Abstract The b 0,+ transporter ( SLC3A1 / SLC7A9 ) imports cystine and selenocystine at the enterocyte apical membrane and defines a selenium-utilization program in the healthy human intestine (He et al., bioRxiv 2026); how this program is altered in disease is unknown. Using a multi-cohort single-cell framework with donor-level statistics, we find that inflammation and cancer remodel a single epithelial redox-currency axis in opposite directions. In adult Crohn’s disease, b 0,+ co-expression in small-intestinal enterocytes was suppressed (median 4.9% vs 34% positive; P = 0.008) but preserved in paediatric IBD; this loss reflected replacement of b 0,+ -high mature enterocytes by a dedifferentiated state rather than transcriptional disruption, with the SLC7A9 - SELENOP coupling intact. Downstream, GPX4 alone was selectively suppressed while the selenocysteine-incorporation machinery was preserved, priming the epithelium for ferroptosis in a severity-graded manner. Inflammatory cytokines (IFNγ, TNF) suppressed the selenium pole in small-intestinal enteroids, and a network knockout indicated that b 0,+ supports the selenoproteome through substrate supply rather than transcriptional control; an independent patient proteome and a Caco-2 polarization model corroborated the axis. Colorectal-cancer colonocytes showed the opposing pole, near-absent b 0,+ with induction of the xCT/thiol program ( P = 5.6 × 10 −5 ) and a ferroptosis-resistant configuration. Thus b 0,+ marks the supply node of a dual-pole selenium-thiol axis: inflammation collapses the selenium pole toward a ferroptosis-prone dedifferentiated state, whereas cancer bypasses it toward an xCT antioxidant pole. The baseline axis state is an exploratory correlate of anti-TNF response and a candidate point of disease-associated vulnerability.

Abstract Selenium is an essential trace element incorporated into selenoproteins as selenocysteine, yet the intestinal cellular programs associated with selenium utilization remain poorly defined. Here, we performed a large-scale single-cell transcriptomic analysis of 169,068 human enterocytes from 105 donors to systematically profile nine candidate selenium transporter systems and their relationship to selenoprotein expression. Among all transporters examined, the b 0+ amino acid transporter system (SLC3A1/SLC7A9) showed the strongest and most maturation-independent association with selenoproteins, including SELENOP (Spearman r = 0.489) and GPX4 ( r = 0.392), with enrichment across 21 of 24 detected selenoproteins in b 0+ -complete versus transporter-negative enterocytes. These associations were robust across individual donors and confirmed by pseudobulk validation, and were only partially explained by enterocyte maturation state, intestinal segment, and sequencing depth after covariate adjustment, indicating both differentiation-dependent and differentiation-independent components. Furthermore, enterocytes carrying the b 0+ complex expressed a non-canonical LRP repertoire (LRP1, LRP5 and LRP6) rather than the canonical SELENOP receptors LRP2 and LRP8; this co-expression was maturation-dependent, nominating these LRPs as candidate intestinal SELENOP-handling receptors. Together, these single-cell data identify b 0+ transporter expression as a marker of a selenoprotein-enriched enterocyte state in the human intestine.

OBJECTIVES:Cystine stones account for 1%‒2% of adult and up to 10% of pediatric kidney stones. They result from cystinuria, an autosomal recessive disorder caused by mutations in solute carrier family 3 member 1 (SLC3A1) and SLC7A9, which encode the renal cystine transporter subunits. These mutations impair cystine reabsorption, raising urinary cystine levels and driving stone formation. Current diagnostic options remain limited in terms of detecting molecular dysfunctions. Thus, we aimed to develop a nonradioactive, cell-based method for the functional assessment of cystine transporters and mutation-specific pathologies. METHODS:Using human embryonic kidney 293 (HEK293) cells transiently co-expressing wild-type or mutant SLC3A1 and SLC7A9, we developed an integrated approach that combined a selenocystine-based fluorescence uptake assay with AlphaFold3-based structural predictions to rapidly and accurately assess cystine transporter function and the molecular impact of genetic mutations. RESULTS:The affinity of the SLC3A1/SLC7A9 complex was comparably apparent for selenocystine (Michaelis constant Km=(156.3±24.2) μmol/L) and cystine (literature Km approximately 200 μmol/L). Using operational thresholds (mild >60%, moderate 20%‒60%, severe <20% residual activity), the assay differentiated the functional impacts of eight clinically characterized variants, including SLC7A9 A70V, A182T, G105R, R333W, V170M, A354T, and P482L, and SLC3A1 M467T, with categorical assignments consistent with previously published radioisotope-based functional data. AlphaFold3 modeling, combined with molecular docking, provides mechanistic interpretations of the dysfunction observed in the P482L and A354T mutants. CONCLUSIONS:The integrated approach employed in this work, which combines a sensitive selenocystine fluorescence assay with artificial intelligence (AI)-powered structural analysis, enables the rapid, precise diagnosis of cystinuria variants. This platform is compatible with standard microplate-reader infrastructure and offers potential utility in variant-interpretation pipelines and future genotype-guided therapeutic decision-making, pending prospective clinical validation.

MEGDHEL syndrome is a severe mitochondrial disorder caused by mutations in the SERAC1 gene, characterized by sensorineural deafness, encephalopathy, hepatopathy, and Leigh-like syndrome. A hallmark feature is neonatal liver failure, often leading to high mortality. There is currently no effective treatment. In this study, we used AAV9-SERAC1 gene therapy to address liver dysfunction and mitochondrial impairments in the Serac1-/- mouse model. Treatment with 4 × 1011 viral genomes led to improvements in liver histology, including reduced fatty degeneration and cholesterol accumulation, as well as enhanced mitochondrial morphology and function. Transmission electron microscopy revealed restored mitochondrial cristae and an increased number of mitochondria in treated mice. Respiratory complex showed activity recovery and mitochondrial DNA content was increased. Behavioral assessments also demonstrated significant improvements in motor coordination, with treated mice showing enhanced grasping strength and balance compared to controls. These findings suggest that AAV9-SERAC1 gene therapy can improve liver function and locomotor abilities in Serac1-/- mice, offering a promising therapeutic strategy for MEGDHEL syndrome.

Background Cystine stones, a rare but recurrent type of kidney stones, primarily result from cystinuria, an inherited disorder caused by mutations in the genes SLC3A1 and SLC7A9 , which encode renal cystine transporters. These mutations impair cystine reabsorption, resulting in elevated urinary cystine concentrations and stone formation. Current diagnostic methods are limited, particularly for detecting molecular-level dysfunctions. We aimed to develop a nonradioactive method for functional assessment of cystine transporters and mutation-specific pathologies. Methods We designed an innovative diagnostic approach combining a selenocystine-based fluorescence assay with structural predictions using AlphaFold to rapidly and accurately assess cystine transporter function and the molecular impacts of genetic mutations. Results Our assay demonstrated comparable transport efficiencies of cystine and selenocystine by the SLC3A1/SLC7A9 complex, and effectively differentiated mild, moderate, and severe functional impairments associated with known clinical mutations, including A354T and P482L. Structural modeling further provided mechanistic insights into mutation-induced dysfunctions. Conclusion This integrated approach—combining a sensitive selenocystine fluorescence assay with AI-powered structural analysis—enables rapid, precise diagnosis of cystinuria variants and delivers mechanistic insights for personalized therapeutic strategies. ### Competing Interest Statement The authors have declared no competing interest. Zhejiang Provincial Natural Science Foundation of China, LMS25H160004 Zhejiang Provincial Medical and Health Science and Technology Programs, 2023KY650, 2021KY080

Proteins, as the primary executors of physiological activity, serve as a key factor in disease diagnosis and treatment. Research into their structures, functions, and interactions is essential to better understand disease mechanisms and potential therapies. DeepMind’s AlphaFold2, a deep-learning protein structure prediction model, has proven to be remarkably accurate, and it is widely employed in various aspects of diagnostic research, such as the study of disease biomarkers, microorganism pathogenicity, antigen-antibody structures, and missense mutations. Thus, AlphaFold2 serves as an exceptional tool to bridge fundamental protein research with breakthroughs in disease diagnosis, developments in diagnostic strategies, and the design of novel therapeutic approaches and enhancements in precision medicine. This review outlines the architecture, highlights, and limitations of AlphaFold2, placing particular emphasis on its applications within diagnostic research grounded in disciplines such as immunology, biochemistry, molecular biology, and microbiology.

Hepatocellular carcinoma (HCC) is a prevalent type of liver cancer, and CD24 gene is reportedly involved in HCC progression. However, the precise regulatory mechanisms of CD24 in HCC remain unclear. In this study, we established a primary HCC mouse model and observed that CD24, induced by inactivation of the Hippo pathway, was highly expressed in HCC. Using a systematic molecular and genomic approach, we identified the Hippo-YAP1-SOX4 pathway as the mechanism through which YAP1 induces CD24 upregulation in HCC cells. CD24 knockdown significantly attenuated YAP1 activation-induced HCC. These findings shed light on the link between CD24 and HCC progression, particularly in the Hippo-inactivated subclass of HCC. Therefore, CD24 may serve as a potential target for specific treatment of this HCC subclass.

Respiratory infectious viruses, including SARS‐CoV‐2, undergo rapid genetic evolution, resulting in diverse subtypes with complex mutations. Detecting and differentiating these subtypes pose significant challenges in respiratory virus surveillance. To address these challenges, we integrated ARMS‐PCR with molecular beacon probes, allowing selective amplification and discrimination of subtypes based on adjacent mutation sites. The method exhibited high specificity and sensitivity, detecting as low as 104 copies/mL via direct fluorescence analysis and ~106 copies/mL using real‐time PCR. Our robust detection approach offers a reliable and efficient solution for monitoring evolving respiratory infections, aiding early diagnosis and control measures. Further research could extend its application to other respiratory viruses and optimize its implementation in clinical settings.

Protein structure prediction is an important research field in life sciences and medicine, and it is also a key application scenario of artificial intelligence in scientific research. AlphaFold2 is a protein structure prediction system developed by DeepMind based on deep learning, capable of efficiently generating the atomic-scale spatial structure of a protein from the amino acid sequence. It has demonstrated superior performance in the prediction of protein structures since its inception, thus attracting much attention and research. This paper introduces the model architecture, highlights, limitations, and application progress of AlphaFold2. Furthermore, it briefs the capabilities, highlights, and limitations of several other types of protein structure prediction models and prospects the future development direction in this field.

Feeding behavior, the most fundamental physiological activity, is controlled by two opposing groups of factors, orexigenic and anorexigenic factors. The sulfakinin family, an insect analogue of the mammalian satiety factor cholecystokinin (CCK), has been shown to suppress food intake in various insects. Nevertheless, the mechanisms through which sulfakinin regulates feeding behavior remain a biological question. This study aimed to elucidate the signaling pathway mediated by the anorexigenic peptide sulfakinin in Bombyx mori. We identified the Bombyx mori neuropeptide G protein-coupled receptor A9 (BNGR-A9) as the receptor for sulfakinin through functional assays. Stimulation with sulfakinin triggered a swift increase in intracellular IP3, Ca2+, and a notable enhancement of ERK1/2 phosphorylation, in a manner sensitive to a Gαq-specific inhibitor. Treatment with synthetic sulfakinin resulted in decreased food consumption and average body weight. Additionally, administering synthetic sulfakinin to silkworms significantly elevated hemolymph trehalose levels, an effect markedly reduced by pre-treatment with BNGR-A9 dsRNA. Consequently, our findings establish the sulfakinin/BNGR-A9 signaling pathway as a critical regulator of feeding behavior and hemolymph trehalose homeostasis in Bombyx mori, highlighting its roles in the negative control of food intake and the positive regulation of energy balance.

Background: Respiratory infectious viruses, such as the novel coronavirus SARS-CoV-2, are highly transmissible and undergo rapid genetic evolution, which leads to the emergence of multiple subtypes with diverse mutation patterns. However, detecting and differentiating between these subtypes present significant challenges in the field of respiratory virus surveillance. Methods: To address these challenges, we developed a novel detection approach that integrates amplification refractory mutation system PCR (ARMS-PCR) with molecular beacon probes. The ARMS-PCR primers were designed to selectively amplify specific subtypes by targeting adjacent mutation sites, while the molecular beacon probes allowed for further discrimination of the amplified products. This combined approach effectively addressed the issues of non-specific binding and improved detection accuracy. Results: Our method demonstrated high specificity and sensitivity in the identification and differentiation of respiratory virus subtypes. Using real-time fluorescence PCR, we achieved a detection limit of approximately 106 copies/mL. Moreover, through the direct analysis of fluorescence signals, we further enhanced the sensitivity to a detection limit of 104 copies/mL. This robust and accurate detection approach is capable of identifying and differentiating between respiratory virus subtypes, including those with complex mutation patterns. Conclusions: The integration of ARMS-PCR and molecular beacon probes is a reliable and efficient solution for the rapid and precise monitoring of evolving respiratory infectious diseases, and it has the potential to facilitate early diagnosis and effective control measures. Further research is needed to expand the application of this detection method to other respiratory viruses and optimize its workflow for clinical and public health settings.

As the understanding of the mechanisms of SARS-CoV-2 infection continues to grow, researchers have come to realize that ACE2 and TMPRSS2 receptors are not the only way for the virus to invade the host, and that there are many molecules that may serve as potential receptors or cofactors. The functionality of these numerous receptors, proposed by different research groups, demands a fast, simple, and accurate validation method. To address this issue, we here established a DnaE intein-based cell-cell fusion system, a key result of our study, which enables rapid simulation of SARS-CoV-2 host cell infection. This system allowed us to validate that proteins such as AXL function as SARS-CoV-2 spike protein receptors and synergize with ACE2 for cell invasion, and that proteins like NRP1 act as cofactors, facilitating ACE2-mediated syncytium formation. Our results also suggest that mutations in the NTD of the SARS-CoV-2 Delta variant spike protein show a preferential selection for Spike-AXL interaction over Spike-LDLRAD3. In summary, our system serves as a crucial tool for the rapid and comprehensive verification of potential receptors, screening of SARS-CoV-2-neutralizing antibodies, or targeted drugs, bearing substantial implications for translational clinical applications.

目的 构建可实时监测的小鼠原发性肝癌(HCC)模型.方法 通过高压水动力学转染技术,PB转座子系统和小鼠活体成像技术建立可实时监测的、由特定癌基因驱动的小鼠原发性肝癌模型.结果 YAP5SA基因诱导表达的小鼠原发性肝癌可以成功模拟Hippo信号通路失活的肝癌亚型;通过每隔1周连续检测,整合YAP5SA-IRES-Fluc的小鼠荧光峰值信号在早期逐渐降低,于3周左右,荧光峰值信号与对照组相比,出现明显差异;PTEN-YAP5SA+模型组的荧光信号峰值与YAP5SA+对照组的荧光信号峰值相比,整体均值偏高;索拉菲尼给药1周后,本模型小鼠的荧光信号停止上升,呈现平稳的趋势.结论 成功构建了一种可实时监测的小鼠原发性肝癌模型,可以进行不同肝癌亚型的分子表征,并以此为基础,为临床筛选肝癌靶向药物提供新方法.

Noble metal nanoclusters (NCs) have emerged as a new class of nanomaterials which have great potential in bioapplications. Due to their ultrasmall size and versatile surface chemistry, they show excellent luminescence, high photostability, good biocompatibility, low toxicity, and a high renal clearance rate. In recent years, much work has been reported toward the bioapplications of metal NCs, especially the Au, Ag, and Cu NCs. In this Review, we first discuss the crucial factors of metal NCs for their use as biomaterials, including the size, surface chemistry, photoluminescent properties, cytotoxicity, and metabolic activity. Then, we highlight some recent advances of metal NCs in bioapplications, such as biosensing, bioimaging, biomedical diagnosis, and therapy. It is noteworthy that the metal NCs perform quite well in cancer treatment. Finally, we have briefly discussed the current challenges and our perspectives on metal NCs in bioapplication research and further transformation into clinical application.

Background S ARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/BA.5 have significant differences in transmission and immune escape ability. Currently, no effective detection methods are available for these subtypes. Routine detection methods are prone to missed detection. Methods In this study, a rapid detection method based on ARMS-PCR and molecular beacon probes was developed for the identification of SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5. Specific primers and probes were designed and validated using gel electrophoresis, real-time fluorescence quantitative PCR, and molecular hybridization. Results ARMS-PCR and molecular beacon probe-based assays can be applied in RT-PCR and fluorescence assays to differentiate SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5. Conclusions In the present study, we developed a simple, rapid and accurate detection method based on ARMS-PCR and molecular beacon probes for rapid genotyping of SARS-CoV-2 subtypes Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, and Omicron BA.4/5. It can be used in real-time fluorescence quantitative PCR and molecular hybridization to identify subtypes of COVID-19, effectively improving the detection rate to provide guidance for disease prevention and treatment.

为解析家蚕AN品系高抗BmNPV的分子机制，采用家蚕抗性品系AN与易感品种C108杂交及多代回交，结合累代BmNPV攻毒选择，构建了高抗BmNPV的近等基因系C108＿AN;在5龄起蚕经口添食BmNPV多角体1×10~8 mL -1 悬浊液浸渍桑叶对C108及C108＿AN攻毒，对感染后12、24、36、48、60 h的中肠组织进行转录组测序发现，C108＿AN样本中检出BmNPV基因个数与表达量均大幅减少，病毒虽然能够侵染但不能完成复制；转录组中编码230 aa的家蚕病毒核衣壳蛋白基因（命名为BmVNCP）在C108＿AN样本中显著高表达，表明与BmNPV抗性可能具有关联性。在BmN细胞中过表达BmVNCP基因可以抑制BmNPV的增殖，在BmN细胞中进行靶向BmVNCP的CRISPR/Cas9基因编辑，未能观察到BmNPV增殖和vp39基因表达的稳定增加；BmN细胞内过表达BmVNCP基因可以明显提高宿主IMD信号通路中Dredd基因的表达水平，对FADD基因转录水平无影响，PGRP-LB和PGRP-LF基因表达量极低以致qRT-PCR无法检出。综合分析上述结果认为，家蚕细胞或组织中BmVNCP基因高水平表达不能够阻止BmNPV侵染，但能够降低BmNPV侵染后病毒基因的表达以及病毒增殖，BmVNCP基因产物可能是通过IMD信号通路触发免疫机制。

RYamides constitute a novel family of neuropeptides newly identified in insects, and play important roles in regulating a variety of physiological processes. However, the signaling characteristics and physiological actions of RYamide signaling system remain largely unknown. In the present study, we cloned the full-length complementary DNA of the RYamide receptor BNGR-A19 from Bombyx mori larvae. After expression in mammalian HEK293T and insect Sf9 cells, functional assays revealed that BNGR-A19 was activated by synthetic RYamide peptides, triggering a significant increase in cAMP-response element controlled luciferase activity and Ca2+ mobilization in a Gq inhibitor-sensitive manner. Upon activation by RYamide peptides, BNGR-A19 elicited ERK1/2 phosphorylation via a Gq -PLC-PKC pathway, and also underwent a rapid internalization from the cell surface to the cytoplasm. Further cross-activity analysis indicated that BNGR-A19 exhibited very weak response upon stimulation by high concentration (1 μM) of Bombyx sulfakinin-1, neuropeptide F-1, and short neuropeptide F-1, and vice versa, Bombyx RYamides also showed slight potency for activating Bombyx NPF receptor (BNGR-A4) and sNPF receptor (BNGR-A11). In addition, the quantitative reverse-transcription polymerase chain reaction results showed that the high-level expression of BNGR-A19 was detected in the hindgut and testis, suggesting that the RYamide signaling is likely involved in the regulation of feeding, water homeostasis and testis development. This study provides the first in-depth information on the insect RYamide signaling system, facilitating the further clarification of its endocrinological roles in insect physiology.

CCHamides are newly identified insect neuropeptides, which are widely occurring in most insects. However, our knowledge about their signaling characteristics and physiological roles is still limited. Here, we cloned two fulllength cDNAs encoding putative CCHamide receptors, Bombyx neuropeptide GPCR A14 (BNGR-A14) and -A15 (BNGR-A15), from the brain of B. mori larvae. Characterization of signaling indicated that Bombyx CCHamide-1 and CCHamide-2 are specific endogenous ligands for BNGR-A15 and BNGR-A14, respectively. Further functional assays combined with specific inhibitors demonstrated that upon activation by CCHamide-2, BNGR-A14 elicited significant increases in CRE-driven luciferase activity, intracellular Ca2+ mobilization and ERK1/2 phosphorylation in a G(q) inhibitor-sensitive manner, while BNGR-A15 was activated by CCHamide-1, thus leading to intracellular accumulation of cAMP, Ca2+ mobilization, and ERK1/2 phosphorylation in a G(s) and G(q) inhibitor-sensitive manner. Based on these findings, we designated the receptors BNGR-A15 and -A14 as BommoCCHaR-1 and -2, respectively. In addition, our results showed that CCHamides are considered to require intrachain disulfide bonds to activate their respective receptor in the physiological concentration range. Moreover, quantitative RT-PCR analysis revealed that CCHamide-1 is more likely to serve as a brain-gut peptide to regulate feeding behavior and growth through BNGR-A15, whereas the CCHamide-2 signaling system might play an important role in the control of multiple physiological processes. Our findings provide in-depth information on CCHamide-1 and -2-mediated signaling, facilitating further elucidation of their endocrinological roles in the regulation of fundamental physiological processes.

Tachykinin signaling system is present in both vertebrates and invertebrates, and functions as neuromodulator responsible for the regulation of various physiological processes. In human, the internalization of G protein-coupled receptors has been extensively characterized; however, the insect GPCR internalization has been rarely investigated. Here, we constructed two expression vectors of Bombyx tachykinin-related peptide receptor (BmTKRPR) fused with Enhanced Green Fluorescent Protein (EGFP) at the C-terminal end for direct visualization of receptor expression, localization, and trafficking in cultured mammalian HEK293 and insect Sf21 cells. Our results demonstrated that agonist-activated BmTKRPR underwent rapid internalization in a dose-and time-dependent manner via a clathrin-dependent pathway in both HEK293 and Sf21 cells. Further investigation via RNAi or specific inhibitors, or co-immunoprecipitation demonstrated that agonist-induced BmTKRPR internalization was mediated by PKC, GRK5 and β-arrestin2/BmKurtz. In addition, we also observed that most of the internalized BmTKRP receptors were recycled to the cell surface via early endosomes upon peptide ligand removal. Our study provides the first in-depth information on mechanisms underlying insect TKRP receptor internalization and perhaps aids in the interpretation of the signaling in the regulation of physiological processes.

Alternative splicing enables G protein-coupled receptor (GPCR) genes to greatly increase the number of structurally and functionally distinct receptor isoforms. However, the functional role and relevance of the individual GPCR splice variants in regulating physiological processes are still to be assessed. A naturally occurring alternative splice variant of Bombyx CAPA-PVK receptor, BomCAPA-PVK-R1-Δ341, has been shown to act as a dominant-negative protein to regulate cell surface expression and function of the canonical CAPA-PVK receptor. Herein, using functional assays, we identify the splice variant Δ341 as a specific receptor for neuropeptide CAPA-PK, and upon activation, Δ341 signals to ERK1/2 pathway. Further characterization demonstrates that Δ341 couples to Gαi/o, distinct from the Gαq-coupled canonical CAPA-PVK receptor, triggering ERK1/2 phosphorylation through Gβγ-PI3K-PKCζ signaling cascade. Moreover, our ELISA data show that the ligand-dependent internalization of the splice variant Δ341 is significantly impaired due to lack of GRKs-mediated phosphorylation sites. Our findings highlight the potential of this knowledge for molecular, pharmacological and physiological studies on GPCR splice variants in the future.