Fibroblast activation protein (FAP) is highly expressed in cancer-associated fibroblasts (CAFs) and has emerged as an important target for tumor imaging and therapy. Although numerous FAP-targeted PET tracers have demonstrated promising clinical performance, their high cost and limited accessibility restrict their widespread clinical application. In contrast, SPECT imaging is a more cost-effective and widely available alternative. Among the developed FAPI derivatives, FAPI-04 and FAPI-46 are two of the most extensively studied compounds with high translational potentials. In this study, we performed a systematic preclinical comparison of [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 to evaluate their imaging performance and provide guidance for optimal tracer selection in clinical SPECT. Bioinformatic analyses were performed to evaluate FAP expression and its association with prognosis and CAF-related markers. [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 were synthesized and assessed for their radiochemical purity and in vitro stability. FAP expression in FAP-HT1080 and HT1080 cells was validated using western blotting and immunofluorescence. Cellular uptake and saturation binding assays were conducted to evaluate the tracer affinity, and blocking experiments were conducted to confirm specificity. Small-animal SPECT/CT imaging and biodistribution studies were performed using FAP-HT1080 xenograft mouse models to assess in vivo tumor targeting. Immunohistochemical staining was used to further validate FAP expression in the tumor tissue. [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 exhibited high radiochemical purity and stability. FAP overexpression in FAP-HT1080 cells was confirmed by western blotting, immunofluorescence, and immunohistochemistry, and both tracers showed a high affinity for FAP. The cellular uptake of FAPI-04 was slightly higher than that of FAPI-46, and blocking experiments confirmed specific binding. SPECT/CT imaging demonstrated strong tumor accumulation with prolonged retention for both tracers, enabling clear visualization even 24 h post-injection. Quantitative analysis revealed no significant differences in tumor uptake or tumor-to-normal tissue ratios, despite the slightly higher tumor uptake of FAPI-04 observed in biodistribution studies. Consistently, comparable T/M, T/B, T/K, and T/L ratios were observed, indicating similar overall imaging performance. Bioinformatic analyses further revealed that FAP was upregulated in multiple tumors and correlated with CAF markers (COL1A1 and ACTA2). Both [99mTc]Tc-HYNIC-FAPI-04 and -46 showed high tumor uptake and clear visualization. Despite differences in pharmacokinetics, a similar tumor-to-normal tissue contrast was observed, supporting the suitability of both tracers for clinical SPECT imaging of FAP-expressing tumors.

The process of male germ cell development is a central determinant of spermatogenesis. Nevertheless, the genetic regulatory mechanisms underlying male germ cell development in mammals remain largely unclear. In this study, employing a germ cell-specific Hnrnpk knockout mouse model combined with multi-omics analyses, we identified hnRNPK as a key factor necessary for maintaining normal development in differentiating spermatogonia. Phenotypically, adult mice with germ cell-specific hnRNPK deletion exhibited infertility, characterized by a near-complete absence of spermatocytes in the seminiferous tubules. Single-cell RNA sequencing (scRNA-seq) analysis revealed that hnRNPK deletion induced cell-cycle dysregulation in differentiating spermatogonia, triggering apoptotic cell death. As a consequence, the population of differentiating spermatogonia in the testes is markedly diminished, and these cells fail to undergo proper maturation or successfully enter meiosis. Mechanistically, cytoplasmic hnRNPK exerts its regulatory function at the post-transcriptional level, regulating the translation efficiency (TE) of genes involved in meiosis, the cell cycle, and transcriptional regulation. Furthermore, hnRNPK interacts with and colocalizes with DAZL at the 40S ribosome, thereby modulating the initiation of target messenger RNA translation. In the nucleus, hnRNPK interacts with splicing factors and participates in the splicing of target genes related to germ cell differentiation and meiosis. Collectively, these findings emphasize the functional role and mechanistic involvement of hnRNPK in differentiating spermatogonia, providing valuable insights into the post-transcriptional regulatory mechanisms that govern male germ cell development.

[This corrects the article DOI: 10.1016/j.bioactmat.2024.03.013.].

BACKGROUND:The epidermal growth factor receptor pathway substrate 8 (EPS-8) is a tumor-associated antigen that is frequently overexpressed in various types of human solid tumors and is associated with aggressiveness and poor prognosis. The role of EPS-8 in cutaneous malignant melanoma and its potential mechanism remains unknown. METHODS:EPS-8 expression, mutation immune infiltration, and the tumor microenvironment in melanoma were analyzed using various databases. Clinical samples were collected and melanoma cell viability, apoptosis and protein levels were detected using cell counting kit-8, colony formation, Hoechst 33258 staining, and Western blot. Meanwhile, xenograft tumor models in nude mice were produced to evaluate the effect of EPS-8 on malignant melanoma in vivo. RESULTS:EPS-8 levels were highly expressed in melanoma and correlated with immune-infiltrating cells, immune-related scores, and immunotherapy. Additionally, in clinical malignant melanoma samples, the EPS-8 level was significantly higher in the malignant melanoma samples compared with adjacent normal tissue, and patients with a high expression of EPS-8 had significantly poor tumor differentiation and a high clinical stage. The overexpression of EPS-8 promoted the proliferation but inhibited the apoptosis of malignant melanoma cells. The knockdown of EPS-8 markedly inhibited the activation of the Hedgehog (Hh) pathway. Notably, the knockdown of Patched-1 (Ptch1) could attenuate the changes in proteins and mRNA level, cell proliferation, apoptosis, and tumor growth induced by the knockdown of EPS-8. CONCLUSION:The overexpression of EPS-8 had impacts on the proliferation and apoptosis of cutaneous malignant melanoma cells. The degradation of Ptch1 contributed to the activation of the Hh pathway induced by EPS-8.

Therapeutic liver grafts (Livergraft, 10 mm x 3 mm) were developed by biocasting bioorthogonal gelatin with in vitro-expanded primary hepatocytes (3 x 107 cells/mL). The bioorthogonal gelatin composed of methyltetrazinemodified gelatin (Gel-T) and norbornene-modified gelatin (Gel-N), which were synthesized by reacting methyltetrazine-NHS ester and cis-5-Norbornene-endo-2,3-dicarboxylic anhydride with the amino groups of gelatin, respectively. Uniform mixing of in vitro-expanded primary hepatocytes with Gel-T and Gel-N triggered an immediate bioorthogonal reaction between norbornene and methyltetrazine, resulting in solidification to form Livergraft within 10 min. The resulting Livergraft exhibited a well-defined 3D architecture with pore sizes of 100-400 mu m. Remarkably, hepatocytes within Livergraft proliferated and self-organized into cell aggregates with an average diameter of 118 mu m after three days of in vitro 3D culture. This strategy was faclie and efficient, requiring neither external triggers (e.g., UV light), Matrigel pre-incubation, nor specialized equipment. Livergraft transplantation effectively compensated liver function in mice undergoing 90 % hepatectomy, with five out of twelve mice surviving long-term. On the 5th day after hepatectomy, the caudate lobe was significantly enlarged, and the liver function indicators (ALT, AST, and ALP) were consistent with those of normal mice. Comparative characterization demonstrated that bioorthogonal gelatin outperformed GelMA in supporting hepatocyte function and tissue formation, demonstrating its potential as a promising material for functional liver tissue fabrication.

Fusobacterium nucleatum (F. nucleatum) has emerged as a potential contributor to ulcerative colitis (UC) pathogenesis, although the specific mechanisms remain incompletely understood. This study demonstrates that F. nucleatum promotes colitis by disrupting intestinal barrier integrity, inducing apoptosis in epithelial cells, and modulating inflammatory pathways. Furthermore, we demonstrate that F. nucleatum promotes STAT3 acetylation at K685, followed by phosphorylation at Y705, thereby enhancing its transcriptional activity and exacerbating colitis severity. Additionally, F. nucleatum-mediated upregulation of acetyl-CoA levels is responsible for STAT3 acetylation, linking metabolic processes to UC pathophysiology. Pharmacological inhibition of acetyl-CoA production effectively mitigates F. nucleatum-induced colitis in experimental models, suggesting potential therapeutic strategies targeting these pathways. These findings unveil a novel regulatory pathway in F. nucleatum-associated UC progression and offer new insights for future UC prevention and treatment.

BACKGROUND:Cilia are specialized microtubule-based organelles that extend from the cell surface and are classified into non-motile and motile types. The assembly and function of cilia are regulated by a complex molecular network that enables motile cilia to generate fluid flow across epithelial surfaces through coordinated beating. These motile cilia are found in the respiratory, nervous, and reproductive systems. In males, motile cilia are found in the efferent ducts and facilitate the transport of sperm from the testis to the epididymis. In females, they are mainly found in the oviducts, where they help to transport, nourish and fertilize eggs, and are also present in the endometrial epithelium. MATERIALS AND METHODS:This review compares the common factors that affect motile cilia in both male and female reproductive tracts, discusses the origin and development of multiciliated cell and cilia within the efferent ducts and oviducts, and enumerates the infertility or related reproductive diseases that may arise due to motile cilia defects. RESULTS AND DISCUSSION:In males, motile cilia in the efferent ducts create turbulence through their beating, which keeps semen suspended and prevents ductal obstruction. In females, motile cilia are distributed on the epithelia of the oviducts and the endometrium. Specifically, motile cilia in the infundibulum of the oviduct aid in capturing oocytes, while cilia in the isthmus region have been found to bind to sperm heads, facilitating the formation of the sperm reservoir. Several common factors, such as miR-34b/c and miR-449, TAp73, Gemc1, and estrogen, etc., have been shown to play crucial regulatory roles in motile cilia within the efferent ducts and oviducts, thereby further influencing fertility outcomes. CONCLUSIONS:Pathogenic mutations that disrupt ciliary function can impair ciliogenesis or alter the structure of sperm flagella, potentially resulting in infertility. Consequently, motile cilia in both the male and female reproductive tracts are crucial for fertility. There are still numerous unresolved mysteries surrounding these cilia that merit further investigation by researchers, as they hold great significance for the clinical diagnosis and treatment of infertility and related reproductive disorders.

BACKGROUND AND AIMS:While metformin has been shown to alleviate dextran sulfate sodium (DSS)-induced colitis in murine models, the mechanisms underlying its anti-inflammatory and barrier-restorative effects remain poorly defined. This study investigates the role of acetyl coenzyme A (acetyl-CoA)-dependent STAT3 acetylation in mediating metformin's therapeutic actions, with the goal of identifying novel molecular targets for ulcerative colitis (UC) treatment. METHODS:Acute colitis was induced in wild-type C57BL/6J mice via oral DSS administration, followed by daily intraperitoneal metformin treatment. Intestinal inflammation, barrier integrity, and STAT3 signaling were assessed using histopathology, western blotting, and transmission electron microscopy. To validate STAT3's critical role in colitis pathogenesis, intestinal epithelium-specific STAT3 knockout mice were employed, enabling targeted investigation of STAT3 acetylation and its regulation by metformin. RESULTS:Metformin attenuated DSS-induced colitis by suppressing pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), reducing epithelial apoptosis, and restoring tight junction proteins (ZO-1, E-cadherin, Occludin). Mechanistically, metformin reduced acetyl-CoA levels, thereby inhibiting STAT3 acetylation and downstream pathway activation. The pivotal role of STAT3 in colitis progression was confirmed using STAT3 knockout mice, as the therapeutic effects of metformin were significantly diminished in the absence of STAT3-mediated inflammatory signaling. CONCLUSION:This study identifies acetyl-CoA-dependent STAT3 acetylation as a novel mechanism through which metformin ameliorates intestinal inflammation and barrier dysfunction. These findings not only advance our understanding of metformin's immunomodulatory properties but also highlight the therapeutic potential of targeting acetyl-CoA metabolism in UC.

Protein and peptide aggregation involves the transition from soluble species to β-sheet-rich aggregates through oligomeric intermediates. While mature aggregates are well-characterized, the mechanism governing the transition from isotropic spherical nuclei to diverse nanostructures remains unclear. Here, we investigate the self-assembly of five amphipathic peptides that form distinct nanostructures. Using oligomer-resolved ion mobility-mass spectrometry and molecular dynamics simulations, we reveal that the oligomerization propensity critically determines the morphological outcomes of peptide assembly. Oligomers formed at lower critical oligomerization concentrations promote nanofiber formation, while those stabilized at higher concentrations lead to nanoribbon assembly. Molecular dynamics simulations confirm that transitional oligomers exhibit minimal solvent-accessible surface areas and maximal hydrogen bonding. These findings establish the oligomerization propensity as a key determinant in directing peptide self-assembly pathways, providing design principles for engineering peptide-based nanostructures with controlled morphologies.

N6-methyladenosine (m6A) and its reader proteins are involved in pre-mRNA processing and play a variety of roles in numerous biological processes. However, much remains to be understood about the regulation of m6A and the function of its specific readers during meiotic processes. Here, this study shows that the potential m6A reader protein hnRNPC is essential for both male and female meiosis in mice. Germ cell-specific knockout of Hnrnpc causes meiotic arrest at pachynema in male mice. Specifically, hnRNPC-deficient males show abnormal meiosis initiation and defective meiotic progression, ultimately leading to meiotic arrest at the pachytene stage. Interestingly, hnRNPC-null females show similar meiotic defects to males. Mechanistically, this study discovers that in male germ cells, hnRNPC works with HuR to directly bind and modulate alternative splicing of meiotic-related genes (e.g., Sycp1, Brca1, and Smc5) in an m6A-dependent manner during spermatogenesis. Collectively, these findings reveal hnRNPC as a critical factor for meiosis and contribute to a mechanistic understanding of the hnRNPC-HuR interaction in alternative splicing of mRNAs during germ cell development.

N6-methyladenosine (m6A) reader proteins have been demonstrated to be involved in numerous biological processes. However, the regulatory mechanism of specific m6A reader proteins during mammalian meiotic processes remains largely elusive. Here, this study identified hnRNPA2B1 as an m6A reader protein that plays a critical role in meiotic pachytene progression using a tamoxifen-induced knockout mouse model. Deletion of hnRNPA2B1 in spermatocytes disrupts homologous recombination and synapsis, with the mislocalization of double-strand break (DSB) repair proteins beyond the chromosome axes in pachytene spermatocytes. Multi-omics analyses revealed extensive dysregulation of the transcriptome and proteome in hnRNPA2B1-deficient spermatocytes, particularly affecting genes involved in chromosome organization, meiotic cell cycle, and DNA damage response, thereby triggering the pachytene checkpoint for cell elimination. In vitro luciferase assays confirmed that hnRNPA2B1 directly targets several meiosis-related transcripts (e.g., Ep400, Rrs1, etc.) in an m6A-dependent manner to regulate their expression. Furthermore, this finding demonstrates that hnRNPA2B1 biologically interacts with mRNA processing regulators and translation factors (e.g., eIF4G3, RPS3, RPL13, DDX5, YTHDC2) and functions as a post-transcriptional factor essential for pachytene progression during male meiosis. Collectively, this study underscores the critical role of the m6A reader hnRNPA2B1 in the pachytene checkpoint and advances our understanding of the regulatory mechanisms underlying male meiosis.

BackgroundThe mitosis-to-meiosis switch during spermatogenesis requires dynamic changes in gene expression. However, the regulation of meiotic transcriptional and post-transcriptional machinery during this transition remains elusive.ResultsWe report that methyltransferase-like protein 16 (METTL16), an N6-methyladenosine (m6A) writer, is required for mitosis-to-meiosis transition during spermatogenesis. Germline conditional knockout of Mettl16 in male mice impairs spermatogonial differentiation and meiosis initiation. Mechanistically, METTL16 interacts with splicing factors to regulate the alternative splicing of meiosis-related genes such as Stag3. Ribosome profiling reveals that the translation efficiency of many meiotic genes is dysregulated in METTL16-deficient testes. m6A-sequencing shows that ablation of METTL16 causes upregulation of the m6A-enriched transcripts and downregulation of the m6A-depleted transcripts, similar to Meioc and/or Ythdc2 mutants. Further in vivo and in vitro experiments demonstrate that the methyltransferase activity site (PP185-186AA) of METTL16 is necessary for spermatogenesis.ConclusionsOur findings support a molecular model wherein the m6A writer METTL16-mediated alternative splicing and translation efficiency regulation are required to control the mitosis-to-meiosis germ cell fate decision in mice, with implications for understanding meiosis-related male fertility disorders.

The development of engineered or modified autologous stem cells is an effective strategy to improve the efficacy of stem cell therapy. In this study, the stemness and functionality of adipose stem cells derived from type 1 diabetic donors (T1DM-ASC) were enhanced by treatment with Cu(II)-baicalein microflowers (Cu-MON). After treatment with Cu-MON, T1DM-ASC showed enhanced expression of the genes involved in the cytokine-cytokine receptor interaction pathway and increased cytokine secretion. Among the top 13 differentially expressed genes between T1DM-ASC and Cu-MON-treated T1DM-ASC (CMTA), some genes were also expressed in HUVEC, Myoblast, Myofibroblast, and Vascular Smooth Muscle cells, inferring the common role of these cell types. In vivo experiments showed that CMTA had the same therapeutic effect as adipose-derived stem cells from non-diabetic donors (ND-ASC) at a 15% cell dose, greatly reducing the treatment cost. Taken together, these findings suggest that Cu-MON promoted angiogenesis by promoting the stemness and functionality of T1DM-ASC and influencing multiple overall repair processes, including paracrine effects.

The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.

Meiosis in males is a critical process that ensures complete spermatogenesis and genetic diversity. However, the key regulators involved in this process and the underlying molecular mechanisms remain unclear. Here, we report an essential role of the m6A methyltransferase METTL16 in meiotic sex chromosome inactivation (MSCI), double-strand break (DSB) formation, homologous recombination and SYCP1 deposition during male meiosis. METTL16 depletion results in a significantly upregulated transcriptome on sex chromosomes in pachytene spermatocytes and leads to reduced DSB formation and recombination, and increased SYCP1 depositioin during the first wave of spermatogenesis. Mechanistically, in pachytene spermatocytes, METTL16 interacts with MDC1/SCML2 to coordinate DNA damage response (DDR) and XY body epigenetic modifications that establish and maintain MSCI, and in early meiotic prophase I, METTL16 regulates DSB formation and recombination by regulating protein levels of meiosis-related genes. Furthermore, multi-omics analyses reveal that METTL16 interacts with translational factors and controls m6A levels in the RNAs of meiosis-related genes (e.g., Ubr2) to regulate the expression of critical meiotic regulators. Collectively, this study identified METTL16 as a key regulator of male meiosis and demonstrated that it modulates meiosis by interacting with MSCI-related factors and regulating m6A levels and translational efficiency (TE) of meiosis-related genes.

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs (ncRNAs) that plays important roles in many biological processes and major cancer diagnosis and treatment, thus becoming a hot research topic. This study aims to provide an in-depth review of computational piRNA-related research, including databases and computational models. Herein, we perform literature analysis and use comparative evaluation methods to summarize and analyze three aspects of computational piRNA-related research: (i) computational models for piRNA-related molecular identification tasks, (ii) computational models for piRNA-disease association prediction tasks, and (iii) computational resources and evaluation metrics for these tasks. This study shows that computational piRNA-related research has significantly progressed, exhibiting promising performance in recent years, whereas they also suffer from the emerging challenges of inconsistent naming systems and the lack of data. Different from other reviews on piRNA-related identification tasks that focus on the organization of datasets and computational methods, we pay more attention to the analysis of computational models, algorithms, and performances that aim to provide valuable references for computational piRNA-related identification tasks. This study will benefit the theoretical development and practical application of piRNAs by better understanding computational models and resources to investigate the biological functions and clinical implications of piRNA.

Regulated cell death (RCD) represents a distinct mode of cell demise, differing from accidental cell death (ACD), characterized by specific signaling cascades orchestrated by diverse biomolecules. The regular process of cell death plays a crucial role in upholding internal homeostasis, acting as a safeguard against biological or chemical damage. Nonetheless, specific programmed cell deaths have the potential to activate an immune–inflammatory response, potentially contributing to diseases by enlisting immune cells and releasing pro-inflammatory factors. Endometriosis, a prevalent gynecological ailment, remains incompletely understood despite substantial progress in unraveling associated signaling pathways. Its complexity is intricately tied to the dysregulation of inflammatory immune responses, with various RCD processes such as apoptosis, autophagic cell death, pyroptosis, and ferroptosis implicated in its development. Notably, limited research explores the association between endometriosis and specific RCD pathways like pyroptosis and cuproptosis. The exploration of regulated cell death in the context of endometriosis holds tremendous potential for further advancements. This article thoroughly reviews the molecular mechanisms governed by regulated cell death and their implications for endometriosis. A comprehensive understanding of the regulated cell death mechanism in endometriosis has the potential to catalyze the development of promising therapeutic strategies and chart the course for future research directions in the field.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.