Loss of UBE2S causes meiosis I arrest with normal spindle assembly checkpoint dynamics in mouse oocytes.

The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.

Discovery of novel biphenyl-sulfonamide analogues as NLRP3 inflammasome inhibitors.

The aberrant activation of NLRP3 inflammasome has been observed in various human diseases. Targeting the NLRP3 protein with small molecule inhibitors shows immense potential as an effective strategy for disease intervention. Herein, a series of novel biphenyl-sulfonamide NLRP3 inflammasome inhibitors were designed and synthesized. The representative compound H28 was identified as potent and specific NLRP3 inflammasome inhibitor with IC50 values of 0.57 µM. Preliminary mechanistic studies have revealed that compound H28 exhibits direct binding to the NLRP3 protein (KD: 1.15 µM), effectively inhibiting the assembly and activation of the NLRP3 inflammasome. The results in a mouse acute peritonitis model revealed that H28 effectively inhibit the NLRP3 inflammasome pathway, demonstrating their anti-inflammatory properties. Our findings strongly support the further development of H28 as potential lead compound for treating NLRP3-related diseases.

HDAC/NAMPT dual inhibitors overcome initial drug-resistance in p53-null leukemia cells.

The occurrence of cancer is closely related to metabolism and epigenetics. Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression as epigenetic regulators, while nicotinamide phosphoribosyltransferase (NAMPT) is significantly involved in maintaining cellular metabolism. In this study, we rationally designed a series of novel HDAC/NAMPT dual inhibitors based on the structural similarity between HDAC and NAMPT inhibitors. The representative compounds 39a and 39h exhibit significant selective inhibitory activity on HDAC1-3 with IC50 values of 0.71-25.1 nM, while displaying modest activity against NAMPT. Compound 39h did not exhibit inhibitory activity against 370 kinases, demonstrating its target specificity. These two compounds exhibit potent anti-proliferative activity in multiple leukemia cell lines with low nanomolar IC50s. It is worth noticing that the dual inhibitors 39a and 39h overcome the primary resistance of HDAC or NAMPT single target inhibitor in p53-null AML cell lines, with the induction of apoptosis-related cell death. NMN recovers the cell death induced by HDAC/NAMPT dual inhibitors, which indicates the lethal effects are caused by the inhibition of NAD biosynthesis pathway as well as HDAC. This research provides an effective strategy to overcome the limitations of HDAC inhibitors in treating p53-null leukemia.

A Synergistic Lipid Nanoparticle Encapsulating mRNA Shingles Vaccine Induces Potent Immune Responses and Protects Guinea Pigs from Viral Challenges.

Shingles is caused by the reactivation of varicella zoster virus (VZV) and manifests as painful skin rashes. While the recombinant protein-based vaccine proves highly effective, it encounters supply chain challenges due to a shortage of the necessary adjuvant. Messenger RNA (mRNA)-based vaccines can be rapidly produced on a large scale, but their effectiveness relies on efficient delivery and sequence design. Here, an mRNA-based VZV vaccine using a synergistic lipid nanoparticle (Syn-LNP) containing two different ionizable lipids is developed. Syn-LNP shows superior mRNA expression compared to LNPs formulated with either type of ionizable lipid and to a commercialized LNP. After encapsulating VZV glycoprotein E (gE)-encoding mRNA, mgE@Syn-LNP induces robust humoral and cellular immune responses in two strains of mice. The magnitude of these responses is similar to that induced by adjuvanted recombinant gE proteins and significantly higher than that observed with live-attenuated VZV. mgE@Syn-LNP exhibits durable humoral responses for over 7 months without obvious adverse effects. In addition, mgE@Syn-LNP protects vaccinated guinea pigs against live VZV challenges. Preliminary studies on the mRNA antigen design reveal that the removal of glycosylation sites of gE greatly reduces its immune responses. Collectively, Syn-LNP encapsulating gE-encoded mRNA holds great promise as a shingles vaccine.

Discovery of Novel 2,3-Dihydro-1H-indene-5-sulfonamide NLRP3 Inflammasome Inhibitors Targeting Colon as a Potential Therapy for Colitis.

The NLRP3 inflammasome is a multiprotein complex that plays a crucial role in the pathophysiology of multiple inflammation-related diseases. In this study, we designed and synthesized a series of novel 2,3-dihydro-1H-indene-5-sulfonamide analogues as NLRP3 inflammasome inhibitors, and then identified compound 15z as a potent and specific inhibitor (IC50: 0.13 µM) with low toxicity. Mechanistic studies indicate that 15z binds directly to NLRP3 protein (KD: 102.7 nM), blocking the assembly and activation of the NLRP3 inflammasome and effectively inhibiting cell pyroptosis. Given the notable distribution of 15z in the colon, the DSS-induced colitis model was employed to evaluate its in vivo effectiveness. 15z significantly impacted NLRP3 inflammasome activation and relieved inflammatory bowel disease symptoms in this model. Acute and subacute toxicity studies suggested that 15z has a favorable safety profile. Our results indicate that 15z has great potential to be further developed as a candidate for the treatment of inflammatory bowel disease.

SRSF2 is required for mRNA splicing during spermatogenesis.

BACKGROUND: RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. RESULTS: Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. CONCLUSIONS: Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

Comprehensive non-coding RNA analysis reveals specific lncRNA/circRNA-miRNA-mRNA regulatory networks in the cotton response to drought stress.

Root and leaf are essential organs of plants in sensing and responding to drought stress. However, comparative knowledge of non-coding RNAs (ncRNAs) of root and leaf tissues in the regulation of drought response in cotton is limited. Here, we used deep sequencing data of leaf and root tissues of drought-resistant and drought-sensitive cotton varieties for identifying miRNAs, lncRNAs and circRNAs. A total of 1531 differentially expressed (DE) ncRNAs was identified, including 77 DE miRNAs, 1393 DE lncRNAs and 61 DE circRNAs. The tissue-specific and variety-specific competing endogenous RNA (ceRNA) networks of DE lncRNA-miRNA-mRNA response to drought were constructed. Furthermore, the novel drought-responsive lncRNA 1 (DRL1), specifically and differentially expressed in root, was verified to positively affect phenotypes of cotton seedlings under drought stress, competitively binding to miR477b with GhNAC1 and GhSCL3. In addition, we also constructed another ceRNA network consisting of 18 DE circRNAs, 26 DE miRNAs and 368 DE mRNAs. Fourteen circRNA were characterized, and a novel molecular regulatory system of circ125- miR7484b/miR7450b was proposed under drought stress. Our findings revealed the specificity of ncRNA expression in tissue- and variety-specific patterns involved in the response to drought stress, and uncovered novel regulatory pathways and potentially effective molecules in genetic improvement for crop drought resistance.

Discovery of a Novel Ubenimex Derivative as a First-in-Class Dual CD13/Proteasome Inhibitor for the Treatment of Cancer.

The CD13 inhibitor ubenimex is used as an adjuvant drug with chemotherapy for the treatment of cancer due to its function as an immunoenhancer, but it has limitations in its cytotoxic efficacy. The proteasome inhibitor ixazomib is a landmark drug in the treatment of multiple myeloma with a high anti-cancer activity. Herein, we conjugated the pharmacophore of ubenimex and the boric acid of ixazomib to obtain a dual CD13 and proteasome inhibitor 7 (BC-05). BC-05 exhibited potent inhibitory activity on both human CD13 (IC50 = 0.13 µM) and the 20S proteasome (IC50 = 1.39 µM). Although BC-05 displayed lower anti-proliferative activity than that of ixazomib in vitro, an advantage was established in the in vivo anti-cancer efficacy and prolongation of survival time, which may be due to its anti-metastatic and immune-stimulating activity. A pharmacokinetic study revealed that BC-05 is a potentially orally active agent with an F% value of 24.9%. Moreover, BC-05 showed more favorable safety profiles than those of ixazomib in preliminary toxicity studies. Overall, the results indicate that BC-05 is a promising drug candidate for the treatment of multiple myeloma.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Insights into the adverse effects of prepubertal chronic ethanol exposure on adult female reproduction.

Heavy drinking in women is known to adversely affect pregnancy and fertility. However, pregnancy is a complex process, and the adverse effects of ethanol on pregnancy does not mean that ethanol will have adverse effects on all stages from gamete to fetal formation. Similarly, the adverse effects of ethanol before and after adolescence cannot be generalized. To focus on the effects of prepubertal ethanol on female reproductive ability, we established a mouse model of prepubertal ethanol exposure by changing drinking water to 20% v/v ethanol. Some routine detections were performed on the model mice, and details such as mating, fertility, reproductive organ and fetal weights were recorded day by day after discontinuation of ethanol exposure. Prepubertal ethanol exposure resulted in decreased ovarian weight and significantly reduced oocyte maturation and ovulation after sexual maturation, however, normal morphology oocytes with discharged polar body showed normal chromosomes and spindle morphology. Strikingly, oocytes with normal morphology from ethanol exposed mice showed reduced fertilization rate, but once fertilized they had the ability to develop to blastocysts. RNA-seq analysis showed that the gene expression of the ethanol exposed oocytes with normal morphology had been altered. These results show the adverse effects of prepubertal alcohol exposure on adult female reproductive health.

Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

With aging, abnormalities during oocyte meiosis become more prevalent. However, the mechanisms of aging-related oocyte aneuploidy are not fully understood. Here we performed Hi-C and SMART-seq of oocytes from young and old mice and reveal decreases in chromosome condensation and disrupted meiosis-associated gene expression in metaphase I oocytes from aged mice. Further transcriptomic analysis showed that meiotic maturation in young oocytes was correlated with robust increases in mevalonate (MVA) pathway gene expression in oocyte-surrounding granulosa cells (GCs), which was largely downregulated in aged GCs. Inhibition of MVA metabolism in GCs by statins resulted in marked meiotic defects and aneuploidy in young cumulus-oocyte complexes. Correspondingly, supplementation with the MVA isoprenoid geranylgeraniol ameliorated oocyte meiotic defects and aneuploidy in aged mice. Mechanically, we showed that geranylgeraniol activated LHR/EGF signaling in aged GCs and enhanced the meiosis-associated gene expression in oocytes. Collectively, we demonstrate that the MVA pathway in GCs is a critical regulator of meiotic maturation and euploidy in oocytes, and age-associated MVA pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

Difluorocarbene-Triggered [1+5] Annulation: Access to Functionalized 1,1-Difluoro-1,9a-dihydropyrido[2,1-c][1,4]thiazine Derivatives.

Difluorocarbene-triggered [1+5] annulation is developed to access 1,1-difluoro-1,9a-dihydropyrido[2,1-c][1,4]thiazine-3,4-dicarboxylate derivatives in satisfactory to good yields via the direct reaction of potassium bromodifluoroacetate and pyridinium 1,4-zwitterionic thiolates under heating. Pyridinium 1,4-zwitterionic thiolates first nucleophilically attack difluorocarbene generated from potassium bromodifluoroacetate followed by an intramolecular nucleophilic addition to pyridiniums. This method provides an expeditious route to introduce the difluoromethyl group into the 1,9a-dihydropyrido[2,1-c][1,4]thiazine ring, even to modify drug molecules.

General Strategy for Incorporation of Functional Group Handles into Covalent Organic Frameworks via the Ugi Reaction.

The library of imine-linked covalent organic frameworks (COFs) has grown significantly over the last two decades, featuring a variety of morphologies, pore sizes, and applications. An array of synthetic methods has been developed to expand the scope of the COF functionalities; however, most of these methods were designed to introduce functional scaffolds tailored to a specific application. Having a general approach to diversify COFs via late-stage incorporation of functional group handles would greatly facilitate the transformation of these materials into platforms for a variety of useful applications. Herein, we report a general strategy to introduce functional group handles in COFs via the Ugi multicomponent reaction. To demonstrate the versatility of this approach, we have synthesized two COFs with hexagonal and kagome morphologies. We then introduced azide, alkyne, and vinyl functional groups, which could be readily utilized for a variety of post-synthetic modifications. This facile approach enables the functionalization of any COFs containing imine linkages.

NAC domain transcription factor gene GhNAC3 confers drought tolerance in plants.

Abiotic stress seriously affects the growth, yield, and fiber quality of cotton. It is of great importance to cultivate drought-resistant and salt-tolerant cotton. NAC (NAM, ATAF1/2, and CUC2) is a plant-specific transcription factor, which is widely involved in the response to abiotic stress. Here, we discovered the GhNAC3 gene isolated from the expression profile of drought stress in cotton and verified its functions in cotton. First, GhNAC3 was strongly induced expression by drought and salt stresses. Gene structure analysis revealed that GhNAC3 had a conserved NAC domain and was homologous to several stress-related NAC transcription factors gene of Arabidopsis. Subcellular localization and transcriptional activation assays revealed that GhNAC3 was a nuclear protein with a C-terminal transcriptional activation domain. Overexpression of GhNAC3 enhanced Arabidopsis tolerance to drought stress with reduced sensitivity to ABA, characterized by increased germination and cotyledon rates under drought stress, and promoted root elongation. VIGS silencing of GhNAC3 reduced cotton tolerance to drought stress as indicated by the low water content of the leaves under drought treatment, significantly faster water loss and lower ABA content in detached leaves, along with the accumulation of more hydrogen peroxide (H2O2) and malondialdehyde (MDA). In conclusion, GhNAC3 plays an important role in the abiotic stress of cotton, which might have great application potential in molecular breeding of cotton varieties with drought resistance.

Microwave-mediated stereocontrolled annulations of diazo(aryl)methyl(diaryl)phosphine oxides with pyridinium 1,4-zwitterionic thiolates.

Chemoselective annulations of phosphoryl carbenes generated from diazo(aryl)methyl(diaryl)phosphine oxides with pyridinium 1,4-zwitterionic thiolates were performed under microwave irradiation, affording 1-diarylphosphoryl-1H-benzo[c]thiopyran derivatives via [3+3] annulation and indolizine derivatives via ([1+5]-1) annulation with P-Cope elimination as the key step. The annuloselectivity was controlled by the steric hindrance of pyridiniums in pyridinium 1,4-zwitterionic thiolates.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

First-in-Class Hydrazide-Based HDAC6 Selective Inhibitor with Potent Oral Anti-Inflammatory Activity by Attenuating NLRP3 Inflammasome Activation.

In this study, we report the first highly selective HDAC6 inhibitor with hydrazide as the zinc-binding group (ZBG), which displays superior pharmacokinetic properties to the current hydroxamic acid inhibitors. Structure-activity relationship study reveals that ethyl group substituent hydrazide-based ZBG and cap group with more substantial rigidity and larger volume increase the HDAC6 selectivity of designed compounds. Representative inhibitor 35m exhibits potent HDAC6 inhibitory activity with an IC50 value of 0.019 µM. To our surprise, 35m establishes significant improvement in the pharmacokinetic property with much higher AUC0-inf (10292 ng·h/mL) and oral bioavailability (93.4%) than hydroximic acid-based HDAC6 inhibitors Tubastatin A and ACY-1215. Low-dose 35m remarkably decreases LPS-induced IL-1ß release both in vitro and in vivo by blocking the activation of NLRP3, indicating that 35m can be a potential orally active therapeutic agent for the treatment of NLRP3-related diseases.

Discovery and validation of tissue-specific DNA methylation as noninvasive diagnostic markers for colorectal cancer.

BACKGROUND: Noninvasive diagnostic markers that are capable of distinguishing patients with colorectal cancer (CRC) from healthy individuals or patients with other cancer types are lacking. We report the discovery and validation of a panel of methylation-based markers that specifically detect CRC. METHODS: This was a large-scale discovery study based on publicly available datasets coupled with a validation study where multiple types of specimens from six cohorts with CRC, other cancer types, and healthy individuals were used to identify and validate the tissue-specific methylation patterns of CRC and assess their diagnostic performance. RESULTS: In the discovery and validation cohort (N = 9307), ten hypermethylated CpG sites located in three genes, C20orf194, LIFR, and ZNF304, were identified as CRC-specific markers. Different analyses have suggested that these CpG sites are CRC-specific hypermethylated and play a role in transcriptional silencing of corresponding genes. A random forest model based on ten markers achieved high accuracy rates between 85.7 and 94.3% and AUCs between 0.941 and 0.970 in predicting CRC in three independent datasets and a low misclassification rate in ten other cancer types. In the in-house validation cohort (N = 354), these markers achieved consistent discriminative capabilities. In the cfDNA pilot cohort (N = 14), hypermethylation of these markers was observed in cfDNA samples from CRC patients. In the cfDNA validation cohort (N = 155), the two-gene panel yielded a sensitivity of 69.5%, specificity of 91.7%, and AUC of 0.806. CONCLUSIONS: Hypermethylation of the ten CpG sites is a CRC-specific alteration in tissue and has the potential use as a noninvasive cfDNA marker to diagnose CRC.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Visible-Light-Induced [1+5] Annulation of Phosphoryl Diazomethylarenes and Pyridinium 1,4-Zwitterionic Thiolates.

Visible-light-induced [1+5] annulation of phosphoryl diazomethylarenes and pyridinium 1,4-zwitterionic thiolates generates various trifunctionalized dialkyl 1-phosphoryl-1,9a-dihydropyrido[2,1-c][1,4]thiazine-3,4-dicarboxylates in good to excellent yields and diastereoselectivities. The annulation involves the nucleophilic attack of thiolates on diazomethylarene-generated carbenes followed by an intramolecular cyclization, is free of catalysts, is atom-economical, and has mild reaction conditions.