diseases, controlled targeting of Treg cells may benefit patients with cancer.MethodsTreg cells from Treg cell-specific heterozygous Cdc42 knockout mice, C57BL/6 mice treated with a Cdc42 inhibitor CASIN, and control mice were examined for their homeostasis and stability by flow cytometry. The autoimmune responses in Treg cell-specific heterozygous Cdc42 knockout mice, CASIN-treated C57BL/6 mice, and control mice were assessed by H&E staining and ELISA. Antitumor T-cell immunity in Treg cell-specific heterozygous Cdc42 knockout mice, CASIN-treated C57BL/6 mice, humanized NSGS mice, and control mice was assessed by challenging the mice with MC38 mouse colon cancer cells, KPC mouse pancreatic cancer cells, or HCT116 human colon cancer cells.ResultsTreg cell-specific heterozygous deletion or pharmacological targeting of Cdc42 with CASIN does not affect Treg cell numbers but induces Treg cell instability, leading to antitumor T-cell immunity without detectable autoimmune reactions. Cdc42 targeting causes an additive effect on immune checkpoint inhibitor anti-programmed cell death protein-1 antibody-induced T-cell response against mouse and human tumors. Mechanistically, Cdc42 targeting induces Treg cell instability and unleashes antitumor T-cell immunity through carbonic anhydrase I-mediated pH changes.ConclusionsRational targeting of Cdc42 in Treg cells holds therapeutic promises in cancer immunotherapy.

Vascular calcification has been considered as a potential therapeutic target in pulmonary hypertension. Mg2+ has a protective role against calcification. This study aimed to investigate whether Mg2+ could alleviate pulmonary hypertension by reducing medial calcification of pulmonary arteries.Monocrotaline (MCT)-induced and chronic hypoxia-induced pulmonary hypertension rats were given an oral administration of 10% MgSO4 (10 ml/kg per day). Additionally, we administered Mg2+ in calcified pulmonary artery smooth muscle cells (PASMCs) after incubating with β-glycerophosphate (β-GP, 10 mmol/l).In vivo, MCT-induced and chronic hypoxia-induced pulmonary hypertension indexes, including right ventricular systolic pressure, right ventricular mass index, and arterial wall thickness, as well as Alizarin Red S (ARS) staining-visualized calcium deposition, high calcium levels, and osteochondrogenic differentiation in pulmonary arteries, were mitigated by dietary Mg2+ intake. In vitro, β-GP-induced calcium-rich deposits stained by ARS, calcium content, as well as the detrimental effects of calcification to proliferation, migration, and resistance to apoptosis of PASMCs were alleviated by high Mg2+ but exacerbated by low Mg2+. Expression levels of mRNA and protein of β-GP-induced osteochondrogenic markers, RUNX Family Transcription Factor 2, and Msh Homeobox 2 were decreased by high Mg2+ but increased by low Mg2+; however, Mg2+ did not affect β-GP-induced expression of SRY-Box Transcription Factor 9. Moreover, mRNA expression and protein levels of β-GP-reduced calcification inhibitor, Matrix GLA protein was increased by high Mg2+ but decreased by low Mg2+.Mg2+ supplement is a powerful strategy to treat pulmonary hypertension by mitigating pulmonary arterial calcification as the calcification triggered physiological and pathological changes to PASMCs.

Chronic itch is a troublesome condition and often difficult to cure. Emerging evidence suggests that the periaqueductal gray (PAG)-rostral ventromedial medulla (RVM) pathway may play an important role in the regulation of itch, but the cellular organization and molecular mechanisms remain incompletely understood. Here, we report that a group of RVM neurons distinctively express the G-protein-coupled estrogen receptor (GPER), which mediates descending inhibition of itch. We found that GPER(+) neurons in the RVM were activated in chronic itch conditions in rats and mice. Selective ablation or chemogenetic suppression of RVM GPER(+) neurons resulted in mechanical alloknesis and increased scratching in response to pruritogens, whereas chemogenetic activation of GPER(+) neurons abrogated itch responses, indicating that GPER(+) neurons are antipruritic. Moreover, GPER-deficient mice and rats of either sex exhibited hypersensitivity to mechanical and chemical itch, a phenotype reversible by the µ type opioid receptor (MOR) antagonism. Additionally, significant MOR phosphorylation in the RVM was detected in chronic itch models in wild-type but not in GPER(−/−) rats. Therefore, GPER not only identifies a population of medullary antipruritic neurons but may also determine the descending antipruritic tone through regulating µ opioid signaling. SIGNIFICANCE STATEMENT Therapeutic options for itch are limited because of an as yet incomplete understanding of the mechanisms of itch processing. Our data have provided novel insights into the cellular organization and molecular mechanisms of descending regulation of itch in normal and pathologic conditions. GPER(+) neurons (largely GABAergic) in the RVM are antipruritic neurons under tonic opioidergic inhibition, activation of GPER promotes phosphorylation of MOR and disinhibition of the antipruritic GPER(+) neurons from inhibitory opioidergic inputs, and failure to mobilize GPER(+) neurons may result in the exacerbation of itch. Our data also illuminate on some of the outstanding questions in the field, such as the mechanisms underlying sex bias in itch, pain, and opioid analgesia and the paradoxical effects of morphine on pain and itch.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31−/− mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

The intervertebral disc has an intrinsic circadian rhythm, elimination of which leads to stress in nucleus pulposus cells (NPCs), contributing to intervertebral disc degeneration (IDD). Disruption or deletion of Bmal1 (a core transcription factor) results in complete loss of circadian rhythms, make mice susceptibility to IDD. However, the underlying mechanism by which Bmal1 mediates IDD is remains enigmatic, and whether there are other upstream factors regulating Bmal1 in NPCs. In our study, we first found that the decrease of Bmal1 was significantly correlated with the grades of IDD. With gain- and loss-of-function, Bmal1 shown a protective effect on NPC viability and functions. Transcriptomic and proteomic landscape reveals the functional contributions of Bmal1, and phosphoproteomic analysis links to autophagy. Bioinformatics analysis identified that a novel miRNA hsa-let-7f-1-3p was directly target Bmal1 3'UTR and negatively correlated with NPC function. Finally, our animal model confirmed the protective role of Bmal1 in rat IDD and this effect could be attenuated by hsa-let-7f-1-3p. The hsa-let-7f-1-3p/Bmal1/autophagy axis provides a potential therapeutic strategy for the clinical treatment of IDD.

The expression of the triggering receptor on myeloid cell-2 (TREM2) knockdown in microglia from the lateral habenula (LHb) reportedly induces depression-like behaviors in mice. However, the key molecular mechanism that mediates major depressive disorder (MDD) pathogenesis remains elusive. We herein show that Nrf2 regulates TREM2 transcription and effects TREM2 mRNA and protein expression. The activation of Nrf2 by sulforaphane (Nrf2 activator) increases the microglial arginase 1

Chinese cordyceps is a well-known fungus-larva complex with medicinal and economic importance. At present the occurrence of Chinese cordyceps has not been fully illuminated. In this study, the microbial diversities of fertilized Thitarodes eggs from sites A (high occurrence rates of Chinese cordyceps), B (low occurrence rates), and C (no Chinese cordyceps) were analyzed using 16S rRNA and ITS gene-sequencing technique. The previous sequencing data of soil from the same sites were conjointly analyzed. The results showed that bacterial communities among the eggs were significantly different. The bacterial diversity and evenness were much higher on site A. Wolbachia was overwhelmingly predominant in the eggs of sites B and C, while Spiroplasma showed preference on site A. The fungal between-group differences in the eggs were not as significant as that of bacteria. Purpureocillium in Cordyceps-related families showed preference on site A. Wolbachia, Spiroplasma, and Purpureocillium were inferred to be closely related to Chinese cordyceps occurrence. Intra-kingdom and inter-kingdom network analyses suggest that closer correlations of microbial communities (especially closer fungal positive correlations) in fertilized eggs might promote Chinese cordyceps occurrence. Besides, metabolic pathway analysis showed that in fertilized eggs or soil the number of bacterial metabolic pathways with significant differences in every comparison between two sites was greater than that of fungi. Collectively, this study provides novel information about the occurrence of Chinese cordyceps, contributing to the large-scale artificial cultivation of Chinese cordyceps.

The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

Long noncoding RNAs (lncRNAs) are expressed in different species and different tissues, and perform different functions, but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone (FSH). In general, we have revealed lncRNA‍‒‍microRNA (miRNA)‍‒‍‍messenger RNA (mRNA) interactions that may play important roles in rat primary pituitary cells. In this study, a new lncRNA was identified for the first time. First, we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization, which indicated that this lncRNA was distributed mainly in the cytoplasm. Next, we used RT-qPCR and enzyme-linked immunosorbent assay (ELISA) to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion. In addition, mothers against decapentaplegic homolog 2 (Smad2) was highly expressed in our sequencing results. We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2. We used RNA immunoprecipitation-qPCR (RIP-qPCR) and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2. Fluorescence in situ hybridization (FISH) showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm. Finally, we determined the relationship among lncRNA-m18as1, miR-18a-5p, and the Smad2/3 pathway. Overall, we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.

Mammalian sperm and oocytes are haploid cells that carry parental genetic and epigenetic information for their progeny. The cytoplasm of oocytes is also capable of reprograming somatic cells to establish totipotency through somatic cell nuclear transfer (SCNT). However, epigenetic barriers seriously counteract SCNT reprogramming. Here, we found that sperm-derived RNAs elevated chromatin accessibility of nuclear donor cells concurrent with the appearance of increased RNA amount and decreased cell proliferation, instead of activating DNA damage response. Additionally, tri-methylation of lysine 9 on histone H3 (H3K9me3) and the H3K9 methyltransferase SUV39H2 were significantly downregulated by the sperm-derived RNA treatment. Our findings thus raise a fascinating possibility that sperm RNA-induced R-loops may activate gene expression and chromatin structure, thereby promoting SCNT reprogramming.

The biogenesis of covalently closed circular DNA (cccDNA) from relaxed circular DNA (rcDNA) is essential for chronic hepatitis B virus (HBV) infection. Different host DNA repair proteins are involved in the conversion of rcDNA to cccDNA. Here, we reported that the DNA repair factor poly(ADP-ribose) polymerase 1 (PARP1) is engaged in HBV cccDNA formation. PARP1 depletion remarkably impaired HBV replication and cccDNA synthesis. Inhibition of PARP1 poly (ADP-ribosylation) activity by olaparib suppressed cccDNA synthesis both in vitro and in vivo. Specifically, the early stage of cccDNA reservoir establishment was more sensitive to olaparib, suggesting that PARP1 participated in de novo cccDNA formation. Furthermore, PARP1 was activated by recognizing the rcDNA-like lesions directly and combined with other DNA repair proteins. The results presented proposed that the DNA damage-sensing protein PARP1 and poly(ADP-ribosylation) modification play a key role in cccDNA formation, which might be the target for developing the anti-HBV drug. IMPORTANCE The biogenesis and eradication of HBV cccDNA have been a research priority in recent years. In this study, we identified the DNA repair factor PARP1 as a host factor required for the HBV de novo cccDNA formation. HBV infection caused PARylation through PARP1 in Huh7-NTCP cells, primary human hepatocytes, and human-liver chimeric mice. We found that PARP1 could directly bind to the rcDNA lesions and was activated, PARylating other DNA repair proteins. We address the importance of PARP1-mediated PARylation in HBV cccDNA formation, which is a potential therapeutic target for chronic hepatitis B.

Background: Subcortical white matter lesions are exceedingly common in cerebral small vessel disease and lead to significant cumulative disability without an available treatment. Here, we tested a rho-kinase inhibitor on functional recovery after focal white matter injury. Methods: A focal corpus callosum lesion was induced by stereotactic injection of N 5 -(1-iminoethyl)-L-ornithine in mice. Fasudil (10 mg/kg) or vehicle was administered daily for 2 weeks, starting one day after lesion induction. Resting-state functional connectivity and grid walk performance were studied longitudinally, and lesion volumes were determined at one month. Results: Resting-state interhemispheric functional connectivity significantly recovered between days 1 and 14 in the fasudil group ( P Conclusions: These data show that delayed fasudil posttreatment improves functional outcomes after a focal subcortical white matter lesion in mice. Future work will aim to elucidate the mechanisms.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) contributes to the pathogenesis of coronavirus disease 2019 (COVID‐19). The strategy used by SARS‐CoV‐2 to evade antiviral immunity needs further investigation. Here, we reported that SARS‐CoV‐2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS‐CoV‐2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS‐CoV‐2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA‐sensing pathways of RIG‐I/MDA5‐MAVS signaling, including RIG‐I, MDA‐5, MAVS, TBK1, and IKKε, rather than IRF3‐5D, which is the active form of IRF3. SARS‐CoV‐2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA‐sensing pathway of TLR3‐TRIF signaling and the adaptor protein of the cytosolic DNA‐sensing pathway of cGAS–STING signaling, respectively. A mechanistic analysis revealed that the SARS‐CoV‐2 ORF9b protein interacted with RIG‐I, MDA‐5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS‐CoV‐2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS‐CoV‐2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS‐CoV‐2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID‐19.

CARD9 is an essential adaptor protein in antifungal innate immunity mediated by C-type lectin receptors. The activity of CARD9 is critically regulated by ubiquitination; however, the deubiquitinases involved in CARD9 regulation remain incompletely understood. In this study, we identified ovarian tumor deubiquitinase 1 (OTUD1) as an essential regulator of CARD9. OTUD1 directly interacted with CARD9 and cleaved polyubiquitin chains from CARD9, leading to the activation of the canonical NF-κB and MAPK pathway. OTUD1 deficiency impaired CARD9-mediated signaling and inhibited the proinflammatory cytokine production following fungal stimulation. Importantly, Otud1–/– mice were more susceptible to fungal infection than wild-type mice in vivo. Collectively, our results identify OTUD1 as an essential regulatory component for the CARD9 signaling pathway and antifungal innate immunity through deubiquitinating CARD9.

The study aimed to characterize a novel vitexin-producing endophytic fungus Fusarium solani G6 from Cajanus cajan, improve its capability for producing vitexin and evaluate its osteoblastic proliferation activity. A total of 153 endophytic fungi, classified into 6 genera, were isolated from C. cajan. Among them, only one strain, endophyte G6 identified as Fusarium solani, was found to produce vitexin. After the optimization of fermentation conditions, the highest vitexin yield (18.72 mg/L) for the strain was observed in PDB liquid medium containing 20.54 g/L of glucose and 8.90 g/L of ammonium sulfate, at an initial medium pH of 5.1 and at 28 °C for 6 days of cultivation. Moreover, the fungal vitexin exhibited notable osteoblastic proliferation stimulating activity. A novel vitexin-producing endophytic fungus F. solani G6 was characterized from C. cajan for the first time. The findings highlighted its potential use for large-scale production of vitexin and might have a promising use as therapeutic agent for osteoporosis.

Infertility caused by male factors is routinely diagnosed by assessing traditional semen parameters. Growing evidence has indicated that the tsRNAs carried in sperm act as epigenetic factors and potential biomarkers for the assessment of sperm quality. We recently demonstrated that tRNAGln-TTG derived small RNAs played notable roles in the first cleavage of a porcine embryo. However, the function of human sperm tRNAGln-TTG derived small RNAs as a diagnostic biomarker and its role in early embryo development remains unclear. In this study, we found that human sperm tRNAGln-TTG derived small RNAs were highly associated with sperm quality. By microinjecting the antisense sequence into human tripronuclear (3PN) zygotes followed by single-cell RNA-sequencing, we found that human sperm tRNAGln-TTG derived small RNAs participated in the development of a human embryo. Furthermore, Gln-TTGs might influence embryonic genome activation by modulating noncoding RNA processing. These findings demonstrated that human sperm tRNAGln-TTG derived small RNAs could be potential diagnostic biomarkers and could be used as a clinical target for male infertility.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) continue to impact countries worldwide. At present, inadequate diagnosis and unreliable evaluation systems hinder the implementation and development of effective prevention and treatment strategies. Here, we conducted a horizontal and longitudinal study comparing the detection rates of SARS-CoV-2 nucleic acid in different types of samples collected from COVID-19 patients and SARS-CoV-2-infected monkeys. We also detected anti-SARS-CoV-2 antibodies in the above clinical and animal model samples to identify a reliable approach for the accurate diagnosis of SARS-CoV-2 infection. Results showed that, regardless of clinical symptoms, the highest detection levels of viral nucleic acid were found in sputum and tracheal brush samples, resulting in a high and stable diagnosis rate. Anti-SARS-CoV-2 immunoglobulin M (IgM) and G (IgG) antibodies were not detected in 6.90% of COVID-19 patients. Furthermore, integration of nucleic acid detection results from the various sample types did not improve the diagnosis rate. Moreover, dynamic changes in SARS-CoV-2 viral load were more obvious in sputum and tracheal brushes than in nasal and throat swabs. Thus, SARS-CoV-2 nucleic acid detection in sputum and tracheal brushes was the least affected by infection route, disease progression, and individual differences. Therefore, SARS-CoV-2 nucleic acid detection using lower respiratory tract samples alone is reliable for COVID-19 diagnosis and study.新型冠状病毒（SARS-CoV-2）及其造成的肺炎（COVID-19）持续严重影响世界各国。稳定可靠的诊断方法和评价系统的欠缺严重阻碍了有效预防和治疗策略的实施和发展。该研究通过横向和纵向研究，比较分析来源于COVID-19患者和SARS-CoV-2感染猴的不同类型样本中SARS-CoV-2的核酸检出率，分析COVID-19患者和SARS-CoV-2感染猴血清中抗SARS-CoV-2抗体的阳性率，以评估SARS-CoV-2感染诊断方法的可靠性。结果显示无论感染者的临床症状如何，痰液和气管刷样品中病毒核酸检出率较高，感染确诊率高，诊断结果稳定。而6.90% COVID-19患者血清中未检测到抗SARS-CoV-2免疫球蛋白M和G。此外，同时采集不同类型样本并结合其核酸检测的结果并不能提高诊断率。另外，与鼻拭子和咽拭子相比，痰和气管刷中SARS-CoV-2病毒载量持续时间较长，动态变化较明显。因此，痰和气管刷中SARS-CoV-2核酸检测受感染途径、疾病进展和个体差异的影响较小，用下呼吸道标本进行SARS-CoV-2核酸检测是SARS-CoV-2感染诊断和研究的可靠依据。.

Circular RNAs (circRNAs) are a new class of covalently closed circular RNA molecules that are involved in many biological processes. However, information about circRNAs in the pineal gland, particularly that of rats, is limited. To establish resources for the study of the rat pineal gland, we performed transcriptome analysis of the pineal glands during the day and night. In this study, 1413 circRNAs and 1989 miRNAs were identified in the pineal gland of rats during the night and day using the Illumina platform. Forty differentially expressed circRNAs and 93 differentially expressed miRNAs were obtained, among which 20 circRNAs and 37 miRNAs were significantly upregulated during the day and 20 circRNAs and 56 miRNAs were significantly upregulated during the night. As circRNAs have been reported to work as miRNA sponges, we predicted 15940 interactions among 40 circRNAs, 93 miRNAs and 400 mRNAs with differential diurnal expression using miRanda and TargetScan to build a ceRNA regulatory network in the rat pineal gland. The diurnal expression profile of circRNAs in the rat pineal gland may provide additional information about the role of circRNAs in regulating changes in melatonin circadian rhythms. The analyzed data reported in this study will be an important resource for future studies to elucidate the altered physiology of circRNAs in diurnal rhythms.

(1) Background: Porcine Parvovirus (PPV) is a single-stranded DNA virus without envelope which causes great harm in relation to porcine reproductive disorders in clinic. Cluster of Differentiation 38 (CD38) is a transmembrane protein widely existing in mammals. Its various functions make it a very popular research object, including in the viral infection field. (2) Methods: Western blotting and an EdU Cell Proliferation Kit were used to evaluate the effect of CD38-deficient cells. Relative quantitative real-time RT-PCR was used to detect the transcription levels of cytokines after PPV infection. The renilla luciferase reporter gene assay was used to verify the activation function of CD38 on downstream factors. The fluorescence probe method was used to detect the level of intracellular reactive oxygen species (ROS). (3) Results: This study found that the loss of CD38 function inhibited the up-regulated state of Toll-like Receptor 9 (TLR9), Interferon-α (IFN-α), and Myxovirus Resistance 1 (Mx1) after PPV infection. The luminescence of the group transfected with both CD38 expression plasmid and TLR9 promoter renilla luciferase reporter plasmid was significantly up-regulated compared with the control, suggesting that CD38 may activate the promoter of TLR9. In addition, CD38 deficiency not only activated the transcription of Sirtuin-1 (SIRT1), but also inhibited ROS level and the transcription of NLR Family Pyrin Domain Containing 3 (NLRP3). (4) Conclusion: (i) CD38 may participate in the TLR9/IFN-α/Mx1 pathway by activating the expression of TLR9 after PPV infected PK-15 cells; (ii) CD38 may activate the NLRP3/CASP1 pathway by increasing ROS level; (iii) CD38 deficiency activates the expression of SIRT1 and can prevent the normal proliferation of PPV.

Non-alcoholic fatty liver disease (NAFLD) has high occurrence in the global world, which poses serious threats to human health. Salvianolic acid B (SalB), an extract of the root of Salvia miltiorrhiza, has the protective effect on metabolic homeostasis. However, the mechanism is still unknown. In this study, we used ob/ob mice, a model of NAFLD, to explore the hepatoprotective effects of SalB. The results showed that SalB significantly reduced the body weights and liver weights, and ameliorated plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), hepatic free fatty acid (FFA), total cholesterol (TC) levels, and hepatic TG and TC levels in ob/ob mice. SalB reduced the number of lipid droplets and inhibited hepatic lipogenesis by regulating peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), stearoyl-Co A desaturase 1 (SCD1), and cluster of differentiation 36 (CD36). Compared to ob/ob mice, the lower expressions of the pro-inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and F4/80, were observed after SalB treatment. Importantly, SalB treatment inhibited the activation of NLRP3 inflammasome and reduced the severity of liver inflammation. Our findings suggested that SalB improved NAFLD pathology in ob/ob mice by reducing hepatic lipid accumulation and NLRP3 inflammasome activation, which might be the potential hepatoprotective mechanism of SalB.

The liver of poultry is the primary site of lipid synthesis. The excessive production of lipids accumulates in liver tissues causing lipid metabolism disorders, which result in fatty liver disease and have a transgenerational effect of acquired phenotypes. However, its specific mechanisms have not yet been fully understood. In this study, the differentially expressed miR-375 as well as its target gene

Mast cells (MCs) are main effector cells in chronic spontaneous urticaria (CSU). Both Fc epsilon RI (FcεRΙ)- and MAS-related G coupled receptor-X2 (MRGPRX2)-mediated MC activations affect CSU course. Leukocyte mono-immunoglobulin-like receptor 3 (CD300f) has been shown to regulate FcεRΙ activation. However, no study has verified CD300f is a target to cure CSU. Therefore this study aimed to verify whether clarithromycin (CLA) regulates FcεRΙ- and MRGPRX2-mediated MC activations via CD300f and shows therapeutic effect on CSU. The target of CLA was verification. CLA inhibited FcεRΙ- and MRGPRX2-mediated MC activations were shown in vivo and in vitro. A single-center, self-comparison study was performed, and CLA-treated CSU was investigated in 28 patients who were not sensitive to the third-generation antihistamines. Serum inflammatory mediators in patients before and after CLA administration were analyzed. CLA effectively inhibited type Ι anaphylactic reactions and pseudo-allergic reactions in mice. Moreover, CLA inhibited FcεRΙ- and MRGPRX2-mediated MC signaling pathway activation. Regulatory effects of CLA were decreased significantly after CD300f knockdown. CLA effectively alleviated the symptoms of wheal and itch and reduced serum cytokine levels in patients. CLA negatively regulated FcεRΙ- and MRGPRX2-mediated MC activation via CD300f and showed significant therapeutic effect on CSU.

Inflammasomes are essential components of the innate immune system and its defense against infections, whereas the dysregulation of inflammasome activation has a detrimental effect on human health. The activation of inflammasomes is subjected to tight regulation to maintain immune homeostasis, yet the underlying mechanism remains elusive. Here, we identify USP3 as a direct deubiquitinating enzyme (DUB) for ASC, the central adapter mediating the assembly and activation of most inflammasomes. USP3 removes the K48-linked ubiquitination on ASC and strengthens its stability by blocking proteasomal degradation. Additionally, USP3 promotes inflammasome activation, and this function was confirmed in mouse models of aluminum (Alum)-induced peritonitis, F. novicida infection and flagellin-induced pneumonia in vivo. Our work unveils that USP3 functions as a key regulator of ASC ubiquitination and maintains the physiological role of ASC in mediating inflammasome activation, and we propose a new mechanism by which the ubiquitination of ASC regulates inflammasome activation.

Psoriasis is a chronic inflammatory disease. Mast cells are significantly increased and activated in the lesions of patients with psoriasis, contributing to psoriatic inflammation. Dermcidin (DCD) is a natural antibacterial peptide secreted by sweat glands and is usually transported to the epidermal surface by sweat. Whether DCD is involved in mast cell activation remains unclear and the mechanisms by which DCD is involved in skin inflammatory reactions require further investigation.We investigated whether dermcidin-derived polypeptides DCD(86-103) activate mast cells and induce skin inflammatory reactions that contribute to psoriasis. Wild-type mice were treated with DCD(86-103) to observe the inflammatory reactions in the skin and cytokine release in vivo. The release of inflammatory mediators by mouse primary mast cells and LAD2 cells was measured in vitro. Molecular docking analysis, molecular dynamics simulation, and siRNA transfection were used to identify DCD(86-103).DCD(86-103) caused a skin inflammatory reaction in wild-type mice via cytokine release. Moreover, DCD(86-103) directly activated mast cells and induced cytokine release in vitro. ST2 may be a key receptor that mediates the activation effect of DCD(86-103) on mast cells leading to cytokine release.DCD(86-103) may have induced an inflammatory reaction and participated in the occurrence and development of psoriasis.

Pesticides are frequently used to control pests in agriculture due to their ease of use and effectiveness, but their use causes serious economic losses to sericulture when their production overlaps with agriculture. However, no suitable internal reference genes (RGs) have been reported in the study of silkworms in response to pesticides. In this study, a standard curve was established to detect the expression levels of seven RGs in different tissues of different silkworm strains after feeding with pesticides using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), including BmGAPDH, BmActin3, BmTBP, BmRPL3, Bm28sRNA, Bmα-tubulin, and BmUBC, and the stability of them was evaluated by using NormFinder, geNorm, Delta CT, BestKeeper, and RefFinder. The results showed that BmGAPDH and Bmα-tubulin were relatively stable in the midgut after feeding with fenvalerate, BmGAPDH and Bmactin3 were relatively stable in the fat body, and Bmα-tubulin and Bmactin3 were relatively stable in the hemolymph, indicating that Bmactin3 was the most suitable RG when evaluating fenvalerate, followed by BmGAPDH and Bmα-tubulin. Besides, BmGAPDH and Bmactin3 were relatively stable in the midgut after treatment with DDVP, BmGAPDH and Bmα-tubulin were relatively stable in the fat body, and BmGAPDH and Bmα-tubulin were relatively stable in the hemolymph, indicating that Bmα-tubulin was the most stable RG when evaluating DDVP, followed by BmGAPDH and Bmactin3. Of note, BmGAPDH was shared by the two pesticides. The results will be valuable for RG selection in studying the pesticide response mechanism of silkworms and other lepidopteran insects.

Sperm are susceptible to excessive reactive oxygen species (ROS). Spermine and spermidine are secreted in large amounts by the prostate and potent natural free radical scavengers and protect cells against redox disorder. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. Seven mature and fertile Duroc boars (aged 15 to 30 mo) were used in this study. In experiment 1, spermine and spermidine (3.6 ± 0.3 and 3.3 ± 0.2 mmol/L, respectively) were abundant in seminal plasma, and the content of polyamine decreased (P0.05) after preservation at 17 °C for 7 d or incubation at 37 °C for 6 h. In experiment 2, using labeling of spermine or spermidine by conjugation with fluorescein isothiocyanate and ultra-high-performance liquid chromatography, we found that the accumulation of spermine or spermidine in sperm was inhibited by quinidine and dl-tetrahydropalmatine (THP, organic cation transporters [OCT] inhibitors, P0.05), but not mildronate and l-carnitine (organic cation/carnitine transporter [OCTN] inhibitors, P0.05). In experiment 3, the addition of spermine or spermidine (0.5 mmol/L) in the extender resulted in higher motility, plasma membrane and acrosome integrity, and lower ROS level after preservation in vitro at 17 °C for 7 d (P0.05). In experiment 4, in the condition of oxidative stress (treatment with H2O2 at 37 °C for 2 h), the addition of spermine (1 mmol/L) or spermidine (0.5 mmol/L) in extender increased activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase; reduced glutathione and oxidized glutathione ratio (P0.05); and alleviate oxidative stress-induced lipid peroxidation, DNA damage, mitochondrial membrane potential (ΔΨm) decline, adenosine triphosphate depletion, and intracellular calcium concentration ([Ca2+]i) overload (P0.05), thereby improving boar sperm motility, the integrity of plasma membrane and acrosome (P0.05) in vitro. These data suggest that spermine and spermidine alleviate oxidative stress via the antioxidant capacity, thereby improving the efficacy of boar semen preservation.Boar semen preservation and artificial insemination are widely used in the pig industry. Although preservation in vitro prolongs sperm lifespan, reactive oxidative species (ROS) also accumulate in sperm with the increased preservation period. ROS over-accumulation would impair motility, the integrity of plasma membrane and acrosome, mitochondrial function, and eventually lead to infertility. Spermine and spermidine are secreted in large amounts by the prostate and are potent natural free radical scavengers. Thus, we used boar sperm as a model to study the polyamines uptake and elucidate whether polyamines protected sperm from ROS stress. We found for the first time that organic cation transporters mediated polyamines uptake in sperm cells, and that extracellular polyamines decreased during preservation in vitro. The addition of polyamines increased the activities of glutathione-related antioxidant enzymes and reduced glutathione and oxidized glutathione ratio, and alleviate oxidative stress-induced mitochondrial dysfunction, lipid peroxidation, and DNA damage, thereby maintaining sperm quality in vitro. These data suggest that spermine and spermidine alleviate oxidative stress, thereby improving the efficacy of boar semen preservation.

Background Cold storage of platelets (PLTs) has the potential advantage of prolonging storage time while reducing posttransfusion infection given the decreased likelihood of bacterial outgrowth during storage and possibly beneficial effects in treating bleeding patients. However, cold storage reduces PLT survival through the induction of complex storage lesions, which are more accentuated when storage is prolonged. Study design and methods Whole blood-derived PLT-rich plasma concentrates from seven PLT pools (n = 5 donors per pool). PLT additive solution was added (67%/33% plasma) and the product was split into 50-mL bags. Split units were stored in the presence or absence of 1 mM of N-acetylcysteine (NAC) under agitation for up to 14 days at room temperature or in the cold and were analyzed for PLT activation, fibrinogen-dependent spreading, microparticle formation, mitochondrial respiratory activity, reactive oxygen species (ROS) generation, as well as in vivo survival and bleeding time correction in immunodeficient mice. Results Cold storage of PLTs for 7 days or longer induces significant PLT activation, cytoskeletal damage, impaired fibrinogen spreading, enhances mitochondrial metabolic decoupling and ROS generation, and increases macrophage-dependent phagocytosis and macrophage-independent clearance. Addition of NAC prevents PLT clearance and allows a correction of the prolonged bleeding time in thrombocytopenic, aspirin-treated, immunodeficient mice. Conclusions Long-term cold storage induces mitochondrial uncoupling and increased proton leak and ROS generation. The resulting ROS is a crucial contributor to the increased macrophage-dependent and -independent clearance of functional PLTs and can be prevented by the antioxidant NAC in a magnesium-containing additive solution.

Long chain unsaturated fatty acids (LCUFAs) are emerging as critical contributors to inflammation and its resolution. Sensitive and accurate measurement of LCUFAs in biological samples is thus of great value in disease diagnosis and prognosis. In this work, a fluorous-derivatization approach for UPLC-MS/MS quantification of LCUFAs was developed by employing a pair of fluorous reagents, namely 3-(perfluorooctyl)-propylamine (PFPA) and 2-(perfluorooctyl)-ethylamine (PFEA). With this method, the LCUFAs in biological samples were perfluoroalkylated with PFPA and specifically retained on a fluorous-phase LC column, which largely reduced matrix interferences-induced quantitation deviation. Moreover, PFEA-labeled LCUFAs standards were introduced as one-to-one internal standards to farthest ensure unbiased results. Application of the proposed method enabled a reliable determination of eight typical LCUFAs with high sensitivity (LLOQ ranged from 30 amol to 6.25 fmol) and low matrix interferences (almost less than 10%). Such a high sensitivity could facilitate the determination of small-volume and low-concentration bio-samples. Further metabolic characterization of these targeted LCUFAs was monitored in OVA-induce asthma mice, requiring only 5 μL serum sample. Our results showed that asthmatic attack led to significant disturbances not only in the concentrations but also in the ratio among these LCUFAs. In view of the favorable advantages in sensitivity and accuracy, the present fluorous-paired derivatization approach will be expected to serve as a new avenue for dissecting the physiological and clinical implications of LCUFAs, thereby shedding light on the management of diseases related to their disturbances.

The pituitary gland functions as a prominent regulator of diverse physiologic processes by secreting multiple hormones. Circular RNAs (circRNAs) are an emerging novel type of endogenous noncoding RNA that have recently been recognized as powerful regulators participating in various biological processes. However, the physiological roles and molecular mechanisms of circRNAs in pituitary remain largely unclear. Herein, we concentrated on expounding the biological function and molecular mechanism of circRNA in rat pituitary. In this study, we identified a novel circRNA in pituitary tissue, circAkap17b, which was pituitary- and stage-specific. Then, we designed circAkap17b siRNA and constructed an overexpression plasmid to evaluate the effect of loss- and gain-of-circAkap17b function on FSH secretion. Interestingly, silencing circAkakp17b significantly inhibited FSH expression and secretion, while overexpression of circAkap17b enhanced FSH expression and secretion. Furthermore, dual luciferase reporter and RNA immunoprecipitation (RIP) assays confirmed that circAkap17b could serve as miR-7 sponge to regulate target genes. Additionally, miR-7b suppressed FSH expression and secretion by directly targeting Fshb through the dual luciferase reporter and RT-qPCR analysis. Additionally, rescue experiments showed that circAkap17b could regulate FSH secretion in pituitary cells through a circAkap17b-miR-7-Fshb axis. Collectively, we demonstrated that circAkap17b could act as a molecular sponge of miR-7 to upregulate expression of the target gene Fshb and facilitate FSH secretion. These findings provide evidence for a novel regulatory role of circRNAs in pituitary.

Background Drug resistance is a major obstacle to effective cancer therapy. In order to detect the change in tumor genomic states under drug selection pressure, we use next-generation sequencing technology to investigate the underlying potential mechanisms of drug resistance. Methods In our study, we presented a bladder cancer patient who had been a bona fide responder to first-line gemcitabine plus cisplatin regimen and second-line pazopanib (tyrosine kinase inhibitor (TKI) for FGFR3-TACC3 fusion) but finally had disease progression as an ideal case for showing genomic alteration during drug resistance. We applied whole-exome sequencing and ultra-deep target sequencing to the patient pre- and post- pazopanib resistance. Protein-protein interaction (PPI) network and Gene Ontology (GO) analyses were used to analysis protein interactions and genomic alterations. Patient-derived xenograft (PDX) model was built to test drug sensitivity. Results Twelve mutations scattered in 12 genes were identified by WES pre- pazopanib resistance, while 63 mutations in 50 genes arose post- pazopanib resistance. PPI network showed proteins from multiple epigenetic regulator families were involved post- pazopanib resistance, including subunits of chromatin remodeler SWI/SNF complex ARID1A/1B and SMARCA4, histone acetylation writers CREBBP, histone methylation writer NSD1 and erasers KDM6A/5A. GO enrichment analysis showed pazopanib resistance genes were prominently tagged for chromatin modification, transcription, as well as gland development, leaving genes with the best adaptive FGFR TKI-coping mechanisms. In addition, significantly elevated tumor mutational burden suggested possible utility of immunotherapy. Intriguingly, PDX model suggested that, sensitivity to original chemotherapy regimen (cisplatin) was restored in patient tumor post-pazopanib. Conclusions Epigenetic regulation may play a role in acquired TKI resistance. Our study traced the complete tumor genomic variation course from chemo-resistant but TKI-sensitive to TKI-resistant but chemo-(re) sensitive, revealing the potential complex dynamic drug-driven mechanisms of resistance.

Compound 6, a novel β-carboline comprising two 1-methyl-9H-β-carboline-3-carboxylic acids and a biotin moiety conjugated together using tris(2-aminoethyl)amine, was synthesized and tested for its c...

Background: Molecular characterization of insulin resistance, a growing health issue worldwide, will help to develop novel strategies and accurate biomarkers for disease diagnosis and treatment. Objective: Integrative analysis of gene expression profiling and gene regulatory network was exploited to identify potential biomarkers early in the development of insulin resistance. Methods: RNA was isolated from livers of animals at three weeks of age, and whole-genome expression profiling was performed and analyzed with Agilent mouse 4×44K microarrays. Differentially expressed genes were subsequently validated by qRT-PCR. Functional characterizations of genes and their interactions were performed by Gene Ontology (GO) analysis and gene regulatory network (GRN) analysis. Results: A total of 197 genes were found to be differentially expressed by fold change ≥2 and P < 0.05 in BKS-db +/+ mice relative to sex and age-matched controls. Functional analysis suggested that these differentially expressed genes were enriched in the regulation of phosphorylation and generation of precursor metabolites which are closely associated with insulin resistance. Then a gene regulatory network associated with insulin resistance (IRGRN) was constructed by integration of these differentially expressed genes and known human protein-protein interaction network. The principal component analysis demonstrated that 67 genes in IRGRN could clearly distinguish insulin resistance from the non-disease state. Some of these candidate genes were further experimentally validated by qRT-PCR, highlighting the predictive role as biomarkers in insulin resistance. Conclusions: Our study provides new insight into the pathogenesis and treatment of insulin resistance and also reveals potential novel molecular targets and diagnostic biomarkers for insulin resistance.

Parkinson's disease (PD) is the second most prevalent central nervous system (CNS) degenerative disease. Oxidative stress is one of key contributors to PD. Nuclear factor erythroid‐2‐related factor 2 (Nrf2) is considered to be a master regulator of many genes involved in anti‐oxidant stress to attenuate cell death. Therefore, activation of Nrf2 signalling provides an effective avenue to treat PD. Ellagic acid (EA), a natural polyphenolic contained in fruits and nuts, possesses amounts of pharmacological activities, such as anti‐oxidant stress and anti‐inflammation. Recent studies have confirmed EA could be used as a neuroprotective agent in neurodegenerative diseases. Here, mice subcutaneous injection of rotenone (ROT)‐induced DA neuronal damage was performed to investigate EA‐mediated neuroprotection. In addition, adult Nrf2 knockout mice and different cell cultures including MN9D‐enciched, MN9D‐BV‐2 and MN9D‐C6 cell co‐cultures were applied to explore the underlying mechanisms. Results demonstrated EA conferred neuroprotection against ROT‐induced DA neurotoxicity. Activation of Nrf2 signalling was involved in EA‐mediated DA neuroprotection, as evidenced by the following observations. First, EA activated Nrf2 signalling in ROT‐induced DA neuronal damage. Second, EA generated neuroprotection with the presence of astroglia and silence of Nrf2 in astroglia abolished EA‐mediated neuroprotection. Third, EA failed to produce DA neuroprotection in Nrf2 knockout mice. In conclusion, this study identified EA protected against DA neuronal loss via an Nrf2‐dependent manner.

First dose prediction is challenging in neonates. Our objective in this proof-of-concept study was to perform a pharmacokinetic (PK) bridging study from juvenile mice to neonates for drugs metabolized by CYP3A. We selected midazolam and clindamycin as model drugs. We developed juvenile mice population PK models using NONMEM. The PK parameters of these two drugs in juvenile mice were used to bridge PK parameters in neonates using different correction methods. The bridging results were evaluated by the fold-error of 0.5- to 1.5-fold. Simple allometry with and without a correction factor for maximum lifespan potential could be used for a bridging of clearance (CL) and volume of distribution (V

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

Prostate cancer (PCa) is the second leading cause of cancer related death among men worldwide. The present study aimed to investigate the role of germ cell-specific gene 2 protein (GSG2), also termed histone H3 phosphorylated by GSG2 at threonine 3, in the development and progression of PCa. GSG2 expression levels in PCa tissues and para carcinoma tissues was detected by immunohistochemistry. The GSG2 knockdown cell model was constructed by lentivirus infec tion, and the knockdown efficiency was verified by qPCR and WB. In addition, the effects of shGSG2 on cell proliferation, colony formation and apoptosis were evaluated by Celigo cell counting assay, Giemsa staining and flow cytometry, respec tively. Tumor development in nude mice was also detected. GSG2 expression was upregulated in PCa tissues and human PCa cell lines PC 3 and DU 145. High expression of GSG2 in tumor samples was associated with progressed tumors. GSG2 knockdown suppressed cell proliferation and colony forma tion, but promoted apoptosis, which was also verified in vivo. The results of the present study revealed that GSG2 upregula tion was associated with PCa progression; GSG2 knockdown inhibited cell proliferation and colony formation and induced apoptosis, and may therefore serve as a potential therapeutic target for PCa therapy.

Stringent regulation of the transcription factor NF-κB signaling is essential for the activation of host immune responses and maintaining homeostasis, yet the molecular mechanisms involved in its tight regulation are not completely understood. In this study, we report that IKK-interacting protein (IKIP) negatively regulates NF-κB activation. IKIP interacted with IKKα/β to block its association with NEMO, thereby inhibiting the phosphorylation of IKKα/β and the activation of NF-κB. Upon LPS, TNF-α, and IL-1β stimulation, IKIP-deficient macrophages exhibited more and prolonged IKKα/β phosphorylation, IκB, and p65 phosphorylation and production of NF-κB–responsive genes. Moreover, IKIP-deficient mice were more susceptible to LPS-induced septic shock and dextran sodium sulfate–induced colitis. Our study identifies a previously unrecognized role for IKIP in the negative regulation of NF-κB activation by inhibition of IKKα/β phosphorylation through the disruption of IKK complex formation.

Hematopoiesis is a finely regulated process in vertebrates under both homeostatic and stress conditions. By whole exome sequencing, we studied the genomics of acute lymphoblastic leukemia (ALL) patients who needed multiple red blood cell (RBC) transfusions after intensive chemotherapy treatment. ARHGEF12, encoding a RhoA guanine nucleotide exchange factor, was found to be associated with chemotherapy-induced anemia by genome-wide association study analyses. A single nucleotide polymorphism (SNP) of ARHGEF12 located in an intron predicted to be a GATA1 binding site, rs10892563, is significantly associated with patients who need RBC transfusion (P=3.469E-03, odds ratio 5.864). A luciferase reporter assay revealed that this SNP impairs GATA1-mediated trans-regulation of ARHGEF12, and quantitative polymerase chain reaction studies confirmed that the homozygotes status is associated with an approximately 61% reduction in ARHGEF12 expression (P=0.0088). Consequently, erythropoiesis was affected at the pro-erythroblast phases. The role of ARHGEF12 and its homologs in erythroid differentiation was confirmed in human K562 cells, mouse 32D cells and primary murine bone marrow cells. We further demonstrated in zebrafish by morpholino-mediated knockdown and CRISPR/Cas9-mediated knockout of arhgef12 that its reduction resulted in erythropoiesis defects. The p38 kinase pathway was affected by the ARHGEF12-RhoA signaling in K562 cells, and consistently, the Arhgef12-RhoA-p38 pathway was also shown to be important for erythroid differentiation in zebrafish as active RhoA or p38 readily rescued the impaired erythropoiesis caused by arhgef12 knockdown. Finally, ARHGEF12-mediated p38 activity also appeared to be involved in phenotypes of patients of the rs10892563 homozygous genotype. Our findings present a novel SNP of ARHGEF12 that may involve ARHGEF12-RhoA-p38 signaling in erythroid regeneration in ALL patients after chemotherapy.