Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

ASD2023: towards the integrating landscapes of allosteric knowledgebase.

Allosteric regulation, induced by perturbations at an allosteric site topographically distinct from the orthosteric site, is one of the most direct and efficient ways to fine-tune macromolecular function. The Allosteric Database (ASD; accessible online at http://mdl.shsmu.edu.cn/ASD) has been systematically developed since 2009 to provide comprehensive information on allosteric regulation. In recent years, allostery has seen sustained growth and wide-ranging applications in life sciences, from basic research to new therapeutics development, while also elucidating emerging obstacles across allosteric research stages. To overcome these challenges and maintain high-quality data center services, novel features were curated in the ASD2023 update: (i) 66 589 potential allosteric sites, covering > 80% of the human proteome and constituting the human allosteric pocketome; (ii) 748 allosteric protein-protein interaction (PPI) modulators with clear mechanisms, aiding protein machine studies and PPI-targeted drug discovery; (iii) 'Allosteric Hit-to-Lead,' a pioneering dataset providing panoramic views from 87 well-defined allosteric hits to 6565 leads and (iv) 456 dualsteric modulators for exploring the simultaneous regulation of allosteric and orthosteric sites. Meanwhile, ASD2023 maintains a significant growth of foundational allosteric data. Based on these efforts, the allosteric knowledgebase is progressively evolving towards an integrated landscape, facilitating advancements in allosteric target identification, mechanistic exploration and drug discovery.

ErbB3 is required for hyperaminoacidemia-induced pancreatic α cell hyperplasia.

Blood amino acid levels are maintained in a narrow physiological range. The pancreatic α cells have emerged as the primary aminoacidemia regulator through glucagon secretion to promote hepatic amino acid catabolism. Interruption of glucagon signaling disrupts the liver - α cells axis leading to hyperaminoacidemia, which triggers a compensatory rise in glucagon secretion and α cell hyperplasia. The mechanisms of hyperaminoacidemia-induced α cell hyperplasia remain incompletely understood. Using a mouse α cell line and in vivo studies in zebrafish and mice, we found that hyperaminoacidemia-induced α cell hyperplasia requires ErbB3 signaling. In addition to mTORC1, another ErbB3 downstream effector STAT3 also plays a role in α cell hyperplasia. Mechanistically, ErbB3 may partner with ErbB2 to stimulate cyclin D2 and suppress p27 via mTORC1 and STAT3. Our study identifies ErbB3 as a new regulator for hyperaminoacidemia-induced α cell proliferation and a critical component of the liver-α cells axis that regulates aminoacidemia.

DeepAlloDriver: a deep learning-based strategy to predict cancer driver mutations.

Driver mutations can contribute to the initial processes of cancer, and their identification is crucial for understanding tumorigenesis as well as for molecular drug discovery and development. Allostery regulates protein function away from the functional regions at an allosteric site. In addition to the known effects of mutations around functional sites, mutations at allosteric sites have been associated with protein structure, dynamics, and energy communication. As a result, identifying driver mutations at allosteric sites will be beneficial for deciphering the mechanisms of cancer and developing allosteric drugs. In this study, we provided a platform called DeepAlloDriver to predict driver mutations using a deep learning method that exhibited >93% accuracy and precision. Using this server, we found that a missense mutation in RRAS2 (Gln72 to Leu) might serve as an allosteric driver of tumorigenesis, revealing the mechanism of the mutation in knock-in mice and cancer patients. Overall, DeepAlloDriver would facilitate the elucidation of the mechanisms underlying cancer progression and help prioritize cancer therapeutic targets. The web server is freely available at: https://mdl.shsmu.edu.cn/DeepAlloDriver.

π-π Stacking at the Perovskite/C60 Interface Enables High-Efficiency Wide-Bandgap Perovskite Solar Cells.

Interface passivation is a key method for improving the efficiency of perovskite solar cells, and 2D/3D perovskite heterojunction is the mainstream passivation strategy. However, the passivation layer also produces a new interface between 2D perovskite and fullerene (C60), and the properties of this interface have received little attention before. Here, the underlying properties of the 2D perovskite/C60 interface by taking the 2D TEA2PbX4 (TEA = C6H10NS; X = I, Br, Cl) passivator as an example are systematically expounded. It is found that the 2D perovskite preferentially exhibits (002) orientation with the outermost surface featuring an oriented arrangement of TEACl, where the thiophene groups face outward. The outward thiophene groups further form a strong π-π stacking system with C60 molecule, strengthening the interaction force with C60 and facilitating the creation of a superior interface. Based on the vacuum-assisted blade coating, wide-bandgap (WBG, 1.77 eV) perovskite solar cells achieved impressive records of 19.28% (0.09 cm2) and 18.08% (1.0 cm2) inefficiency, respectively. This research not only provides a new understanding of interface processing for future perovskite solar cells but also lays a solid foundation for realizing efficient large-area devices.

Functional analysis of a rice 12-oxo-phytodienoic acid reductase gene (OsOPR1) involved in Cd stress tolerance.

BACKGROUND: The accumulation of cadmium (Cd) in plants may compromise the growth and development of plants, thereby endangering human health through the food chain. Understanding how plants respond to Cd is important for breeding low-Cd rice cultivars. METHODS: In this study, the functions of 12-oxo-phytodienoic acid reductase 1 (OsOPR1) were predicted through bioinformatics analysis. The expression levels of OsOPR1 under Cd stress were analyzed by using qRT-PCR. Then, the role that OsOPR1 gene plays in Cd tolerance was studied in Cd-sensitive yeast strain (ycf1), and the Cd concentration of transgenic yeast was analyzed using inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: Bioinformatics analysis revealed that OsOPR1 was a protein with an Old yellow enzyme-like FMN (OYE_like_FMN) domain, and the cis-acting elements which regulate hormone synthesis or responding abiotic stress were abundant in the promoter region, which suggested that OsOPR1 may exhibit multifaceted biological functions. The expression pattern analysis showed that the expression levels of OsOPR1 were induced by Cd stress both in roots and roots of rice plants. However, the induced expression of OsOPR1 by Cd was more significant in the roots compared to that in roots. In addition, the overexpression of OsOPR1 improved the Cd tolerance of yeast cells by affecting the expression of antioxidant enzyme related genes and reducing Cd content in yeast cells. CONCLUSION: Overall, these results suggested that OsOPR1 is a Cd-responsive gene and may has a potential for breeding low-Cd or Cd-tolerant rice cultivars and for phytoremediation of Cd-contaminated in farmland.

Antibiotic-free production of sucrose isomerase in Bacillus subtilis by genome integration.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose to form isomaltulose, a valuable functional sugar widely used in the food industry. However, the lack of safe and efficient heterologous expression systems hinders SIase production and application. In this study, we achieved antibiotic-free SIase expression in Bacillus subtilis through genome integration. Using CRISPR/Cas9 system, SIase expression cassettes were integrated into various genomic loci, including amyE and ctc, both individually and in combination, resulting in single-copy and muti-copy integration strains. Engineered strains with a maltose-inducible promoter effectively expressed and secreted SIase. Notably, multi-copy strain exhibited enhanced SIase production, achieving 4.4 U/mL extracellular activity in shake flask cultivations. Furthermore, crude enzyme solution from engineered strain transformed high concentrations sucrose into high yields of isomaltulose, reaching a maximum yield of 94.6%. These findings demonstrate antibiotic-free SIase production in B. subtilis via genome integration, laying the foundation for its industrial production and application.

Bioinformatics and Functional Analysis of OsASMT1 Gene in Response to Abiotic Stress.

The study aimed to elucidate the functional characteristics of OsASMT1 gene under copper (Cu) or sodium chloride (NaCl) stress. Bioinformatics scrutiny unveiled that OsASMT1 is situated on chromosome 9. Its protein architecture, comprising dimerization and methyltransferase domains, showed significant similarities to OsASMT2 and OsASMT3. High expression in roots and panicles, along with abiotic stress putative cis-regulatory elements in the promoter, indicated potential stress responsiveness. Real-time quantitative PCR confirmed OsASMT1 induction under Cu and NaCl stress in rice. Surprisingly, yeast expressing OsASMT1 did not exhibit enhanced resistance to abiotic stresses. The results of subcellular localization analysis indicated that OsASMT1 plays a role in the cytoplasm. While OsASMT1 responded to Cu and NaCl stress in rice, its heterologous expression in yeast failed to confer abiotic stress resistance, highlighting the need for further investigation of its functional implications.

Synthesis and Antimicrobial Activity Evaluation of Pyridine Derivatives Containing Imidazo[2,1-b][1,3,4]Thiadiazole Moiety.

Four series of novel pyridine derivatives (17 a-i, 18 a-i, 19 a-e, and 20 a-e) were synthesized and their antimicrobial activities were evaluated. Of all the target compounds, almost half target compounds showed moderate or high antibacterial activity. The 4-F substituted compound 17 d (MIC=0.5 µg/mL) showed the highest antibacterial activity, its activity was twice the positive control compound gatifloxacin (MIC=1.0 µg/mL). For fungus ATCC 9763, the activities of compounds 17 a and 17 d are equivalent to the positive control compound fluconazole (MIC=8 µg/mL). Furthermore, compounds 17 a and 17 d showed little cytotoxicity to human LO2 cells, and did not show hemolysis even at ultra-high concentration (200 µM). The results indicate that these compounds are valuable for further development as antibacterial and antifungal agents.

A Label-Free Optical Biosensor Based on an Array of Microring Resonators for the Detection of Human Serum Albumin.

We introduced a label-free sensing system based on an array of microring resonators (MRRs) which was successfully employed for human serum albumin (HSA) detection. The sensing-ring surface was functionalized to immobilize anti-HSA, facilitating HSA binding. Our refractive index sensing system demonstrates high sensitivity at 168 nm/RIU and a low limit of detection (LOD) of 63.54 ng/mL, closely comparable to current HSA detection methods. These findings confirm the potential of MRRs as biocompatible sensors for HSA detection. This system holds great promise as an innovative platform for the detection of HSA, carrying significant importance in medical diagnostics.

Computer-Vision-Based Sensing Technologies for Livestock Body Dimension Measurement: A Survey.

Livestock's live body dimensions are a pivotal indicator of economic output. Manual measurement is labor-intensive and time-consuming, often eliciting stress responses in the livestock. With the advancement of computer technology, the techniques for livestock live body dimension measurement have progressed rapidly, yielding significant research achievements. This paper presents a comprehensive review of the recent advancements in livestock live body dimension measurement, emphasizing the crucial role of computer-vision-based sensors. The discussion covers three main aspects: sensing data acquisition, sensing data processing, and sensing data analysis. The common techniques and measurement procedures in, and the current research status of, live body dimension measurement are introduced, along with a comparative analysis of their respective merits and drawbacks. Livestock data acquisition is the initial phase of live body dimension measurement, where sensors are employed as data collection equipment to obtain information conducive to precise measurements. Subsequently, the acquired data undergo processing, leveraging techniques such as 3D vision technology, computer graphics, image processing, and deep learning to calculate the measurements accurately. Lastly, this paper addresses the existing challenges within the domain of livestock live body dimension measurement in the livestock industry, highlighting the potential contributions of computer-vision-based sensors. Moreover, it predicts the potential development trends in the realm of high-throughput live body dimension measurement techniques for livestock.

Study on the Relationship between Particulate Methane Monooxygenase and Methanobactin on Gold-Nanoparticles-Modified Electrodes.

(1) Background: Particulate methane monooxygenase (pMMO) has a strong dependence on the natural electron transfer path and is prone to denaturation, which results in its redox activity centers being unable to transfer electrons with bare electrodes directly and making it challenging to observe an electrochemical response; (2) Methods: Using methanobactin (Mb) as the electron transporter between gold electrodes and pMMO, a bionic interface with high biocompatibility and stability was created. The Mb-AuNPs-modified functionalized gold net electrode as a working electrode, the kinetic behaviors of pMMO bioelectrocatalysis, and the effect of Mb on pMMO were analyzed. The CV tests were performed at different scanning rates to obtain electrochemical kinetics parameters. (3) Results: The values of the electron transfer coefficient (α) and electron transfer rate constant (ks) are relatively large in test environments containing only CH4 or O2. In contrast, in the test environment containing both CH4 and O2, the bioelectrocatalysis of pMMO is a two-electron transfer process with a relatively small α and ks; (4) Conclusions: It was inferred that Mb formed the complex with pMMO. More importantly, Mb not only played a role in electron transfer but also in stabilizing the enzyme structure of pMMO and maintaining a specific redox state. Furthermore, the continuous catalytic oxidation of natural substrate methane was realized.

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OBJECTIVES: To study the intervention effect of narrative therapy on non-suicidal self-injury (NSSI), as well as anxiety and depression symptoms in adolescents with depressive disorder. METHODS: Sixty adolescents with depressive disorder and NSSI were randomly assigned to either the intervention group or the control group using coin flipping. The control group received conventional psychological support, while the intervention group received individual narrative therapy in addition to the conventional psychological support (twice a week, 60 minutes per session, for a total of 3 weeks). Assessment of treatment efficacy was conducted using the Adolescent Self-Harm Questionnaire, Children's Depression Inventory, and Children's Anxiety and Mood Scale before the intervention, at the end of the intervention, and one month after the intervention for both groups. RESULTS: A total of 26 adolescents in the intervention group and 29 adolescents in the control group completed the entire study. At the end of the intervention and one month after the intervention, the intervention group showed a significant reduction in the NSSI frequency score, NSSI level, anxiety score, and depression score compared to before the intervention (P<0.017). Moreover, at the end of the intervention and one month after the intervention, the intervention group exhibited significantly lower NSSI frequency score, NSSI severity score, NSSI level, anxiety score and depression score compared to the control group (P<0.05). CONCLUSIONS: Narrative therapy is effective in reducing NSSI frequency and alleviating NSSI severity, as well as anxiety and depression symptoms in adolescents with depressive disorder.

Disruption of the glucagon receptor increases glucagon expression beyond α-cell hyperplasia in zebrafish.

The glucagon receptor (GCGR) is a potential target for diabetes therapy. Several emerging GCGR antagonism-based therapies are under preclinical and clinical development. However, GCGR antagonism, as well as genetically engineered GCGR deficiency in animal models, are accompanied by α-cell hyperplasia and hyperglucagonemia, which may limit the application of GCGR antagonism. To better understand the physiological changes in α cells following GCGR disruption, we performed single cell sequencing of α cells isolated from control and gcgr-/- (glucagon receptor deficient) zebrafish. Interestingly, beyond the α-cell hyperplasia, we also found that the expression of gcga, gcgb, pnoca, and several glucagon-regulatory transcription factors were dramatically increased in one cluster of gcgr-/- α cells. We further confirmed that glucagon mRNA was upregulated in gcgr-/- animals by in situ hybridization and that glucagon promoter activity was increased in gcgr-/-;Tg(gcga:GFP) reporter zebrafish. We also demonstrated that gcgr-/- α cells had increased glucagon protein levels and increased granules after GCGR disruption. Intriguingly, the increased mRNA and protein levels could be suppressed by treatment with high-level glucose or knockdown of the pnoca gene. In conclusion, these data demonstrated that GCGR deficiency not only induced α-cell hyperplasia but also increased glucagon expression in α cells, findings which provide more information about physiological changes in α-cells when the GCGR is disrupted.

Cloning, Expression, and Functional Characterization of FUT1, a Key Gene for Histo-Blood Group Antigens Synthesis in Crassostrea gigas.

Histo-blood group antigens (HBGAs) comprise a family of cell-surface carbohydrates that are considered norovirus-specific binding receptors or ligands. HBGA-like molecules have also been detected in oysters as common norovirus carriers, although the pathway involved in the synthesis of these molecules in oysters has yet to be elucidated. We isolated and identified a key gene involved in the synthesis of HBGA-like molecules, FUT1, from Crassostrea gigas, named CgFUT1. Real-time quantitative polymerase chain reaction analysis showed that CgFUT1 mRNA was expressed in the mantle, gill, muscle, labellum, and hepatopancreatic tissues of C. gigas, with the hepatopancreas exhibiting the highest expression level. A recombinant CgFUT1 protein with a molecular mass of 38.0 kDa was expressed in Escherichia coli using a prokaryotic expression vector. A eukaryotic expression plasmid was constructed and transfected into Chinese hamster ovary (CHO) cells. The expression of CgFUT1 and membrane localization of type H-2 HBGA-like molecules in CHO cells were detected using Western blotting and cellular immunofluorescence, respectively. This study indicated that CgFUT1, expressed in C. gigas tissues, can synthesize type H-2 HBGA-like molecules. This finding provides a new perspective for analyzing the source and synthetic pathway of HBGA-like molecules in oysters.

The novel CDK9 inhibitor, XPW1, alone and in combination with BRD4 inhibitor JQ1, for the treatment of clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a highly lethal malignancy with few therapeutic options. Cyclin­dependent kinase 9 (CDK9), a potential therapeutic target of many cancers, has been recently observed to be upregulated in ccRCC patients. Therefore, we aimed to investigate the therapeutic potential of CDK9 in ccRCC and develop a novel CDK9 inhibitor with low toxicity for ccRCC treatment. METHODS: The expression of CDK9 in ccRCC was checked using the online database and tissue microarray analysis. shRNA-mediated CDK9 knockdown and CDK inhibitor were applied to evaluate the effect of CDK9 on ccRCC. Medicinal chemistry methods were used to develop a new CDK9 inhibitor with drugability. RNA-seq and ChIP-seq experiments were conducted to explore the mechanism of action. MTS, western blotting, and colony formation assays were performed to evaluate the anti-ccRCC effects of CDK9 knockdown and inhibition in vitro. The in vivo anti-tumour efficacy was evaluated in a xenograft model. RESULTS: CDK9 is overexpressed and associated with poor survival in ccRCC. Knockdown or inhibition of CDK9 significantly suppressed ccRCC cells. XPW1 was identified as a new potent and selective CDK9 inhibitor with excellent anti-ccRCC activity and low toxicity. In mechanism, XPW1 transcriptionally inhibited DNA repair programmes in ccRCC cells, resulting in an excellent anti-tumour effect. CDK9 and BRD4 were two highly correlated transcriptional regulators in ccRCC patients, and the BRD4 inhibitor JQ1 enhanced XPW1's anti-ccRCC effects in vitro and in vivo. CONCLUSIONS: This work provides valuable insights into the therapeutic potential of CDK9 in ccRCC. The CDK9 inhibitor XPW1 would be a novel therapeutic agent for targeting ccRCC, alone or in rational combinations.

Crispr-Cas9 mediated complete deletion of glucagon receptor in mice display hyperglucagonemia and α-cell hyperplasia.

Glucagon receptor plays an important role in the regulation of glucose metabolism. Studies have revealed that glucagon receptor antagonism is a potential effective treatment for diabetes. However, the functions of GCGR have not been fully illustrated. Although two Gcgr truncation knockout mice models have been widely used for GCGR function studies, truncated gene may remain neomorphic and/or dominant-negative function. In this study, we took the advantages of Crispr-Cas9 technique and generated a novel allele of GCGR in the mouse that yields complete loss of GCGR protein. Our studies reveal that complete deletion of Gcgr results in hyperglucagonemia, α-cell hyperplasia, improvement of glucose tolerance. These results are similar to the Gcgr-truncated mutation in mice. Hence, we provide a novel strain of GCGR knockout mice for the GCGR function studies.

Expression of OsHARBI1-1 enhances the tolerance of Arabidopsis thaliana to cadmium.

BACKGROUND: As one of the major food crops in the world, rice is vulnerable to cadmium (Cd) pollution. Understanding of the molecular mechanisms of Cd uptake, transport and detoxification in rice is essential for the breeding of low-Cd rice. However, the molecular mechanisms underlying the response of rice to Cd stress remains to be further clarified. RESULTS: In this study, a novel Cd-responsive gene OsHARBI1-1 was identified in the rice genome and its expression pattern and function were characterized. Bioinformatics analysis showed that the promoter region of OsHARBI1-1 had multiple cis-acting elements in response to phytohormones and stress, and the expression of OsHARBI1-1 was induced by phytohormones. OsHARBI1-1 protein was targeted to the nucleus. qRT-PCR analysis results showed that the expression of OsHARBI1-1 in the roots was repressed while the expression in the shoots was increased under Cd stress. Heterologous expression of OsHARBI1-1 in yeast conferred tolerance to Cd and reduced Cd content in the cells. Meanwhile, the expression of OsHARBI1-1 in Arabidopsis thaliana (A. thaliana) enhanced the tolerance of A. thaliana to Cd stress. In addition, compared with the wild type plants, the POD activity of transgenic plants was increased, while the SOD and CAT activities were decreased. Interestingly, the accumulation of Cd in the roots of A. thaliana expressing OsHARBI1-1 was significantly increased, whereas the Cd accumulation in the shoots was slightly decreased. Compared to the WT plants, the expression of genes related to Cd absorption and chelation was upregulated in transgenic A. thaliana under Cd stress, while the expression of genes responsible for the translocation of Cd from the roots to the shoots was downregulated. Moreover, the expression of phytohormone-related genes was significantly influenced by the expression of OsHARBI1-1 with and without Cd treatment. CONCLUSIONS: Findings of this study suggest that OsHARBI1-1 might play a role in the response of plants to Cd response by affecting antioxidant enzyme activities, Cd chelation, absorption and transport, and phytohormone homeostasis and signaling.

4*10 Gbps WDM communication system based on a tunable V-cavity semiconductor laser.

This paper is about the V-cavity tunable semiconductor laser with a 1550 nm band used as a transmitter to build a wavelength division multiplexing (WDM) optical fiber communication link. In the experiment, a 20 km optical fiber communication link with a reasonable eye diagram and low bit error rate (BER) transmitted by 40 Gbps can be established. The experimental results show that a single laser can achieve a wavelength tuning range of 25 nm, reach 32 channels at a 100 GHz frequency interval, and the average side mode suppression ratio (SMSR) is above 39 dB. The advantages and application potential of V-cavity tunable semiconductor laser (VCL) for wavelength routing in optical communication networking are verified by experiments.