Acta Biochimica et Biophysica Sinica

The activation of thermogenesis in brown adipose tissue (BAT) represents a pivotal target for ameliorating disorders of glucose and lipid metabolism. This study sought to elucidate the regulatory effects of quercetin on thermogenesis and glucose-lipid metabolism within brown adipocytes, alongside its underlying molecular mechanisms. The findings demonstrated that quercetin markedly upregulated the expression of uncoupling protein 1 (UCP1), a critical thermogenic protein in brown adipocytes, thereby enhancing cellular thermogenic capacity and effectively mitigating glucose and lipid metabolism disorders. Subsequent mechanistic investigations confirmed that quercetin activated the COX2-PGE2-EP4-UCP1 signaling axis by augmenting the stability of cyclooxygenase 2 (COX2) protein, thus mediating its thermogenic-promoting and metabolism-improving effects. This study identifies quercetin as a potential therapeutic agent for the improvement of glucose and lipid metabolism disorders, uncovers a novel molecular mechanism through which quercetin regulates brown adipocyte thermogenesis, and provides a theoretical and experimental foundation for the application of quercetin in the prevention and treatment of obesity and related metabolic diseases.

Human papillomavirus 18 (HPV18) preferentially infects cervical stem cell-like cells and is strongly associated with adenocarcinoma. However, the mechanisms underlying differentiation into cervical adenocarcinoma remain unclear due to the lack of appropriate experimental models. We aimed to establish a model of HPV18-associated cervical adenocarcinoma and elucidate its molecular and cellular differentiation mechanisms. HPV18 E6/E7 were introduced into induced pluripotent stem cell-derived reserve cell-like cells (iRCs) to generate tumor models. Spatial transcriptomics and single-cell multi-omics analyses were performed to integrate histological and molecular data. A distinct component (Gland_A) exhibited morphological and immunohistochemical features of cervical adenocarcinoma and was efficiently induced in iRC-18 tumors. Gland_A showed increased chromatin accessibility and elevated expression of FOXA1, FOXA2, and ALDH1A1. Analysis of clinical samples confirmed enrichment of ALDH1A1 in HPV-associated adenocarcinomas. This model recapitulates key features of HPV18-associated cervical adenocarcinoma and provides insights into its differentiation mechanisms.

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

BackgroundHepatic stellate cells (HSC) are Vitamin A storing non-parenchymal cells of the liver. During injury and inflammation, HSCs are the major contributors of excessive extracellular matrix (ECM) leading to Liver Fibrosis (LF). Emerging evidence suggests a fibrosis-independent role of these cells as key regulators of liver homeostasis and liver regeneration, emphasising on the dual role of HSCs in liver. HSCs are known to secrete several growth factors through which they largely execute their functions. However, the role of secretome (exosomes) from early activated or undifferentiated HSCs in a fibrotic milieu nor its composition are completely understood. MethodsLX-2 cells were cultured in low to no serum conditions and their isolated exosomes were transplanted into fibrotic severe combined immune deficient (SCID) mice livers, followed by post-transplantation analysis of the liver tissue and compared to the untreated controls. Total proteomic profiling of cell and exosomal cargo was performed using mass spectrometry and the data analysed and compared with the total HSC cell proteome. ResultsSignificant reduction in collagen in the transplanted mice livers compared to untreated fibrotic controls was observed with both the cells and exosomes transplantation. Comparative analysis revealed distinct enrichment of proteins and signaling pathways associated with extracellular matrix regulation, cellular communication, and metabolism in exosomes. Notably, these pathways are prominently represented in the exosomal fraction, suggesting a selective packaging of functional mediators. ConclusionThis study suggests the potential role of HSCs in regulating the complex liver homeostasis via exosomal network of proteins that contribute significantly to liver repair by ECM remodelling and growth factor-mediated signalling to regulate metabolism, fibrosis and liver regeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/721862v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@99bbf4org.highwire.dtl.DTLVardef@1029dd0org.highwire.dtl.DTLVardef@c6f578org.highwire.dtl.DTLVardef@1dba81_HPS_FORMAT_FIGEXP M_FIG C_FIG

Multipotent mesenchymal stem cells (MSCs) including bone marrow stromal cells (BMSCs) have shown analgesic efficacy in recent years. Studies suggested that the therapeutic effect of MSCs was mediated by their secreted small extracellular vesicles (sEVs) mainly exosomes. The present study evaluated the antihyperalgesic effect of BMSC-related sEVs in a mouse model of neuropathic pain involving chronic constriction injury of the infraorbital nerve (CCI-ION). Our separation protocol generated EV particles mostly sized in the range of exosomes (30-170 nm) and express exosome marker proteins CD9, CD81, and Tsg101, suggesting their endosome origin. We show that intravenous injection of BMSC-related sEVs attenuated pain hypersensitivity induced by CCI-ION as indicated by decreased mechanical hypersensitivity (von Frey test) and reduced aversion to noxious stimulation (conditioned place avoidance test). The antihyperalgesic effect of sEVs was observed in both female and male animals, and the effect was dose-dependent. sEVs from NAIVE serum-treated BMSC cultures produced short-lasting antihyperalgesia in male but not female mice, suggesting a subtle sex difference. The antihyperalgesia of sEVs from BMSC culture was blocked by the pretreatment of the culture with GM4869, the antagonist of exosome secretion, suggesting that the effect was not related to other co-isolated soluble mediators but mediated by MSC-derived exosomes. Interestingly, the prior injury condition in which sEVs were isolated favors the pain-relieving effect of sEVs. sEVs isolated from the serum of BMSC-treated animals receiving tendon ligation (TL) injury attenuated hyperalgesia for 24 h, while sEVs from the serum of BMSC-treated NAIVE animals only attenuated hyperalgesia at 3 h after injection. sEVs from the BMSC culture treated with the serum of TL rats were antihyperalgesic, but sEVs from the BMSC culture treated with the serum of naive animals were ineffective. Our results indicate that BMSC-related sEVs produced antihyperalgesia similar to that produced by BMSCs. The results suggest that the interactions between BMSCs and injury conditions are crucially important for producing efficacious sEVs/exosomes and support that the effect of sEVs could be optimized by priming BMSCs with injury-related conditions.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

The axon initial segment (AIS) undergoes structural plasticity and refines neuronal excitability, yet the underlying mechanisms remain unclear. We here developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector introduced via electroporation and employed this approach to seek molecules that regulate the developmental shortening of AIS in the chicken nucleus magnocellularis. We have targeted fourteen molecules associated with microtubules and found that knockouts of glycogen synthase kinase 3{beta} (GSK3{beta}) and Tau disabled the AIS shortening. Conversely, overexpression of constitutively active form of GSK3{beta} facilitated the AIS shortening in vivo. This extensive shortening was replicated in slice cultures, which was occluded by stabilization of microtubules. These results suggested that microtubule remodeling by GSK3{beta} activity contributed to the AIS shortening. This study thus provides a genetic approach suitable for genetic screening that allows identifying regulators of the AIS plasticity in the chicken brain.

In wheat, weak seed dormancy (SD) is related to an increased tendency for pre-harvest sprouting (PHS), which reduces yield and quality. However, the molecular mechanism underlying SD remains elusive. Here, we identified a wheat R2R3-MYB transcription factor (TaMYB83-7B) related to SD. Expression analysis showed that TaMYB83-7B was highly expressed in wheat seeds, and was more highly expressed in strong-dormancy varieties than in weak-dormancy varieties. Sequence and association analysis indicated that T/C mutations at -907 bp and -1133 bp in the TaMYB83-7B promoter were significantly associated with wheat SD, with C at both sites related to strong dormancy. Dual-luciferase reporter assays demonstrated that the transcriptional activity of the TaMYB83-7B promoter was significantly higher in strong-dormancy varieties than in weak-dormancy varieties. Further analyses indicated that TaMYB83-7B functions as a transcriptional inhibitor. Germination experiments revealed that overexpression of TaMYB83-7B significantly enhanced SD, while its loss-of-function reduced SD. Finally, TaMYB83-7B was found to regulate SD by influencing the balance between abscisic acid (ABA) and gibberellin (GA) in wheat seeds. Overall, the results of this study enhance our understanding of the complex regulatory mechanism underlying SD, and provide gene targets and molecular markers for the genetic improvement of PHS resistance in wheat.

Background Liver fibrosis (LF) represents a pivotal pathological phase in the advancement of chronic liver disorders toward cirrhosis. Amino acid metabolism reprogramming plays a pivotal role in its pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Methods Integrating three public datasets (GSE14323, GSE84044, and GSE136103) with amino acid metabolism-related gene sets, we performed consensus clustering, machine learning algorithms, functional enrichment analysis, immune microenvironment composition, regulatory network construction, and drug prediction. Results Fibrotic samples were classified into two amino acid metabolism-related subtypes with distinct immune landscapes and functional phenotypes. Through integrated analysis of differentially expressed genes (DEGs) common to both subtypes, fibrotic versus control comparisons, and amino acid metabolism-related gene sets, four biomarkers, GSTP1, LDHB, OXCT1, and PTGDS, were identified. These biomarkers were enriched in pathways related to epithelial-mesenchymal transition, interferon responses, and TNF/NF-{kappa}B signaling. Notably, GSTP1 and LDHB positively correlated with M1 macrophage infiltration and negatively with regulatory T cell abundance. Single-cell transcriptomic analysis revealed that cholangiocytes expressed all four biomarkers with elevated levels in fibrosis and interacted with macrophages/mesenchymal cells via MIF-CD74/CXCR4. Regulatory network analysis highlighted key modulators, including MALAT1, hsa-miR-3163, OXCT1, SMAD4, and RELA. Furthermore, 5-fluorouracil was predicted as a multi-target compound, with the strongest predicted binding affinity for OXCT1. In vitro validation confirmed the upregulation of GSTP1 and LDHB, aligning with the bioinformatics findings. Conclusion This study identified four amino acid metabolism-related biomarkers, revealing immune heterogeneity and cholangiocyte-centered intercellular communication in LF. These findings establish a foundation for biomarker-based diagnosis, subtype-guided patient stratification, and the development of cell-type-specific therapeutic strategies in LF.

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

Obtaining tomato plants with firm and intact fruit is one of the main goals in tomato breeding programs. Achieving these goals through conventional breeding is time-consuming and can lead to the loss of unwanted traits. In other hand, consumers are concerned about the presence of transgenic elements in plants acquired through RNA interference. The use of CRISPR/Cas9 technology has made it possible to overcome the above-mentioned shortcomings. In this study, the {beta}-D-N-acetylhexosaminidase ({beta}-hex) gene, which is involved in tomato fruit ripening, was knocked out using CRISPR/Cas9. In the resulting mutant plant genome, an indel mutation was found in exons 1 and 2 of the {beta}-hex gene. Plants with a mutation in their genome were observed to have increased fruit firmness and shelf life compared to control plants without affecting fruit quality.

Prenylated isoflavonoids are widely distributed specialized metabolites within the Fabaceae and contribute to various characteristic biological activities for both plants and humans. Several aromatic prenyltransferases (PTs) have been identified in Glycyrrhiza species, which are the most widely consumed crude drugs in traditional Chinese medicine. However, these enzymes do not sufficiently explain the structural diversity of prenylated flavonoids produced in the Glycyrrhiza genus. To identify additional novel PTs, we used elicited cultured Glycyrrhiza glabra roots as source material, in which elicitor treatment of cultured roots increased the accumulation of multiple prenylated flavonoids. To identify the responsible enzyme, PT candidates were screened using G. uralensis transcriptomes, currently the sole publicly available transcriptomic resource within the genus, and a homolog designated GgBSPT1 (BSPT; a broad-substrate prenyltransferase) was subsequently isolated from elicited cultured G. glabra roots. GgBSPT1 differed from previously identified Glycyrrhiza PTs in both amino acid sequence and enzymatic properties. GgBSPT1 catalyzed 3'-prenylation of isoliquiritigenin and 6-prenylation of five flavonoids, i.e., this PT displayed broad substrate acceptance across 20 distinct flavonoid structures. Overall, elicited cultured G. glabra roots enabled the identification of a previously unrecognized PT that is functionally distinct from earlier reported Glycyrrhiza PTs. This study provides a new insight into the metabolic plasticity of Glycyrrhiza species and expands the enzymatic toolkit for future metabolic engineering of prenylated phytochemicals by the unusually broad substrate specificity of GgBSPT1. Main conclusionUsing cultured Glycyrrhiza glabra roots, we identified a new prenyltransferase involved in the formation of a variety of flavonoids, thereby revealing novel prenylated isoflavonoid pathways in licorice.

Lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), is the most common primary lung cancer worldwide. Despite advancements in early detection and treatment, up to 39% of patients develop recurrent tumors following complete resection. Currently, no widely available models exist for reliably predicting early recurrence of LUAD, which is a significant prognostic factor of post-recurrence survival. Models leveraging deep learning (DL) techniques have demonstrated notable utility in cancer recurrence prediction, particularly when used in combination with both clinical and genomic data. We developed a DL-based model, Predicting Lung Adenocarcinoma recurrence via Selective Multimodal Attention (PLASMA), to predict early recurrence using clinical, mRNA expression, and mutation data from patients with primary stage I-III LUAD. Trained on The Cancer Genome Atlas (TCGA) dataset, PLASMA outperformed traditional machine learning models in predicting early recurrence in both the TCGA test set and an external validation set (TRACERx Lung), achieving area under the receiver operating characteristic curve (AUROC) scores of 85.0% and 76.5%, respectively. Our results support the potential of multimodal DL for early LUAD recurrence prediction and risk stratification.

Estrogen is an essential hormone that critically impacts bodily and brain functions, supporting learning, memory, and motor activities. A decrease in estrogen levels is associated with cognitive decline and motor dysfunction, such as muscle weakness. While conventional hormone replacement treatments (HRT) exist, those have limitations and potentially severe side effects. NAP (davunetide) is the smallest neuroprotective peptide site of activity-dependent neuroprotective protein (ADNP), a master regulator of cognition, essential for brain formation. It is known that NAP restores ADNP activity in cases of deficiency and it has already shown potential in preventing cognitive impairment, protecting against tauopathy, and improving motor function in various animal models and in clinical trials. Based on the dynamic regulation of ADNP by the estrous cycle and its involvement in steroidogenic pathways, we hypothesize that NAP may restore ADNP activity and thus serve as an alternative to conventional hormonal treatments. To test this, 3-month-old female ICR mice underwent bilateral ovariectomy (OVX) or Sham surgery and received daily intranasal administration of NAP, estrogen, or vehicle. Results showed a significant reduction in weight-normalized forelimb grip strength in the OVX model. Daily administration of NAP or estrogen resulted in intermediate grip strength levels that did not statistically differ from either the Sham control or untreated OVX groups. Interestingly, grip strength was the only test that yielded significant results, and no significant differences were observed in the Novel Object Recognition (NOR) test or computed tomography (CT) scans. These findings suggest that NAP may effectively prevent the loss of physical force production typically seen following ovarian hormone depletion, presenting a viable, non-hormonal candidate strategy for managing musculoskeletal symptoms. We hypothesize that the lack of significance in other parameters was due to soy-derived phytoestrogens in the diet, which may have exerted a systemic estrogenic effect that masked the expected physiological phenotypes typically observed in OVX models. Future replication using phytoestrogen-deficient food is required to isolate the specific neuroprotective and musculoskeletal effects of NAP from dietary influence and clarify the broader therapeutic benefits of NAP.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

Pregnancy is a period of extensive metabolic rewiring. Insulin secreting {beta}-cells respond to the metabolic challenges of pregnancy by increasing their mass and size and by altering secretory patterns to maintain glucose homeostasis. If glucose metabolism is not tightly controlled, gestational diabetes may develop. Most studies on {beta}-cell adaptation during pregnancy are derived from rodent models, making translation to the vastly different human gestational setting challenging. In this work, we performed an extensive characterization of pancreatic adaptations throughout porcine pregnancy. Pigs have a long gestational period (114 days) and share a similar size and metabolism to humans, making them an ideal model to bridge the knowledge gap between rodents and humans. By analyzing pancreatic samples from early and late gestational ages, we captured the full trajectory of endocrine remodeling. We observed pregnancy-driven remodeling of endocrine cell types, marked by preferential expansion of pancreatic polypeptide-secreting cells. Proteomic characterization of the pancreas from early and late gestation showed a downregulation of SLC20A2 and ZCCHC7, identifying new protein targets involved in physiological endocrine cell adaptation. Overall, our comprehensive characterization of pancreatic adaptations in the pig model helps bridge the translational gap between rodents and humans and highlights previously unrecognized proteins with therapeutic potential for gestational diabetes.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

The bone marrow (BM) vascular network plays crucial roles in driving bone development and supporting hematopoiesis, yet the mechanisms governing its specialized architecture, particularly sinusoidal morphogenesis, remain inadequately characterized. We show in this study that TIE2 (Tek) was highly expressed by BM sinusoidal endothelial cells (SEC) and the endothelial Tek excision led to BM sinusoidal capillarization. Particularly, the BM sinusoids displayed thinner vessel diameter with the aberrant mural cell coverage in the Tek mutants. Mechanistically, TIE2 insufficiency led to a dramatic decrease of VEGFR3 in BM-SECs while its expression in hepatic sinusoids was not obviously altered. The RNA-seq analysis showed that GO terms enriched for the downregulated genes were related to the biological processes including sinusoidal development while pathways related to arterial ECs and angiogenesis were upregulated in the bone marrow of Tek mutants. The alteration of sinusoidal VEGFR3 expression occurred within 48 h after the induced endothelial deletion of Tek. Consistently, the defective BM sinusoidal formation was validated with the induced Tek deletion in VEGFR3+ SECs. The insufficiency of TIE2 ligand ANGPT1 also led to reduced sinusoidal VEGFR3, accompanied by similar BM sinusoidal defects. Furthermore, disruption of sinusoidal morphogenesis was observed in mutant mice with the endothelial excision of Nr2f2 (COUP-TFII), displaying a decreased expression of BM sinusoidal TIE2 and VEGFR3. These findings suggest that ANGPT1/TIE2 and COUP-TFII form a reciprocal regulatory loop to coordinate BM sinusoidal specification via regulating VEGFR3.

PUF60 is a splicing factor related to the polypyrimidine-tract binding protein U2AF2. PUF60 is deleted in developmental disorders such as Verheij syndrome and amplified in approximately 8% of cancers. Thus, both increases and decreases in PUF60 expression can have profound physiological effects. However, little is known about how changes in PUF60 expression impact global splicing patterns. Here, we created a model system of CRISPRa/i in mouse stem cells (mESCs) to transcriptionally upregulate or downregulate Puf60. Our results uncovered extensive transcriptional, post-transcriptional, and post-translational regulation of Puf60 protein expression. We observed that Puf60 protein levels in normal mESCs drop dramatically at a critical cell density, leading to cell death. Puf60 is very essential in stem cells, and its repression causes cell death and impacts specific splicing events, including its own splicing autoregulation, providing valuable insights into the functional consequences of PUF60 dysregulation. Analysis of phosphoprotein data revealed phosphorylation of threonine at the N-terminus of PUF60. Our results showed that mutating threonine to glutamate downregulates the protein and alters its localization. Thus, our study reveals a novel regulatory mechanism of Puf60 phosphorylation that mediates its function and may be related to its frequent overexpression in cancer cells.