BACKGROUND:Heat stress can induce skeletal muscle injury. Typical characteristics of heat-exposed muscle tissues include apoptosis, oxidative stress and autophagy. The understanding of molecular mechanisms underlying heat stress-induced muscle injury is limited, especially at the single-cell transcription level. METHODS:We collected skeletal muscles from 12-week-old female C57BL/6J mice in control (NC) and four heat stress groups. The experimental scheme comprised five groups: NC (25.5°C ± 0.5°C), HS0 (after ~3-h heat exposure, 41.5°C ± 0.5°C), HS8, HS16 and HS24 group (recovery at 25.5°C ± 0.5°C for 8, 16 and 24 h, respectively). Skeletal muscles were subjected to HE staining (n = 6), TUNEL staining (n = 3) and transmission electron microscopy (n = 3). Transcripts were measured at the tissue (n = 5 or 6) and single-nucleus levels (n = 2). RESULTS:Histologically, myofibrillar structure deformation, mitochondrial swelling and fusion, intramuscular triglyceride accumulation and autophagy occurred in the muscles subjected to heat stress. At the tissue level, a gene cluster associated with the response to heat exhibited an increasing trend of transcription in the HS0 versus NC groups, and the levels decreased to those in the NC group after 16 h. At the single-cell level, 134 320 high-quality myonuclei were collected from the muscles and annotated as seven cell types, including myonuclei, muscle stem cells (MuSCs) and immune cells. We identified the larger number of differentially expressed genes in the myonuclei. After heat stress, new cell clusters appeared in type IIa/IIx (HS8 group) and IIb (HS0 group) myonuclei but not in type I myonuclei. Generally, immediate early genes, highly expressed genes and transcription factor regulons identified in new cell clusters induced by heat were related to responses to heat, heat shock, oxidative stress and antioxidative stress. To repair the injured muscles, MuSCs highly expressed the development-related genes, such as Atp2a1, Ckm, Myh1, Aldoa, Pde4d and Pdlim5. Analysis of cell-cell communication showed that Dag1 and Egf signals associated with myonuclei were the underlying pathways that participated in repair tissues. CONCLUSIONS:We constructed the largest transcriptomic dataset, to date, for heat-exposed skeletal muscles. At tissue resolution, the response of muscles to heat stress was eliminated after a recovery period of 16 h. At single-nucleus resolution, the myofibre was the most heat-sensitive cell type, and type IIb myonuclei were the most heat-sensitive subtype of myonuclei. To our knowledge, this is the first study to reveal the molecular mechanisms underlying heat-induced muscle injury and repair at the single-cell transcriptional level.

Aging is frequently associated with a progressive loss of physiological integrity, with immunosenescence and chronic inflammation playing pivotal roles in this process. While natural compounds like Dihydromyricetin (DHM) exhibit significant anti-aging potential, its precise upstream immunomodulatory targets and cross-species conservation remain largely elusive. Through a combination of transcriptomic analyses and cross-species assays, we demonstrated that DHM systematically downregulates basal antimicrobial peptide expression to resolve chronic inflammaging in Drosophila, while simultaneously maintaining robust acute pathogen clearance. Furthermore, DHM significantly suppressed the senescence-associated secretory phenotype (SASP) in a mammalian H₂O₂-induced stress-induced premature senescence (SIPS) model. Crucially, genetic silencing revealed that the immune-regulatory kinase TAK1 (and its mammalian homolog MAP3K7) is fundamentally required for the lifespan-extending benefits of DHM in Drosophila and mediates the suppression of key SASP components, such as IL-1β and IL-8, in mammalian fibroblasts. These findings suggest that the TAK1/MAP3K7 axis serves as a crucial conserved node mediating a significant portion of DHM's cross-species anti-aging effects. This study underscores the therapeutic potential of targeting the TAK1 axis with natural compounds to combat age-related immune dysregulation.

Cold stress poses significant challenges to livestock production; however, the regulatory mechanisms underlying porcine responses to chronic cold stress remain inadequately understood. To address this gap, we characterized the transcriptional responses of five metabolism-related tissues to chronic cold stress via bulk RNA sequencing. Our findings revealed tissue-specific transcriptional responses to cold stress, with skeletal muscle-particularly the longissimus dorsi muscle (LDM)-exhibiting the most pronounced changes. Furthermore, single-nucleus RNA sequencing analysis revealed cellular heterogeneity and metabolic remodeling in the LDM, which were induced by chronic cold stress. Specifically, chronic cold stress alters the proportions of key cell types-especially myonuclei, fibroblasts, and immune cells-enhances metabolic processes such as glucose metabolism and oxidative phosphorylation, and reduces intercellular communication related to growth signaling, particularly within the IGF pathway. Notably, we identified a distinct subset of oxidative metabolizing fibroblasts in skeletal muscle, potentially regulated by transcription factors such as CREB3L1, EGR1, EGR2, BHLHE41, and KLF5, as well as the CXCL signaling pathway, in response to chronic cold stress. Moreover, increased energy intake enhances cold stress-induced metabolic strategies without allocating energy to growth, adipose storage, or immune responses. These findings elucidate the adaptive transcriptional and metabolic remodeling that occurs in pigs under prolonged cold stress, offering valuable insights into the cold adaptation mechanisms and energy allocation strategies.

BACKGROUND:Musk, secreted by the musk gland of adult male musk-secreting mammals, holds significant pharmaceutical and cosmetic potential. However, understanding the molecular mechanisms of musk secretion remains limited, largely due to the lack of comprehensive multiomics analyses and available platforms for relevant species, such as muskrat (Ondatra zibethicus Linnaeus) and Chinese forest musk deer (Moschus berezovskii Flerov). RESULTS:We generated chromosome-level genome assemblies for the 2 species of muskrat (Ondatra zibethicus Linnaeus) and musk deer (Moschus berezovskii Flerov), along with 168 transcriptomes from various muskrat tissues. Comparative analysis with 11 other vertebrate genomes revealed genes and amino acid sites with signs of adaptive convergent evolution, primarily linked to lipid metabolism, cell cycle regulation, protein binding, and immunity. Single-cell RNA sequencing in muskrat musk glands identified increased acinar/glandular epithelial cells during secretion, highlighting the role of lipometabolism in gland development and evolution. Additionally, we developed MuskDB (http://muskdb.cn/home/), a freely accessible multiomics database platform for musk-secreting mammals. CONCLUSIONS:The study concludes that the evolution of musk secretion in muskrats and musk deer is likely driven by lipid metabolism and cell specialization. This underscores the complexity of the musk gland and calls for further investigation into musk secretion-specific genetic variants.

In the original publication [...].

Mosquitoes of the Culex (Cx.) pipiens complex are vectors of severe diseases including West Nile fever by West Nile virus, Japanese encephalitis by Japanese encephalitis virus, and Lymphatic filariasis by filarial nematode Wuchereria bancrofti. As a major portion of mosquito immune system, the Toll pathway implicates in response against infections of mosquito-borne pathogens and biocontrol agents. The genetic diversity of immune-related molecules is expected to be a feasible and effective introduction to expand our knowledge of the mosquito-microbe interplay. However, a comprehensive description is currently lacking regarding the genetic characteristic of the Toll pathway molecules in Cx. pipiens complex mosquitoes. In the present study, genetic changes in Cx. pipiens complex MyD88 (Myeloid differentiation primary response protein 88) were analyzed as a precedent for the Toll pathway molecules in this taxon. MyD88 is a critical adaptor of the pathway transducing signals from TIR-containing receptors to downstream death domain-containing molecules. Our results revealed that adaptive selection has influenced the genetic changes of the molecule, giving rise to acceleration of diversity at a number of amino acid sites. The adaptively selected sites lie in the death domain, intermediate domain, and C-terminal extension. The characteristics of the genetic changes shed insights into the prominent molecular-level structural basis and the involvement strategy of the adaptor in the arms race against exogenous challenges. This finding would be beneficial for further exploration and deeper understanding of the mosquitoes' vectorial capacity and facilitating the effectiveness and sustainability of the biocontrol agents.

Global warming has intensified the threat of heat stress in pig husbandry, with the intestine emerging as a primary target of heat- induced injury. This perspective reviews recent advances in our understanding of the complex effects of heat stress on porcine intestinal health. We highlight current intervention strategies implemented in pig farming to enhance resilience against heat stress and mitigate associated intestinal damage. These strategies offer promising avenues to preserve pig health and improve productivity under rising environmental temperatures.

Ageing is an evolutionarily conserved and irreversible biological process in different species. Numerous studies have reported that taking medicine is an effective approach to slow ageing. Lemon extract (LE) is a natural extract of lemon fruit that contains a variety of bioactive phytochemicals. Various forms of LE have been shown to play a role in anti-ageing and improving ageing-related diseases. However, studies on the molecular mechanism of LE in Drosophila ageing have not been reported. In this study, we found that 0.05 g/L LE could significantly extend Drosophila lifespan and greatly improve antioxidative and anti-heat stress abilities. Furthermore, transcriptome and metabolome analyses of 10 d flies between the LE-fed and control groups suggested that the differentially expressed gene ppo1 (Prophenoloxidase 1) and metabolite L-DOPA (Levodopa) were co-enriched in the tyrosine metabolism pathway. Overall, our results indicate that affecting metabolism was the main reason for LE extending Drosophila lifespan.

A long noncoding RNA (lncRNA) is longer than 200 bp. It regulates various biological processes mainly by interacting with DNA, RNA, or protein in multiple kinds of biological processes. Adenosine monophosphate-activated protein kinase (AMPK) is activated during nutrient starvation, especially glucose starvation and oxygen deficiency (hypoxia), and exposure to toxins that inhibit mitochondrial respiratory chain complex function. AMPK is an energy switch in organisms that controls cell growth and multiple cellular processes, including lipid and glucose metabolism, thereby maintaining intracellular energy homeostasis by activating catabolism and inhibiting anabolism. The AMPK signalling pathway consists of AMPK and its upstream and downstream targets. AMPK upstream targets include proteins such as the transforming growth factor β-activated kinase 1 (TAK1), liver kinase B1 (LKB1), and calcium/calmodulin-dependent protein kinase β (CaMKKβ), and its downstream targets include proteins such as the mechanistic/mammalian target of rapamycin (mTOR) complex 1 (mTORC1), hepatocyte nuclear factor 4α (HNF4α), and silencing information regulatory 1 (SIRT1). In general, proteins function relatively independently and cooperate. In this article, a review of the currently known lncRNAs involved in the AMPK signalling pathway is presented and insights into the regulatory mechanisms involved in human ageing and age-related diseases are provided.

In recent years, the prevalence of extremely high-temperature climates has led to an increase in cases of acute heat stress (HS), which has been identified as a contributing factor to various kidney diseases. Fructose, the end product of the polyol pathway, has been linked to kidney conditions such as kidney stones, chronic kidney disease, and acute kidney injury. However, the relationship between acute HS and kidney injury caused by endogenous fructose remains unclear. The study found that acute HS triggers the production of reactive oxygen species, which in turn activate the Nrf-2 and Akr1b3 leading to an increase in endogenous fructose levels in kidney cells. It was further demonstrated that the elevated levels of endogenous fructose play a crucial role in causing damage to kidney cells. Moreover, inhibiting Nrf-2 effectively mitigated kidney damage induced by acute HS by reducing endogenous fructose levels. These findings underscore the detrimental impact of excessive fructose resulting from acute stress on kidney function, offering a novel perspective for future research on the prevention and treatment of acute HS-induced kidney injury.

Aging is a gradual process of natural change that occurs after reaching sexual maturity. It is also a known risk factor for many chronic diseases. Recent research has shown that senolytics can extend the lifespans and health spans of model organisms, and they have also been demonstrated effective in treating age-related diseases. In this study, we conducted a high-throughput screening of 156 drugs that targeted the PI3K/AKT/mTOR pathway to identify potential senolytic medications. Among these drugs, PF-04691502 was selected for further investigation to understand its molecular mechanism of action. Our findings indicate that PF-04691502, a dual inhibitor of PI3K/AKT and mTOR, specifically eliminates senescent cells. It reduces the expression levels of key markers of cellular senescence, such as SA-β-Gal, senescence-associated secretory phenotypes (SASPs) and p16INK4a. Additionally, PF-04691502 inhibits the phosphorylation of S6K and AKT, leading to the apoptosis of senescent cells. These results suggest that PF-04691502 holds promise as a new senolytic drug. This paper provides important insights into the potential application of PF-04691502 in the study of cell senescence.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

The proteins that coordinate the complex transcriptional networks of aging have not been completely documented. Protein 14-3-3zeta is an adaptor protein that coordinates signaling and transcription factor networks, but its function in aging is not fully understood. Here, we showed that the protein expression of 14-3-3zeta gradually increased during aging. High levels of 14-3-3zeta led to shortened lifespan and imbalance of intestinal immune homeostasis in Drosophila, but the decrease in 14-3-3zeta protein levels by RNAi was able to significantly promote the longevity and intestinal immune homeostasis of fruit flies. Importantly, we demonstrate that adult-onset administration of TIC10, a compound that reduces the aging-related AKT and extracellular signal-regulated kinase (ERK) signaling pathways, rescues the shortened lifespan of 14-3-3zeta-overexpressing flies. This finding suggests that 14-3-3zeta plays a critical role in regulating the aging process. Our study elucidates the role of 14-3-3zeta in natural aging and provides the rationale for subsequent 14-3-3zeta-based antiaging research.

BACKGROUND:Neurodegenerative disease is a collective term for a category of diseases that are caused by neuronal dysfunction, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Circular RNAs (circRNAs) are a class of non-coding RNAs without the 3' cap and 5' poly(A) and are linked by covalent bonds. CircRNAs are highly expressed in brain neurons and can regulate the pathological process of neurodegenerative diseases by affecting the levels of various deposition proteins.AIMS:This review is aiming to suggest that the majority of circRNAs influence neurodegenerative pathologies mainly by affecting the abnormal deposition of proteins in neurodegenerative diseases.METHODS:We systematically summarized the pathological features of neurodegenerative diseases and the regulatory mechanisms of circRNAs in various types of neurodegenerative diseases.RESULTS:Neurodegenerative disease main features include intercellular ubiquitin-proteasome system abnormalities, changes in cytoskeletal proteins, and the continuous deposition of insoluble protein fragments and inclusion bodies in the cytoplasm or nucleus, resulting in impairment of the normal physiological processes of the neuronal system. CircRNAs have multiple mechanisms, such as acting as microRNA sponges, binding to proteins, and regulating transcription. CircRNAs, which are highly stable molecules, are expected to be potential biomarkers for the pathological detection of neurodegenerative diseases such as AD and PD.CONCLUSIONS:In this review, we describe the regulatory roles and mechanisms of circRNAs in neurodegenerative diseases and aim to employ circRNAs as biomarkers for the diagnosis and treatment of neurodegenerative diseases.

tRNA-derived small RNAs (tsRNAs) are derived from tRNA and include tRNA halves (tiRNAs) and tRNA fragments (tRFs). tsRNAs have been implicated in a variety of important biological functions, such as cell growth, transcriptional regulation, and apoptosis. Emerging evidence has shown that Ago1-guided and Ago2-guided tsRNAs are expressed at 3 and 30 days in Drosophila and that tRF biogenesis in fruit flies affects tRNA processing and tRNA methylation. However, a wide analysis of tsRNA patterns in different ages of Drosophila have not been reported via the small RNA sequencing method. In the present study, tsRNAs of young (7 days) and old (42 days) Drosophila were sequenced and their expression characteristics were analysed. Then, a specific tRF (named tRF-Trp-CCA-014) was determined and was found to be conserved in fruit flies, mice, and humans. The expression patterns of tRF-Trp-CCA-014 in different tissues and stages of fruit flies and mice, and mouse NIH/3T3 cells were detected. Furthermore, mouse embryonic fibroblast NIH/3T3 cells were used as a model to analyse the function and targets of tRF-Trp-CCA-014. The RNA-seq data of six groups (Mimics, Mimic NC, Inhibitors, Inhibitor NC, Aging (adriamycin), and Control (Normal)) in mouse NIH3T3 cells were analysed. The results showed that the number of tsRNAs at 42 days (417) was more than at 7 days (288); thus, it was enriched with age. tRFs-1 were the most enriched, followed by 5′-tRFs and 3′-tRFs. Twenty-one differentially expressed tsRNAs were identified between 7 days and 42 days. Then, the conserved tRF tRF-Trp-CCA-014 was identified and found to accumulate in aged fruit flies and aged mouse NIH3T3 cells. RNA-seq data showed that most differentially expressed genes were involved in the immune system, cancer: overview, and signal translation. Furthermore, tRF-Trp-CCA-014 was found to bind to the 3′UTR of H3C4 in a dual-luciferase reporter gene assay. tRF-Trp-CCA-014 and H3C4 were detected in the cytoplasm of aged NIH3T3 cells by RNA in situ hybridization. These results suggest that the H3C4 gene is the target of tRF-Trp-CCA-014. This study will advance the current understanding of tRF roles and their implication in Drosophila and mouse studies.

Using an adult female miniature pig model with diet-induced weight gain/weight loss, we investigated the regulatory mechanisms of three-dimensional (3D) genome architecture in adipose tissues (ATs) associated with obesity. We generated 249 high-resolution in situ Hi-C chromatin contact maps of subcutaneous AT and three visceral ATs, analyzing transcriptomic and chromatin architectural changes under different nutritional treatments. We find that chromatin architecture remodeling underpins transcriptomic divergence in ATs, potentially linked to metabolic risks in obesity development. Analysis of chromatin architecture among subcutaneous ATs of different mammals suggests the presence of transcriptional regulatory divergence that could explain phenotypic, physiological, and functional differences in ATs. Regulatory element conservation analysis in pigs and humans reveals similarities in the regulatory circuitry of genes responsible for the obesity phenotype and identified non-conserved elements in species-specific gene sets that underpin AT specialization. This work provides a data-rich tool for discovering obesity-related regulatory elements in humans and pigs.

Nutrition during the developmental stages has long-term effects on adult physiology, disease and lifespan, and is termed nutritional programming. However, the underlying molecular mechanisms of nutritional programming are not yet well understood. In this study, we showed that developmental diets could regulate the lifespan of adult Drosophila in a way that interacts with various adult diets during development and adulthood. Importantly, we demonstrated that a developmental low-yeast diet (0.2SY) extended both the health span and lifespan of male flies under nutrient-replete conditions in adulthood through nutritional programming. Males with a low-yeast diets during developmental stages had a better resistance to starvation and lessened decline of climbing ability with age in adulthood. Critically, we revealed that the activity of the Drosophila transcription factor FOXO (dFOXO) was upregulated in adult males under developmental low-nutrient conditions. The knockdown of dFOXO, with both ubiquitous and fat-body-specific patterns, can completely abolish the lifespan-extending effect from the larval low-yeast diet. Ultimately, we identify that the developmental diet achieved the nutritional programming of the lifespan of adult males by modulating the activity of dFOXO in Drosophila. Together, these results provide molecular evidence that the nutrition in the early life of animals could program the health of their later life and their longevity.

As Drosophila is an extensively used genetic model system, understanding of its regulatory networks has great significance in revealing the genetic mechanisms of ageing and human diseases. Competing endogenous RNA (ceRNA)-mediated regulation is an important mechanism by which circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) regulate ageing and age-related diseases. However, extensive analyses of the multiomics (circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA) characteristics of adult Drosophila during ageing have not been reported. Here, differentially expressed circRNAs and microRNAs (miRNAs) between 7 and 42-day-old flies were screened and identified. Then, the differentially expressed mRNAs, circRNAs, miRNAs, and lncRNAs between the 7- and 42-day old flies were analysed to identify age-related circRNA/miRNA/mRNA and lncRNA/miRNA/mRNA networks in ageing Drosophila. Several key ceRNA networks were identified, such as the dme_circ_0009500/dme_miR-289-5p/CG31064, dme_circ_0009500/dme_miR-289-5p/frizzled, dme_circ_0009500/dme_miR-985-3p/Abl, and XLOC_027736/dme_miR-985-3p/Abl XLOC_189909/dme_miR-985-3p/Abl networks. Furthermore, real-time quantitative PCR (qPCR) was used to verify the expression level of those genes. Those results suggest that the discovery of these ceRNA networks in ageing adult Drosophila provide new information for research on human ageing and age-related diseases.

Slow lorises are small arboreal and nocturnal primates. Due to the illegal trade, a large number of slow lorises were confiscated into wildlife sanctuaries or rescue centers. The re-release has been considered a preferable approach for alleviating the captive pressure, but inappropriate and long-term confinement make it difficult to achieve this goal. In this study, we investigated and compared the fecal and oral microbiome of Bengal slow lorises (Nycticebus bengalensis) under long-term captivity (LC) and short-term captivity (SC) groups based on 16s rRNA high-throughput gene sequencing. The oral microbiome displayed higher Chao1 richness but lower Shannon and Simpson indices than the fecal microbiome. The Bengal slow lorises under long-term captivity had abundant pathogenic genera in both gut and oral microbiomes, such asDesulfovibrio,Actinomyces,Capnocytophaga,Neisseria, andFusobacterium, while some specific bacterial taxa associated with intestinal balance were more enriched in the SC group. Due to the plant gum scarcity in the diet, both groups had a low abundance ofBifidobacterium. Function profile prediction indicated that the LC group was enriched with genetic information processing and metabolism pathways due to the stable food intake. The increased membrane transport and xenobiotic metabolism and degradation functions in the SC group could be explained by the function of the host microbiome in facilitating adaptation to changing environments and diets. The results demonstrated that the oral microbiome had the potential to be used as a regular surveillance tool. Also, current captive management should be improved to ensure reintroduction success.