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.
