Testicular aging, a key feature of late-onset hypogonadism (LOH), is closely associated with Sertoli cells dysfunction. Emerging evidence implicates lipid droplet (LD) accumulation as a hallmark of aging in Sertoli cells, but its role in Sertoli cells senescence and the associated molecular mechanisms are unknown. We found that aging and obesity drove progressive LD accumulation in Sertoli cells, accompanied by mitochondrial dysfunction and ROS overproduction. Palmitic Acid (PA)-induced LD overload in vitro replicated these aging phenotypes, triggering ROS overproduction that provoked ribosome collisions and caused decreased protein synthesis globally. Moreover, LD-driven ROS disrupted mRNA translation, particularly at GA-rich sequences encoding aspartate and glutamate. Collided ribosomes activated the ZAKα-p38 axis in Sertoli cells, causing cellular senescence and impairing the blood-testis barrier. ZAKα inhibitor Nilotinib attenuated testicular atrophy, restored testosterone levels, and mitigated Sertoli cells dysfunction in aged mice. Targeting this pathway with ZAKα inhibitor offers a therapeutic strategy for age-related gonadal decline, bridging lipid metabolism dysfunction, and reproductive aging.

KRAS inhibitors (KRASi) have emerged as promising new cancer therapeutics for KRAS-mutant cancers; however, resistance remains a potential clinical challenge. Here, we show that reactivation of ERK is a hallmark of KRASi-resistant colorectal cancers (CRCs) and further demonstrate that enhancer remodeling rewires cholesterol biosynthesis through the mevalonate (MVA) pathway to confer this resistance. Mechanistically, enhancer remodeling activates MVA pathway, which facilitates the trafficking of KRAS to the membrane and sustains the MAPK signaling despite KRAS inhibition. Pharmacological inhibition of the MVA pathway with statins effectively blocks KRAS localization to the cell membrane, overcoming KRASi resistance in CRC. Together, these findings identify epigenetic-metabolic coupling of cholesterol biosynthesis as a mechanism of KRASi resistance and highlight targetable metabolic vulnerability in KRAS-mutant CRC.

Maladaptive interactions between adipocytes and immune cells drive obesity-associated inflammation, yet pharmacological interventions targeting this mechanism remain scarce. Here, we identify a disease-emergent, spatially organized, and conserved immunometabolic module within the obese white adipose tissue (WAT) microenvironment, reflecting the coordinated crosstalk between adipocytes and macrophages. By integrating single-nucleus RNA sequencing, spatial transcriptomics, and bulk RNA-seq across 247,406 nuclei from 489 individuals, we identified a transcriptionally distinct population of adipocytes with high end-of-trajectory signature (hEOS). This population, characterized by impaired adipokine secretion and defective insulin signaling, re-emerges in obesity and contributes to pathogenic WAT remodeling. These hEOS cells preferentially colocalize with a conserved macrophage subset (Mac3) within discrete niches, forming a co-adapted spatial unit that contributes to maladaptive inflammation. Mechanistically, HIF1A activation in hEOS under hypoxic conditions is associated with increased expression of the extracellular matrix protein LAMA4, which contribute to niche remodeling and is spatially associated with Mac3 macrophages. Our data further suggest that LAMA4 signals through an ITGB1-mediated pathway to promote NF-κB activation in Mac3 cells. The overall activity of this axis correlates with BMI, HbA1c, and insulin resistance in humans. Importantly, pharmacological intervention with the IKKε/TBK1 inhibitor amlexanox improved metabolic dysfunction in vivo, and this was accompanied by reduced adipocyte Lama4 expression and suppressed pro-inflammatory macrophage activation, consistent with attenuation of the hEOS-Mac3 module. Together, these findings define hEOS adipocytes as a disease-emergent, pharmacologically responsive population that is implicated in maladaptive adipocyte-macrophage crosstalk and prioritize the LAMA4-integrin-NF-κB axis as a potential therapeutic target.

Objective: To investigate anti-gastric cancer effects of gingerenone A (GA) and explore its association with the PI3K/Akt/FOXO1 signaling pathway, autophagy, and apoptosis. Methods: Cell viabilities were detected via CCK-8 assay. Proliferation, migration, invasion, and apoptosis were assessed by colony formation, EdU, Transwell assays, and flow cytometry. Autophagy, mitochondrial dysfunction, and apoptosis were detected via Western blot, immunofluorescence, and staining assays. Inhibitors and siFOXO1 were used for molecular mechanism studies. An MKN-45 xenograft model was used to evaluate in vivo efficacy and safety. Results: GA reduced gastric cancer cell proliferation, migration, and invasion, and promoted apoptosis, with MKN-45 cells being most sensitive. GA induced autophagy-related changes, mitochondrial dysfunction, and apoptosis-related changes, with suppression of PI3K/Akt signaling and reduced inhibitory phosphorylation of FOXO1; these effects were attenuated by the pharmacological and genetic interventions. In vivo, GA inhibited tumor growth without notable histopathological damage to major organs at the tested doses. Conclusions: GA may exert anti-gastric cancer effects via PI3K/Akt/FOXO1 signaling pathway, with autophagy-related changes and apoptosis-related responses. It could be a candidate compound for gastric cancer therapy.

Fibrotic diseases involve increased mechanical stress due to extracellular matrix deposition, significantly altering cellular metabolism. However, a systematic understanding of how mechanical force regulates lipid metabolism across different molecular layers remains limited. We employed an integrated multi-omics approach, including transcriptomics, Ribo-seq, proteomics, and lipidomics, to comprehensively assess the effects of mechanical stretch on lipid metabolism in human cavernous fibroblasts. Our analysis revealed that while mechanical force elicits complex multi-layered responses, post-translational regulation predominantly drives this lipid metabolic reprogramming. Lipidomics identified a significant reduction in fatty acids and an upregulation of ganglioside. Key genes central to this mechanosensitive metabolic shift were pinpointed. This study demonstrates that mechanical force hierarchically reprograms lipid metabolism. The shift from fatty acids to ganglioside underscores a key metabolic adaptation in fibroblasts. Targeting the identified critical genes may offer a promising therapeutic strategy for fibrosis-related diseases.

CDK4/6 inhibitors (CDK4/6i) have shown striking clinical potential in hormone receptor-positive breast cancers (BC) and endometrial cancers (EC), whereas resistance hinders their clinical utilization. The role of epigenetic alterations in CDK4/6i resistance remains poorly elucidated. Herein, through a comprehensive analysis of transcriptomic and chromatin profiles in 16 EC tissues and cell-based resistance models, we delineate the super-enhancer (SE) landscape and identify aldehyde dehydrogenase 1 family member A1 (ALDH1A1) as a SE-driven gene closely associated with CDK4/6i resistance. ALDH1A1 inhibition increases susceptibility to CDK4/6i in multiple EC and BC models. Mechanistically, we demonstrate that ALDH1A1 promotes vitamin A (vitA) metabolism and induces the accumulation of its downstream metabolite, retinoic acid (RA), in resistant cells. In return, the elevated RA potentiates the interaction between retinoic acid receptor alpha (RARα) and estrogen receptor alpha (ERα) in the nucleus and facilitates RARα/ERα-occupied SE-driven transcriptional activation of ALDH1A1, establishing a positive feedback loop that promotes CDK4/6i resistance. These findings highlight the crucial role of vitA metabolism in the epigenetic transcriptional program associated with CDK4/6i resistance, suggesting that avoiding high vitA intake or inhibiting the ALDH1A1-RA axis may be effective strategies to overcome this challenge.

During meiosis, ZMM proteins play essential roles in stabilizing the recombination intermediates and promoting crossover (CO) formation. In mice, shortage in chiasmata 1 (SHOC1) forms a trimeric complex with the other two ZMM proteins, SPO16 and TEX11, to bind recombination intermediates after strand invasion. Although genetic variants of SHOC1 are clinically associated with male infertility, their conserved functions in human gametogenesis remain enigmatic. Here, we delineated species-specific divergences between human and mouse SHOC1 complex and identified a missense variant within the XPF-like domain in SHOC1 (p.Q590R). This variant impaired DNA double-strand breaks repair by compromising its ability to bind branched DNA structures and the recruitment of crucial proteins to recombination intermediates, ultimately abolishing CO formation. Furthermore, the variant disrupted dynamic chromatin structure in pachytene spermatocytes and induced synapsis defects. Importantly, the XPF-like domain in SHOC1 was revealed to prevent autosome intrusion into the sex body compartment, thereby protecting critical autosomal loci from meiotic silencing of unsynapsed chromatin (MSUC). Overall, our study underscores the critical role of the XPF-like domain in human SHOC1 in CO formation and in protecting autosomes from MSUC.

During meiosis, a group of evolutionarily conserved ZMM proteins plays essential roles in stabilizing the recombination intermediates and promoting crossover (CO) formation. In mice, SHOC1 forms a trimeric complex with the other two ZMM proteins, SPO16 and TEX11, to bind recombination intermediates after strand invasion. Although genetic variants of SHOC1 are clinically associated with meiotic arrest and male infertility, their precise molecular mechanisms and evolutionarily conserved functions in human gametogenesis remain enigmatic. Here, we delineated species-specific divergences between human and mouse SHOC1 complex, and identified a missense variant within the XPF-like domain in SHOC1 (c.A1769G:p.Q590R) that was associated with meiotic arrest and non-obstructive azoospermia (NOA). The disorder of the XPF-like domain in SHOC1 impaired DNA double-strand breaks repair by compromising its ability to bind branched DNA structures and the recruitment of M1AP, REDIC1, and ZMM factors to recombination intermediates, ultimately abolishing CO formation. Furthermore, the variant disrupted dynamic 3D chromatin structure in pachytene spermatocytes and induced defects in homologous chromosome synapsis. More importantly, the XPF-like domain in SHOC1 was revealed to prevent autosome intrusion into the sex body compartment, thereby safeguarding critical autosomal loci from meiotic silencing of unsynapsed chromatin (MSUC). Overall, our study demonstrated that the XPF-like domain in SHOC1 is required for homologous recombination and safeguarding autosome from MSUC in meiosis. ### Competing Interest Statement The authors have declared no competing interest. National Key Research and Development Program of China, 2022YFC2702701, 2022YFC2703000 National Natural Science Foundation of China, 82401869, 82371616, 82371607, 82171590, 82401868 Inner Mongolia Academy of Medical Sciences Public Hospital Joint Science and Technology Project, 2023GLLH0045 Scientific Research Startup Funding for High-Level Talents of Taizhou School of Clinical Medicine Fujian Provincial Natural Science Foundation of China, 2023J05271 Scientific Research Startup Funding for High-Level Talents of Taizhou School of Clinical Medicine, TZKY2023RC01

Erectile dysfunction (ED) is an important cause of reduced quality of life for men and their partners. A common pathological feature across various types of ED, including diabetes mellitus-induced ED (DMED) and bilateral cavernous nerve injury-induced ED (CNIED), is the loss of endothelial cells (ECs) and smooth muscle cells (SMCs) in the corpus cavernosum (CC). Stem cell-based therapies have garnered attention due to their potential to differentiate into specialized cell types, offering promise for the treatment of ED. Fibroblasts (FBs), the most abundant cell type in the CC, have raised considerable interest in recent years. However, the functional role of FBs in the progression of ED remains unclear. We established DMED and CNIED animal models and performed single-cell RNA sequencing (scRNA-seq) to analyze cell subsets within the pathological environments of these two ED types. To further investigate the cellular landscape, we combined spatial transcriptomics with scRNA-seq and multiplexed immunofluorescence to identify specific FB subsets in the CC. scRNA-seq revealed a distinct subset of FBs that overexpress both Sca1 and PDGFRa. CytoTRACE analysis and Gene Set Enrichment Analysis (GSEA) indicated that PDGFRa + Sca1 + FBs may be associated with angiogenesis and possess the potential to differentiate into ECs and SMCs. Immunofluorescence analysis confirmed that PDGFRa + Sca1 + FBs were localized to the vessel walls, with co-localization of Sca1 and PDGFRa observed with markers for SMCs and ECs. Our findings shed light on the role of PDGFRa + Sca1 + FBs in the CC, demonstrating their involvement in angiogenesis and vascular repair. The depletion of these FBs in disease conditions may contribute to the exhaustion of ECs and SMCs, providing new insights into the pathogenesis of ED. These results open potential avenues for novel therapeutic strategies aimed at targeting PDGFRa + Sca1 + FBs to restore vascular function in ED.

Leydig cells’ (LCs’) senescence is an important reason for the decline of testicular function in elderly men. Cellular communication network factor 5 (CCN5) regulates lipid metabolism and cellular fibrosis through multiple mechanisms. However, its role in LCs’ aging and the underlying molecular mechanisms remain unclear. This study aimed to elucidate the effects and molecular mechanisms by which CCN5 drives aging phenotypes in LCs and to evaluate the potential of targeting CCN5 as a therapeutic strategy for testicular aging. CCN5 expression was located in LCs and elevated in aged testis. Overexpression of CCN5 led to LCs’ aging and testis dysfunction. Extracellularly, CCN5 activated β-catenin and SMAD2/3 phosphorylation, promoting the expression of fibrosis-related genes. Intracellularly, CCN5 did not affect de novo cholesterol synthesis-related genes but changed the balance of cholesterol transporters. CCN5 bound to and reduced ring finger protein 213 (RNF213) protein levels. RNF213 knockdown activated forkhead box O, p16, and p21, resulting in SA-β-gal activation, reduced cell proliferation, and lipid droplet loss. In aged mice, CCN5 knockdown improved testicular atrophy, restored lipid droplet content and testosterone synthesis, and enhanced physical endurance and sexual behavior. In summary, CCN5 drives LCs’ aging and testicular dysfunction maybe via promoting fibrosis and lipid droplet loss. Targeting CCN5 offers a promising strategy to treat testicular aging and associated reproductive endocrine disorders.

Rhabdomyolysis is a severe condition that commonly leads to acute kidney injury (AKI), with limited targeted treatments for rhabdomyolysis-induced AKI (RIAKI) adding to the challenge. Emerging evidence implicates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) in the pathological processes of various kidney diseases, but its role in RIAKI remains unclear. We applied renal tubular epithelial cell (RTEC)-specific NOX4 knockout and the NOX4 inhibitor GKT137831 to treat RIAKI in vivo and in vitro. We found that genetic and pharmacological inhibition of NOX4 protected against glycerol-induced renal dysfunction, mitigated inflammatory responses and attenuated apoptotic rates. Additionally, NOX4 blockade suppressed the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), and enhanced the activities of antioxidant enzymes. Furthermore, NOX4 inhibition reduced the expression of endoplasmic reticulum stress (ERS)-associated proteins at both the RNA and protein levels. Collectively, these findings demonstrate that genetic and pharmacological suppression of NOX4 protects against RIAKI by reducing ROS generation, boosting antioxidant defense and inhibiting ERS activation. NOX4 inhibition may offer a potential approach for developing new treatment options for RIAKI.

Testicular somatic cells play an important role in supporting spermatogenesis. Leydig cells (LCs) and peritubular myoid cells (PTMs) originate from a common progenitor population and show similar expression signatures in adulthood, making it difficult to distinguish and isolate the two in vitro. In this study, new surface markers for identifying adult LCs (ALCs) and PTMs were discovered by reanalyzing testicular single-cell dataset. Differential expressions of ITGA9 and NGFR were confirmed through immunofluorescence staining of human testes. A novel Fluorescence activated Cell Sorting (FACS) protocol is established for the isolation of ALCs and PTMs based on the two markers. Long-term culture of both cells were performed and their characteristics were characterized and explored. ITGA9+ /NGFR + cells were positive for markers of PTMs (SMA, CNN1) and negative for markers of ALCs (HSD3B, STAR), and were able to form tubular and spheroid structures in vitro. In contrast, ITGA9-/NGFR + cells were positive for ALC markers and negative for PTM markers, and showed a capacity of testosterone production in vitro. Also, both cells were negative for Sertoli cell marker SOX9. When the two cells were cultured, they can expand for more than 15 passages. Our study established a novel and efficient method for identifying and isolating human ALCs and PTMs, which provides a great potential for researches of the two cell types in human.

Mitochondrial fatty acid β-oxidation (FAO) is essential for energy production and cellular homeostasis, yet its role in sperm function has remained unclear. Through whole-exome sequencing (WES) of 800 patients with asthenozoospermia, we identified biallelic Testis-Expressed Protein 44 (TEX44) variants in six individuals, all of whom exhibited defective mitochondrial sheath assembly and impaired sperm motility. Using Tex44 knockout mice, we show that TEX44 interacts with carnitine palmitoyltransferase 1B (CPT1B) to form a mitochondrial glue, anchoring adjacent mitochondria and facilitating the assembly of the sperm-specific mitochondrial sheath. In vitro, we show that purified TEX44 protein can modulate CPT1B enzymatic activity, limiting the conversion of long-chain fatty acids such as palmitic acid and myristic acid into acyl-carnitines, thereby reducing reactive oxygen species (ROS) production. Loss of TEX44 disrupts this regulatory mechanism, leading to unregulated FAO, excessive ROS generation, and severe oxidative damage to sperm DNA and flagellar structure. Additionally, germ cell-specific Cpt1b knockout mice exhibit phenotypes similar to TEX44 deficiency, including mitochondrial sheath defects and reduced sperm motility. These findings reveal a sperm-specific mechanism by which TEX44 regulates CPT1B activity to balance FAO and ROS generation, providing critical insights into energy metabolism, mitochondrial integrity, and male infertility.

Background:Peyronie's disease (PD) is a relatively common clinical disorder of the penis that causes curvature and erectile dysfunction. However, the pathophysiological processes of PD are not well understood in current animal models and there exists limited clinical treatment options, which significantly impedes translational research. Aim:This study aimed to develop a novel rat model of PD induced by local surgical tunica albuginea trauma and compare it with the TGF-β-induced model to elucidate the scientific soundness and feasibility of the local surgical tunica albuginea trauma-induced PD model. Methods:A total of 24 male standard deviation rats were randomly allocated into three groups: sham group, surgical trauma group, and TGF-β group. The sham group received a skin incision only, whereas the surgical trauma group and the TGF-β group underwent PD model establishment via microsurgical tunica albuginea trauma and TGF-β injection, respectively. Six weeks post-modeling, penile blood perfusion, degree of curvature and erectile function were quantified. Penile tissues were subsequently harvested for histological analysis and Western blotting was used to evaluate tunica albuginea fibrosis. Outcomes:PD model of surgical tunica albuginea trauma was successfully established and exhibited more pronounced fibrotic phenotypes in the penile tunica albuginea. Results:Compared with TGF-β-induced models, laser speckle imaging revealed significantly reduced penile blood perfusion in surgical trauma group, accompanied by more severe penile curvature with corresponding angular and curvature alterations. HE and Masson's trichrome staining demonstrated marked local thickening and significantly increased collagen deposition in the penile tunica albuginea of rats in the surgical trauma group. Sirius red staining revealed a marked increase in collagen I and collagen III content. Immunofluorescence staining and Western blot analysis revealed that the surgical trauma group exhibited more pronounced alterations in the expression levels of fibrosis-related markers (Fibronectin, α-SMA, Collagen I, and Collagen III) in penile tissue. Clinical Implications:The rat model of tunica albuginea surgical trauma provides a promising option for preclinical PD research. Strengths and Limitations:The tunica albuginea surgical trauma-induced PD model established in our study has been scientifically validated. However, the precise pathogenesis of the model requires further investigation. Conclusion:The tunica albuginea surgical trauma-induced PD model was successfully established and demonstrates a more pronounced fibrotic phenotype in the penile tunica albuginea, potentially better recapitulating the pathophysiological processes of PD.

INTRODUCTION:Low-intensity pulsed ultrasound (LIPUS) is a type of specific ultrasound that is delivered at low intensity and generated in pulsed wave mode. Previous studies have demonstrated the therapeutic applications of LIPUS in the healing or regeneration of several tissues. However, research on LIPUS therapy for erectile dysfunction (ED) is still limited. OBJECTIVES:This literature review aims to summarize the clinical trials, clinical applications, and the molecular mechanisms of LIPUS in ameliorating ED. METHODS:A comprehensive literature search was conducted in PubMed and MEDLINE using the keywords: "low-intensity pulsed ultrasound," "erectile dysfunction," "treatment," "clinical trials," and "mechanism." RESULTS:LIPUS significantly improved erectile function in patients with mild-to-moderate ED in all of the clinical trials. Notably, no adverse events were reported in any of the studies. In addition, LIPUS was shown to promote erectile function mainly via the regeneration of cavernous nerves, the corpus cavernosum endothelium or smooth muscles, as well as via the activation of stem cells. CONCLUSION:LIPUS has demonstrated safety and efficacy in the treatment of ED based on clinical trials, and the mechanisms that are involved in these effects have been elucidated via basic studies. Although most clinical studies have lacked placebo-controlled groups and have included limited sample sizes, LIPUS therapy is still a promising area in ED research. However, it should be noted that the studies were conducted in China, which may limit the generalizability of the findings.

AIMS:While previous studies showed that micro-energy acoustic pulse (MAP) therapy restores urethral structure and function in adult stress urinary incontinence (SUI) rats, the underlying mechanisms remain unclear. This study aimed to explore the effect of MAP therapy in a rat model of vaginal birth injury-induced SUI at single cell resolution. METHODS:Rat model of SUI was established using vaginal balloon dilation plus ovariectomy combined with β-aminopropionitrile treatment. Subsequently, MAP therapy was administered twice weekly for a total of 4 weeks. At the end of the treatment, single-cell sequencing was performed to analyze changes of cell heterogeneity and signaling in the urethral tissue microenvironment. Functional evaluations, including leak point pressure (LPP) measurements, electromyography, and electrophysiological studies, were conducted to confirm the effects of MAP therapy on urethral repair and muscle reinnervation. RESULTS:MAP enhanced the transcription levels of various cell proliferation markers, including Pcna, Mki67, and Ccne2. The MAP treatment group exhibited higher muscle fiber content and structural integrity in the urethral sphincter compared to the untreated SUI model group. MAP also improved LPP, reduced sudden jumps in the CMAP value, and activated pathways such as "ribosome" in muscle cells. Additionally, MAP reduced senescence-associated beta-galactosidase levels in the urethral epithelium and influenced the expression of multiple transcription factors. CONCLUSIONS:MAP therapy effectively promotes urethral repair by modulating cellular aging and improving muscle reinnervation. These findings provide valuable insights into urethral repair mechanisms and lay the groundwork for developing novel therapies for SUI. CLINICAL TRIAL REGISTRATION:This study is based on an animal model and does not require a clinical trial registration.

BACKGROUND:Para-aortic lymph node metastasis (PALNM) is a rare occurrence in colorectal cancer (CRC), and the high risk of radical lymphadenectomy leads to persistent debate about the best treatment strategy. This study aims to evaluate the predictor for PALNM and the clinical value of para-aortic lymph node dissection (PALND) in CRC patients with radiologically suspected synchronous PALNM. METHODS:Patients who have synchronous radiologically suspected PALNM and underwent primary tumor resection were included. Logistic regression and receiver operating characteristic curve analysis were used to assess the predictive value of lymph node short axis in preoperative CT, identifying the optimal cut-off value. Propensity score matching and Cox regression explored factors affecting overall and disease-free survival, while Kaplan-Meier curves and decision tree models identified patient characteristics suitable for synchronous para-aortic lymph node dissection. RESULTS:A total of 578 patients were enrolled, and 125 patients received synchronous PALND. We found that simultaneous PALND significantly improved overall survival (HR, 0.56; 95% CI, 0.35-0.91; P = .019) in multivariate analysis, while disease-free survival showed no significant difference (P = .41). The short axis diameter of PALN on preoperative CT is a crucial predictor of PALNM (P < .001, AUC = 0.759) with a threshold of > 7 mm. N-stage and distant metastasis were included as independent predictors in the diagnostic model to enhance accuracy. A larger short axis diameter of PALN correlated with advanced tumor stage and poorer prognosis. Subgroup analysis revealed that PALND offers survival benefits for colorectal cancer patients at all stages with a short axis diameter >10 mm on preoperative CT (P = .037) and for stage III patients with a diameter between 7 to10 mm (P < .001, AUC = 0.810). CONCLUSION:Synchronous PALND can improve overall survival in CRC patients with suspected PALNM, with the maximum short axis diameter of PALN serving as a key criterion for selecting patients for surgery.