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Background: Testosterone deficiency (TD) occurs most frequently in older men and can cause many health problems. Testosterone replacement therapy (TRT) is widely used to treat TD, but this regimen can lead to a series of side effects. Stem cell therapy has been wildly studied in vitro. However, due to the multidirectional differentiation potential and heterogeneity of stem cells, it is difficult to achieve the good efficiency and reproducibility in basic research and clinical applications. This study aims to identify a new strategy for the treatment of TD. Methods: Bone marrow-derived mesenchymal stromal cells (BMSCs) were enucleated by Ficoll density gradient centrifugation. The organelles and cellular functions of enucleated BMSCs were analyzed by immunofluorescence staining and flow cytometry. Extracellular vesicles (EVs) were isolated by ultracentrifugation and characterized. For the animal studies, enucleated BMSCs were labelled with Mitotracker and injected into ethane dimethanesulfone (EDS)-treated rats. Testosterone production and spermatogenesis were detected at different time points through various tests. To determine the mechanism of efferocytosis, we analysed the number of macrophages by immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR). Results: The injection of enucleated BMSCs (Cargocytes) into the testes of EDS-treated rats restored the levels of serum testosterone, increased the number of Leydig cells (LCs), and improved spermatogenesis. We found that enucleated BMSCs underwent apoptosis earlier than BMSCs did. Subsequently, testicular interstitial macrophages phagocytosed apoptotic enucleated BMSCs through efferocytosis. Efferocytosis promoted macrophage polarization from the M1 to the M2 phenotype, reduced the expression of proinflammatory cytokines, and decreased the levels of inflammation and oxidative stress. Conclusions: In summary, this study pioneered the application of stromal cell enucleation technology to repair tissue damage in the reproductive system, explored the potential of cell burial in the treatment of reproductive system diseases and provided a new approach for the clinical treatment of male infertility. [ABSTRACT FROM AUTHOR]

Introduction: Type 2 diabetes mellitus (T2DM) is a disease that involves autoimmunity. However, how immune cells function in the peripheral blood remains unclear. Exploring T2DM biomarkers via single-cell RNA sequencing (scRNA-seq) could provide new insights into the underlying molecular mechanisms. Methods: The clinical trial registration number is ChiCTR2100049613. In this study, we included three healthy participants and three T2DM patients. The observed clinical indicators included weight and fasting blood glucose (FBG), glycosylated haemoglobin A1c (HbA1c) and fasting insulin levels. Direct separation and purification of peripheral blood mononuclear cells (PBMCs) were performed via the Ficoll density gradient centrifugation method. Immune cell types were identified via scRNA-seq. The differentially expressed genes, biological functions, cell cycle dynamics, and correlations between blood glucose indicators and genes in different cell types were analysed. Results: There were differences between the healthy and T2DM groups in terms of FBG and HbA1c (p<0.05 or p<0.01). We profiled 13,591 cells and 3188 marker genes from PBMCs. B cells, T cells, monocytes, and NK cells were grouped into 4 subclusters from PBMCs. CD4+ T cells are mainly in the memory activation stage, and CD8+ T cells are effectors. Monocytes include mainly CD14+ monocytes and FCGR3A+ monocytes. There were 119 differentially expressed genes in T cells and 175 differentially expressed genes in monocytes. Gene set enrichment analysis revealed that the marker genes were enriched in HALLMARK_ INTERFERON_GAMMA_RESPONSE and HALLMARK_TNFA_SIGNALING_VIA_ NFKB. Moreover, TNFRSF1A was identified as the core gene involved in network interactions in T cells. Discussion: Our study provides a transcriptional map of immune cells from PBMCs and provides a framework for understanding the immune status and potential immune mechanisms of T2DM patients via scRNA-seq. Clinical trial registration: http://www.chictr.org.cn , identifier ChiCTR2100049613. [ABSTRACT FROM AUTHOR]

The article published in Nature Communications discusses the oligoclonality of TRBC1 and TRBC2 in T cell lymphomas as a primary mechanism of resistance to TRBC-directed CAR T cell therapies. The distinction between TRBC1 and TRBC2 is crucial for targeting T cell lymphomas effectively, as they differ by only two amino acids. The study challenges the binary assumption of restricted TRBC1 or TRBC2 expression in peripheral T cell lymphomas and highlights the importance of clonal heterogeneity in treatment outcomes. Further research is needed to understand the mechanisms of primary resistance and improve the efficacy of TRBC-directed therapies. [Extracted from the article]

Background: Staphylococcus aureus, a known contributor to non-healing wounds, releases vesicles (SAVs) that influence the delicate balance of host-pathogen interactions. Efferocytosis, a process by which macrophages clear apoptotic cells, plays a key role in successful wound healing. However, the precise impact of SAVs on wound repair and efferocytosis remains unknown. Methods: Filtration, ultracentrifugation, and iodixanol density gradient centrifugation were used to purify the bacterial vesicles. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB) were used to characterize the vesicles. Macrophage efferocytosis efficiency was assessed using flow cytometry and confocal microscopy, while efferocytosis at wound sites was analyzed through WB, FACS, and TUNEL staining. Hematoxylin and eosin (H&E) staining and wound size measurements were used to evaluate the wound healing process. Phosphorylation of signaling pathways was detected by WB, and efferocytosis receptor expression was measured using RNA sequencing, qPCR, and flow cytometry. siRNA and pathway inhibitors were used to investigate the roles of key receptors and signaling pathways in efferocytosis. Results: We identified SAVs at infected wound sites, linking them to delayed healing of wounds. SAVs inhibit efferocytosis by activating the TLR2-MyD88-p38 MAPK signaling pathway, which regulates efferocytosis receptor genes. This activation promoted cleavage and shedding of MerTK, a crucial receptor for macrophage-driven efferocytosis. Notably, selective inhibition of p38 MAPK prevented MerTK shedding, restored efferocytosis and accelerated wound healing significantly, offering a promising therapeutic approach for chronic, non-healing wounds. Conclusion: These findings uncover a novel mechanism in S. aureus-infected wounds, highlighting how the disruption of efferocytosis via the TLR2-MyD88-p38 MAPK-MerTK axis becomes a key force behind impaired healing of wounds. Targeting this pathway could open up a new therapeutic avenue facilitating the treatment of chronic, non-healing skin injuries. [ABSTRACT FROM AUTHOR]

Objective: To evaluate whether the ZyMōt™ Multi 850 μl sperm separation device (SSD) effectively recovers motile spermatozoa from cryopreserved ejaculates and compare its effect on key embryology outcomes including fertilization, cleavage stage, and total and top-quality blastocyst formation rates to the traditional Density Gradient Centrifugation (DGC) method. Methods: In this prospective, single-center, controlled study, we used fresh sibling donor oocytes and non-donor cryopreserved ejaculates. In total, 150 couples participated in this study. At least eight MII donor oocytes were allocated to each couple split into two arms. One arm underwent ICSI with the control DGC-processed sample, and the other arm processed with SSD. Results: No significant difference on fertilization and cleavage stage embryo rates was observed between the two techniques. We observed a significant increase in the percentage of total (SSD: 74.03 ± 23.47% vs. DGC: 67.86 ± 23.92%; p = 0.016) and top-quality (SSD: 66.38 ± 24.94% vs. DGC: 60.98 ± 24.40%; p = 0.035) blastocysts formed post-SSD processing. Sub-analysis showed that this increase remained significant for the WHO-normal group (n = 118), but not for the WHO-abnormal group (n = 32). Conclusion: The SSD was successfully applied in all 150 cases, providing adequate numbers of spermatozoa to undergo ICSI. Additionally, SSD significantly improved blastocyst development rates; however, this was of limited clinical impact considering the minor improvement on the average number of top-quality blastocysts. It can be hypothesized that this positive contribution may be stronger and clinically significant when a larger number of oocytes is used or in homologous oocyte ICSI cycles, where the repair mechanisms of the oocytes may insufficient for promoting healthy embryo development. [ABSTRACT FROM AUTHOR]

Objective: Rheumatoid arthritis (RA) is an autoimmune condition that causes severe joint deformities and impaired functionality, affecting the well-being and daily life of individuals. Consequently, there is a pressing demand for identifying viable therapeutic targets for treating RA. This study aimed to explore the molecular mechanisms of osteoclast differentiation in PBMC from patients with RA through transcriptome sequencing and bioinformatics analysis.Blood samples were collected from 20 patients with RA, including 15 females and 5 males. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Osteoclast differentiation was induced using a medium containing RANKL and M-CSF for 14 days, with medium changes every 2 days. After 14 days, osteoclasts were identified by TRAP staining, and multinucleated TRAP-positive cells were counted as osteoclasts. Subsequently, transcriptome sequencing was performed using the Illumina Novaseq 6000 platform, and differential expression analysis was conducted using the DESeq2 package in R. Differentially expressed genes were selected with a significance threshold of p < 0.05 and a fold change ≥ 2 (|Log2FC|≥ 1). Bioinformatics analysis was performed using R, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.TRAP staining showed successful induction of PBMCs into osteoclasts. Transcriptome sequencing revealed a significant number of differentially expressed genes (DEGs) in the induced groups compared with the control group. GO analysis showed that these DEGs were predominantly associated with biological processes related to the transmission of chemokine signals, reactions to living organisms, and bolstering neutrophil-driven defense mechanisms. KEGG analysis showed that these DEGs were enriched by primary signaling pathways, including interactions between cytokines and their receptors, chemokine signaling pathway, cell cycle regulation, neutrophil extracellular trap formation, and TNF signaling pathway.Osteoclast differentiation of PBMC from patients with RA involves various gene alterations, multiple biological processes, and signaling pathways, providing insight into the potential mechanism of PBMC osteoclast differentiation in RA.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Methods: Rheumatoid arthritis (RA) is an autoimmune condition that causes severe joint deformities and impaired functionality, affecting the well-being and daily life of individuals. Consequently, there is a pressing demand for identifying viable therapeutic targets for treating RA. This study aimed to explore the molecular mechanisms of osteoclast differentiation in PBMC from patients with RA through transcriptome sequencing and bioinformatics analysis.Blood samples were collected from 20 patients with RA, including 15 females and 5 males. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Osteoclast differentiation was induced using a medium containing RANKL and M-CSF for 14 days, with medium changes every 2 days. After 14 days, osteoclasts were identified by TRAP staining, and multinucleated TRAP-positive cells were counted as osteoclasts. Subsequently, transcriptome sequencing was performed using the Illumina Novaseq 6000 platform, and differential expression analysis was conducted using the DESeq2 package in R. Differentially expressed genes were selected with a significance threshold of p < 0.05 and a fold change ≥ 2 (|Log2FC|≥ 1). Bioinformatics analysis was performed using R, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.TRAP staining showed successful induction of PBMCs into osteoclasts. Transcriptome sequencing revealed a significant number of differentially expressed genes (DEGs) in the induced groups compared with the control group. GO analysis showed that these DEGs were predominantly associated with biological processes related to the transmission of chemokine signals, reactions to living organisms, and bolstering neutrophil-driven defense mechanisms. KEGG analysis showed that these DEGs were enriched by primary signaling pathways, including interactions between cytokines and their receptors, chemokine signaling pathway, cell cycle regulation, neutrophil extracellular trap formation, and TNF signaling pathway.Osteoclast differentiation of PBMC from patients with RA involves various gene alterations, multiple biological processes, and signaling pathways, providing insight into the potential mechanism of PBMC osteoclast differentiation in RA.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Results: Rheumatoid arthritis (RA) is an autoimmune condition that causes severe joint deformities and impaired functionality, affecting the well-being and daily life of individuals. Consequently, there is a pressing demand for identifying viable therapeutic targets for treating RA. This study aimed to explore the molecular mechanisms of osteoclast differentiation in PBMC from patients with RA through transcriptome sequencing and bioinformatics analysis.Blood samples were collected from 20 patients with RA, including 15 females and 5 males. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Osteoclast differentiation was induced using a medium containing RANKL and M-CSF for 14 days, with medium changes every 2 days. After 14 days, osteoclasts were identified by TRAP staining, and multinucleated TRAP-positive cells were counted as osteoclasts. Subsequently, transcriptome sequencing was performed using the Illumina Novaseq 6000 platform, and differential expression analysis was conducted using the DESeq2 package in R. Differentially expressed genes were selected with a significance threshold of p < 0.05 and a fold change ≥ 2 (|Log2FC|≥ 1). Bioinformatics analysis was performed using R, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.TRAP staining showed successful induction of PBMCs into osteoclasts. Transcriptome sequencing revealed a significant number of differentially expressed genes (DEGs) in the induced groups compared with the control group. GO analysis showed that these DEGs were predominantly associated with biological processes related to the transmission of chemokine signals, reactions to living organisms, and bolstering neutrophil-driven defense mechanisms. KEGG analysis showed that these DEGs were enriched by primary signaling pathways, including interactions between cytokines and their receptors, chemokine signaling pathway, cell cycle regulation, neutrophil extracellular trap formation, and TNF signaling pathway.Osteoclast differentiation of PBMC from patients with RA involves various gene alterations, multiple biological processes, and signaling pathways, providing insight into the potential mechanism of PBMC osteoclast differentiation in RA.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Conclusions: Rheumatoid arthritis (RA) is an autoimmune condition that causes severe joint deformities and impaired functionality, affecting the well-being and daily life of individuals. Consequently, there is a pressing demand for identifying viable therapeutic targets for treating RA. This study aimed to explore the molecular mechanisms of osteoclast differentiation in PBMC from patients with RA through transcriptome sequencing and bioinformatics analysis.Blood samples were collected from 20 patients with RA, including 15 females and 5 males. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Osteoclast differentiation was induced using a medium containing RANKL and M-CSF for 14 days, with medium changes every 2 days. After 14 days, osteoclasts were identified by TRAP staining, and multinucleated TRAP-positive cells were counted as osteoclasts. Subsequently, transcriptome sequencing was performed using the Illumina Novaseq 6000 platform, and differential expression analysis was conducted using the DESeq2 package in R. Differentially expressed genes were selected with a significance threshold of p < 0.05 and a fold change ≥ 2 (|Log2FC|≥ 1). Bioinformatics analysis was performed using R, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.TRAP staining showed successful induction of PBMCs into osteoclasts. Transcriptome sequencing revealed a significant number of differentially expressed genes (DEGs) in the induced groups compared with the control group. GO analysis showed that these DEGs were predominantly associated with biological processes related to the transmission of chemokine signals, reactions to living organisms, and bolstering neutrophil-driven defense mechanisms. KEGG analysis showed that these DEGs were enriched by primary signaling pathways, including interactions between cytokines and their receptors, chemokine signaling pathway, cell cycle regulation, neutrophil extracellular trap formation, and TNF signaling pathway.Osteoclast differentiation of PBMC from patients with RA involves various gene alterations, multiple biological processes, and signaling pathways, providing insight into the potential mechanism of PBMC osteoclast differentiation in RA.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways.Key Points• A total of 1841 DEGs were obtained between the induced group and the normal group.• These DEGs were involved in multiple biological processes and signaling pathways. [ABSTRACT FROM AUTHOR]

Background Sperm selection from the population of processed spermatozoa cells after density gradient centrifugation (DGC) can assist embryologists in selecting high-quality sperm. Sperm selection of low-quality and chromatin-damaged spermatozoa is inevitable in severe teratozoospermia semen specimens. This study was conducted to evaluate whether sperm selection at ×400 magnification enables embryologists to select a population of spermatozoa with low DNA fragmentation and high sperm telomere length (STL) in semen samples with severe teratozoospermia. Methods A total of 23 infertile men characterized by severe teratozoospermia were selected. Sperm DNA fragmentation (SDF) and relative STL (r-STL) were evaluated at three stages: specimen collection, after DGC, and during the single selection of spermatozoa at ×400 magnification (single selection). The 23 patients were divided into two groups, including 14 with normal morphology ≤1% and nine with normal morphology of 2%. SDF and r-STL were compared between the two groups at three stages. Results The results of this study showed that although SDF decreased remarkably after DGC and single selection (F=64.327, P-value=0.000), the DNA fragmentation index obtained for each semen sample was more than the cutoff point of 18% based on the Halo sperm test. No statistically significant differences were observed in r-STL after DGC and single selection (F=1.978, P-value=0.163). Meanwhile, the pairwise comparison of r-STL showed that in the 2% normal morphology group, the mean relative telomere length was significantly higher in the selected spermatozoa compared to the semen specimen (P=0.014). This increase can be attributed to DGC and single selection by the embryologist. Also, there was no correlation between SDF and r-STL in the semen samples with severe teratozoospermia (r=0.01, P-value=0.964). Conclusions This study suggests that investing more time in sperm selection can decrease SDF, but r-STL of spermatozoa selected by the embryologist does not increase in severe teratozoospermia semen samples with morphology ≤1%., Competing Interests: Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd issued approval IR.SSU.MEDICINE.REC.1400.308, dated December 18, 2021. Informed consent was obtained from the infertile couples referred to the Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2025, Shakeri et al.)

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)