Human aging is associated with testosterone decline and spermatogenic defects, impacting fertility and health, yet the underlying cellular mechanisms remain poorly understood. This study aimed to establish a regulatory cell atlas of Leydig cells in aging rat testes and characterize intercellular interactions. Using single-cell RNA sequencing and immunohistochemistry, interstitial cells from young (3-month) and aged (21-month) BN rat testes were analyzed. Among 8 identified interstitial cell types, aging was not synchronous, but with remarkable heterogeneity and stochasticity. Mesenchymal cells exhibited reduced expression of genes involved in detoxification and extracellular matrix maintenance, alongside increased immune regulation genes. Endothelial cells, the most communicatively active population, showed decreased angiogenesis and cell adhesion/migration but enhanced immune responses and leukocyte chemotaxis. In immune populations, dendritic cells decreased while lymphocytes increased, with no significant change in macrophages. Transcriptomic shifts indicated a transition from innate to adaptive immunity. The loss of dendritic cells and gain of lymphocytes may be linked to CCL7 and CXCL9-12, as receptors Ccr1/Ccr5 and Cxcr3 were upregulated in lymphocytes but downregulated in dendritic cells. Overall, inflammatory activation and functional losses occurred in both immune and non-immune cells during testicular aging. This innate-to-adaptive immune shift may further compromise non-immune cell functions, accelerating testicular aging.

Postoperative peritoneal adhesion is a common complication of abdominal and pelvic surgeries, often leading to chronic pain, bowel obstruction, and increased reoperation rates. Despite clinical efforts, effective preventive strategies remain limited. In this study, we develope a sprayable, biomimetic nanotherapeutic system - designated Apo-Q - composed of phosphatidylserine (PS)-exposing liposomes encapsulating quercetin (Que@PSLs), inspired by the biological features of apoptotic bodies and prepared by a thin-film hydration-extrusion method. The obtained Apo-Q with the particle size about 140 nm and PS-functionalized surface can be selectively recognized and internalized by macrophages, thereby promoting targeted modulation of the postoperative inflammatory microenvironment. In a mouse model of peritoneal adhesion, Apo-Q promotes macrophage polarization from the pro-inflammatory M1 phenotype to the reparative M2 phenotype, suppresses inflammatory response, and also prevents pathological accumulation of GATA6+ macrophages at injury sites, significantly reducing adhesion formation and severity. These findings demonstrate that Apo-Q exerts potent anti-inflammatory and anti-fibrotic effects probably via a similar mechanism to macrophage efferocytosis, offering a minimally invasive and translationally promising strategy for preventing postoperative peritoneal adhesions.

Nanobodies (Nbs), the antigen-binding single-domain fragments derived from camelid heavy-chain antibodies (Abs), have rapidly become a focus of biomedical research due to their compact size, high stability, strong antigen affinity, and ease of molecular engineering. This review systematically outlines their structural and functional features, current strategies for acquisition, screening, optimization, and large-scale production, and comprehensively discusses their wide-ranging applications in therapeutics, diagnostics, and basic research. Specifically, Nbs have shown outstanding efficacy in tumor, toxin, infectious, and cardiovascular disease treatments, while serving as versatile tools for molecular imaging, biosensing, protein purification, structural analysis, and intracellular regulation. The challenges of immunogenicity, off-target effects, and industrial-scale manufacturing are also critically examined. Furthermore, the integration of artificial intelligence in structure prediction, de novo design, and immunogenicity assessment has opened powerful new avenues for rational Nb engineering. Combined with emerging technologies such as gene therapy, nanomaterial delivery, and multispecific architectures, these advances promise to accelerate clinical translation. Overall, Nb technology is poised to become a cornerstone of next-generation precision medicine and biotechnology, offering innovative solutions for disease diagnosis, targeted therapy, and molecular discovery.

Periodontitis-induced destruction of periodontal tissues and tooth loss remain major clinical challenges. Although periodontal regenerative therapies aim to reconstruct damaged structures, particularly the repair of alveolar bone defects, current biomaterials have limited capacity to simultaneously promote cell adhesion and osteogenic differentiation. Amelogenin (Am) plays a key role in mineralized tissue development through its highly conserved N-terminal and C-terminal regions, but studies have shown that Am has certain limitations in promoting cell adhesion. In contrast, the arginine-glycine-aspartic acid (RGD) domain of fibronectin (FN) effectively mediates cell-matrix adhesion. Based on these properties, we developed a novel recombinant fusion protein (rtAmR) by combining the conserved regions of Am with the RGD domain of FN. In vitro, rtAmR significantly promoted the adhesion and spreading of human stem cells from the apical papilla (hSCAPs) compared with the control group. Quantitative analysis showed that the number of adherent cells and the cell spreading area in the rtAmR group were 1.9-fold and 2.1-fold higher than those in the rhAm group, respectively. In osteogenic differentiation assays, rtAmR exhibited activity comparable to that of rhAm and even outperformed rhAm in terms of alkaline phosphatase (ALP) activity, collagen type I (COL I) expression, and calcium nodule formation. In a Sprague-Dawley (SD) rat alveolar bone defect model, rtAmR treatment significantly promoted bone regeneration, achieving superior bone volume/total volume (BV/TV) values compared to the rhAm and rhFN groups. Immunohistochemistry revealed that rtAmR did not obviously increase neutrophils, mast cells, or M2 macrophages versus control, confirming its biosafety and suggesting M2-independent osteogenesis. These findings suggest that rtAmR is a promising bifunctional bioactive protein for periodontal bone regeneration.

The onset of pregnancy is marked by the formation of a zygote, while the culmination of gestation is manifested by the delivery of a fetus. Meanwhile, a successful pregnancy entails a meticulously coordinated sequence of events from embryo implantation to sustained decidualization of the uterus to placental development and childbirth. The decidual reaction, a pivotal process occurring within the endometrium during pregnancy, is finely regulated by sex steroids and cytokines. Notably, fibroblast growth factors (FGFs), particularly FGF2, play a critical role in this physiological cascade. Dysregulated FGF expression may trigger inadequate decidualization, precipitating a spectrum of adverse pregnancy outcomes, including preeclampsia, recurrent implantation failure, and miscarriage. Furthermore, the human decidua, distinct from most mammalian species and similar to great apes, undergoes regular cycles of formation and shedding, independent of the presence of the embryo in the endometrium. This process is also tightly controlled by various FGFs. In this review, we comprehensively compare diverse research decidualization models, delineate the trend of endometrial FGFs during the menstrual cycle, and provide a synopsis of endometrial diseases triggered by FGF dysregulation.

Fibroblast growth factors (FGFs) play a crucial role in various biological processes, including tissue development, metabolic regulation, and injury repair. Previous studies have shown that certain peptides can exhibit similar biological functions to FGFs, whether they are fragments extracted from natural FGF molecules or derived peptides designed based on the structural characteristics of FGFs and their receptor molecules. These FGF-derived peptides have shown significant application potential in fields including tissue repair and regeneration, cancer therapy, metabolic regulation, neural recovery, and biological delivery. This article reviews the sources, bioactive functions, molecular mechanisms, and application prospects of FGF-derived peptides, aiming to provide new research ideas for subsequent structural optimization, drug delivery system development, and clinical translation of these peptides.

Inflammatory skin diseases comprise a group of skin conditions characterized by damage to skin function due to overactive immune responses. These disorders not only impair the barrier function of the skin but also deteriorate the quality of life and increase the risk of psychiatric issues. Here, a low-modulus phosphatidylserine-exposing microvesicle (deformed PSV, D-PSV) was produced, characterized, and evaluated for its potential therapeutic function against skin diseases. Compared to conventional PSVs (C-PSVs), D-PSVs exhibited a more robust and longer-lasting inhibitory effect on the inflammatory response triggered by lipopolysaccharides and interferon-γ in a primary bone marrow-derived macrophage model. Transcriptome analysis indicated that the inhibitory effect of D-PSVs was mainly achieved by modulating inflammation-related signaling pathways, leading to a reduction in the expressions of pro-inflammatory genes. In an imiquimod-induced psoriatic dermatitis mouse model, topical application of D-PSVs effectively mitigated inflammation in the skin microenvironment and reduced lesion severity. These improvements were attributed to the superior skin permeability and more persistent adhesion of D-PSVs to macrophages compared with C-PSVs. In summary, this macrophage-targeted microvesicle offers a promising non-invasive approach to managing inflammatory skin diseases by persistently inhibiting M1 macrophage polarization and restoring immune microenvironment balance.

BACKGROUND:The frequent occurrence of non-specific amplification (NSA) in Loop-mediated Isothermal Amplification (LAMP) has significantly constrained its widespread adoption. When the amount of template is limited or absent, NSA products can be randomly generated, leading to unreliable and inaccurate detection results. The mechanisms underlying NSA remain poorly elucidated, resulting in mitigation strategies that inadequately address the fundamental causes and are therefore suboptimal. RESULTS:Non-template (primers-only) real-time LAMP assays were conducted with three published and three routinely designed primer sets. All reactions demonstrated detectable NSA, thereby supporting the hypothesis that primers constitute a principal source of NSA. Comprehensive analyses of two representative primer sets included single-primer reactions and various multi-primer combinations (ranging from two to six primers) under non-template conditions, a process referred to as primer "SCAN" analysis. These systematic investigations consistently revealed the existence of at least one initiator inner primer (IIP) within each set that was capable of independently initiating NSA. The IIP was consistently identified in the most efficient NSA-amplifying combinations and acted as the primary driver of unwanted amplification events. Importantly, the introduction of unamplified oligonucleotide Blockers specifically targeting F1c/B1c sequences of the IIP significantly inhibited NSA while preserving the original binding specificity, thereby demonstrating the effectiveness and selectivity of targeted inhibition strategies. Adding only one or two Blockers could significantly inhibit NSA. The -3 and -6 shortened Blocker achieve an optimal trade-off between inhibiting NSA and preserving the efficiency of the desired reaction via extending the NSA-free window. In some -6 Blocker groups, the limit of detection (LOD) was improved, greatly enhancing the accuracy of detecting low viral load samples in early stages of infection. SIGNIFICANCE:This study confirms that certain primers, particularly IIP, play a key role in initiating NSA in LAMP. Blockers have the potential to serve as a universal strategy to effectively inhibit NSA in LAMP diagnostics. By postpone the times to positive (TTP) of primer-initiated NSA, the probabilities of false positives during a conventional detection time window (30-60 min) are reduced. In particular, some Blockers at suitable length achieved this at only minor cost of sensitivity. Blocker-LAMP approach represent a targeted strategy different from existing general additives or amplicon discrimination ones. It can be applied individually or jointly with other methods to mitigate NSA problem and improve the overall performance of LAMP assays.

Excessive dietary fat intake is closely associated with an increased risk of obesity, type 2 diabetes, cardiovascular disease, gastrointestinal diseases, and certain types of cancer. The administration of multi-strain probiotics has shown a significantly beneficial effect on the mitigation of obesity induced by high-fat diets (HFDs). In this study, Amuc_1100, an outer membrane protein of Akkermansia muciniphila, was fused with green fluorescent protein and LPXTG motif anchor protein and displayed on the surface of Lactobacillus rhamnosus (pLR-GAA) and Lactobacillus plantarum (pLP-GAA), respectively. The localization of the fusion protein on the bacterial cell surface was confirmed via fluorescence microscopy and Western blotting. Both recombinant strains demonstrated the capacity to ameliorate hyperglycemia and decrease body weight gain in a dose-dependent manner. Moreover, daily oral supplementation of pLR-GAA or pLP-GAA suppressed the HFD-induced intestinal permeability by regulating the mRNA expressions of tight junction proteins and inflammatory cytokines, thereby reducing gut microbiota-derived lipopolysaccharide concentration in serum and mitigating damage to the gut, liver, and adipose tissue. Compared with Lactobacillus rhamnosus treatment, high-dose pLR-GAA restored the expression level of anti-inflammatory factor interleukin-10 in the intestine. In conclusion, our approach enables the maintenance of intestinal health through the use of recombinant probiotics with surface-displayed functional protein, providing a potential therapeutic strategy for HFD-induced obesity and associated metabolic comorbidities.

The forest musk deer (Moschus berezovskii) is an endangered artiodactyl species. Males have a musk gland that secretes musk. Musk is the raw material for many medicines and has high economic value. In recent years, because of the impacts of illegal trade and habitat fragmentation, the wild forest musk deer has nearly been driven to extinction, and it has been listed as a key protected animal by many countries. Since 2002, the Chinese government has listed wild forest musk deer populations as first-class nationally protected animals and has conducted many artificial breeding studies. In this article, we review and summarise the biological characteristics and protective measures of wild forest musk deer, the musk synthesis mechanisms and the factors influencing musk yield. We also discuss the problems facing forest musk deer conservation and the development of musk-related medicines.

The effect of heavy metal cadmium (Cd) on testicular function is recognized. However, the mechanism involved is not well-established. In the present study, we analyzed the testicular transcriptomic changes induced by acute Cd exposure of adult rats with and without supplementation of antioxidants selenium (Se) and/or coenzyme Q10 (CoQ). Cd significantly decreased serum testosterone and two steroidogenic proteins SCARB1 and STAR. RNA-Seq analyses of testicular RNAs revealed specific activation of oxidative stress-, inflammation-, MAPK- and NF-κB-related signaling molecules. In addition, Cd treatment down-regulated gene for I, III and IV complexes of mitochondrial electron transport chain and up-regulated genes for NADPH-oxidase, major cascade in ROS production. The decrease in steroidogenesis and increase in inflammation may result from oxidative stress since supplementation of Se and CoQ, but not with either alone, almost completely prevented these changes, including overall alterations in transcriptome. Cd exposure induced total of 1192 differentially expressed genes (DEGs), which was reduced to 29 without considering confounding factors associated with Se/CoQ, a 97.6% protection rate. In conclusion, Cd exposure inhibited Leydig cell steroidogenesis by down-regulating SCARB1 and STAR through increasing oxidative stress and inflammation, but Se plus CoQ synergistically prevented all the changes induced by the Cd exposure.

Collagen is the functional protein of the skin, tendons, ligaments, cartilage, bone, and connective tissue. Due to its extraordinary properties, collagen has a wide range of applications in biomedicine, tissue engineering, food, and cosmetics. In this study, we designed a functional fragment of human type I collagen (rhLCOL-I) and expressed it in Escherichia coli (E. coli) BL21(DE3) PlysS containing a thermal-induced plasmid, pBV-rhLCOL-I. The results indicated that the optimal expression level of the rhLCOL-I reached 36.3% of the total protein at 42 °C, and expressed in soluble form. In a 7 L fermentation, the yield of purified rhLCOL-I was 1.88 g/L. Interestingly, the plasmid, pBV220-rhLCOL-I, was excellently stable during the fermentation process, even in the absence of antibiotics. Functional analyses indicated that rhLCOL-I had the capacity to promote skin cell migration and adhesion in vitro and in vivo. Taken together, we developed a high-level and low-cost approach to produce collagen fragments suitable for medical applications in E. coli.

Among the artiodactyls, male animals belonging to the Family Moschidae have a unique tissue, the musk gland, with the capability of musk synthesis. However, the genetic basis of musk gland formation and musk production are still poorly understood. Here, musk gland tissues from two juvenile and three adult Chinese forest musk deer (Moschus berezovskii) were utilized to analyze genomic evolution events, evaluate mRNA profiles and investigate cell compositions. By performing genome reannotation and comparison with 11 ruminant genomes, three expanded gene families were identified in the Moschus berezovskii genome. Transcriptional analysis further indicated that the musk gland displayed a prostate-like mRNA expression pattern. Single-cell sequencing revealed that the musk gland is composed of seven distinguishable cell types. Among them, sebaceous gland cells and luminal epithelial cells play important roles in musk synthesis, while endothelial cells master the regulation of cell-to-cell communication. In conclusion, our study provides insights into musk gland formation and the musk-synthesizing process.

Correction to: Asian Journal of Andrology 2022; 24 (4): 335-44. Doi: 10.4103/aja202193. In the published article by Li et al.,1 an error occurred in the Acknowledgments. The funding agency should be “the Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515010947)”, not “the Natural Science Foundation of Guangdong Province (No. 2021A1515010947)”. The authors sincerely apologize for the error.

Engineered nanosystems offer a promising strategy for macrophage-targeted therapies for various diseases, and their physicochemical parameters including surface-active ligands, size and shape are widely investigated for improving their therapeutic efficacy. However, little is known about the synergistic effect of elasticity and surface-active ligands. Here, two kinds of anti-inflammatory N-acetylcysteine (NAC)-loaded macrophage-targeting apoptotic-cell-inspired phosphatidylserine (PS)-containing nano-liposomes (PSLipos) were constructed, which had similar size and morphology but different Young's modulus (E) (H, ~ 100 kPa > Emacrophage vs. L, ~ 2 kPa < Emacrophage). Interestingly, these PSLipos-NAC showed similar drug loading and encapsulation efficiency, and in vitro slow-release behavior of NAC, but modulus-dependent interactions with macrophages. Softer PSLipos-L-NAC could resist macrophage capture, but remarkably prolong their targeting effect period on macrophages via durable binding to macrophage surface, and subsequently more effectively suppress inflammatory response in macrophages and then hasten inflammatory lung epithelial cell wound healing. Especially, pulmonary administration of PSLipos-L-NAC could significantly reduce the inflammatory response of M1-like macrophages in lung tissue and promote lung injury repair in a bleomycin-induced acute lung injury (ALI) mouse model, providing a potential therapeutic approach for ALI. The results strongly suggest that softness may enhance ligand-directed macrophage-mediated therapeutic efficacy of nanosystems, which will shed new light on the design of engineered nanotherapeutics.

Expression of sufficient quantities of soluble protein for structural biology and other applications is often a very difficult task, especially when multimilligram quantities are required. In order to improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single- or multi-domain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. Several methods have been described employing aspects of directed evolution to the recombinant expression of challenging proteins. These combine the construction of a random library of genetic constructs of a target with a screening or selection process to identify solubly expressing protein fragments. Here we review several datasets from the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technology to provide a view on its capabilities. Firstly, we demonstrate how it functions using the well-characterised NF-kappaB p50 transcription factor as a model system. Secondly, application of ESPRIT to the challenging PB2 subunit of influenza polymerase has led to several novel atomic resolution structures; here we present an overview of the screening phase of that project. Thirdly, analysis of the human kinase TBK1 is presented to show how the ESPRIT technology rapidly addresses the compatibility of challenging targets with the Escherichia coli expression system.

Background: aFGF content in serum and cerebrospinal fluid is increased in Alzheimer's disease (AD) patients and attenuates the activation of astrocytes.Extracellular vesicles (EVs) are a major mediator in astrocyte-neuron communications.Since excessive or persistent reactive astrocytes lead to chronic inflammation and neuronal dysfunction, and the activation of astrocytes can be inhibited by aFGF, we proposed that the cargoes of astrocyte-derived EVs (AEVs) might be modified by aFGF stimulation, playing an important role in AD progression.However, the mechanisms underlying the role of aFGF remain unclear.Methods: AEVs were isolated from damaged astrocytes, treated with or without aFGF in Aβ-loading condition, and were intranasally administered to AD mice.We determined the ability of AEVs to enter the brain, ameliorate cognitive behavior deficits, alleviate the Aβ burden in the brain, and improve synapse ultrastructure.Subsequently, the miRNAs enriched in AEVs were sequenced to identify the key molecules specifically modified by aFGF.Finally, we explored the protective effects of miR-206-3p inhibition on cognitive deficiency and its regulatory mechanism and determined its role as a specific biomarker for potential AD diagnosis.Results: AEVs stimulated by aFGF (defined as AEVs-A β+H ) had favorable neuroprotection in AD pathology by enhancing neurite growth and reduction of Aβ loading on neurons in vitro.Following intranasal administration, AEVs-A β+H ameliorated cognitive behavior deficits, promoted synaptic plasticity, and alleviated brain Aβ burden in the APP/PS1 and Aβ brain-injected mice.AEVs-A β+H showed beneficial effects on AD similar to AEVs produced in normal situations (AEVs-Ctrl).aFGF stimulation modified the cargoes in EVs derived from Aβ damaged astrocytes, the most significant of which being the down-regulation of miR-206-3p.The miR-206-3p level was specifically high in the plasma of AD mice and patients, and miR-206-3p antagomir reversed the Alzheimer phenotype in AD mice.The brain-derived neurotrophic factor (BDNF) gene was negatively regulated by miR-206-3p and upregulated by AEVs-A β+H and miR-206-3p antagomir in AD mice.AEVs-A β+H inhibited δ-secretase (Asparagine endopeptidase, AEP) activation via the miR-206-3p/BDNF axis to alleviate Aβ burden in the AD brain.Conclusion: Our findings highlight the role of aFGF in the modification of AEVs cargoes, especially miR-206-3p that can potentially serve as a biomarker for AD diagnosis and therapeutic target.

Spermatogenesis is an efficient, complex, and highly organized proliferation and differentiation process that relies on multiple factors including testosterone produced by the Leydig cells. Although the critical role played by testosterone in spermatogenesis is well recognized, the mechanism by which it works is still not completely understood, partially due to the inability to specifically and precisely monitor testosterone-dependent changes within developing germ cells. Here we present single-cell RNA sequencing data from10,983 adult rat testicular cells after the rats were treated with ethanedimethanesulfonate, which temporarily eliminates Leydig cells. The elimination and recovery of Leydig cells represented a complete testosterone depletion and restoration cycle. The dataset, which includes all developing germ cells from spermatogonia to spermatozoa, should prove useful for characterizing developing germ cells, their regulatory networks, and novel cell-specific markers. The dataset should be particularly useful for exploring the effects of the androgen environment on the regulation of spermatogenesis. As this is the first single-cell RNA-Seq dataset for rat testes, it can also serve as a reference for future studies.

Stem Leydig cells (SLCs) play a critical role in the development and maintenance of the adult Leydig cell (ALC) population. SLCs also are present in the adult testis. Their identification, characteristics, and regulation in the adult testis remain uncertain. Using single-cell RNA-seq, we found that the mesenchymal stromal population may be involved in ALC regeneration. Upon ALC elimination, a fraction of stromal cells begins to proliferate while a different fraction begins to differentiate to ALCs. Transcriptomic analysis identified five stromal clusters that can be classified into two major groups representing proliferation and differentiation populations. The proliferating group represents stem cells expressing high levels of CD90, Nes, Lum, Fn and Gap43. The differentiating group represents a progenitor stage that is ready to form ALCs, and specifically expresses Vtn, Rasl11a, Id1 and Egr2. The observation that the actively dividing cells after ALC loss were not those that formed ALCs suggests that stem cell proliferation and differentiation are regulated separately, and that the maintenance of the stromal stem cell pool occurs at the population level. The study also identified specific markers for the major interstitial cell groups and potential paracrine factors involved in the regulation of SLCs. Our data suggest a new theory about SLC identity, proliferation, differentiation, and regulation.

Testosterone production by Leydig cells (LCs) plays a crucial role in male reproduction. The functional degeneration of LCs can cause testosterone deficiency, ultimately resulting in primary male hypogonadism. Transplantation of exogenous LCs with the ability to produce testosterone in response to the regulation of the hypothalamus–pituitary–gonad axis could be a promising alternative option to treat male primary hypogonadism. Recent studies have shown that it is possible to generate Leydig-like cells from stem cells by various approaches. In addition, somatic cells, such as embryonic or adult fibroblasts, have also been successfully reprogrammed into Leydig-like cells. In this review, we summarized the recent advances in the generation of Leydig-like cells, with an emphasis on comparing the effectiveness and safety of different protocols used and the cells generated. By further analyzing the characteristics of Leydig-like cells generated from fibroblasts based on small signaling molecules and regulatory factors, we found that although the cells may produce testosterone, they are significantly different from real LCs. For future in vivo applications, it is important that the steroidogenic cells generated be evaluated not only for their steroidogenic functions but also for their overall cell metabolic state by proteomics or transcriptomic tools.