Objective:Testis-specific actin-like 7A (ACTL7A) is an acrosome/perinuclear-theca-related protein implicated in male infertility. In this exploratory study, we assessed ACTL7A transcript levels in a clinical assisted reproductive technology (ART) setting and performed preliminary protein-level validation in a subset of samples, together with sperm chromatin integrity assays, to clarify potential associations with sperm quality. Materials and Methods:For this preliminary case-control study, twenty-seven men were included: 11 infertile intracytoplasmic sperm injection (ICSI) candidates and 16 fertile preimplantation genetic testing (PGT) candidates. ACTL7A mRNA levels were quantified in all samples by quantitative real time polymerase chain reaction (qRTPCR). ACTL7A protein expression was evaluated by Western blot in a small feasibility-limited subset (n=3 per group) and interpreted as exploratory protein-level support for the mRNA findings. Localization was assessed by immunostaining. Classical semen parameters and sperm functional assays including DNA fragmentation (SCSA), histone retention (aniline blue), and protamine deficiency (CMA3) were performed. Clinical outcomes were also documented. Results:ACTL7A mRNA levels were significantly reduced in infertile men compared with fertile controls (qPCR: P=0.020). Preliminary Western blot analysis in the subset also suggested lower protein expression in the infertile group (P=0.010), providing supportive evidence for the transcript findings. Infertile men also showed lower sperm viability and poorer chromatin integrity (P=0.040 and P=0.020, respectively). ACTL7A expression positively correlated with sperm viability (r=0.40, P=0.040) and inversely correlated with DNA fragmentation (r=-0.43, P=0.020). Exploratory receiver operating characteristic (ROC) analysis showed a moderate discriminatory ability for ACTL7A expression [area under the curve-ROC (AUC-ROC=0.727, P=0.048], with 62.5% sensitivity and 81.8% specificity. Conclusion:Reduced ACTL7A expression in human sperm is linked to impaired chromatin quality and decreased viability, supporting ACTL7A as a candidate research biomarker rather than a clinically validated predictor. Given the small cohort and limited protein-level testing, these findings are exploratory and emphasize the need for validation in larger independent populations to confirm clinical utility.
Objective:RMonocarboxylate transporter protein 4 (MCT4), encoded by the SLC16A3 gene, mediates lactate efflux into the extracellular microenvironment. This study aimed to characterize the MCT4 overexpression in tumor-associated macrophages (TAMs) at the single-cell level in hepatocellular carcinoma (HCC) and to investigate its role in HCC progression through metabolic reprogramming of TAMs. Materials and Methods:In this experimental study, R software and multiple public databases, including UALCAN, TIMER2.0 and TISCH2, were used to analyze SLC16A3 expression at the single-cell level and its association with HCC prognosis. In vitro experiments were conducted and SLC16A3 knockdown was achieved using shRNAs. TAM polarization was evaluated by Western blot, quantitive real time-polymerase chain reaction (qRT-PCR) and flow cytometry. Additional bioinformatic analyses, including protein-protein interaction (PPI) network analysis, were conducted using STRING to investigate the association between SLC16A3 expression and key glycolysis genes. Results:Analysis of the GEO database GSE166635 demonstrated that MCT4 expression was significantly higher in macrophages than in other immune cell populations in HCC. Immunofluorescence (IF) revealed co-localization of MCT4 with the M2 TAM marker CD163. Western blot demonstrated that SLC16A3 knockdown reduced CD163 expression at protein levels to 84.1% ± 5.0% (KD-1) and 44.6% ± 8.4% (KD-3) compared to control. Flow cytometry further demonstrated that SLC16A3 knockdown increased the proportion of CD86-positive cells from approximately 13.5% (Con sh) to 43.3% (KD-1) or 43.8% (KD-3), whereas the proportion of CD163-positive cells decreased to approximately 38.2% (KD-1) or 33.4% (KD-3). Survival analysis was performed on HCC patients from TIMER2.0, including 371 HCC patients with complete survival information, further indicated that high SLC16A3 expression combined with increased M2 TAM infiltration was associated with poorer prognosis (P=0.008). Conclusion:High MCT4 expression in TAMs may be associated with metabolic reprogramming toward glycolysis, and could promote M2 polarization of TAMs, thereby contributing to HCC progression and poor clinical outcomes.
Objective:Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. ADCYAP1 encodes pituitary adenylate cyclase activating polypeptide (PACAP), which can exert neuroprotective effects in neurodegenerative diseases. This study aims to probe the specific function of ADCYAP1 in AD. Materials and Methods:In this experimental study, brain tissue samples from AD patients and healthy controls were collected to assess ADCYAP1 expression. APP/PS-1 transgenic mice were utilised as an AD mouse model along with amyloid beta (Aβ)-induced PC12 cells as an AD cell model. ADCYAP1 expression in the AD mice was determined by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. An IF assay was employed to test Aβ deposition and microtubule-associated protein 2 (MAP2) expression (a neuron marker). Behavioural tests, including the Morris water maze (MWM) and footprint tests, were performed to evaluate cognitive impairment and motor ability. Cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL) assay. Western blot was utilised to test the expression of autophagy-related genes, including LC3 and beclin1. The expressions of transcription factors involved in neuronal differentiation was assessed by RTqPCR. Results:ADCYAP1 expression was significantly downregulated in the brain tissues of AD patients and mice. ADCYAP1 overexpression reduced Aβ deposition and enhanced MAP2 expression in the AD mice. ADCYAP1 overexpression effectively improved cognitive impairment and mobility decline in the AD mice. ADCYAP1 upregulation inhibited neuronal apoptosis and autophagy both in vivo and in vitro. ADCYAP1 upregulated the expression of neuronal differentiationrelated factors [transforming growth factor β (TGFβ), early growth response 1 (Egr1), brain-derived neurotrophic factor (BDNF), fibroblast growth factor 21 (FGF21), and insulin-like growth factor 1 (Igf1)] in the AD models. Conclusion:ADCYAP1 inhibits neuronal apoptosis and autophagy to improve AD progression in APP/PS-1 mice and Aβ-induced P12 cells.
Objective:Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint inflammation, disability, and reduced quality of life. Current mesenchymal stem cell (MSC) therapies face limitations due to cell variability and lack of standardization, highlighting the need for effective, cell-free alternatives that replicate MSCs' immunomodulatory functions. This study aimed to enhance MSC secretion of regulatory factors to modulate inflammation in an RA model. Materials and Methods:In this experimental study, MSCs were isolated from mouse bone marrow and characterized by CD73 expression and lack of CD45/CD80. MSCs were treated with Muramyl dipeptide (MDP; 0 and 10 μg/mL) for 24 hours, followed by washing and further culture for 48 hours to obtain conditioned medium (CM). The resulting CM from untreated MSCs and MDP-primed MSCs (MDP-CM) was collected for subsequent in vivo administration. RA was induced in male Wistar rats (160-180 g) via intradermal injection of Freund's complete adjuvant. Treatments with CM derived from either untreated MSCs or MDP-pulsed MSCs (MDP-CM) were initiated on day 7 post-induction and repeated after 5 days via intraperitoneal injection (n=10 per group). Results:MDP did not affect MSC viability but significantly altered the CM's immunological profile: MDP-CM had lower levels of IFN-γ, IL-6, and IL-1β, and higher levels of indoleamine 2,3-dioxygenase, TGF-β, and IL-10 compared to control CM. in vivo administration of MDP-CM significantly attenuated RA severity and promoted body weight recovery more effectively than CM alone. In addition, MDP-CM significantly reduced systemic inflammatory markers , such as TNF-α, IL-1β, myeloperoxidase, nitric oxide, and C-reactive protein (CRP), and more strongly modulated T-cell- associated gene expression in joint tissue, evidenced by upregulation of FOXP3 and downregulation of T-bet, Gata3, and Rorc compared with RA rats receiving CM. Conclusion:The CM derived from appropriately primed MSCs may represent a more potent and standardized "off-theshelf" alternative to direct stem cell transplantation.
In the article published in Cell J, Vol 23, No 3, 2021, pages 273-287, Figure 2H panel (S2-EC/CD31-DAPI) inadvertently contains an image that originally appeared in a 2016 Biomaterials paper previously published by this team (doi: 10.1016/j.biomaterials.2016.01.025). The correct figure is provided below. The authors sincerely apologize for this inconvenience.
Objective:Methamphetamine (METH) is a highly addictive psychostimulant that alters gene expression in brain reward circuits. This study aimed to identify METH-associated transcriptional changes in the nucleus accumbens (NAc) and explore potential pharmacological interventions. Materials and Methods:We conducted an in silico analysis of publicly available microarray data (GSE46717) from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified using limma and analyzed for functional enrichment via EnrichR. Protein-protein interaction (PPI) networks were constructed using STRING to identify hub genes, validated in silico with jackknife resampling. Adult male Wistar rats were injected with METH (10 mg/ kg, followed by 2.5 mg/kg after one month), and expression of selected hub genes was measured in NAc tissue using quantitative polymerase chain reaction (qPCR). Connectivity mapping was applied to identify candidate drugs reversing METH-induced transcriptional changes. Results:We identified 280 DEGs (210 upregulated, 70 downregulated). Upregulated pathways included caffeine metabolism, long-term potentiation, and cocaine addiction, whereas GABAergic and glutamatergic synapse genes were downregulated. Network analysis highlighted Fos, Crh, Oprl1, and Slc17a6 as hub genes, validated both computationally and experimentally. Connectivity mapping identified D-64131 and Mebendazole as potential therapeutics. Conclusion:METH induces substantial transcriptional alterations in the NAc, affecting synaptic signaling and addiction pathways. Integrating in silico network analysis with experimental validation identified robust hub genes and suggested candidate compounds for therapeutic intervention.
Chronic kidney disease (CKD) significantly affects kidney function, often leading to end-stage kidney disease (ESKD). Due to its high rates of morbidity, mortality, and economic effects, CKD is becoming the ninth most important risk factor for mortality worldwide, presenting serious healthcare issues. Regulatory T lymphocytes (Tregs) play a role in regulation, particularly in transplant tolerance and autoimmune conditions. Recent research has highlighted the potential of Tregderived vehicles (EVs) to modulate responses and promote graft tolerance during kidney transplantation. These EVs can suppress effector T lymphocytes (T effect), trigger apoptosis, and modify cytokine production to maintain balance and prevent graft rejection. Moreover, Treg-EVs have emerged as potential biomarkers for diagnosing CKD, paving the way for new diagnostic and therapeutic strategies. This study explores how Treg-derived EVs influence processes underlying CKD and kidney transplantation, highlighting their significance as useful tools.
A study demonstrated that increased miR-129-5p expression is associated with reduced Fndc5 expression in adipose tissue of a type 2 diabetes mellitus (T2DM) mouse model, suggesting a post-transcriptional regulatory mechanism contributing to metabolic dysfunction. This Letter discusses the strengths and limitations of these findings. Although the dual-luciferase assay provided strong evidence of a direct interaction between miR-129-5p and the Fndc5 3'UTR, the absence of in vivo loss-of-function or gain-of-function experiments limits causal interpretation. In addition, downstream metabolic consequences of reduced FNDC5/irisin signaling, including insulin signaling pathways, GLUT4 regulation, and thermogenic activity, were not comprehensively evaluated. Methodological concerns, including sample size and the lack of reported blinding procedures, may also affect reproducibility. Future studies integrating miRNA manipulation strategies, circulating irisin measurement, and pathway-based metabolic analyses are needed to strengthen the biological and translational relevance of the miR-129-5p-FNDC5 axis in T2DM.
Breast cancer remains a leading cause of cancer-related deaths among women globally, presenting significant challenges due to its heterogeneity and complex biology. While traditional treatments such as surgery, chemotherapy, and targeted therapies offer some benefit, the lack of universal treatment strategies underscores the need for innovative approaches. A total of 255 articles were identified through PubMed, Scopus, Web of Science, and Google Scholar up to 1 December 2025, and 30 articles were selected for inclusion in this narrative review. This narrative review focuses on cancer-testis antigens (CTAs) as potential therapeutic targets in breast cancer. The objective of this narrative review is to evaluate the therapeutic role and immunogenic characteristics of CTAs in breast cancer, with emphasis on their potential in diagnosis, prognosis, and immunotherapy. CTAs are tumor-associated antigens that are aberrantly expressed in various cancers, including breast cancer, while their expression in normal tissues is restricted to the testis. Their unique immunogenic properties make them ideal candidates for both early diagnostic tools and targeted immunotherapy. This article discusses the potential of CTAs in improving breast cancer screening, prognosis, and treatment strategies, with particular emphasis on their roles in immunotherapy and vaccine development. Furthermore, the challenges of CTA-based treatments, including antigen heterogeneity and immune evasion, are outlined, along with insights into future research directions aimed at overcoming these obstacles and improving clinical outcomes.
Objective:The 1-carbon metabolic cycle is essential for cellular growth, biomolecule synthesis, and epigenetic regulation. This cycle depends on B-complex vitamins, particularly folate (B9) and cobalamin (B12), which act as cofactors. Deficiencies in these vitamins can disrupt homocysteine metabolism, impair spermatogenesis, and increase oxidative stress, thereby compromising male fertility. To investigate the impact of dietary deficiencies in vitamins B12 and B9 on trans-sulfuration enzymes [cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE)], sperm function, oxidative stress, and DNA methylation in a mouse model. Materials and Methods:In this experimental study, male mice were fed either a standard chow diet or a diet deficient in vitamins B12 and B9. Sperm parameters, chromatin integrity [via acridine orange (AO) and aniline blue (AB)], lipid peroxidation, and intracellular reactive oxygen species (ROS; assessed using DCFH-DA and BODIPY C11) were evaluated. Sperm DNA methylation was measured using immunofluorescence. Serum levels of folate, vitamin B12, and testosterone were quantified. Expression of CBS, CSE, and HO-1 were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and Western blot. Results:Mice on the vitamin B deficient (VBD) exhibited significantly decreased sperm concentration and motility, along with increased morphological abnormalities, DNA damage, histone retention, and lipid peroxidation. Intracellular ROS levels did not differ significantly from controls. The VBD group also showed lower serum folate and B12, elevated homocysteine, and reduced testosterone levels. DNA methylation intensity in sperm was significantly decreased. Histological analysis revealed impaired testicular architecture and reduced spermatogenic indices. CSE gene expression was significantly downregulated, whereas CBS and Heme oxygenase 1 (HO-1) expression remained unchanged. Protein levels of CBS, CSE, and HO-1 showed no significant differences. Conclusion:Deficiencies in folate and vitamin B12 negatively affect sperm quality and testicular function in male mice, likely through disrupted homocysteine metabolism and altered gene expression in the trans-sulfuration pathway. These findings underscore the importance of adequate B-vitamin intake for male reproductive health.
Objective:Central venous lines (CVLs) are indispensable in pediatric hematopoietic stem cell transplantation (HSCT) but carry a high risk of complications. This study aimed to compare the clinical, microbiological, and outcome profiles between pediatric HSCT recipients who developed infectious versus non-infectious CVL complications. Materials and Methods:In this single-center, retrospective study, from a total of 228 pediatric HSCT recipients (2015-2024), this analysis focused on comparing the 76 patients who developed CVL-related infectious complications with the 74 patients who had non-infectious complications. Demographic, clinical, and transplant-related variables were analyzed. Bivariate analyses (Chi-square/Fisher's exact tests) were used to explore unadjusted associations between these variables and complication type. Results:In this study, infection was the most common complication (50.7% of all events). Patients with infectious complications more frequently had a non-malignant underlying disease (63.2% vs. 40.5%; OR=2.51, P=0.006) and had undergone allogeneic transplantation (89.5% vs. 58.1%; OR=6.33, P<0.001) compared to those with non-infectious complications. A higher proportion of patients with infectious complications had received a reduced-intensity conditioning (RIC) regimen (48.7% vs. 32.4%), although this difference did not reach statistical significance after multiple-comparison adjustment (P=0.031). Conclusion:This within-group comparative study characterizes a subgroup of pediatric HSCT recipients with infectious CVL complications, defined by non-malignant underlying disease, allogeneic transplantation, and RIC. These exploratory findings describe clinically relevant patterns among complicated cases and provide a hypothesis-generating basis for future analytical studies to investigate causal risk factors.
Objective:Chronic tramadol (TRA) exposure has been reported to induce inflammatory responses in testicular tissue, whereas exercise training exerts immunomodulatory effects within the testes. Accordingly, this study investigated whether exercise training protocols (ETPs) during the post-withdrawal period modulate testicular inflammation via the TLR4/NF-κB-mediated inflammatory signaling pathway. Materials and Methods:In this experimental study, adult Wistar rats (n=36; sex: male; age: 8 weeks; body weight: 180-220 g) were randomly assigned to a control group and tramadol-treated groups. The control group received normal saline. TRA was administered at a dose of 40 mg/kg, via intraperitoneal route, for 60 days. TRA withdrawal was induced by stopping TRA administration after 60 days and maintained for an additional 60 days. Following withdrawal, tramadol-exposed rats were allocated to a withdrawal-only group or to low-, moderate-, or high-intensity continuous exercise training groups. At the end of the experimental period, testicular tissue samples were collected. The expression or levels of IL-6, IL-10, COX-II, TLR4, NF-κB, TNF-α, and iNOS parameters were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence staining, and Western blotting methods. Results:The TRA administration increased the expression of TLR4, NF-κB, IL-6, and TNF-α. These elevations were reduced after withdrawal and were more markedly attenuated in exercise-trained groups. IL-10 levels were decreased in the tramadol-only group but were restored during withdrawal with exercise training. TRA exposure was associated with increased numbers of iNOS+ and COX-II+ somatic and germ cells; these alterations were reversed after withdrawal, particularly in exercised animals. Despite TRA cessation, residual immune dysregulation persisted, characterized by elevated TLR4, NF-κB, IL-6, and TNF-α expression. Conclusion:Exercise training following TRA withdrawal could restore inflammatory balance by suppressing TLR4 and NF-ΚB signaling, normalizing pro- and anti-inflammatory cytokine profiles, and possibly attenuating the iNOS/NO/COXII pathway in Wistar rats.
Objective:Gastric cancer (GC) is one of the most common malignancies worldwide. Since the PI3K/AKT/mTOR signalling pathway plays a crucial role in tumour growth and survival, this study aims to evaluate the anti-proliferative and pro-apoptotic effects of Vipera raddei kurdistanica venom on human GC cells (C615). Materials and Methods:In this an experimental study, C615 cancer cells and normal fibroblasts were treated with 0.63-10 μg/ml of venom for 24, 48, 72, and 96 hours. Cell viability was analysed using MTT and trypan blue staining, and cytotoxicity was assessed by the lactate dehydrogenase (LDH) assay. Apoptosis at the half-maximal inhibitory concentration (IC50) concentration was evaluated using the TUNEL, annexin V/PI flow cytometry, and diphenylamine DNA fragmentation assays. Mitochondrial membrane potential (JC-1), cytosolic cytochrome c levels, and the expressions of apoptosis-related and PI3K/ AKT/mTOR pathway genes were measured. All experiments were performed in three independent biological replicates. Results:Venom treatment significantly reduced cell viability in a time- and concentration-dependent manner. The IC50 values in fibroblasts being approximately 51-59-fold higher than in C615 cells across time points. At the IC50 dose, venom treatment produced a very large effect on apoptosis induction, increasing early and late apoptosis by approximately 84-fold and 463-fold, respectively, compared with controls. DNA fragmentation (15.7-fold) were significantly elevated (P<0.001). Venom exposure exerted a reducing effect on mitochondrial membrane potential (37%, P=0.032) and increased cytochrome c release (2.31-fold, P=0.001). Gene expression analysis showed upregulation of FAS (1.67-fold; P<0.001), FASL (1.28 fold; P=0.005), BAX (2.34 fold; P<0.001), BAK (1.29 fold; P=0.004), and BIM (1.4 fold; P<0.001) and downregulation of BCL-2 (0.44 fold; P<0.001), BCL-XL (0.47 fold; P<0.001), PI3K (P<0.001), AKT (P<0.001), and MTOR (P<0.001). Conclusion:Vipera raddei kurdistanica venom may exert selective cytotoxic effects against gastric cancer cells and may induce apoptosis through both intrinsic and extrinsic pathways, potentially via inhibition of the PI3K/AKT/mTOR signalling axis.
Objective:This study intends to determine whether exercise-based interventions improve haemoglobin A1c (HbA1c) levels in adults with type 1 diabetes (T1D) and examines how different types of exercise influence this association. Materials and Methods:A search of the appropriate electronic databases revealed 18 eligible studies from initial 685 records. The effect of exercise training on HbA1c was examined in two parts: i. Exercise training intervention compared to a T1D control group (between-group analysis) and ii. Pre/post comparison of exercise training intervention on T1D patients (within-group analysis). Results:The between-group results showed that exercise training had a statistically significant reduction in HbA1c [mean difference (MD), confidence interval (CI)]: -0.39 (-0.64, -0.15), P=0.002, I2=0%). Subgroup analysis based on the types of exercise training showed a statistically significant effect of high-intensity interval training (HIIT) on HbA1c levels in both the within-group analyses [MD: -0.24 (-0.45, -0.02), P=0.03, I2=0%] and between-group analyses [MD: -0.39 (-0.64, -0.15), P=0.002, I2=0%]. Conclusion:The meta-analysis and meta-regression results suggest that HIIT may be a supportive intervention in T1D patients; however, there was insufficient evidence to confirm these findings. Data from more randomised controlled trials (RCTs) are needed to compare the effect of different types of exercise on HbA1c levels in T1D patients [registration: PROSPERO (CRD42023414580)].
Objective:Prenatal prediction of CD34+ adequacy supports cord-blood banking by reducing expenses on low-yield units and reserving capacity for clinically promising grafts. We have developed and evaluated a prenatal machine learning model that predicts whether an umbilical cord blood (UCB) unit will meet a clinically supported adequacy threshold (≥1.5×105 CD34+ cells/kg recipient) before collection for single-unit grafts. Materials and Methods:In this retrospective study, we analysed 126,406 records from the Royan Stem Cell Technology Company (RSCT; Tehran, Iran), which included routinely available maternal, neonatal, and family-history variables. A pipeline of imputation (IterativeImputer numeric; SimpleImputer+OrdinalEncoder categorical), feature selection (Extra Trees), and hyperparameter tuning using Bayesian optimisation with model training/evaluation was performed within cross-validation folds. Decision Tree (DT), K-Nearest Neighbours (KNN), Random Forest (RF), Support Vector Machine (SVM), and Multilayer Perceptron (MLP) classifiers were tuned via Bayesian optimisation. Models were ranked by the Technique for Order Preference by Similarity to Ideal Solution (TOPSIS). Majority voting (MV) ensembles were constructed from the top-k models. Model interpretability used SHapley Additive exPlanations (SHAP). Results:The MV (top-4: RF, KNN, DT, MLP) ensemble achieved an area under the receiver operating characteristic curve (ROC-AUC)=0.808 and an area under the precision-recall curve (PR-AUC)=0.744 on the held-out test set, with an accuracy=0.757, precision=0.726, recall/sensitivity=0.804, F1=0.762, specificity=0.716, and Brier score=0.181. SHAP highlighted history of hepatitis C, birth place, hyperthyroidism, history of anaemia, oral fungus, and rheumatism among the most influential features. Conclusion:Prenatal prediction of UCB CD34+ adequacy using an interpretable MV ensemble is feasible and accurate to support pre-collection triage and can potentially improve banking efficiency. The resultant model offers a non-invasive tool to enhance the efficiency of cord blood banking by prioritising units with higher transplantation potential.
Objective:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the most effective treatment for patients with high-risk acute myeloid leukemia (AML), but relapse remains a major challenge. Immunotherapy is considered a promising approach for reducing the risk of relapse. Natural killer (NK) cells exert cytotoxic effects against malignant cells, and their activation with interleukin-15 (IL-15) enhances anti-leukemic immune responses. This study evaluated the safety and feasibility of in vitro IL-15 activation of CD56+ cells and assessed the safety of their infusion in AML patients following allo-HSCT. Materials and Methods:In this phase I clinical trial study, CD56+ cells were isolated from non-mobilized donors using a one-step CD56 enrichment protocol to obtain NK and NKT cells. CD56+ cells were activated by overnight incubation with IL-15. A cytotoxicity assay was performed against K562 cells. Three escalating doses of CD56+ cells consisting of 1×106, 3×106, and 5×106 cells/kg of patient bodyweight were infused to the three patients on days +7, +14, and +21 post-allo-HSCT. Patients were observed during and up to 4 hours after the infusion for immediate adverse events and were monitored for 21 days to detect delayed events. Results:Activation with IL-15 increased the expression of activating receptors, including CD25, CD69, NKp30, NKp46, and NKG2D decreased the expression of the inhibitory receptor NKG2A. The cytotoxicity of IL-15-activated CD56+ cells was higher than that of non-activated CD56+ cells. This method was safe and no intervention-related complications were observed. Conclusion:We identified a feasible method to activate CD56+ cells and evaluate the safety of their infusion in patients. Despite the value of activating CD56+ cells with IL-15, further studies with larger sample sizes are needed to confirm and validate the current hypothesi (registration number: IRCT20230801058996N2).
Objective:Cardiovascular diseases remain a leading cause of global mortality and morbidity, despite advances in prevention, diagnosis, and treatment. Cardiac tissue engineering (CTE) offers a promising approach to repairing damaged myocardium through bioengineered constructs that closely mimic native cardiac tissue. The integration of biomaterials that enhance angiogenesis, exhibit antioxidant activity, and support cellular proliferation can further improve regenerative outcomes. This study aimed to assess the potential of a sulfated alginate (S-Alg)/cerium-doped 45S5 bioactive glass (Ce-BG) scaffold as a cardiac patch for tissue engineering applications. Materials and Methods:In this experimental study, novel porous scaffolds composed of S-Alg and Ce-BG were fabricated using a freeze-drying technique. The scaffolds underwent comprehensive physicochemical characterization and cytocompatibility evaluation. Their angiogenic potential was assessed by quantifying vascular endothelial growth factor receptor 2 (VEGFR2) expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) over a seven-day period. Additionally, scaffold antioxidant activity was assessed using the DPPH assay. Results:Scanning electron microscopy (SEM) revealed an interconnected porous network with an average pore diameter of 100-300 μm. Incorporation of BG particles led to a reduction in tensile strength and Young's modulus. The ion release profile demonstrated sustained release of bioactive ions. MTT assays and cell morphology analyses confirmed scaffold cytocompatibility, supporting adhesion and proliferation of human umbilical vein endothelial cells (HUVECs). qRT-PCR showed significant upregulation of VEGFR2 in HUVECs cultured on Ce-containing scaffolds over seven days. Moreover, the addition of Ce enhanced the antioxidant activity of BG-containing scaffolds, effectively scavenging DPPH radicals over time, highlighting their potential for cardiac patch applications. Conclusion:The S-Alg/Ce-BG composite scaffold exhibits favorable properties for CTE, including cytocompatibility, enhanced angiogenic potential, and antioxidant activity. These features highlight the scaffold as a highly promising candidate for advancing cardiac tissue regeneration strategies.
Objective:The PI3K/Akt signaling pathway plays a central role in regulating cell growth, survival, and metabolism, and its dysregulation is a hallmark of many cancers. The PIK3CA gene, which encodes the alpha catalytic subunit of PI3K, is altered in approximately 30% of breast cancers. Among its mutations, c.3140A>G (p.His1047Arg) in the kinase domain is the most prevalent, producing a constitutively active enzyme with oncogenic potential. Here, we engineered a population of MCF7 cells carrying the PIK3CA c.3140A>G mutation using CRISPR-Cas9 with precise single-nucleotide editing, and evaluated its impact on cellular characteristics. Materials and Methods:In this experimental study, nearly homogeneous populations of PIK3CA H1047R mutant MCF7 cells were generated using CRISPR-Cas9-mediated genome editing followed by hierarchical single-cell isolation. Editing efficiency was validated through allele-specific polymerase chain reaction (PCR) and multiple rounds of Sanger sequencing. Cell cycle distribution and proliferation were analyzed using flow cytometry and cell count assays, respectively. Gene expression changes were assessed by quantitative real-time PCR to evaluate the mutation's impact on cell cycle-related genes. Results:Tracking of insertions, deletions, and recombination events (TIDER) analysis showed approximately 60% homology-directed repair (HDR) efficiency in the edited population. Flow cytometry revealed a 5% increase in the G2/M cell population in the edited clone compared with unedited controls (P<0.001). Proliferation assays demonstrated significantly accelerated growth (1.30 fold) under low fetal bovine serum (FBS) conditions (P=0.029). Quantitative real-time PCR confirmed upregulation of cell cycle-promoting genes, with CCND1 and MYC expression increasing by 1.62-fold (P<0.001) and 1.23-fold (P<0.001), respectively, relative to controls. Conclusion:The genetically edited cell lines represent robust and well-defined experimental models that enable direct assessment of the functional consequences of oncogenic driver mutations on cellular behavior and signaling pathways. Our findings demonstrate that targeted genetic alterations induce measurable changes in proliferation, cell-cycle regulation, and gene expression, thereby providing mechanistic insight into tumorigenesis and the specific contribution of driver mutations to cancer-related cellular phenotypes.
There is a high prevalence of infertility worldwide (17.5% of the adult population), with male factors comprising 40% of cases. Therefore, research is vital for addressing the challenges faced by infertile men. Innovative methods, diagnosis, and treatment are necessary for male infertility, and stem cell science has brought about significant breakthroughs in its treatment. Spermatogonial stem cells (SSCs) play a crucial role in maintaining reproductive function in males, and advancements in their in vitro culture show promising developments. An array of specific growth factors and compounds that include epidermal growth factor (EGF), leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), vitamin A/retinol, testosterone, and follicle stimulating hormone (FSH) are applied to natural or artificial surfaces to mimic the testicular niche to preserve SSCs viability and induce their differentiation. These media should maintain self-renewal ability, continuously generate daughter spermatogonia, and differentiate into spermatozoa. However, several limitations and challenges exist despite the significant progress in SSC culture techniques. One limitation is the difficulty in fully replicating the intricate in vivo testicular niche. This complex microenvironment that includes the dynamic interplay of somatic cells, hormones, growth factors, and external matrix components is crucial for supporting SSC self-renewal and differentiation. Currently, in vitro systems do not adequately replicate this microenvironment, resulting in suboptimal SSC maintenance and inconsistent differentiation outcomes. This might be a key challenge for achieving complete and functional spermatogenesis in vitro. We review the foundational concepts and recent breakthroughs in in vitro spermatogenesis, focusing particularly on studies in human systems from the past twenty years. As a conclusion from this review article, developing a defined culture system that accurately mimics the in vivo niche is crucial for maintaining the functional properties of spermatogonial stem cells. The establishment of such a reliable platform is an essential prerequisite for translating in vitro spermatogenesis into clinical applications for treating infertility.
Objective:Neuroinflammation plays a crucial role in neuropathic pain, of which toll-like receptor 4 (TLR4) is a key mediator. Virtual screening identified Drospirenone as a potential TLR4 inhibitor. This study aims to evaluate the in vitro effects of Drospirenone on TLR4 signalling in U87-MG astrocytoma cells under different inflammatory conditions. Materials and Methods:In this experimental study, three treatment approaches with Drospirenone were employed to model various stages of neuroinflammation: co-incubation with lipopolysaccharides (LPS) to assess preventive effects; treatment after inflammation to evaluate its impact on sustained inflammation; and delayed treatment after LPS removal to investigate its role in reducing persistent inflammation. Expression levels of TLR4, myeloid differentiation primary response 88 (MyD88), nuclear factor-κB (NF-κB) p65, and interleukin-1 beta (IL-1β) were assessed by Western blotting, while nitric oxide (NO) secretion was measured by ELISA. Results:The potential ability of Drospirenone to inhibit early-stage inflammation was shown by significant reductions in TLR4, MyD88, NF-κBp65, IL-1β, and NO. Treatment after inflammation showed that Drospirenone reduced NO secretion but did not significantly affect TLR4 and other inflammatory markers, which could indicate its potential efficacy in controlling sustained inflammation. In the delayed treatment approach, inflammation persisted after LPS removal, and Drospirenone did not return the inflammatory state to baseline. Conclusion:Drospirenone may exhibit potential as a prophylactic agent in vitro during the early phases of neuroinflammation, though its efficacy appears limited in models of chronic or prolonged inflammation. These preliminary findings require in vivo validation, and future studies could explore possible synergistic effects with other treatments or alternative dosing strategies for neuropathic pain management.
