T-cell receptor-engineered T (TCR-T) cell therapy is considered highly promising for treating solid tumors. However, it still has significant limitations; one is exogenous-endogenous TCR chain mispairing, which could substantially compromise cell surface expression and the efficacy of the engineered TCR while raising clinical safety concerns. To address this obstacle, we developed a disulfide-substituted TCR (DSS-TCR), in which the native disulfide bond is replaced with artificially designed disulfide bonds within constant domains to increase the fidelity and functionality of TCR pairing. Our study demonstrated that ablation of the native interchain disulfide bond significantly reduces mispairing but severely impairs tumor-killing activity. Using structure-guided computational prediction, we designed nine pairs of artificial interchain disulfide bond-forming sites within TCR constant domains de novo. When introduced into native disulfide-deficient TCRs, four pairs reversed the decrease in cell surface expression. Notably, compared with the wild-type human TCR, two of them significantly enhanced both TCR surface expression and cytotoxic activity. By further combining different pairs of mutations and incorporating hydrophobic substitutions in the α-chain transmembrane domain, DSS-TCRs achieved superior pairing efficiency and antitumor efficacy that were comparable to those of TCRs incorporating mouse-derived constant regions. DSS-TCR significantly decreases the TCR mismatching rate while theoretically reducing immunogenicity. This superior optimization effect was also confirmed for other TCR-based receptors. Therefore, this engineering approach offers a safer and more potent paradigm for TCR-based T-cell therapeutics.

Anisodus tanguticus (Maxim.) Pascher (A. tanguticus), a cold-tolerant perennial herb in the Solanaceae family, is distributed across China's Qinghai-Tibet Plateau and extends to Nepal, Bhutan, Sikkim, and India. As a Tibetan medicinal plant, it is used to treat pain, ulcers, etc.; its roots yield antispasmodic and anesthetic compounds, and other parts are used as a feed additive for yaks to enhance cold resistance in northwest Sichuan. In this study, samples were collected from Seda County (northwestern Sichuan, China) for sequencing. Using PacBio HiFi sequencing and Hi-C scaffolding, a high-quality chromosome-scale genome assembly was generated, with a genome size of 1599.64 Mb, a scaffold N50 of 62.01 Mb, and a contig N50 of 38.51 Mb. A total of 24 superscaffolds (93.65% of the genome) were anchored to 24 chromosomes. Compared with previously reported assemblies of A. tanguticus and A. acutangulus, this assembly shows improved scaffold length and completeness. Genome annotation identified 64.95% repetitive elements and 45,930 protein-coding genes, and comparative analysis of four Anisodus genomes revealed conserved patterns of gene density, GC content, LTR, and LINE elements. This study provides the first high-quality chromosome-scale genome resource of A. tanguticus from the Qinghai-Tibet Plateau, supporting studies on phylogeny, genetic diversity, and breeding, as well as further exploration of its genomic basis of high-altitude adaptation.

Dental caries poses significant therapeutic challenges due to resilient biofilms and uncontrolled demineralization. To address these challenges, by leveraging the broad antimicrobial activity and strong phosphate-binding capability of arginine peptides, we developed dental adhesive nanonets to effectively capture and kill S. mutans strain for caries management. FmocFFRRR was selected from a two-round structural screening, and was co-assembled with sodium monofluorophosphate (MFP) to construct multifunctional nanonets (F@MFP). Specifically, F@MFP nanonets adhere robustly to tooth enamel, capture S. mutans via electrostatic interactions, and kill S. mutans by disrupting bacterial membrane integrity, inducing oxidative stress, and suppressing metabolic activities. F@MFP nanonets not only inhibit biofilm formation but also effectively dismantle mature biofilms by downregulating biofilm-associated virulence genes. Furthermore, F@MFP nanonets also serve as a fluoride reservoir to inhibit demineralization and promote enamel remineralization. In vivo experiments reveal that F@MFP exhibits superior caries-inhibiting efficacy. This multifunctional platform integrates dental adhesion, a capture-and-kill antibacterial mechanism, anti-biofilm properties, and enamel repair capacity, offering a novel paradigm for targeted caries therapy.

Modifying the light formula is a central strategy for improving the yield and quality of fruits and vegetables in agriculture. While light signals have long been acknowledged as primary factors in regulating plant growth and development, their role in reprogramming metabolic networks is not well understood. Using tomato as a model, we demonstrate that supplementation with red or blue light induces metabolic shifts in tomato fruit. Through the creation of the Tomato Light-induced Expression Database (TomLED), we identified extensive transcriptomic and metabolic changes in tomato fruit under varying light conditions. Notably, the induction of key master regulators and metabolic genes is mediated by increased genome-wide DNA demethylation, facilitated by SlDML2. Additionally, we show that SlHY5, a central regulator in the light signaling pathway, directly induces the expression of SlDML2. This study reveals the molecular mechanisms by which light regulates the plant epigenome and establishes a direct link between light signals and plant metabolism.

Eukaryotes harbor both Sm-type and Lsm-type heteroheptameric rings, which are essential in RNA metabolism. Despite their similar subunits and evolutionary ties, they interact with RNA in distinct ways, functioning as scaffolds and chaperones, respectively. However, the mechanistic basis of their evolutionary divergence remains unclear. Using the Sm ring (D1-D2-F-E-G-D3-B) and the Lsm2-8 ring, both of which form the cores of distinct spliceosomal snRNPs, as model systems, we investigated the feasibility and mechanisms of their interconversion. We found that the interactions among subcomplexes (SCs) 1-3 in the Sm ring (D1/D2, F/E/G, and D3/B) differ from those in Lsm2-8 (Lsm2/3, Lsm6/5/7, and Lsm8/4), implying the formation of distinct assembly intermediates. By strengthening the SC1-SC3 interaction, we achieved the conversion of the Sm ring into an Lsm-type ring. Conversely, increasing the SC2-SC3 affinity did not yield a successful conversion. Furthermore, by weakening the SC1-SC3 interaction and introducing mutations in the RNA-binding regions of SC1 and SC2, we converted Lsm2-8 into a Sm-type ring. These findings provide mechanistic insights into how similar protein components can assemble into functionally distinct heteroheptameric rings, a principle likely applicable to Lsm1-7 and the U7 snRNP core, and offer deep insights into spliceosome and eukaryotic evolution.

Flavonoids are polyphenolic secondary metabolites in tomato fruit with important roles in nutritional quality. Dissecting the transcriptional regulatory network modulating flavonoid metabolism is the first step to improve the nutritional quality of tomato fruits through molecular breeding technology. In this study, we identified a transcription factor SlbHLH95 as a key regulator in flavonoid metabolism through analysis of the MicroTom Metabolic Network (MMN) data set. Functional analyses revealed that knockout of SlbHLH95 increased the accumulation of naringenin, while the levels of rutin and nictoflorin decreased. Conversely, overexpression of SlbHLH95 resulted in an opposite pattern of accumulation of flavonoids. Transactivation assays showed that SlbHLH95 positively activated the expression of SlF3H and SlFLS, two key enzyme-encoding genes in the flavonoid pathway, while repressing the expression of SlCHS1. Electrophoretic mobility shift assays (EMSA) demonstrated that SlbHLH95 could directly bind to the promoters of SlF3H and SlFLS, although it could not bind to the promoter of SlCHS1. Furthermore, SlbHLH95 interacted with the transcription factor SlMYB12 and coordinately regulated the expression of SlF3H and SlFLS. Beyond its role in flavonoid metabolism, SlbHLH95 positively regulated the grey mould resistance in tomato fruits by repressing SlBG10. Overall, our findings revealed the important role of bi-functional SlbHLH95 in flavonoid metabolism and grey mould resistance in tomato fruits by acting as both a transcriptional activator and a repressor. This study provides new insights into strategies for improving fruit quality and enhancing fruit disease resistance through targeted genetic modulation.

The assembly of most spliceosomal snRNP cores involves seven Sm proteins (D1/D2/F/E/G/D3/B) forming a ring around snRNA, typically requiring essential assembly chaperones like the SMN complex, associated with spinal muscular atrophy (SMA). Strikingly, in budding yeast, snRNP core assembly only involves Brr1, a nonessential homolog of Gemin2. Here, we reveal two distinct pathways in budding yeast: an inefficient chaperone-mediated pathway involving Brr1 and a novel factor, Lot5, and a direct pathway. Lot5 binds D1/D2/F/E/G to form a heterohexameric ring (6S). Brr1 binds D1/D2/F/E/G and 6S but cannot displace Lot5 to facilitate assembly. Disruption of BRR1 and LOT5 genes caused mild growth retardation, but LOT5 overexpression substantially impeded growth. The direct pathway uniquely involves F/E/G as a trimer and a stable D1/D2/F/E/G intermediate complex, explaining the non-essentiality of chaperones. These findings unveil a unique snRNP core assembly mechanism, illuminate the evolution of assembly chaperones, and suggest avenues for studying SMA pathophysiology.

6-phosphogluconate dehydrogenase (6PGDH) is an essential enzyme in energy metabolism and redox reactions, and represents a potential drug target for the development of therapies targeting trypanosomes, plasmodium, or other pathogens. Tuberculosis, caused by Mycobacterium tuberculosis, is a contagious disease that severely affects human health, with approximately one-third of the world's population infected. However, the protein structure, exact oligomeric state, and catalytic mechanism of 6PGDH in Mycobacterium tuberculosis (Mt6PGDH) have remained largely unknown. In this study, we successfully purified and determined the structure of Mt6PGDH, revealing its function as a tetramer in both solution and crystal states. Through structural comparisons, we clarified the tetramer formation mechanism and the oligomeric organization of short-chain 6PGDHs. Additionally, we identified key residues for coenzyme recognition and catalytic activity. This work not only deepens our understanding of the enzymatic function of Mt6PGDH but also lays a foundation for the development of drugs targeting this enzyme.

Kiwifruit ripening is a complex and highly coordinated process that occurs in conjunction with the formation of fruit edible quality. The significance of epigenetic changes, particularly the impact of N6-methyladenosine (m6A) RNA modification on fruit ripening and quality formation, has been largely overlooked. We monitored m6A levels and gene expression changes in kiwifruit at four different stages using LC-MS/MS, MeRIP, RNA-seq, and validated the function of AcALKBH10 through heterologous transgenic expression in tomato. Notable m6A modifications occurred predominantly at the stop codons and the 3 ' UTRs and exhibited a gradual reduction in m6A levels during the fruit ripening process. Moreover, these m6A modifications in the aforementioned sites demonstrated a discernible inverse relationship with the levels of mRNA abundance throughout the ripening process, suggesting a repression effect of m6A modification in the modulation of kiwifruit ripening. We further demonstrated that AcALKBH10 rather than AcECT9 predominantly regulates m6A levels in ripening-related genes, thereby exerting the regulatory control over the ripening process and the accumulation of soluble sugars and organic acids, ultimately influencing fruit ripening and quality formation. In conclusion, our findings illuminate the epi-regulatory mechanism involving m6A in kiwifruit ripening, offering a fresh perspective for cultivating high-quality kiwifruit with enhanced nutritional attributes.

3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key rate-limiting enzyme that catalyzes the synthesis of the shikimate, which is an important metabolic intermediate in plants and animals. However, the function of SlDQD/SDH family genes in tomato (Solanum lycopersicum) fruit metabolites are still unknown. In the present study, we identified a ripening-associated SlDQD/SDH member, SlDQD/SDH2, that plays a key role in shikimate and flavonoid metabolism. Overexpression of this gene resulted in an increased content of shikimate and flavonoids, while knockout of this gene by CRISPR/Cas9 mediated gene editing led to a significantly lower content of shikimate and flavonoids by downregulation of flavonoid biosynthesis-related genes. Moreover, we showed that SlDQD/SDH2 confers a resistance against Botrytis cinerea attack in postharvest tomato fruit. Dual-luciferase reporter and EMSA assays indicated that SlDQD/SDH2 is a direct target of the key ripening regulator SlTAGL1. In general, this study provided a new insight into the biosynthesis of flavonoid and B. cinerea resistance in fruit tomato.

3-Dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key rate-limiting enzyme that catalyzes the synthesis of the shikimate, which is an important metabolic intermediate in plants and animals. However, the function of SlDQD/SDH family genes in tomato (Solanum lycopersicum) fruit metabolites is still unknown. In the present study, we identified a ripening-associated SlDQD/SDH member, SlDQD/SDH2, that plays a key role in shikimate and flavonoid metabolism. Overexpression of this gene resulted in an increased content of shikimate and flavonoids, while knockout of this gene by CRISPR/Cas9 mediated gene editing led to a significantly lower content of shikimate and flavonoids by downregulation of flavonoid biosynthesis-related genes. Moreover, we showed that SlDQD/SDH2 confers resistance against Botrytis cinerea attack in post-harvest tomato fruit. Dual-luciferase reporter and EMSA assays indicated that SlDQD/SDH2 is a direct target of the key ripening regulator SlTAGL1. In general, this study provided a new insight into the biosynthesis of flavonoid and B. cinerea resistance in fruit tomatoes.

The spliceosomal snRNP cores, each comprised of a snRNA and a seven-membered Sm ring (D1/D2/F/E/G/D3/B), are assembled by twelve chaperoning proteins in human. However, only six assembly-assisting proteins, ICln and the SMN complex (SMN/Gemin2/Gemin6-8), have been found in Schizosaccharomyces pombe (Sp). Here, we used recombinant proteins to reconstitute the chaperone machinery and investigated the roles of these proteins systematically. We found that, like the human system, the assembly in S. pombe requires ICln and the SMN complex sequentially. However, there are several significant differences. For instance, h_F/E/G forms heterohexamers and heterotrimers, while Sp_F/E/G only forms heterohexamers; h_Gemin2 alone can bind D1/D2/F/E/G, but Sp_Gemin2 cannot. Moreover, we found that Sp_Gemin2 is essential using genetic approaches. These mechanistic studies reveal that these six proteins are necessary and sufficient for Sm core assembly at the molecular level, and enrich our understanding of the chaperone systems in species variation and evolution.

GO@LM-SP-FA was constructed by connecting linear maltodextrin polymer and folic acid to the surface of graphene oxide nanoparticles. Doxorubicin hydrochloride was loaded into GO@LM-SP-FA to form GO@LM-SP-FA/DOX, and controlled release of anticancer drugs was realized.

Respiratory syncytial virus (RSV) is a nonsegmented, negative strand RNA virus that has caused severe lower respiratory tract infections of high mortality rates in infants and the elderly, yet no effective vaccine or antiviral therapy is available. The RSV genome encodes the nucleoprotein (N) that forms helical assembly to encapsulate and protect the RNA genome from degradation, and to serve as a template for transcription and replication. Previous crystal structure revealed a decameric ring architecture of N in complex with the cellular RNA (N-RNA) of 70 nucleotides (70-nt), whereas cryo-ET reconstruction revealed a low-resolution left-handed filament, in which the crystal monomer structure was docked with the helical symmetry applied to simulate a nucleocapsid-like assembly of RSV. However, the molecular details of RSV nucleocapsid assembly remain unknown, which continue to limit our complete understanding of the critical interactions involved in the nucleocapsid and antiviral development that may target this essential process during the viral life cycle. Here we resolve the near-atomic cryo-EM structure of RSV N-RNA that represents roughly one turn of the helical assembly that unveils critical interaction interfaces of RSV nucleocapsid and may facilitate development of RSV antiviral therapy.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a significant impact on global health. To address the urgent need for plasmids containing SARS-CoV-2 sequences in research, we have developed a high-throughput FastCloning platform for the construction of associated plasmids. Our platform uses a FastCloning method to construct a plasmid library from 29 ORFs of the virus and 20 commonly used vectors in the lab. The library contains 536 recombinant vectors, with a highly positive clone success rate of 92.4%. Our study provides a rapid and efficient approach to constructing a large plasmid library for SARS-CoV-2 research.

GSK3 alpha and GSK30 are two GSK3 isoforms with 84% overall identity and 98% identity in their catalytic domains. GSK30 plays important roles in the pathogenesis of can-cer, while GSK3 alpha has long been considered a functionally redundant protein of GSK30. Few studies have specifically investigated the functions of GSK3 alpha. In this study, unex-pectedly, we found that the expression of GSK3 alpha, but not GSK30, was significantly correlated with the overall sur-vival of colon cancer patients in 4 independent cohorts. To decipher the roles of GSK3 alpha in colon cancer, we profiled the phosphorylation substrates of GSK3 alpha and uncovered 156 phosphosites from 130 proteins specifically regulated by GSK3 alpha. A number of these GSK3 alpha-mediated phos-phosites have never been reported before or have been incorrectly identified as substrates of GSK30. Among them, the levels of HSF1S303p, CANXS583p, MCM2S41p, POGZS425p, SRRM2T983p, and PRPF4BS431p were signifi-cantly correlated with the overall survival of colon cancer patients. Further pull-down assays identified 23 proteins, such as THRAP3, BCLAF1, and STAU1, showing strong binding affinity to GSK3 alpha. The interaction between THRAP3 and GSK3 alpha was verified by biochemical experi-ments. Notably, among the 18 phosphosites of THRAP3, phosphorylation at S248, S253, and S682 is specifically mediated by GSK3 alpha. Mutation of S248 to D (S248D), which mimics the effect of phosphorylation, obviously increased cancer cell migration and the binding affinity to proteins related to DNA damage repair. Collectively, this work not only discloses the specific function of GSK3 alpha as a kinase but also suggests GSK3 alpha as a promising therapeutic target for colon cancer.

The current monkeypox outbreak has caused over 64,000 global cases, but the effective treatments are very limited. The dual specific phosphatase (H1) from monkeypox antagonizes the immune response and is crucial for viral replication, making it an attractive antiviral target. Here we determined a 1.8-Å crystal structure of H1, which forms a domain swapped dimer resembling a butterfly. Each active site, which consists of a Cys-Arg-Asp triad, captures a phosphate ion. The observed conformation mimics the final step of catalysis prior to product release. The crystal structure provides a strong foundation for the discovery of new antivirals against this emerging worldwide pathogen.

The phytohormone ethylene is well known for its important role in the ripening of climacteric fruit, such as tomato (Solanum lycopersicum). However, the role and mode of action of other plant hormones in climacteric fruit ripening regulation are not fully understood. Here, we showed that exogenous GA treatment or increasing endogenous gibberellin content by overexpressing the gibberellin synthesis gene SlGA3ox2 specifically in fruit tissues delayed tomato fruit ripening, whereas treatment with the GA biosynthesis inhibitor paclobutrazol (PAC) accelerated fruit ripening. Moreover, exogenous ethylene treatment cannot completely reverse the delayed fruit ripening phenotype. Furthermore, exogenous GA treatment of ethylene signalling mutant Never ripe (Nr) or SlEBF3-overexpressing lines still delayed fruit ripening, suggesting that GA involved in fruit ripening partially depends on ethylene. Transcriptome profiling showed that gibberellin affect the ripening of fruits by modulating the metabolism and signal transduction of multiple plant hormones, such as auxin and abscisic acid, in addition to ethylene. Overall, the results of this study provide new insight into the regulation of gibberellin in fruit ripening through mediating multiple hormone signals.