BACKGROUND & AIMS:Radiotherapy confers clinical benefits to hepatocellular carcinoma (HCC) patients across all stages, yet its clinical efficacy is limited by radioresistance. This study aimed to identify key regulators of HCC radiosensitivity through genome-wide functional screening. METHODS:A genome-wide CRISPR-Cas9 screen in Huh7 cells identified radiosensitivity regulators, with SRSF6 validated by siRNA knockdown and γ-H2AX assessment. Stable shRNA-mediated SRSF6 knockdown was established in Huh7 and HepG2 cells, followed by clonogenic, EdU incorporation, apoptosis, micronucleus, and comet assays. Mechanistically, RNA-seq, western blotting, mRNA stability assays, RIP-qPCR, and RAD51 overexpression rescue assays were performed. The therapeutic potential of the SRSF6 inhibitor indacaterol was evaluated using MTS assays, HCC xenograft mouse models (BALB/c-nu/nu, N=28), and HCC patient-derived organoids (PDOs) (N=3). In addition, SRSF6 expression and its correlation with patient survival were analyzed using data from The Cancer Genome Atlas and a tissue microarray (N=14 HCC and 14 paired adjacent non-tumorous liver samples). RESULTS:We identified the RNA-binding protein SRSF6 as a driver of HCC radioresistance. SRSF6 depletion enhanced the radiosensitivity of HCC cells (p<0.05-0.0001) by post-transcriptionally destabilizing the mRNAs of critical DNA repair genes (p<0.05-0.0001), thereby impairing radiation-induced DNA damage repair. The radiosensitizing effect of SRSF6 depletion was partially abrogated by ectopic overexpression of the core DNA repair protein RAD51 (p<0.05-0.001). Indacaterol exhibited cytotoxic effects on HCC cells (p<0.05-0.0001) and enhanced the antitumor efficacy of radiation in vivo (p<0.05-0.0001), as further validated across multiple HCC patient-derived organoids (p<0.05-0.0001). CONCLUSIONS:SRSF6 is a key regulator of HCC radioresistance through its post-transcriptional control of DNA repair capacity, and represents a novel therapeutic target to sensitize HCC to radiotherapy. IMPACT AND IMPLICATIONS:In this study, we performed a genome-wide CRISPR-Cas9 knockout library screen to dissect the molecular determinants governing HCC radiosensitivity, and identified RNA-binding protein SRSF6 as a driver of HCC radioresistance. We demonstrate that SRSF6 depletion disrupts the post-transcriptional stability of key DNA repair gene mRNAs and enhances HCC radiosensitivity. These findings are important for radiation oncologists and translational researchers, as they identify SRSF6-dependent RNA regulation as a critical determinant of radiotherapy response in HCC. Practically, we show that the clinically approved bronchodilator indacaterol suppresses SRSF6 function and enhances the antitumor efficacy of radiotherapy, offering a readily repurposable pharmacological strategy to overcome radioresistance. These implications are based on preclinical evidence across multiple models; however, future clinical trials are needed to validate the safety and efficacy of indacaterol-based radiosensitization in patients with HCC.

The extreme environmental conditions of the Qinhai–Tibetan Plateau (QTP) challenge livestock survival and productivity, yet little is known about how high-altitude environments impact the gut microbiota of dairy cows. To fill this gap, we systematically investigated the differences in the hindgut microbiome between 87 plateau Holstein cows and 72 plain Holstein cows using 16S rRNA gene sequencing. Our analysis revealed that the hindgut microbiota of the plateau group exhibited lower species richness but higher evenness than that in the plain group. Additionally, significant separation in hindgut microbiota composition between the two groups was observed based on altitude, while parity, days in milk, and age did not show a comparable impact. Moreover, altitude had a lasting impact on bacterial communities and their co-occurrence networks, resulting in reduced microbial interactions and lower modularity in the plateau group. Furthermore, we identified four key microbial taxa, the Bacteroidaceae and Rikenellaceae families, as well as the Prevotella and Treponema genera, which were associated with the regulation of carbohydrate digestion and energy metabolism and might help the Holstein cows adapt to the plateau environment. Our findings provide insights into strategies for enhancing the adaptability of dairy cows to high-altitude environments through microbiota modulation, which could ultimately contribute to improving livestock management and sustainability in these extreme environments.

BACKGROUND:Heat stress seriously affects the production and health of dairy cows and is a key factor limiting the sustainable development of the dairy industry. DNA methylation serves as an important epigenetic regulatory mechanism closely associated with an animal's response to heat stress. However, the specific molecular mechanism of DNA methylation in cows' heat stress response is not fully understood. RESULTS:In this study, whole genome bisulfite sequencing analysis of blood identified 49861 specific differentially methylated regions corresponding to 7613 differentially methylated genes between spring and summer dairy cows. Among them, 4069 the promoter region of differentially methylated genes were significantly enriched in key biological pathways such as substance transport, reactive oxygen species metabolism, signal transduction, and energy metabolism. By integrating the expression data of 4069 promoter differentially methylated genes, 157 genes were further screened, and their DNA methylation levels were negatively correlated with gene expression. The changes in DNLZ, GNAS, and SMAD5 genes were most significant, and network analysis showed that DNLZ gene has high connectivity in the protein-protein interaction network, indicating its potential key function in heat stress response. Experimental verification shows that under heat stress conditions, the methylation level of CpG islands in the promoter region of DNLZ gene significantly increases, and its methylation level is significantly negatively correlated with gene expression level. The Dual-luciferase reporter assays using constructs containing the DNLZ promoter reporter gene experiment further confirms that promoter methylation significantly inhibits DNLZ transcriptional activity, and the higher the degree of methylation, the stronger the inhibitory effect. CONCLUSIONS:The research results provide new insights into the mechanism of heat stress-related DNA methylation in dairy cows, clarify the key roles of genes such as DNLZ, and provide potential target genes and epigenetic markers for the cultivation of heat-resistant dairy cows.

The high diversity and complexity of the eukaryotic transcriptome make it difficult to effectively detect specific transcripts of interest. Current targeted RNA sequencing methods often require complex pre-sequencing enrichment steps, which can compromise the comprehensive characterization of the entire transcriptome. Here we describe programmable full-length isoform transcriptome sequencing (PROFIT-seq), a method that enriches target transcripts while maintaining unbiased quantification of the whole transcriptome. PROFIT-seq employs combinatorial reverse transcription to capture polyadenylated, non-polyadenylated and circular RNAs, coupled with a programmable control system that selectively enriches target transcripts during sequencing. This approach achieves over 3-fold increase in effective data yield and reduces the time required for detecting specific pathogens or key mutations by 75%. We applied PROFIT-seq to study colorectal polyp development, revealing the intricate relationship between host immune responses and bacterial infection. PROFIT-seq offers a powerful tool for accurate and efficient sequencing of target transcripts while preserving overall transcriptome quantification, with broad applications in clinical diagnostics and targeted enrichment scenarios. Zhang, Hou, Ma et al. present PROFIT-seq, a sequencing strategy that involves adaptive sampling of transcriptome libraries to enrich genes of interest and allows unbiased quantification of the whole transcriptome.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with high incidence and mortality, accounting for approximately 90% of liver cancer.The development of HCC is a complex process involving the abnormal activation or inactivation of multiple signaling pathways.Transforming growth factor-β (TGF-β)/Small mothers against decapentaplegic (SMAD) signaling pathway regulates the development of HCC.TGF-β activates intracellular SMADs protein through membrane receptors, resulting in a series of biological cascades.Accumulating studies have demonstrated that TGF-β/SMAD signaling plays multiple regulatory functions in HCC.However, there is still controversy about the role of TGF-β/SMAD in HCC.Because it involves different pathogenic factors, disease stages, and cell microenvironment, as well as upstream and downstream relationships with other signaling pathways.This review will summary the regulatory mechanism of the TGF-β/SMAD signaling pathway in HCC, involving the regulation of different pathogenic factors, different disease stages, different cell populations, microenvironments, and the interaction with microRNAs.In addition, we also introduced small molecule inhibitors, therapeutic vaccines, and traditional Chinese medicine extracts based on targeting the TGF-β/SMAD signaling pathway, which will provide future research direction for HCC therapy targeting the TGF-β/SMAD signaling pathway.

Rationale: Sma mothers against decapentaplegic homologue 4 (Smad4) is a key mediator of the transforming growth factor beta (TGF-beta) pathway and plays complex and contradictory roles in hepatocellular carcinoma (HCC). However, the specific role of Smad4 in hepatocytes in regulating hepatocarcinogenesis remains poorly elucidated. Methods: A diethylnitrosamine/carbon tetrachloride-induced HCC model was established in mice with hepatocyte-specific Smad4 deletion (Alb(Smad 4-/-)) and liver tumorigenesis was monitored. Immune cell infiltration was examined by immunofluorescence and fluorescence activated cell sorting (FACS). Cytokine secretion, glycolysis, signal pathway, and single-cell RNA sequencing were analysed for mechanism. Results: Alb(Smad4-/-) mice exhibited significantly fewer and smaller liver tumor nodules, less fibrosis, reduced myeloid-derived suppressor cell infiltration and increased CD8(+ )T cell infiltration. Smad4 deletion in hepatocytes enhanced C-X-C motif ligand 10 (CXCL10) secretion, promoting tumor necrosis factor-alpha (TNF-alpha) production in CD8(+) T cells. The loss of Smad4 activated the CXCL10/mammalian target of rapamycin (mTOR)/lactate dehydrogenase A (LDHA) pathway, which increased glycolytic activity in CD8(+ )T cells. HCC patients with high Smad4 expression exhibited decreased CD8(+) T cell infiltration and altered glycolysis. Conclusion: Our results demonstrate that Smad4 in hepatocytes promotes hepatocarcinogenesis and is a potential and candidate target for the prevention and therapy of HCC.

Circular RNAs (circRNAs) have important roles in regulating developmental processes and disease progression. As most circRNA sequences are highly similar to their cognate linear transcripts, the current short-read sequencing-based methods rely on the back-spliced junction signal for distinguishing circular and linear reads, which does not allow circRNAs’ full-length structure to be effectively reconstructed. Here we describe a long-read sequencing-based protocol, CIRI-long, for the detection of full-length circular RNAs. The CIRI-long protocol combines rolling circular reverse transcription and nanopore sequencing to capture full-length circRNA sequences. After poly(A) tailing, RNase R treatment, and size selection of polymerase chain reaction products, CIRI-long achieves an increased percentage (6%) of circular reads in the constructed library, which is 20-fold higher compared with previous Illumina-based strategies. This method can be applied in cell lines or tissue samples, enabling accurate detection of full-length circRNAs in the range of 100–3,000 bp. The entire protocol can be completed in 1 d, and can be scaled up for large-scale analysis using the nanopore barcoding kit and PromethION sequencing device. CIRI-long can serve as an effective and user-friendly protocol for characterizing full-length circRNAs, generating direct and convincing evidence for the existence of detected circRNAs. The analytical pipeline offers convenient functions for identification of full-length circRNA isoforms and integration of multiple datasets. The assembled full-length transcripts and their splicing patterns provide indispensable information to explore the biological function of circRNAs.

The signal adaptor myeloid differentiation primary response 88(MyD88)of Toll-like receptor(TLR)signaling is universally expressed in immune cells and non-immune cells,and myeloid cells play a significant role in modulating colitis.Macrophages are myeloid lineage cells which is important for maintaining intestinal homeostasis in inflammation.1 And macrophages can recognize invading pathogens through pattern recognition receptors(PRRs),such as TLRs and the nucleotide oligomerization domain(NOD)-like receptor family,pyrin domain-containing 3(NLRP3),and quickly infiltrate the injured site leading to inflammation.

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 ± 1.5% (**p < 0.01), 57.49 ± 2.4% (**p < 0.01), and 20.17 ± 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 ± 0.5% (**p < 0.01) and 15.0 ± 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis.

Objective Fetal growth restriction (FGR) is a devastating pregnancy complication that increases the risk of perinatal mortality and morbidity. This study aims to determine the combined and relative effects of genetic and intrauterine environments on neonatal microbial communities and to explore selective FGR-induced gut microbiota disruption, metabolic profile disturbances and possible outcomes. Design We profiled and compared the gut microbial colonisation of 150 pairs of twin neonates who were classified into four groups based on their chorionicity and discordance of fetal birth weight. Gut microbiota dysbiosis and faecal metabolic alterations were determined by 16S ribosomal RNA and metagenomic sequencing and metabolomics, and the long-term effects were explored by surveys of physical and neurocognitive development conducted after 2~3 years of follow-up. Results Adverse intrauterine environmental factors related to selective FGR dominate genetics in their effects of elevating bacterial diversity and altering the composition of early-life gut microbiota, and this effect is positively related to the severity of selective FGR in twins. The influence of genetic factors on gut microbes diminishes in the context of selective FGR. Gut microbiota dysbiosis in twin neonates with selective FGR and faecal metabolic alterations features decreased abundances of Enterococcus and Acinetobacter and downregulated methionine and cysteine levels. Correlation analysis indicates that the faecal cysteine level in early life is positively correlated with the physical and neurocognitive development of infants. Conclusion Dysbiotic microbiota profiles and pronounced metabolic alterations are associated with selective FGR affected by adverse intrauterine environments, emphasising the possible effects of dysbiosis on long-term neurobehavioural development.

水资源及其有效利用,关系到粮食安全和农业可持续发展.本文基于粮食主产区农户调查数据,构建反事实分析框架考察灌溉技术采用的节水效应,在此基础上,从水资源稀缺性视角检验节水效应的异质性,并进一步对灌溉水回弹效应展开讨论.结果 表明:水资源短缺、技术认知充分、家庭资本充裕以及参与合作社将提高农户灌溉技术采用的概率;从节水效应来看,高效灌溉技术可实现单位面积粮食生产节水22％～62％,但是灌溉面积扩张与种植结构变动会导致灌溉水的回弹效应,若不加以限制,节水效应将被新增用水完全抵消;此外,不同水资源禀赋水平下,灌溉技术采用对粮食生产用水的影响存在明显异质性,在水资源短缺尚未严重制约农业发展的阶段,预先推广高效灌溉技术的节水效果更优.据此,本文从政府、地区和农户3个层面提出粮食生产节水利用的对策和政策建议.

Liver fibrosis is a wound-healing response caused by the abnormal accumulation of extracellular matrix, which is produced by activated hepatic stellate cells (HSCs). Most studies have focused on the activated HSCs themselves in liver fibrosis, and whether hepatocytes can modulate the process of fibrosis is still unclear. Sma mothers against decapentaplegic homologue 4 (Smad4) is a key intracellular transcription mediator of transforming growth factor-β (TGF-β) during the development and progression of liver fibrosis. However, the role of hepatocyte Smad4 in the development of fibrosis is poorly elucidated. Here, to explore the functional role of hepatocyte Smad4 and the molecular mechanism in liver fibrosis, a CCl4-induced liver fibrosis model was established in mice with hepatocyte-specific Smad4 deletion (Smad4Δhep). We found that hepatocyte-specific Smad4 deficiency reduced liver inflammation and fibrosis, alleviated epithelial-mesenchymal transition, and inhibited hepatocyte proliferation and migration. Molecularly, Smad4 deletion in hepatocytes suppressed the expression of inhibitor of differentiation 1 (ID1) and the secretion of connective tissue growth factor (CTGF) of hepatocytes, which subsequently activated the p38 and p65 signaling pathways of HSCs in an epidermal growth factor receptor-dependent manner. Taken together, our results clearly demonstrate that the Smad4 expression in hepatocytes plays an important role in promoting liver fibrosis and could therefore be a promising target for future anti-fibrotic therapy.

This article reviews China’s agricultural development history and prospects China’s agricultural development by 2050 considering the implications of global agricultural development trends on China’s agriculture. The transformations of the overall economic structure and the rural economy should be combined to enhance agricultural labor productivity and promote the rapid development of agriculture. By 2050, the share of agriculture GDP in China’s total economy and the share of agricultural employment in China's total employment will reach convergency gradually. China’s food self-sufficiency rate will drop from the current 95% to about 90% in 2035 and will further drop by 2050. High-value agricultural products and multifunctional agriculture are the major driving forces for future agricultural growth and income raise of farmers. The strategic priorities for realizing China’s agricultural modernization by 2050 include: (1) improving agricultural productivity and ensuring national food security; (2) maximizing the comparative advantages of agricultural products; (3) promoting green, high-efficiency, and high-value agriculture; (4) guaranteeing sustainable utilization of agricultural water and soil resources; and (5) guiding modern agricultural development through institutional, policy, and investment reforms. Salient policy recommendations include: (1) increasing investment in technology and infrastructure to improve agricultural productivity; (2) enhancing the development of advantageous agriculture and agriculture with large demand potentials based on ensuring food security; (3) improving the market environment by rectifying market failures to support the development of high-value agriculture; (4) maintaining the sustainable development of agriculture; and (5) optimizing the allocation of land, labor, capital, and other agricultural production factors.

Dear Editor,Recent advances in military technology have led to the development of sophisticated and intelligent weapons, which increase mortality and morbidity. Since the advent of highexplosion weapons, "shock wave" has surpassed "shrapnel" and become the most important injurious component in conventional combat. Traumatic brain injury（TBI） is a leading cause of disability and death among military personnel and civilians during wartime[1]. Clinically, its symptoms and indications vary depending on the location and severity of the injury. The most frequently observed symptoms included altered consciousness and local dysfunction. To avoid

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl 4 -induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAP MyD88−/− mice) or aHSCs (α-SMA MyD88−/− mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.

Myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in the Toll-like receptors (TLRs) signalling pathway, is expressed in various liver cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs). And yet, the functional role of MyD88 in HSCs is poorly elucidated in alcoholic fatty liver (AFL). Here, to study the functional role of MyD88 in HSCs and the molecular mechanism related to the development of AFL, chronic-binge ethanol mouse models were established in mice with specific MyD88 knockout in quiescent (MyD88GFAP−KO) and activated HSCs (MyD88SMA−KO), respectively. Our results clearly showed an elevated expression of MyD88 in liver tissues of ethanol treated mouse model which harbours the wild type. Intriguingly, ethanol treatment profoundly inhibited inflammation in both MyD88GFAP−KO and MyD88SMA−KO mice, but the suppression of lipogenesis was only observed in MyD88GFAP−KO mice. Molecularly, our study indicated that MyD88 induced osteopontin (OPN) secretion in HSCs, which consequently resulted in activation of AKT signalling pathway and accumulation of fat in hepatocytes. Additionally, our data also suggested that OPN promoted inflammation by activating p-STAT1. Thus, targeting MyD88 may be a potentially represent a promising strategy for the prevention and treatment of AFL.

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.

Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.

为研究县域农业生产集聚形成的影响因素,采用空间杜宾模型,基于县域视角,运用环渤海区域5省市县域农业生产数据,对该区域农业生产空间集聚形成的影响因素进行分析.结果表明:第一,农业生产空间集聚具有正的空间溢出效应,表现为邻近县域农业生产集聚对本县集聚具有促进作用;第二,农业生产要素投入对本地区集聚具有促进作用,邻近县域农业资本投入对本县具有抑制作用;第三,市场需求、地区经济发展水平和交通条件对农业生产集聚具有显著影响.