Cell Cycle

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

MYC oncoprotein binding at promoters and enhancers influences RNA polymerase II (RNAPII)-driven gene expression. Numerous genes also bind MYC near their transcriptional end sites (TESs). This often allows direct promoter-TES contact via looping and further regulates total and 'read-through' transcription that extends beyond standard termination sites. We aimed here to better clarify the rules governing TES associated MYC and/or RNAPII binding cross-talk in human and murine cells. Using ChIPseq and RNAseq datasets from the ENCODE portal and elsewhere, MYC and RNAPII binding profiles were found to differ around TESs and transcriptional start sites (TSSs). Variations in E box flanking sequences likely accounted for the somewhat lower affinities of MYC for TES-associated sites. Motifs for numerous other transcription factors were also observed to cluster non-randomly and in close proximity to MYC and RNAPII binding site peak summits. On average, genes with TES-proximal MYC or RNAPII sites were more highly expressed than those without, although co-binding tended to be suppressive. Both normal and neoplastic proliferative stimuli altered the MYC and RNAPII binding patterns of many genes, indicating that 'category switching' was common, subject to disparate external signals and often reversible. Functionally related gene sets with high levels of read-through transcription were uniformly marked by significant amounts of TES-associated MYC and/or RNAPII binding. These findings indicate that, both independently and together, MYC and RNAPII binding near TESs dynamically impact total and read-through transcription while also coordinating the expression of many common purpose gene sets.

BackgroundHeterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty and Orly that are transcribed in testis and have known functions in male fertility. Of these Ssty and Sly encode proteins - yet all the transcripts are not translated. To investigate the roles of these Y-heterochromatic transcripts further, we analyzed them. MethodsMice with 2/3rd deletion of the Y-chromosome (XYRIIIqdel) and its wild type (XYRIII) were used in this study. Bioinformatic approaches, small RNA northern blots, Electrophoretic Mobility Shift Assays, Luciferase reporter assays, dPCR analysis, RT-qPCR assays and western blotting techniques were used to identify piRNAs that regulate autosomal genes. ResultsWe demonstrate that the multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. We observed sequences homologous to these piRNAs in the UTRs of a few autosomal genes, which are differentially expressed in the sperms of XYRIIIqdel mice. Furthermore, the Endogenous Retrovirus Element (ERV) LTR, found in the Orly1 transcript identified piRNAs in the database, showed homology to UTRs and associated genomic regions of a few autosomal genes. Orly1 showed a reduction in genomic copy number by digital PCR in XYRIIIqdel mice. One of the four autosomal genes containing the ERV segment in their UTRs, showed a differential testicular protein expression in the mutant mice. ConclusionsThus, we further elucidate that different classes of repeats from Y-chromosome regulate autosomal gene expression via piRNAs. Besides, this study also identified novel roles for a Y-derived ERV in autosomal gene regulation in testis.

The Src homology 2 (SH2) domain-containing non-transmembrane protein-tyrosine phosphatases 1 and 2 (Shp1 and Shp2) have been implicated in regulating signaling from a variety of receptors and cell types, including the thrombopoietin (Tpo) receptor Mpl in megakaryocytes (MKs) and platelets. We previously showed that deletion of Shp1 and Shp2 in the MK/platelet lineage in mice using the Pf4-Cre transgene/loxP system impairs megakaryopoiesis and thrombopoiesis. However, we also observed unexpected phenotypes including a motheaten-like phenotype in Shp1-deficient mice and severe myelofibrosis in mice lacking both phosphatases. To determine whether these were lineage-specific effects, we utilized the Gp1ba-Cre transgenic mouse to delete loxP-flanked Shp1 and Shp2 in mice. Bone marrow-derived MKs from these mice expressed approximately 20-25% of Shp1 and Shp2, whereas platelets contain 5-10% of each phosphatase compared with controls. Minor MK/platelet defects were observed in mice lacking either Shp1 or Shp2 alone, however mice lacking both Shp1 and Shp2 exhibited macrothrombocytopenia, mild bleeding following tail injury, and impaired GPVI-mediated platelet aggregation and Syk phosphorylation, associated with reduction GPVI and integrin 2 subunit expression. Reduced Shp1 and Shp2 expression resulting in a significant reduction in ploidy, a block in MK maturation and proplatelet-producing MKs. Tpo-mediated Ras/MAPK signaling was reduced in Shp1/2-deficient MKs. Treatment of MKs with structurally distinct Shp2 allosteric inhibitors recapitulated key aspects of the Shp2-deficient phenotype, including aberrant megakaryopoiesis and reduced Mpl signaling. Our study highlights the synergistic functions of Shp1 and Shp2 in the MK/platelet lineage, and identifies Shp2 as a potential therapeutic target in myeloproliferative neoplasms. Key PointsO_LIDeletion of Shp1 and Shp2 in the MK/platelet lineage in mice results in macrothrombocytopenia and minor effects on platelet function. C_LIO_LIDefects can be partially explained by reduced Mpl signaling and aberrant megakaryopoiesis in the absence of Shp2 activity. C_LI

The early onset of labour increases mortality and developmental risks for a human newborn. Key genes in human labour have been investigated using multiple modalities, but their regulation by non-coding RNA (e.g. lncRNA and miRNA) remains incomplete. This study explores the three-way relationship between labour-associated transcription factors (TFs), miRNA and lncRNA suggested by the competing endogenous RNA (ceRNA) hypothesis, to understand the underlying regulatory framework. Experimentally validated miRNA-lncRNA interactions are modelled using five distinct machine learning (ML) architectures to predict 20469 labour-linked miRNA-lncRNA interactions. Known mRNA-ncRNA interactions from databases were included to construct a tripartite network, and a subset of 9989 labour-linked network motifs containing TFs were isolated and analysed. Gene enrichment of nodes in TF-lncRNA-miRNA network, as well as validation from public myometrial datasets indicate high significance in contractile pathways including immune signalling. Experimentally unconfirmed tripartite network motifs have been found, and we elaborate on their potential regulation in labour using 8 TF-lncRNA-miRNA network motifs. A unified ncRNA-TF regulatory atlas in labour has been synthesized, and a complete summary of the tripartite network motifs can be accessed and visualised using the user-friendly, public database.

BackgroundMethyltransferase-like 1 (METTL1) is highly expressed in organs like the pancreas but less so in the brain. The METTL1-WDR4 complex catalyzes N7-methylguanosine (m7G) methylation in tRNA, miRNA, mRNA, and rRNA, which impacts RNA stability and function. These modifications affect mRNA translation and tRNA functionality, influencing protein production and cellular activities. Such modifications can regulate tumor growth, invasion, and metabolism by selectively controlling protein expression. MethodGene expression data from public databases were analyzed to compare METTL1 expression in normal and tumor tissues. Western blot (WB) and immunohistochemistry (IHC) were used to quantify METTL1 levels in glioma samples and assess their prognostic significance. Cell viability, migration, invasion, and proliferation were evaluated using Cell Counting Kit-8 (CCK-8), wound healing, Transwell, cell cycle analysis, and colony formation assays. RNA immunoprecipitation PCR (RIP-PCR) identified m7G methylation sites on EPHA2 mRNA, and RNA stability was assessed with actinomycin D. ResultsBioinformatics analysis revealed that METTL1 is overexpressed in gliomas, correlating with poor prognosis. Knockdown of METTL1 significantly affected cell proliferation, migration, and invasion. RNA sequencing (RNA-seq) and m7G analysis identified EPHA2 as a downstream target, influencing the cell cycle via the AKT pathway. RIP and methylated RNA immunoprecipitation (MeRIP) confirmed two m7G sites on EPHA2 mRNA regulated by METTL1. Small interfering RNA (siRNA)-mediated METTL1 knockdown in EPHA2 mutants affected mRNA stability. Rescue experiments restored cell proliferation and AKT pathway gene expression. ConclusionMETTL1 methylates EPHA2 mRNA, enhancing its stability and expression, which activates the AKT signaling pathway and influences glioma cell proliferation. METTL1 could be a potential therapeutic target in glioma treatment.

BackgroundPremature ovarian insufficiency (POI) affects approximately 1% of women under 40 and is characterized by elevated levels of gonadotropins, reduced estradiol, impaired folliculogenesis, and infertility. Bone marrow-derived mesenchymal stem cell (BM-MSC)-based therapy has emerged as a promising regenerative strategy in preclinical POI models. This systematic review and meta-analysis evaluated BM-MSC-based interventions, including cell transplantation and secretome/extracellular vesicle administration, in animal models of POI. MethodsA systematic review and meta-analysis was conducted following PRISMA guidelines. PubMed, Web of Science, Scopus, ScienceDirect, and the Cochrane Library were searched from inception to February 19, 2025. Preclinical studies assessing BM-MSC-based interventions in animal models of POI were included. ResultsThirty-four studies comprising 1,357 animals were included. Compared with controls, BM-MSC-based therapy increased serum estradiol (standardized mean difference [SMD] 3.11; 95% confidence interval [CI] 2.38-3.84) and anti-Mullerian hormone (SMD 1.86; 95% CI 1.03-2.69), while reducing follicle-stimulating hormone (SMD -3.54; 95% CI -4.37 to -2.71) and luteinizing hormone (SMD -3.44; 95% CI -5.17 to -1.70). Follicular counts increased across developmental stages, with fewer atretic follicles. Reproductive outcomes improved, including normal estrous cycles (risk ratio [RR] 7.80; 95% CI 3.15-19.34), pregnancy occurrence (RR 3.72; 95% CI 2.14-6.44), and offspring number (SMD 1.57; 95% CI 1.04-2.09). ConclusionBM-MSC-based therapy consistently improved hormonal, follicular, and reproductive outcomes in preclinical POI models. More well-designed, standardized, and adequately controlled studies to confirm these findings are warranted. Systematic review registration: CRD42023449053

Senescence human bone marrow mesenchymal stem cells (BMSCs), vulnerable to age-related defects, is poor in tissue regeneration. Cells in bone marrow accumulated senescent contributing to the development of metabolic energy regulation hold prospects for therapeutic advances. This study aimed to evaluate energy metabolic changes in male bone marrow mesenchymal stem cells senescence process. Our research established cell specific surface marker and enzymes expression level changes, as well as ECAR and OCR resonance. Notably, CD14, HLA-DRB1 and CD90 upregulated, glycolysis-related genes are increased, tricarboxylic acid cycle-related genes are decreased. We firstly identified links between time-dependent cell aging process and energy metabolism in BMSCs.

The most prevalent RNA alteration in the mammalian genome is N-6-methylenediosine (m6A). There is mounting evidence linking dysregulation of m6A regulatory factors and alterations in m6A levels to the development, course, or prognosis of ovarian cancer. Genes having prognostic value were screened using the univariate, multifactorial, and Least Absolute Shrinkage Selection Operator (LASSO) Cox regression analyses. Important genes' m6A expression in clinical material was verified by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). In present study, all 23 regulators were significantly differentially expressed in ovarian cancer tissues. LASSO regression analysis screened for 10 key genes associ-ated with both autophagy and m6A. A risk score was constructed and nomogram was developed to forecast the prognosis of ovarian cancer patients. Additionally, individuals with ovarian cancer were classified as high-risk or low-risk; and the low-risk group might be more likely to benefit from im-munotherapy. RT-qPCR was used for the bioinformatics study of human ovarian cancer and normal tissues. Lastly, PLK2 and LEPR were confirmed to be associated with tumorigenesis in scRNA-seq. The risk score established by m6A and autophagy can be used to predict prognosis and susceptibility to anticancer drugs in patients with ovarian cancer.

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

Oocytes arrested at the dictyate stage of meiosis I must maintain genomic integrity for prolonged periods to preserve female fertility. During this extended arrest, DNA lesions arising from endogenous and exogenous sources threaten oocyte survival, yet the molecular mechanisms coordinating DNA repair in dormant oocytes remain poorly understood. Here, we identify cyclin-dependent kinase 1 (CDK1) as a critical regulator of the oocyte DNA damage response and homologous recombination (HR) repair under genotoxic stress. Using cisplatin-induced DNA damage models in fetal goat ovaries and neonatal mouse ovaries, we investigated repair mechanisms operating within the ovarian reserve. Label-free proteomic profiling revealed significant enrichment of DNA damage response pathways following cisplatin exposure, with CDK1 emerging as one of the most prominently upregulated kinases. Pharmacological inhibition of CDK1 had little effect on follicle survival under physiological conditions but aggravated oocyte and follicle loss following DNA damage, indicating a stress-dependent role for CDK1 in preserving ovarian follicle pool integrity. Mechanistically, DNA damage activated a Chk2-dependent signaling pathway that promoted p63 phosphorylation and altered the WEE1-CDK1 regulatory axis, resulting in reduced inhibitory CDK1 phosphorylation (Thr14/Tyr15) and increased activating phosphorylation (Thr161). Activated CDK1 was associated with enhanced RAD51 phosphorylation and accumulation at DNA damage foci, supporting homologous recombination (HR)-mediated repair in dictyate-arrested oocytes. In contrast, CDK1 inhibition reduced phospho-RAD51 levels, impaired RAD51 localization, increased persistent {gamma}H2AX accumulation, and elevated oocyte apoptosis. Notably, suppression of CDK1 was accompanied by increased expression of the non-homologous end joining (NHEJ) marker Ku80 and the nucleotide excision repair (NER) factor XPA, suggesting increased engagement of alternative DNA repair pathways. Furthermore, inhibition of Chk2 abolished the DNA damage-associated CDK1 activation signature and restored WEE1 expression, supporting a model in which CDK1 functions downstream of Chk2 signaling during the oocyte DNA damage response. Collectively, our findings identify a previously unrecognized Chk2-CDK1-RAD51 signaling axis that coordinates homologous recombination repair in dormant oocytes and safeguards ovarian follicular pool integrity under genotoxic stress. These findings provide new mechanistic insight into how dictyate-arrested oocytes maintain genome stability during prolonged meiotic arrest.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

Anoxia is a major stress for most vertebrates and frequently accompanies harsh winter conditions, particularly in species that spend much of the season frozen solid. North American freeze-tolerant wood frogs (Rana sylvatica) can survive several months without oxygen and endure whole-body freezing for up to eight months of the year, with [~]70% of total body water frozen as extracellular ice, yet revive when temperatures rise in spring. Survival depends on multiple adaptations, including tolerance of prolonged oxygen deprivation while frozen, when breathing and circulation are halted. A key strategy involves hepatic glycogen mobilization, producing large amounts of glucose that are distributed to tissues where it functions both as a cryoprotectant and as a substrate for anaerobic ATP production. The present study examines the role of histone lysine methylation and demethylation in regulating liver proteins under anoxic conditions. Relative protein expression of seven histone methyltransferases (ASH2L-S, ASH2L-L, RBBP5, SETD8, SMYD2, ESET, SETD1), six lysine demethylases (KDM1A, KDM3B, KDM4A, KDM4B, KDM5A, KDM5C), and eight histone marks (H3K4me1, H3K4me2, H3K9me3, H3K27me3, H3K36me3, H3K79me3, H4K20me1, H4K20me3) were evaluated in wood frog liver under control, 4-hour, and 24-hour anoxia exposures. The data indicate that histone lysine methylation and demethylation contribute significantly to transcriptional regulation under anoxia. Specifically, H3K4, H3K36, and H3K79 methylation were associated with transcriptional activation, whereas H3K9, H3K27, and H4K20 methylation correlated with transcriptional repression. These findings highlight the dynamic role of epigenetic regulation in supporting hypometabolism and stress adaptation in freeze-tolerant wood frogs.

The heterotrimeric G-protein transducin (Gt) is among the key proteins mediating phototransduction in rod and cone cells of the vertebrate retina. Even though this protein has been extensively characterized in mammals, little is known about its expression patterns in migratory songbirds. Here we characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. The mechanism underlying magnetoreception is not fully understood, but one well-supported hypothesis involves a radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors. Specifically, we combined single cell RNA sequencing and immunohistochemistry, and tested for protein interaction by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. We show that genes for G-protein subunits GNB1 and GNB3 (coding for Gt{beta}1 and Gt{beta}3, respectively) are predominantly expressed in rods and cones. Among {gamma}-subunits, GNGT2 (coding for Gt{gamma}T2) was the principal isoform in cones, whereas GNG11 (coding for Gt{gamma}11) was associated with rods. In contrast, we did not detect GNG10 (coding for Gt{gamma}10) expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. Furthermore, the absence of GNG10 expression in rods and cones does not support a role of this {gamma}-subunit in photoreceptor-based magnetoreception.

BackgroundThe fission yeast stress-activated protein kinase (SAPK) pathway includes a conserved mitogen-activated protein (MAP) kinase cascade that regulates multiple cellular processes and is activated by several types of external stress. Understanding how Sty1, the MAP kinase in the SAPK pathway, controls these processes is complicated by the fact that different stressors can have stressor-specific effects that may be difficult to separate from the effects of Sty1 activation itself. Moreover, upon stress, Sty1 activation is usually short-lived. Previously, we developed a fission yeast strain, SISA, in which Sty1 kinase activity can be switched on in a sustained manner in the absence of external stress. This required combining multiple mutations in the SAPK pathway, including an analog-sensitive version of Sty1. When SISA cells are grown in the presence of analog-sensitive kinase inhibitors, Sty1 is inhibited, but when inhibitor is removed, Sty1 becomes hyperactive. While this strain was useful, it had several limitations. ResultsHere we describe and validate a more rationally-designed strain, SISA4, that retains the features of the original SISA strain while overcoming its limitations. SISA4 is more stable genetically than SISA, easier to use in genetic crosses, and easy to identify by phenotype or genotyping. We show that analog-sensitive kinase inhibitors 4-Amino-1-tert-butyl-3-(1-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NM-PP1) and 4-Amino-1-tert-butyl-3-(3-bromobenzyl)pyrazolo[3,4-d]pyrimidine (3-BrB-PP1) are equally potent for inhibiting analog-sensitive Sty1 in vivo, and we determine optimal inhibitor concentrations for converting SISA4 cells from a Sty1-inhibited state to a Sty1-hyperactive state. We also find that both 1-NM-PP1 and 3-BrB-PP1 have measurable off-target effects in wild-type cells, although these are modest and generally do not affect interpretation of experiments. Finally, using SISA4, we show that the Sty1-activated transcription factor Atf1 plays an unexpected role in maintaining cell-polarity disruption after Sty1 hyperactivation. ConclusionsSISA4 will be useful for investigating how SAPK pathway activation regulates diverse cellular processes.

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts.

CCCTC-binding factor (CTCF) is frequently mutated in endometrial cancer, resulting in genetic haploinsufficiency that contributes to tumour progression. We previously showed that depletion of CTCF disrupted cell polarity in KLE endometrial cancer spheroids; however, the implications for gene dysregulation and endometrial cancer pathophysiology remains poorly understood. ZNF185, an actin-associated and LIM domain-containing protein involved in cytoskeletal remodelling, was identified as a dysregulated target following CTCF haploinsufficiency. In this study, shRNA-mediated knockdown of CTCF was used to model haploinsufficiency in endometrial cancer cells, leading to the identification of a previously unrecognised isoform of ZNF185, named ZNF185B. Unlike the full-length protein, ZNF185B lacked co-localisation with F-actin and exhibited a diffuse cytoplasmic distribution, and ZNF185B was significantly upregulated in CTCF-depleted endometrial cancer cells and in an auxin-inducible degron model in a dose-dependent manner. Functional studies demonstrated that depletion of ZNF185 expression reduced endometrial cancer cell proliferation and clonogenic potential. Together, these findings identify ZNF185B as a novel isoform negatively regulated by CTCF protein dosage and establish ZNF185 as a requirement for endometrial cancer cell proliferation. Our results suggest that dysregulated ZNF185 expression is a crucial downstream consequence of CTCF haploinsufficiency and may contribute to tumour progression in endometrial cancer.

BackgroundReactive Oxygen Species (ROS) are the small, unstable and highly reactive species, having DNA oxidizing ability. Oxidation of the DNAs purine and pyrimidine bases can lead to single or double strands in this macromolecule. In this situation, the ATM molecule, a serine-threonine kinase, targets several proteins for phosphorylation, which causes the cell cycle to stop and the DNA damage repair begins. It has previously been proven that natural polyphenols have the cancer inhibiting properties due to their high efficacy and low side effects. Silibinin is the main herbal and medical ingredient in Milk Thistle (Silybum marianum) is a polyphenol flavonolignan, which has been widely considered as an antioxidant and anticancer agent. The purpose of the present study was to investigate the ATM gene expression and measurement of reactive oxygen species (ROS) in SKBR3 cell line, treated with Silibinin. Materials and MethodsAt first, the SKBR3 cell line was cultured in RPMI1640 culture medium and MTT assay was carried out to evaluate the Silibinin cytotoxicity. Flow Cytometry was carried out for cell cycle analysis, apoptotic induction, and ROS detection. While, Real Time PCR was used to evaluate the ATM gene expression in the Silibinin-treated and un-treated SKBR3 cells. ResultsPresent results have shown that 150 {micro}M Silibinin had the most significant cytotoxicity and apoptotic induction influence after the treatment period of 48 h. Flow cytometry data have shown that Silibinin induced considerable amount of apoptosis and caused cell cycle arrest at G1/S phase and induced production of ROS. Real-time PCR results have revealed that Silibinin increased the ATM expression in SKBR3 cell line. ConclusionSilibinin causes increased ATM gene expression by inducing ROS production, which initiates cell cycle arrest and apoptotic induction in SKBR3 cells line.

Cryopreservation of ram semen induces structural and functional alterations that compromise sperm fertility. Since seminal plasma contributes to the regulation and preservation of sperm function, increasing attention has been directed toward seminal plasma extracellular vesicles (EVs) that are involved in sperm physiology. EVs act as carriers of proteins that are involved in sperm membrane organization and capacitation, suggesting that they may contribute to the maintenance of sperm stability during cryopreservation.. Thus, the aim of this study was to evaluate the effect of seminal plasma-derived EVs on post-thaw functional parameters of ram sperm. Semen was cryopreserved in the presence or absence of EVs isolated by ultracentrifugation that have been characterized by nanoparticle tracking analysis (NTA) and Western blotting (WB). Post-thaw sperm quality was assessed by evaluating viability, membrane lipid disorder, reactive oxygen species production, protein phosphorylation, acrosome status, intracellular calcium levels, and sperm motility. Sperm cryopreserved with an extender containing EVs showed a significant reduction in membrane lipid disorder and lower intracellular calcium levels compared to control samples (p < 0.05). CASA analysis revealed that EV supplementation did not affect total or progressive motility but modified sperm kinematic patterns, with increased linearity and straightness, indicating improved trajectory efficiency without induction of hyperactivated motility. No differences were detected in viability, ROS content, phosphorylation of proteins in residuous tyrosine (pY) or PKA or acrosome status. These results provide the first evidence that seminal plasma derived extracellular vesicles exert a protective effect during ram semen cryopreservation, preserving membrane organization and calcium homeostasis and improving sperm functional quality after thawing. Highlights- Seminal EVs protect ram sperm during cryopreservation. - EVs reduce membrane lipid disorder and intracellular Ca2+ levels. - EVs modify kinematics, increasing linearity and straightness. - No effects on viability, ROS, phosphorylation or acrosome status. - EVs improve post-thaw sperm functional quality and stability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/732841v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1f8a9org.highwire.dtl.DTLVardef@11c3d6aorg.highwire.dtl.DTLVardef@104124forg.highwire.dtl.DTLVardef@4e355f_HPS_FORMAT_FIGEXP M_FIG C_FIG