Diagnosis of Angioimmunoblastic T Cell Lymphoma After Receiving First Dose of Pfizer/BioNTech (BNT162b2) Vaccine: A Case Report.

Pfizer/BioNTech (BNT162b2) is a messenger RNA (mRNA) vaccine that is highly effective in preventing the most severe outcomes of COVID-19 infection. Nucleoside-modified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines induce effective stimulation of T follicular helper (TFH) cells, leading to a robust germinal center B cell response. Side effects from the BNT162b2 vaccination, including significant lymphadenopathy, have been reported previously. Here, we present a case of angioimmunoblastic lymphoma (AITL), a rare, peripheral T-cell lymphoma with RHOA-G17v-mutated gene developing in a patient following BNT162B2 vaccine with a plausible explanation. A 60-year-old Asian female received her first dose of Pfizer BNT162B2 mRNA vaccine in August 2021. Right after her vaccination, she developed right axillary lymphadenopathy. She received her second vaccine dose in September 2021. Thereafter, she developed lymph node (LN) enlargement in her neck and groin. She underwent left posterior cervical and left groin LN excisional biopsy in April 2022 due to persistent palpable lymphadenopathy. Biopsy results then demonstrated benign follicular hyperplasia. For progressive B symptoms, a right axillary LN biopsy was done, which demonstrated AITL, with molecular studies revealing mutation in TET-2, IDH-2, and RHOA-G17v genes. Progression of AITL following BNT162B2 mRNA vaccine is limited in literature. Our case demonstrates a plausible correlation between the diagnosis of AITL following mRNA vaccination due to the malignant transformation of the TFH cells in patients who have a predisposing mutation of RHOA-17v. Given the rarity of AITL and the heterogeneity of molecular findings, more studies are needed to establish such an association.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Migrasomes from adipose derived stem cells enrich CXCL12 to recruit stem cells via CXCR4/RhoA for a positive feedback loop mediating soft tissue regeneration.

BACKGROUND: Adipose-derived stem cells (ASCs) represent the most advantageous choice for soft tissue regeneration. Studies proved the recruitment of ASCs post tissue injury was mediated by chemokine CXCL12, but the mechanism by which CXCL12 is generated after tissue injury remains unclear. Migrasomes are newly discovered membrane-bound organelles that could deliver CXCL12 spatially and temporally in vivo. In this study, we sought to investigate whether migrasomes participate ASC-mediated tissue regeneration. METHODS: Discrepant and asymmetrical soft tissue regeneration mice model were established, in which HE staining, immunofluorescent staining, western blot and qPCR were conducted to confirm the role of CXCL12 and migrasomes in ASC-mediated tissue regeneration. Characterization of ASC-derived migrasomes were carried out by confocal microscopy, scanning electron microscopy, transmission electron microscopy as well as western blot analysis. The function and mechanism of migrasomes were further testified by assisting tissue regeneration with isolated migrasomes in vivo and by in vitro transwell combined with co-culture system. RESULTS: Here, we show for the first time that migrasomes participate in soft tissue regeneration. ASCs generate migrasomes enriched with CXCL12 to mediate tissue regeneration. Migrasomes from ASCs could promote stem cells migration by activating CXCR4/RhoA signaling in vivo and in vitro. Chemoattracted ASCs facilitate regeneration, as demonstrated by the upregulation of an adipogenesis-associated protein. This positive feed-back-loop creates a favorable microenvironment for soft tissue regeneration. Thus, migrasomes represent a new therapeutic target for ASC-mediated tissue regeneration. CONCLUSIONS: Our findings reveal a previously unknown function of ASCs in mediating tissue regeneration by generating migrasomes. The ASC-derived migrasomes can restore tissue regeneration by recruiting stem cells, which highlighting the potential application of ASC-derived migrasomes in regenerative medicine.

The Roles of RhoA/ROCK/NF-κB Pathway in Microglia Polarization Following Ischemic Stroke.

Ischemic stroke is the leading cause of death and disability worldwide. Nevertheless, there still lacks the effective therapies for ischemic stroke. Microglia are resident macrophages of the central nervous system (CNS) and can initiate immune responses and monitor the microenvironment. Microglia are activated and polarize into proinflammatory or anti­inflammatory phenotype in response to various brain injuries, including ischemic stroke. Proinflammatory microglia could generate immunomodulatory mediators, containing cytokines and chemokines, these mediators are closely associated with secondary brain damage following ischemic stroke. On the contrary, anti-inflammatory microglia facilitate recovery following stroke. Regulating the activation and the function of microglia is crucial in exploring the novel treatments for ischemic stroke patients. Accumulating studies have revealed that RhoA/ROCK pathway and NF-κB are famous modulators in the process of microglia activation and polarization. Inhibiting these key modulators can promote the polarization of microglia to anti-inflammatory phenotype. In this review, we aimed to provide a comprehensive overview on the role of RhoA/ROCK pathway and NF-κB in the microglia activation and polarization, reveal the relationship between RhoA/ROCK pathway and NF-κB in the pathological process of ischemic stroke. In addition, we likewise discussed the drug modulators targeting microglia polarization.

Exome sequencing implicates ancestry-related Mendelian variation at SYNE1 in childhood-onset essential hypertension.

Childhood-onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 (SYNE1), a cardiovascular candidate gene, in 3 of 16 families with early-onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1, we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.

Effect of cryopreservation on Odc1 and RhoA genes expression in diapausing mouse blastocysts.

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re-activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non-diapausing embryos before and after freeze-thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non-diapausing in vivo-derived embryos continued their development in vitro after freezing-thawing as evidenced by blastocoel re-expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non-diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze-thaw procedures. Future studies may attempt to facilitate the re-activation of diapausing embryos, for example frozen-thawed ones, specifically targeting Odc1 or Rho/ROCK system.

Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef.

Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

Urotensin II receptor deficiency ameliorates ligation-induced carotid intimal hyperplasia partially through the RhoA-YAP1 pathway.

Intimal hyperplasia (IH) is a common pathological feature of vascular proliferative diseases, such as atherosclerosis and restenosis after angioplasty. Urotensin II (UII) and its receptor (UTR) are widely expressed in cardiovascular tissues. However, it remains unclear whether the UII/UTR system is involved in IH. Right unilateral common carotid artery ligation was performed and maintained for 21 days to induce IH in UTR knockout (UTR-/-) and wild-type (WT) mice. Histological analysis revealed that compared with WT mice, UTR-deficient mice exhibited a decreased neointimal area, angiostenosis and intima-media ratio. Immunostaining revealed fewer smooth muscle cells (SMCs), endothelial cells and macrophages in the lesions of UTR-/- mice than in those of WT mice. Protein interaction analysis suggested that the UTR may affect cell proliferation by regulating YAP and its downstream target genes. In vitro experiments revealed that UII can promote the proliferation and migration of SMCs, and western blotting also revealed that UII increased the protein expression of RhoA, CTGF, Cyclin D1 and PCNA and downregulated p-YAP protein expression, while these effects could be partly reversed by urantide. To evaluate the translational value of UTRs in IH management, WT mice were also treated with two doses of urantide, a UTR antagonist, to confirm the benefit of UTR blockade in IH progression. A high dose of urantide (600 µg/kg/day), rather than a low dose (60 µg/kg/day), successfully improved ligation-induced IH compared with that in mice receiving vehicle. The results of the present study suggested that the UII/UTR system may regulate IH partly through the RhoA-YAP signaling pathway.

Genomic and Pathologic Profiling of Very Well-Differentiated Gastric Adenocarcinoma of Intestinal Type: A Study With Emphasis on Diffuse-Type Transformation.

Very well-differentiated adenocarcinoma of intestinal type is a distinct subtype of gastric cancer characterized by anastomosing glands with a hand-in-hand pattern and low-grade cytologic atypia resembling intestinal metaplasia. This is a slow-growing neoplasm with an indolent clinical course; however, a subset demonstrates transformation into adenocarcinoma with higher-grade histology, typically diffuse-type carcinoma, and behaves aggressively. This study aimed to better characterize the genomic and pathologic features, with a focus on factors associated with diffuse-type transformation. A total of 58 cases with (n=31) and without (n=27) diffuse-type transformation were analyzed for molecular and pathologic features. First, comprehensive deep DNA sequencing was conducted in 18 cases (discovery cohort), followed by a digital droplet polymerase chain reaction of hot spot RHOA mutations in 40 cases (validation cohort). In total, RHOA mutations were the most common alteration (34%), followed by loss of ARID1A (12%), p53 alterations (10%), and CLDN18 :: ARHGAP26/6 fusions (3.4%). FGFR2 amplification was identified in an advanced case with a p53 alteration. Altered p53 expression was recognized only in higher-grade components and was significantly associated with advanced disease ( P =0.0015) and diffuse-type transformation ( P =0.026). A mixed mucin phenotype was also strongly correlated with advanced disease ( P <0.001) and diffuse-type transformation ( P <0.001). Decreased E-cadherin expression was frequently observed (74%) in poorly cohesive components. This study demonstrated that a subset of RHOA -mutant diffuse-type gastric cancers develops through the transformation of very well-differentiated adenocarcinoma of intestinal type. Our observations suggest a mixed mucin phenotype as a risk factor and alterations in p53 and E-cadherin as drivers of diffuse-type transformation.

Dynamic light-responsive RhoA activity regulates mechanosensitive stem cell fate decision in 3D matrices.

The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment. Specifically, we temporally controlled the activity of Ras homolog family member A (RhoA), which is known to have a central role in mechanotransduction, using our previously developed Arabidopsis thaliana cryptochrome-2-based optoactivation system. Interestingly, pulsed RhoA activation induced Egr1 upregulation in stiff 3D gels only, whereas static light stimulation induced an increase in Egr1 expression across a wide range of 3D gel stiffnesses. Actin assembly inhibition limited Egr1 upregulation upon RhoA activation, implying that RhoA signaling requires an actin-involved process to upregulate Egr1. Consistently, static-light RhoA activation rather than pulsed-light activation restricted neurogenesis in soft gels. Our findings indicate that the dynamics of RhoA activation influence Egr1-mediated stem cell fate within 3D matrices in a matrix stiffness-dependent manner.

Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-ß-cyclodextrin (MßCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

RVG29 Peptide-Modified Exosomes Loaded with Mir-133b Mediate the RhoA-ROCK Pathway to Improve Motor and Neurological Symptoms in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Drug delivery to the brain through the blood-brain barrier (BBB) is a significant challenge in PD treatment. Exosomes, which can efficiently traverse the BBB, which many drugs cannot penetrate, are ideal natural carriers for drug delivery. In this study, the BBB shuttle peptide was modified on the exosome surfaces. Three types of exosomes were constructed, each modified with a distinct peptide (RVG29, TAT, or Ang2) and loaded with miR-133b. The safety and brain-targeting capabilities of these peptide-modified exosomes were then evaluated. Finally, the mechanism by which RVG29-Exo-133b regulates the RhoA-ROCK signaling pathway was investigated. The findings indicate that the three peptide-modified exosomes were adequately tolerated, safe, and effectively assimilated in vivo and ex vivo, with RVG29 exhibiting superior targeting to the brain. Furthermore, RVG29-Exo-133b decreased the phosphorylation level of the Tau protein by targeting the RhoA-ROCK signaling pathway. It also enhanced the motor function in mice with PD, thereby reducing the degree of depression, improving dopaminergic neuron function, and attenuating 6-OHDA-induced nerve damage. In this study, we developed a stable drug delivery mechanism that targets the intracerebral region using exosomes. Furthermore, a novel strategy was developed to manage PD and can potentially serve as a preclinical basis for utilizing exosomes in the diagnosis and treatment of neurodegenerative conditions.

CTNNAL1 promotes the structural integrity of bronchial epithelial cells through the RhoA/ROCK1 pathway.

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1 ‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1 ‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin ß1, and integrin ß4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 pathway.

Interplay of RAP2 GTPase and the cytoskeleton in Hippo pathway regulation.

The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo signaling pathway activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.

Targeting osteopontin alleviates endometriosis and inflammation by inhibiting the RhoA/ROS axis and achieves non-invasive in vitro detection via menstrual blood.

STUDY QUESTION: How does osteopontin (OPN) in endometriosis ectopic stromal cells (EESCs) participate in the pathogenesis of endometriosis and achieve non-invasive detection in vitro? SUMMARY ANSWER: Targeted OPN regulates endometriosis's necroptosis and inflammatory state by inhibiting the RhoA/reactive oxygen species (ROS) axis, thereby alleviating endometriosis and enabling non-invasive detection of menstrual blood in vitro. WHAT IS KNOWN ALREADY: Endometriosis is a chronic inflammatory disease. Recent studies have shown that OPN plays an important role in disease progression by regulating cell death and inflammation. STUDY DESIGN, SIZE, DURATION: The study included 20 patients diagnosed with endometriosis (confirmed by laparoscopy and histology) and 10 controls without endometriosis. Endometriotic stromal cells were isolated from endometrial samples, while menstrual blood endometrial cells (MESCs) were isolated from menstrual blood. These cells were then cultured in vitro and utilized in subsequent experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: OPN expression in EESCs was assessed using inflammatory factor sequencing, immunohistochemical staining (IHC), quantitative real-time PCR (qRT-PCR) analysis, and Western blotting (WB). The biological behavior of OPN and its effects on inflammatory factors were examined using EdU, wound-healing, Transwell, and ELISA assays. Necroptosis in EESCs and its impact on inflammatory factors were detected through qRT-PCR, WB, and Calcein-AM/PI fluorescence assays. The examination of mitochondrial stress in EESCs involved the use of the Mitochondrial Membrane Potential (ΔΨm) Assay, ROS detection, and Calcein-AM Loading/cobalt chloride Quenching. qRT-PCR, WB, and other experiments were conducted to verify the regulation of necroptosis and inflammatory factor levels in EESCs by OPN through the RhoA/ROS axis. Knockdown of OPN and its inhibitory effect on endometriosis lesion size were confirmed using AAV9 virus, IHC, qRT-PCR, WB, and other experiments. Additionally, OPN expression in MESCs was detected using transcriptome sequencing, RT-PCR, WB, and other experiments. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro assays demonstrated a significant upregulation of OPN in EESCs, and the knockdown of OPN effectively inhibited necroptosis and the release of inflammatory factors. OPN inhibited necroptosis and inflammatory factor release by mediating RhoA-dependent ROS production and blocking mixed lineage kinase domain-like protein phosphorylation at the cell membrane. In vivo, targeting of OPN can inhibit the growth of endometriosis lesions. Clinically, OPN was also significantly upregulated in the menstrual blood of patients with endometriosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to limitations in obtaining surgical specimens, our study primarily involved collecting endometriosis tissues from women during the proliferative and secretory phases of the menstrual cycle. We observed a significant overexpression of OPN in the samples used for our investigation. However, the expression of OPN in endometriosis tissues during the intermenstrual phase remains unknown. WIDER IMPLICATIONS OF THE FINDINGS: Our findings highlight the pivotal role of the OPN/RhoA/ROS axis in the regulation of necroptosis and the release of inflammatory factors. OPN knockdown exerts a therapeutic effect in vivo, and the high expression detection of OPN in menstrual blood in vitro. In summary, targeting OPN provides possibilities for the treatment and detection of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071626), the Zhejiang Province Public Welfare Technology Application Research Project (LGF21H040010), and the Clinical Research project of the Second Affiliated Hospital of Wenzhou Medical University (1010293). The authors have no conflicts of interest.

Kisspeptin/KISS1R Signaling Modulates Human Airway Smooth Muscle Cell Migration.

Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle (ASM) cell mass and upregulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of Kp (kisspeptin) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF [platelet-derived growth factor])-induced human ASM cell proliferation in vitro and airway remodeling in vivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of promigration proteins like CDC42 (cell division control protein 42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The antimigratory effect of KISS1R was abolished by PKA (protein kinase A)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of CREB (cAMP-response element binding protein) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

The antiangiogenic effect of digitoxin is dependent on a ROS-elicited RhoA/ROCK pathway activation.

We previously showed that digitoxin inhibits angiogenesis and cancer cell proliferation and migration and these effects were associated to protein tyrosine kinase 2 (FAK) inhibition. Considering the interactions between FAK and Rho GTPases regulating cell cytoskeleton and movement, we investigated the involvement of RhoA and Rac1 in the antiangiogenic effect of digitoxin. Phalloidin staining of human umbilical vein endothelial cells (HUVECs) showed the formation of stress fibers in cells treated with 10 nM digitoxin. By Rhotekin- and Pak1- pull down assays, detecting the GTP-bound form of GTPases, we observed that digitoxin (10-25 nM) induced sustained (0.5-6 h) RhoA activation with no effect on Rac1. Furthermore, inhibition of HUVEC migration and capillary-like tube formation by digitoxin was counteracted by hindering RhoA-ROCK axis with RhoA silencing or Y-27632 treatment. Digitoxin did not decrease p190RhoGAP phosphorylation at Tyr1105 (a site targeted by FAK), suggesting that RhoA activation was independent from FAK inhibition. Because increasing evidence points to a redox regulation of RhoA, we measured intracellular ROS and found that digitoxin treatment enhanced ROS levels in a concentration-dependent manner (1-25 nM). Notably, the flavoprotein inhibitor DPI or the pan-NADPH oxidase (NOX) inhibitor VAS-2870 antagonized both ROS increase and RhoA activation by digitoxin. Our results provide evidence that inhibition of HUVEC migration and tube formation by digitoxin is dependent on ROS production by endothelial NOX, which leads to the activation of RhoA/ROCK pathway. Digitoxin effects on proteins regulating cytoskeletal organization and cell motility could have a wider impact on cancer progression, beyond the antiangiogenic activity.

Advancements in the Research of GEF-H1: Biological Functions and Tumor Associations.

Guanine nucleotide exchange factor H1 (GEF-H1) is a unique protein modulated by the GDP/GTP exchange. As a regulator of the Rho-GTPase family, GEF-H1 can be activated through a microtubule-depended mechanism and phosphorylation regulation, enabling it to perform various pivotal biological functions across multiple cellular activities. These include the regulation of Rho-GTPase, cytoskeleton formation, cellular barrier, cell cycle, mitosis, cell differentiation, and vesicle trafficking. Recent studies have revealed its crucial effect on the tumor microenvironment (TME) components, promoting tumor initiation and progress. Consequently, an in-depth exploration of GEF-H1's biological roles and association with tumors holds promise for its potential as a valuable molecular target in tumor treatment.

Constraint-Induced Movement Therapy Promotes Myelin Remodeling and Motor Function by Mediating Sox2/Fyn Signals in Rats With Hemiplegic Cerebral Palsy.

OBJECTIVE: Hypoxic-ischemic brain injury in infants often leads to hemiplegic motor dysfunction. The mechanism of their motor dysfunction has been attributed to deficiencies of the transcription factor sex-determining region (SRY) box 2 (Sox2) or the non-receptor-type tyrosine kinase Fyn (involved in neuronal signal transduction), which causes a defect in myelin formation. Constraint-induced movement therapy (CIMT) following cerebral hypoxia-ischemia may stimulate myelin growth by regulating Sox2/Fyn, Ras homolog protein family A (RhoA), and rho-associated kinase 2 (ROCK2) expression levels. This study investigated how Sox2/Fyn regulates myelin remodeling following CIMT to improve motor function in rats with hemiplegic cerebral palsy (HCP). METHODS: To investigate the mechanism of Sox2 involvement in myelin growth and neural function in rats with HCP, Lentivirus (Lenti)-Sox2 adeno-associated virus and negative control-Lenti-Sox2 (LS) adeno-associated virus were injected into the lateral ventricle. The rats were divided into a control group and an HCP group with different interventions (CIMT, LS, or negative control-LS [NS] treatment), yielding the HCP, HCP plus CIMT (HCP + CIMT), HCP + LS, HCP + LS + CIMT, HCP + NS, and HCP + NS + CIMT groups. Front-limb suspension and RotaRod tests, Golgi-Cox staining, transmission electron microscopy, immunofluorescence staining, western blotting, and quantitative polymerase chain reaction experiments were used to analyze the motor function, dendrite/axon area, myelin ultrastructure, and levels of expression of oligodendrocytes and Sox2/Fyn/RhoA/ROCK2 in the motor cortex. RESULTS: The rats in the HCP + LS + CIMT group had better values for motor function, dendrite/axon area, myelin ultrastructure, oligodendrocytes, and Sox2/Fyn/RhoA/ROCK2 expression in the motor cortex than rats in the HCP and HCP + NS groups. The improvement of motor function and myelin remodeling, the expression of oligodendrocytes, and the expression of Sox2/Fyn/RhoA/ROCK2 in the HCP + LS group were similar to those in the HCP + CIMT group. CONCLUSION: CIMT might overcome RhoA/ROCK2 signaling by upregulating the transcription of Sox2 to Fyn in the brain to induce the maturation and differentiation of oligodendrocytes, thereby promoting myelin remodeling and improving motor function in rats with HCP. IMPACT: The pathway mediated by Sox2/Fyn could be a promising therapeutic target for HCP.

Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway.

BACKGROUND: Obesity has emerged as a global epidemic. Recent research has indicated that diet-induced obesity can be prevented by promoting lacteal junction zippering. Berberine, which is derived from natural plants, is found to be promising in weight reduction, but the underlying mechanism remains unspecified. PURPOSE: To determine whether berberine protects against obesity by regulating the lacteal junction and to explore potential molecular mechanisms. METHODS: Following the induction of the diet-induced obese (DIO) model, mice were administered low and high doses of berberine for 4 weeks. Indicators associated with insulin resistance and lipid metabolism were examined. Various methods, such as Oil Red O staining, transmission electron microscopy imaging, confocal imaging and others were used to observe the effects of berberine on lipid absorption and the lacteal junction. In vitro, human dermal lymphatic endothelial cells (HDLECs) were used to investigate the effect of berberine on LEC junctions. Western Blot and immunostaining were applied to determine the expression levels of relevant molecules. RESULTS: Both low and high doses of berberine reduced body weight in DIO mice without appetite suppression and ameliorated glucolipid metabolism disorders. We also found that the weight loss effect of berberine might contribute to the inhibition of small intestinal lipid absorption. The possible mechanism was related to the promotion of lacteal junction zippering via suppressing the ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) signaling pathway. In vitro, berberine also promoted the formation of stable mature junctions in HDLECs, involving the same signaling pathway. CONCLUSION: Berberine could promote lacteal junction zippering and ameliorate diet-induced obesity through the RhoA/ROCK signaling pathway.