Proliferation Index Research Articles

Epigenetic mechanisms of Nsd1-mediated histone methylation modifications in chondrocyte ferroptosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a degenerative joint disease characterized by pain, stiffness, and impaired mobility, with current therapies offering limited efficacy. This study investigates the epigenetic role of nuclear receptor-binding SET domain protein 1 (NSD1) in KOA pathogenesis. A KOA mouse model was established, and adenoviral vectors were employed to upregulate Nsd1 and inhibit SRY-box transcription factor 9 (Sox9), followed by histopathological assessments. We examined changes in cell morphology, proliferation, viability, and ferroptosis-related markers. The expression of Nsd1, Sox9, and acyl-CoA synthetase long-chain family member 4 (Acsl4) was analyzed, along with the enrichment of Nsd1 and dimethylated lysine 36 of histone 3 (H3K36me2) on the Sox9 promoter and Sox9 on the Acsl4 promoter. Additionally, the binding relationship between Sox9 and the Acsl4 promoter sequence was analyzed. Our results revealed that Nsd1 expression was reduced in KOA mouse tissues and interleukin (IL)-1β-stimulated chondrocytes. Nsd1 upregulation alleviated KOA, promoted chondrocyte proliferation and viability, and inhibited ferroptosis. Mechanistically, Nsd1 enhanced H3K36me2 to upregulate Sox9 expression, which in turn suppressed Acsl4 expression and ferroptosis. Sox9 inhibition partially reversed the protective effect of Nsd1 overexpression. In summary, Nsd1 upregulation mitigates chondrocyte ferroptosis and ameliorates KOA by modulating H3K36me2 to upregulate Sox9 and downregulate Acsl4 expression.

Establishing and Characterizing the Molecular Profiles, Cellular Features, and Clinical Utility of a Patient-Derived Xenograft Model Using Benign Prostatic Tissues

Benign prostatic hyperplasia (BPH) is a common condition marked by the enlargement of the prostate gland, which often leads to significant urinary symptoms and a decreased quality of life. The development of clinically relevant animal models is crucial for understanding the pathophysiology of BPH and improving treatment options. This study aims to establish a patient-derived xenograft (PDX) model using benign prostatic tissues to explore the molecular and cellular mechanisms of BPH. PDXs were generated by implanting fresh BPH (transition zone) and paired normal (peripheral zone) prostate tissue from 8 patients under the renal capsule of immunodeficient male mice. Tissue weight, architecture, cellular proliferation, apoptosis, prostate-specific marker expression, and molecular profiles of PDXs were assessed after 1 week and 1, 2, or 3 months of implantation by immunohistochemistry, enzyme-linked immunosorbent assay, transcriptomics, and proteomics. Responses to finasteride, a standard-of-care therapy, were evaluated. PDXs maintained histologic and molecular characteristics of the parental human tissues. BPH, but not normal PDXs, demonstrated significant increases in weight and cellular proliferation, particularly at 1 month. Molecular profiling revealed specific gene and protein expression patterns correlating with BPH pathophysiology. Specifically, an increased immune and stress response was observed at 1 week, followed by increased expression of proliferation markers and BPH-specific stromal signaling molecules, such as BMP5 and CXCL13, at 1 month. Graft stabilization to preimplant characteristics was apparent between 2 and 3 months. Treatment with finasteride reduced proliferation, increased apoptosis, and induced morphologic changes consistent with therapeutic responses observed in human BPH. Our PDX model recapitulates the morphologic, histologic, and molecular features of human BPH, offering a significant advancement in modeling the complex interactions of cell types in BPH microenvironments. These PDXs respond to therapeutic intervention as expected, providing a valuable tool for preclinical testing of new therapeutics that will improve the well-being of BPH patients.

Paracrine signalling in breast cancer: Insights into the tumour endothelial phenotype

Tumour endothelial cells (TECs) are genetically and phenotypically distinct from their normal, healthy counterparts and provide various pro-tumourigenic effects. This study aimed to investigate the impact of conditioned media (CM) from non-tumourigenic MCF-12A breast epithelial cells as well as from MCF-7 and MDA-MB-231 breast cancer cells on human umbilical vein endothelial cells (HUVECs). Significant increases in cell viability were observed across all breast CM groups compared to controls, with notable differences between the MCF-12A, MCF-7, and MDA-MB-231 groups. Despite increased viability, no significant differences in MCM2 expression, a marker of cell proliferation, were detected. Morphological changes in HUVECs, including elongation, lumen formation, and branching, were more pronounced in breast cancer CM groups, especially in the MDA-MB-231 CM group. qPCR and Western blot analyses showed increased expression of TEC markers such as MDR1, LOX, and TEM8 in HUVECs treated with MCF-12A CM. The MCF-7 CM group significantly enhanced HUVEC migratory activity compared to MCF-12A CM, as evidenced by a scratch assay. These findings underscore distinct angiogenic responses elicited by non-tumourigenic and tumourigenic breast epithelial cells, with tumourigenic cells inducing a hyperactivated angiogenic response. The study highlights the differential effects of breast cancer cell paracrine signalling on endothelial cells and suggests the need for further investigation into TEC markers' role in both physiological and tumour angiogenesis.

Injectable dual thermoreversible hydrogel for sustained intramuscular drug delivery

Herein, we reported novel docetaxel-decorated solid lipid nanoparticle (DCT-SLN)-loaded dual thermoreversible system (DCT-DRTS) for intramuscular administration with reduced burst effect, sustained release and improved antitumor efficacy. The optimized DCT-DRTs was subjected to in-vitro and in-vivo analyses. Antitumor evaluation of the DCT-DRTS was executed and compared with DCT-hydrogel, and DCT-suspension trailed by the histopathological and immune-histochemical analyses. The DCT-SLN gave a mean particle size of 157 nm and entrapment efficiency of 93 %. It was a solid at room temperature, and changed to liquid at physiological temperature due to its melting point of about 32 °C. Unlikely, poloxamer mixture remained liquefied at 25-27 °C, however converted to gel at physiological temperature. This behavior demonstrated opposed reversible property of the DCT-SLN and poloxamer hydrogel in DCT-DRTS system, making it ideal for intramuscular administration and quick gelation inside the body. The DCT-DRTS sustained the drugs release and unlike DCT-hydrogel, the preliminary plasma concentration of DCT-DRTS was significantly reduced, overcoming the burst release. A meaningfully enhanced antitumor efficacy and improved survival rate was observed from DCT-DRTS in tumor cell xenograft athymic nude mice. Additionally, increased apoptotic and reduced proliferation markers were observed in DCT-DRTS treated tumor masses. It was concluded that DCT-DRTS may be a suitable choice for intramuscular administration of DCT with sustained release, improved bioavailability, reduced toxicity and enhanced antitumor effects.

Effects of selection for litter size variability on ovarian folliculogenesis, ovarian cell proliferation, apoptosis, and production of regulatory peptides in rabbits

The present study aimed to identify the novel mechanisms regulating rabbit fecundity. For this purpose, the association between litter size variability, fecundity, ovarian morphology, and markers of proliferation, apoptosis, steroidogenesis, and the presence of regulatory proteins were examined in ovarian cells from two rabbit lines divergently selected for low (LL) and high (HL) variability in litter size throughout sixteen generations. Ovaries and uteri were isolated from multiparous non-lactating female rabbits from each line at sixteen generations of selection. One part of the ovary was subjected to histomorphometric analysis of folliculogenesis. From the rest of ovary, ovarian granulosa cells (OGCs) were isolated, cultured, and cell viability, proliferation (accumulation of PCNA and of cyclin B1, and BrdU-positive cells), and apoptosis (accumulation of caspase 3, bax and DNA fragmentation) were evaluated by the Trypan blue exclusion test and BrdU, quantitative immunocytochemistry, and cell death detection assays. Furthermore, OGCs were subjected to proteomic analysis by using the nano HPLC-Chip-MS/MS method. The release of progesterone and oestradiol was measured by ELISA. The LL had more than one kit per litter than the HL (7.6 kits vs. 6.5 kits, p ≤ 0.05). No differences were found in the diameter of primordial and primary ovarian follicles, theca, and granulosa thickness, but the diameter of oocytes in the primary and secondary follicles was higher in the LL than in the HL (88.49 µm in the LL vs. 77.86 µm in the HL for oocytes of primary follicles, p ≤ 0.05; 122.10 µm in the LL vs. 109.87 µm in the HL for oocytes of secondary follicles, p ≤ 0.05). Preovulatory follicles were presented only in the ovaries of the LL. The LL had higher incorporation of BrdU and reduced accumulation of bax within OGCs (0.92% in the LL vs. 0.44% in the HL for incorporation of BrdU, p ≤ 0.05; 41% in the LL vs. 48% in the HL for accumulation of bax, p ≤ 0.05). Ovarian fragments from the LL produced less progesterone and oestradiol than those of the HL (12 ng/mg tissue/day in the LL vs, 45 ng/mg tissue/day in the HL for progesterone, p ≤ 0.05; 11 ng/mg tissue/day in the LL vs. 30 ng/mg tissue/day in the HL for oestradiol, p ≤ 0.05). Besides, the OGCs from the LL produced a higher number of specific regulatory proteins involved in cell differentiation, proliferation, and adhesion than the HL (50 vs 38, p ≤ 0.05). In conclusion, higher prolificacy in the LL line would be caused by: (1) the selection of growing primordial ovarian follicles; (2) better transformation to preovulatory follicles; (3) increased cytoplasmic maturation of oocytes; (4) increased DNA synthesis and decreased cytoplasmic apoptosis in OGCs; (5) changes in ovarian steroidogenesis; and (6) changes in the number of peptides involved in cell differentiation, proliferation, and adhesion. HIGHLIGHTS Assessing fecundity in two divergently rabbit lines for variability in litter size. Fecundity and its variability are related to the diameter and quality of oocytes. Selection and better growth of preovulatory follicles have been related to fecundity.

Pronounced early differentiation underlies zebra finch gonadal germ cell development

The diversity of germ cell developmental strategies has been well documented across many vertebrate clades. However, much of our understanding of avian primordial germ cell (PGC) specification and differentiation has derived from only one species, the chicken (Gallus gallus). Of the three major classes of birds, chickens belong to Galloanserae, representing less than 4% of species, while nearly 95% of extant bird species belong to Neoaves. This represents a significant gap in our knowledge of germ cell development across avian species, hampering efforts to adapt genome editing and reproductive technologies developed in chicken to other birds. We therefore applied single-cell RNA sequencing to investigate inter-species differences in germ cell development between chicken and zebra finch (Taeniopygia castanotis), a Neoaves songbird species and a common model of vocal learning. Analysis of early embryonic male and female gonads revealed the presence of two distinct early germ cell types in zebra finch and only one in chicken. Both germ cell types expressed zebra finch Germline Restricted Chromosome (GRC) genes, present only in songbirds among birds. One of the zebra finch germ cell types expressed the canonical PGC markers, as did chicken, but with expression differences in several signaling pathways and biological processes. The second zebra finch germ cell cluster was marked by proliferation and fate determination markers, indicating beginning of differentiation. Notably, these two zebra finch germ cell populations were present in both male and female zebra finch gonads as early as HH25. Using additional chicken developmental stages, similar germ cell heterogeneity was identified in the more developed gonads of females, but not males. Overall, our study demonstrates a substantial heterochrony in zebra finch germ cell development compared to chicken, indicating a richer diversity of avian germ cell developmental strategies than previously known.

Tiron enhances the anti-cancer activity of doxorubicin in DMBA-induced breast cancer: Role of Notch signaling/apoptosis/autophagy/oxidative stress

Existing work intended to investigate the outcomes of the localized mitochondrial antioxidant tiron (TR) alone or in combination with doxorubicin (DOX) in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in rats and the mechanistic pathways behind these effects. Also, to examine the preventive role of TR against DOX-related cardiotoxicity. 64 female Sprague-Dawley rats were randomly assigned into 8 groups: CTRL, DOX, TR, DMBA, DMBA + DOX, DMBA + TR100, DMBA + TR200, and DMBA + DOX + TR200. Rats received TR (100 and 200 mg/kg), DOX (2mg/kg), and DMBA (7.5 mg/kg) for four consecutive weeks. TR alone or combined with DOX not only inhibited oxidative status-related parameters and Notch pathway proteins but also attenuated proliferation markers, and enhanced apoptosis, and autophagy-related genes. Consistently, the histopathological analysis showed better scores in mammary tissues isolated from groups treated with TR only or combined with DOX. Additionally, TR dramatically decreased relative heart weight, myocardial injury biomarkers, and heart oxidative stress parameters while maintaining the myocardial histological integrity. Here we provided evidence that TR acts via modulating Notch signaling/apoptosis/autophagy/oxidative stress to elicit anti-tumor activity and combination with DOX revealed a higher efficacy as a novel anticancer strategy. Moreover, TR could be a potential cardio-protective candidate during DOX-chemotherapy, possibly via its antioxidant activity.

The effects of Atoh8 on postnatal murine neurogenesis.

Introduction Basic helix-loop-helix (bHLH) transcription factors are expressed in various organs and are involved in diverse developmental processes. The mouse atonal homolog 8 (Atoh8), a bHLH transcription factor plays a crucial role in various developmental processes, especially as a regulator of neurogenesis in the retina. Besides, Atoh8 expression has been observed in the central nervous system. The function of Atoh8 during the postnatal neurogenesis is still unclear. Methods This study focuses on elucidating the impact of Atoh8 on postnatal neurogenesis in the brain, particularly in selected regions; the subventricular zone (SVZ), rostral migration stream (RMS) and olfactory bulb (OB), across different life stages, using male homozygous Atoh8-knockout (M6KO) mice. Our morphometric analysis is based on immunohistochemically labeled markers for neuroblasts (Doublecortin) and proliferation (Phospho-Histone H3, PHH3) as well as pan neuronal markers. Results In Atoh8-/- mice alteration in the postnatal neurogenesis can be observed. Immunohistochemical analysis revealed a significant reduction in doublecortin-positive neuroblasts within the SVZ of neonatal M6KO mice compared to wildtype (WT) mice. Interestingly, no differences in cell number and distribution was observed in the subsequent migration of neuroblasts through the rostral migration stream (RMS) to the olfactory bulb (OB). Proliferating PHH3-positive neuronal progenitor cells were significantly diminished in proliferation rate in both the SVZ and RMS of neonatal and young M6KO mice. Furthermore, in the glomerular layer of the OB significantly fewer neurons were detected in the neonatal stage. Conclusion In conclusion, Atoh8 emerges as a positive regulator of postnatal neurogenesis in the brain. Its role encompasses the promotion of neuroblast formation, modulation of proliferation rates, differentiation, and maintenance of mature neurons. Understanding the intricacies of Atoh8 function provides valuable insights into the complex regulatory mechanisms governing neurogenesis.

Prognostic and Clinical Significance of the Proliferation Marker MCM7 in Breast Cancer.

Minichromosome maintenance complex component 7 (MCM7) plays an essential role in proliferation and DNA replication of cancer cells. However, the expression and prognostic significance of MCM7 in breast cancer (BC) remain to be defined. In this study, we aimed to evaluate the role of MCM7 in BC. We conducted immunohistochemistry staining of MCM7 in 1,156 operable early-stage BC samples and assessed MCM7 at the transcriptomic levels using publicly available cohorts (n = 13,430). MCM7 expression was evaluated and correlated with clinicopathological parameters including Ki67 labelling index and patient outcome. At the transcriptomic level, there was a significant association between high MCM7 mRNA levels and shorter patient survival in the whole cohort and in luminal BC class but not in the basal-like molecular subtype. High MCM7 protein expression was detected in 43% of patients and was significantly associated with parameters characteristic of aggressive tumour behaviour. MCM7 was independently associated with shorter survival, particularly in oestrogen receptor-positive (luminal) BC. MCM7 stratified luminal tumours with aggressive clinicopathological features into distinct prognostic groups. In endocrine therapy-treated BC patients, high MCM7 was associated with poor outcome, but such association disappeared with administration of adjuvant chemotherapy. Patients with high expression of Ki67 and MCM7 showed worst survival, while patients with double low expression BC showed the best outcome compared with single expression groups. The current findings indicate that MCM7 expression has a prognostic value in BC and can be used to identify luminal BC patients who can benefit from adjuvant chemotherapy.

The expression of Ki-67 and clinicohistopathological characteristics of breast cancer in Southern Sumatra, Indonesia

Background: Breast cancer is the most frequently diagnosed cancer in the world, approximately 2.3 million new cases (11.7% of all cancer cases) per year in 2020. The Ki-67 expression is clinically used to classify molecular subtypes of breast cancer into luminal A and luminal B groups.Objectives: This study aimed to investigate relationships between clinicohistopathological characteristics (ages, histopathological types, histopathological grades, molecular subtypes) of breast carcinoma patients and the Ki-67 proliferation index at dr. Moh. Hoesin General Hospital (RSMH) Palembang in 2019–2021.Method: This study applied an observational analytic study with a cross-sectional approach. 521 samples were included in this study based on inclusion and exclusion criteria. The samples in this study included invasive breast carcinoma patients who underwent histopathological examination and immunohistochemistry (IHC), recorded in the medical records at RSMH Palembang for 2019–2021 period. Then a statistical analysis was performed by using the chi-square test, which was analysed in the SPSS application.Results: The correlation between Ki-67 proliferation index and histopathological grades was statistically significant (p=0.018). The Ki-67 proliferation index was also statistically significantly associated with the molecular subtypes (p=0.000). Neither age (p=0.315) nor histopathological types (p=0.417) were significantly associated with the Ki-67 proliferation index.Conclusion: The Ki-67 expression was significantly associated with histopathological value and molecular subtype in breast carcinoma patients at RSMH Palembang in 2019–2021. The Ki-67 expression was not associated with the clinic-histopathological characteristics of ages and histopathological types.

Aberrant p53 immunostaining patterns in breast carcinoma of no special type strongly correlate with presence and type of TP53 mutations

Recent studies have revealed an association between TP53 mutations and endocrine resistance in hormone receptor-positive, HER2-negative breast cancer (HR + HER2 −BC). Aberrant p53 immunostaining (IHC) patterns may provide a surrogate marker for TP53 mutations. Building upon a ternary algorithm of aberrant staining patterns, this study evaluates the reliability of p53 IHC as screening tool for TP53 mutations in BC (NST). Furthermore, it describes the histopathological and molecular characteristics of TP53-mutated cases, along with the mutational status of PIK3CA. This study comprised 131 early-stage, node-negative BCs with available core biopsies and resection specimens. Cases were categorized as follows: HR + HER2 − (85 cases), HER2 + (21 cases) and triple negative (TN, 25 cases). Aberrant IHC staining patterns for p53 were defined as overexpression (OE), complete absence (CA) and cytoplasmic (CY). In addition, targeted sequencing of TP53 and PIK3CA genes was performed. TP53 mutations were identified in 53 of 126 cases (42.1%). Within HR + HER2 − cases, TP53 mutations were found in 17 of 80 cases (21.3%). IHC accurately predicted TP53 mutation in 96.2% of cases with a specificity of 100%. Additionally, there was a significant agreement between missense mutations and OE, as well as between truncating mutations and CA (κ 73% and 76%). CY was observed in two TN cases with truncating mutations within the nuclear localization signalling domain of p53. TP53-mutated cases exhibited higher grade, greater nuclear pleomorphism and higher Ki-67 proliferation index and were associated with the PIK3CA wild-type status (p < 0.001). p53 IHC may provide a useful screening tool for identifying TP53-mutated BC of NST.

Molecular markers of proliferation, DNA repair, and immune infiltration defines high-risk subset of resectable retroperitoneal sarcomas

IntroductionFor retroperitoneal sarcomas (RPS), aggressive surgical resection offers the only chance for a cure; however, 5-year survival remains below 65%. Therefore, there is a critical need to identify drivers of poor clinical outcomes. Materials and methodsTo identify biomarkers of tumors likely to recur following curative intent resection, we performed genomic and transcriptomic sequencing for 47 and 34 patients, respectively, with non-metastatic RPS at a single, high-volume sarcoma center. ResultsAt the DNA level, alterations in TERT were associated with poor disease-free survival (DFS) and overall survival (OS). Increased RNA expression of gene sets related to growth signaling and DNA repair were associated with poor DFS and OS. Infiltration of CD8+ T-Cells and activated dendritic cells were associated with poor DFS and OS. ConclusionThese findings may help to better identify and treat non-metastatic, high-risk RPS.

Additive genotoxic effects in cord blood cells upon indirect exposure to chemotherapeutic compounds crossing an in vitro placental barrier

Prenatal exposure to toxins can adversely affect long-term health outcomes of the offspring. Though chemotherapeutics are now standard of care for treating cancer patients during pregnancy, certain compounds are known to cross the placenta and harm placental tissue. The consequences for the fetus are largely unexplored. Here we examined the responses of newborn cord blood mononuclear cells in tissue culture to two chemotherapeutic drugs, cyclophosphamide and epirubicin, when either directly exposed to these drugs, or indirectly after crossing a placenta trophoblast bilayer barrier. Cord blood mononuclear cells exposed to the conditioned media obtained from cyclophosphamide-exposed trophoblast barriers showed a significant 2.4-fold increase of nuclear ROS levels compared to direct exposure to cyclophosphamide. Indirect exposure to epirubicine-exposed trophoblast barriers not only enhanced nuclear ROS levels but also significantly increased the fraction of cord blood cells with double strand breaks, relative to directly exposed cells. Neither apoptosis nor proliferation markers were affected in cord mononuclear blood cells upon direct or indirect exposure to cyclophosphamide or epirubicin. Our data suggests that trophoblast cells exposed to cyclophosphamide or epirubicine may induce an indirect ‘bystander’ effect and can aggravate genotoxicity in the fetal compartment.

EEF1B2 regulates the proliferation and apoptosis of human spermatogonial stem cell lines through TAF4B

BackgroundSpermatogonial stem cells (SSCs) are essential for male fertility, maintaining sperm production throughout life. While mouse SSCs have been studied extensively, the mechanisms regulating human SSCs are less understood. ObjectivesTo investigate the role of EEF1B2 in regulating human SSC proliferation and apoptosis. Material and methodsSingle cell RNA sequencing (scRNA-seq) analysis was utilized to investigate the differentially expressed genes of SSC. The distribution of EEF1B2 in the human testis was examined using immunofluorescence and immunohistochemistry techniques. Cell proliferation, DNA replication, and self-renewal were analyzed using CCK8, EdU, Western blot, and flow cytometry. RNA sequencing was employed to analyze the downstream target molecules and signaling pathways of EEF1B2. ResultsIn this study, we analyzed single-cell sequencing data from human testicular samples and identified EEF1B2 as a protein highly expressed in SSCs, with expression decreasing during development. Immunohistochemistry and immunofluorescence confirmed this pattern and showed co-localization with the proliferation marker KI67. Knockdown of EEF1B2 in human SSC lines impaired proliferation and viability, reducing self-renewal proteins like PLZF and CCNE1. RNA sequencing revealed decreased TAF4B following EEF1B2 knockdown, which could be rescued by replenishing TAF4B. Testicular SSCs from non-obstructive azoospermia (NOA) patients also showed reduced EEF1B2. Discussion and conclusionOur findings reveal a novel regulatory mechanism involving EEF1B2 and TAF4B in human SSCs, suggesting EEF1B2 deficiency may contribute to male infertility.

Divergent Immediate and Delayed Effects of Juvenile Exposure to Doxorubicin on the Thymus in C57BL/6 Mice.

The understanding of alterations within the immune system following doxorubicin (DOX) chemotherapy, and subsequent restoration, in childhood cancer survivors remains limited. This investigation endeavors to elucidate the immediate and delayed changes in thymic immune cell populations and their phenotypes in response to clinically relevant low doses of DOX in a juvenile mouse model. Male mice underwent a regimen of repeated low-dose DOX intraperitoneal injections at 4 mg/kg/week for three consecutive weeks. One week after the last dose of DOX, a subset of mice was euthanized to assess the immediate effects of DOX administration. A second subset of mice was euthanized five weeks after the last DOX dose to evaluate the delayed effects. Thymic samples were collected for multiparameter flow cytometry analysis to evaluate alterations in immune cell composition and phenotype. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure gene expression of- cytokines and senescence markers. One week following DOX administration, DOX treatment resulted in significant decline in thymus weight, with notable alterations in immune cell subpopulations. Reduced frequencies of mature CD3+CD4+ and CD3+CD8+ T cells were observed, along with changes in proliferation and exhaustion markers. Gene expression analysis revealed upregulation of Foxn, Pax1, Ifnγ, and Il7 alongside decreased Il6 and Il17 expression. Furthermore, Cdkn1a (p21Cip1) expression was elevated, suggesting immunosenescence. Five weeks following DOX administration, delayed effects of DOX treatment manifested in rebound increase in thymus weight and altered frequencies of CD4+ and CD8+ T cell subsets, with distinct patterns of proliferation and exhaustion observed. Notably, central memory CD4+ T cells exhibited significant decrease in frequency, while naive and effector memory CD4+ T cells showed reduced proliferation (Ki67+) and PD1 expression. Similar trends were observed in CD8+ T cell subsets, indicating selective effects of DOX on T cell differentiation and function. Although expression of thymus-related genes was normalized, p21Cip1 gene expression remained elevated. DOX treatment elicits a multifaceted influence on immune cell subsets and thymic weight. Immediate effects included thymic atrophy and reductions in mature T cell populations, while delayed effects showed rebound thymic hyperplasia and selective changes in CD4+ and CD8+ T cell subsets. Notably, both central memory and effector memory T cells exhibited reduced proliferation and exhaustion, suggesting unique impacts of DOX on immune cell function. The enduring elevation in p21Cip1 gene expression 5 weeks after DOX treatment suggests an immunosenescent phenotype. These observations collectively illuminate the formidable task of preserving immune competence and overall well-being in childhood cancer survivors subjected to DOX therapy.

TDP-43 ameliorates aging-related cartilage degradation through preventing chondrocyte senescence

Senescent chondrocytes or signaling mechanisms leading to senescence are promising new therapeutic approaches for ameliorating cartilage degradation. Herein, we show that the transactive response DNA/RNA-binding protein (TDP-43) regulates chondrocyte senescence and ameliorates cartilage degradation. First, a significant decrease in TDP-43 was observed in 16-month-old mice compared with younger mice. Immunohistochemistry (IHC) analysis of mouse articular cartilage showed that p21, p16, p53, and matrix metalloprotein-13 (MMP13) were increased, but laminB1 and Collagen type II alpha1 1 chain (Col2a1) were decreased in 16-month-old mice. Furthermore, TDP-43 levels were decreased in vivo following D-galactose (D-gal) induction. Therefore, we investigated the role of TDP-43 in the senescent chondrocytes. ATDC5 cells were induced to overexpress TDP-43. Western blot analysis showed increased expression of laminB1, Ki67, and PCNA but decreased expression of p21, p16, p53, and MMP13. Senescence-associated-β-galactosidase (SA-β-Gal) assay, γH2AX staining, and EdU were performed to assess changes in chondrocytes, showing weaker SA-β-Gal and γH2AX staining but stronger EdU and Alican Blue staining. However, TDP-43 deficiency had opposing effects, and similar to D-gal stimulation results. Taken together, our data verified that TDP-43 negatively correlated with senescence markers, positively correlated with cell proliferation markers, and could alleviate cartilage degradation induced by D-gal. This may be an essential mechanism of cellular senescence and cartilage degradation.

Inhibitor of Myom3 inhibits proliferation and promotes differentiation of sheep myoblasts

Skeletal muscle quality and yield are important production traits in livestock, and improving skeletal muscle quality while increasing its yield is an important goal of economic breeding. The proliferation and differentiation process of sheep myoblasts directly affects the growth and development of their muscles, thereby affecting the yield of mutton. Myomesin 3 (Myom3), as a functional gene related to muscle growth, currently lacks research on its function in myoblasts. This study aims to investigate the effect of the Myom3 gene on the proliferation and differentiation of sheep myoblasts and its potential molecular mechanisms. The results showed that inhibitor of Myom3 in the proliferation phase of myoblasts resulted in significant downregulation of the proliferation marker gene paired box 7 (Pax7) and myogenic regulatory factors (MRFs; Myf5, Myod1, Myog, P < 0.01), a significant decrease in the EdU-positive cell rate (P < 0.05), and a significant increase in the cell apoptosis rate (P < 0.01), which inhibited the proliferation of myoblasts and promoted their apoptosis. During the differentiation phase of myoblasts, the inhibitor of Myom3 resulted in significant downregulation of the Pax7 gene, upregulation of MRFs (Myod1, Myog, P < 0.05), and a significant increase in fusion index (P < 0.05), promoting the differentiation of myoblasts. Further transcriptome sequencing revealed that differentially expressed genes in the Myom3 interference group were mainly enriched in the MAPK signaling pathway, TNF signaling pathway, and IL-17 signaling pathway. In summary, the inhibitor of Myom3 inhibits myoblast proliferation and promotes myoblast differentiation. Therefore, Myom3 has a potential regulatory effect on the growth and development of sheep muscles, and in-depth functional research can be used for molecular breeding practices in sheep.

Altered memory CCR6+ Th17-polarised T-cell function and biology in people with HIV under successful antiretroviral therapy and HIV elite controllers

Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6+ Th17-polarised CD4+ T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied. mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6+CD4+ T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and invitro functional assays. Decreased Th17cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production invitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6+ T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6+ T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6+ T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67. These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17cells. This study was funded by the Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Pogostemon cablin Extract Promotes Wound Healing through OR2AT4 Activation and Exhibits Anti-Inflammatory Activity.

Skin healing occurs through an intricate process called wound healing which comprises four phases: coagulation and hemostasis, inflammation, cellular proliferation, and remodeling. Chronic wounds often arise because of prolonged or excessive inflammation, which hinders the healing process and wound closure. Despite the recognized efficacy of Pogostemon cablin (patchouli) in wound healing, the precise mechanism of action of Pogostemon cablin extract (PCE) on inflammation and wound healing remains poorly understood. In this study, we investigated the effects of PCE on cell proliferation and wound healing, as well as its anti-inflammatory activity, using in vitro experiments. We found that PCE increased cell proliferation and expression of the cell proliferation marker Ki67 and accelerated wound healing in human keratinocytes through the activation of OR2AT4. Furthermore, PCE exhibited anti-inflammatory effects by decreasing the levels of pro-inflammatory cytokines interleukin-6 and -8 in lipopolysaccharide-treated and TNF-α-exposed THP-1 and HaCaT cells, respectively. Overall, these findings suggest that PCE holds therapeutic potential by promoting cell proliferation, facilitating wound healing, and exerting anti-inflammatory effects.

Fast-acting antidepressant-like effects of ketamine in aged male rats

BackgroundThe aging process causes anatomical and physiological changes that predispose to the development of late-life depression while reduces the efficacy of classical antidepressants. Novel fast-acting antidepressants such as ketamine might be good candidates to be explored in the context of aging, especially given the lack of previous research on its efficacy for this age period. Thus, the aim of the present study was to characterize ketamine’s effects in older rats.MethodsThe fast-acting (30 min) and repeated (7 days) antidepressant-like effects of ketamine (5 mg/kg, ip) were evaluated in 14-month-old single-housed rats through the forced-swim and novelty-suppressed feeding tests. In parallel, the modulation of neurotrophic-related proteins (i.e., mBDNF, mTOR, GSK3) was assessed in brain regions affected by the aging process, prefrontal cortex and hippocampus, as well as possible changes in hippocampal cell proliferation.ResultsAcute ketamine induced a fast-acting antidepressant-like response in male aged rats, as observed by a reduced immobility in the forced-swim test, in parallel with a region-specific increase in mBDNF protein content in prefrontal cortex. However, repeated ketamine failed to induce antidepressant-like efficacy, but decreased mBDNF protein content in prefrontal cortex. The rate of hippocampal cell proliferation and/or other markers evaluated was not modulated by either paradigm of ketamine.ConclusionsThese results complement prior data supporting a fast-acting antidepressant-like effect of ketamine in rats, to further extend its efficacy to older ages. Future studies are needed to further clarify the lack of response after the repeated treatment as well as its potential adverse effects in aging.