Acetylation coordinates the crosstalk between carbon metabolism and ammonium assimilation in Salmonella enterica.

Enteric bacteria use up to 15% of their cellular energy for ammonium assimilation via glutamine synthetase (GS)/glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in response to varying ammonium availability. However, the sensory mechanisms for effective and appropriate coordination between carbon metabolism and ammonium assimilation have not been fully elucidated. Here, we report that in Salmonella enterica, carbon metabolism coordinates the activities of GS/GDH via functionally reversible protein lysine acetylation. Glucose promotes Pat acetyltransferase-mediated acetylation and activation of adenylylated GS. Simultaneously, glucose induces GDH acetylation to inactivate the enzyme by impeding its catalytic centre, which is reversed upon GDH deacetylation by deacetylase CobB. Molecular dynamics (MD) simulations indicate that adenylylation is required for acetylation-dependent activation of GS. We show that acetylation and deacetylation occur within minutes of "glucose shock" to promptly adapt to ammonium/carbon variation and finely balance glutamine/glutamate synthesis. Finally, in a mouse infection model, reduced S. enterica growth caused by the expression of adenylylation-mimetic GS is rescued by acetylation-mimicking mutations. Thus, glucose-driven acetylation integrates signals from ammonium assimilation and carbon metabolism to fine-tune bacterial growth control.

Viral and host factors related to the clinical outcome of COVID-19.

In December 2019, coronavirus disease 2019 (COVID-19), which is caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in Wuhan (Hubei province, China)1; it soon spread across the world. In this ongoing pandemic, public health concerns and the urgent need for effective therapeutic measures require a deep understanding of the epidemiology, transmissibility and pathogenesis of COVID-19. Here we analysed clinical, molecular and immunological data from 326 patients with confirmed SARS-CoV-2 infection in Shanghai. The genomic sequences of SARS-CoV-2, assembled from 112 high-quality samples together with sequences in the Global Initiative on Sharing All Influenza Data (GISAID) dataset, showed a stable evolution and suggested that there were two major lineages with differential exposure history during the early phase of the outbreak in Wuhan. Nevertheless, they exhibited similar virulence and clinical outcomes. Lymphocytopenia, especially reduced CD4+ and CD8+ T cell counts upon hospital admission, was predictive of disease progression. High levels of interleukin (IL)-6 and IL-8 during treatment were observed in patients with severe or critical disease and correlated with decreased lymphocyte count. The determinants of disease severity seemed to stem mostly from host factors such as age and lymphocytopenia (and its associated cytokine storm), whereas viral genetic variation did not significantly affect outcomes.

OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG.

Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl ß-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.

Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.

In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved ß flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.

The global nitrogen regulator GlnR is a direct transcriptional repressor of the key gluconeogenic gene pckA in actinomycetes.

In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei. Next, we proved that the pckA gene was essential for A. mediterranei gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes (Streptomyces coelicolor and Mycobacterium smegmatis). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses. IMPORTANCE: The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA, the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.

Zymograph profiling reveals a divergent evolution of sirtuin that may originate from class III enzymes.

Sirtuins are a group of NAD+-dependent deacylases that conserved in three domains of life and comprehensively involved in the regulation of gene transcription, chromosome segregation, RNA splicing, apoptosis, and aging. Previous studies in mammalian cells have revealed that sirtuins not only exist as multiple copies, but also show distinct deacylase activities in addition to deacetylation. However, the understanding of sirtuin zymographs in other organisms with respect to molecular evolution remains at an early stage. Here, we systematically analyze the sirtuin activities in representative species from archaea, bacteria, and eukaryotes, using both the HPLC assay and a 7-amino-4-methylcoumarin-based fluorogenic method. Global profiling suggests that the deacylase activities of sirtuins could be divided into three categories and reveals undifferentiated zymographs of class III sirtuins, especially for those from bacteria and archaea. Nevertheless, initial differentiation of enzymatic activity was also observed for the class III sirtuins at both paralog and ortholog levels. Further phylogenetic analyses support a divergent evolution of sirtuin that may originate from class III sirtuins. Together, this work demonstrates a comprehensive panorama of sirtuin zymographs and provides new insights into the cellular specific regulation and molecular evolution of sirtuins.

A Noncoding A-to-U Kozak Site Change Related to the High Transmissibility of Alpha, Delta, and Omicron VOCs.

Three prevalent SARS-CoV-2 variants of concern (VOCs) emerged and caused epidemic waves. It is essential to uncover advantageous mutations that cause the high transmissibility of VOCs. However, viral mutations are tightly linked, so traditional population genetic methods, including machine learning-based methods, cannot reliably detect mutations conferring a fitness advantage. In this study, we developed an approach based on the sequential occurrence order of mutations and the accelerated furcation rate in the pandemic-scale phylogenomic tree. We analyzed 3,777,753 high-quality SARS-CoV-2 genomic sequences and the epidemiology metadata using the Coronavirus GenBrowser. We found that two noncoding mutations at the same position (g.a28271-/u) may be crucial to the high transmissibility of Alpha, Delta, and Omicron VOCs although the noncoding mutations alone cannot increase viral transmissibility. Both mutations cause an A-to-U change at the core position -3 of the Kozak sequence of the N gene and significantly reduce the protein expression ratio of ORF9b to N. Using a convergent evolutionary analysis, we found that g.a28271-/u, S:p.P681H/R, and N:p.R203K/M occur independently on three VOC lineages, suggesting that coordinated changes of S, N, and ORF9b proteins are crucial to high viral transmissibility. Our results provide new insights into high viral transmissibility co-modulated by advantageous noncoding and nonsynonymous changes.

Quantitative measurement of cell-surface displayed proteins based on split-GFP assembly.

BACKGROUND: Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells. RESULTS: In this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively. CONCLUSIONS: Our study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.

Creating a functional single-chromosome yeast.

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.

Comparative efficacy of sequential treatment and open abdomen approaches for corrosive abdominal hemorrhage due to inadequate drainage of duodenal leakage: a cohort study.

BACKGROUND: Intra-abdominal bleeding resulting from inadequate drainage of duodenal leakage (DL) is typically caused by the corrosiveness of duodenal fluid. Open abdomen (OA) treatment addresses both the drainage and bleeding simultaneously. However, a sequential treatment (ST) approach involving hemostasis through transcatheter arterial embolization (TAE) followed by percutaneous drainage of source control has emerged as an alternative method. This study aimed to evaluate the prognosis of ST in cases of DL-induced intra-abdominal bleeding. METHODS: This retrospective cohort study included 151 participants diagnosed with DL-induced intra-abdominal bleeding from January 2004 to December 2010, and January 2013 to December 2021. The ST and OA groups were established based on the treatment method applied. Propensity score-matching (PSM) matched patients in the ST group with those in the OA group. RESULTS: Among the 151 patients, 61 (40.4%) died within 90 days after the bleeding episode. ST was associated with a lower mortality rate (28.2% vs. 51.3% adjusted odds ratio [OR] = 0.34; 95% confidence interval [CI] 0.17-0.68; P = 0.003) compared to OA. Following PSM, ST remained the only factor associated with reduced mortality (OR = 0.32; 95% CI 0.13-0.75; P = 0.009). Moreover, ST demonstrated a higher rate of initial hemostasis success before (90.1% [64/71] vs. 77.5% [62/80]; adjusted OR = 2.84; 95% CI 1.07-7.60; P = 0.04) and after PSM (94.4% [51/54] vs. 77.8% [42/54], adjusted OR = 3.85; 95% CI 2.15-16.82; P = 0.04). Additionally, ST was associated with a lower incidence of rebleeding within 90 days after the initial bleeding, before (7 vs. 23; adjusted OR 0.41; 95% CI 0.18-0.92; P = 0.03) and after PSM (5 vs. 14; adjusted OR 0.37; 95% CI 0.15-0.93; P = 0.03). CONCLUSIONS: Applying ST involving TAE and subsequent percutaneous drainage might be superior to OA in lowering the mortality in DL-induced intra-abdominal hemorrhage.

Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence.

Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from ß-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.

The use of amino acids and their derivates to mitigate against pesticide-induced toxicity.

Exposure to pesticides induces oxidative stress and deleterious effects on various tissues in non-target organisms. Numerous models investigating pesticide exposure have demonstrated metabolic disturbances such as imbalances in amino acid levels within the organism. One potentially effective strategy to mitigate pesticide toxicity involves dietary intervention by supplementing exogenous amino acids and their derivates to augment the body's antioxidant capacity and mitigate pesticide-induced oxidative harm, whose mechanism including bolstering glutathione synthesis, regulating arginine-NO metabolism, mitochondria-related oxidative stress, and the open of ion channels, as well as enhancing intestinal microecology. Enhancing glutathione synthesis through supplementation of substrates N-acetylcysteine and glycine is regarded as a potent mechanism to achieve this. Selection of appropriate amino acids or their derivates for supplementation, and determining an appropriate dosage, are of the utmost importance for effective mitigation of pesticide-induced oxidative harm. More experimentation is required that involves large population samples to validate the efficacy of dietary intervention strategies, as well as to determine the effects of amino acids and their derivates on long-term and low-dose pesticide exposure. This review provides insights to guide future research aimed at preventing and alleviating pesticide toxicity through dietary intervention of amino acids and their derivates.

Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate neuropathic pain.

(Switching from the microglial M1 phenotype to the M2 phenotype is a promising therapeutic strategy for neuropathic pain (NP). This study aimed to investigate the potential use of stigmasterol for treating NP. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, chronic constriction injury (CCI) group, CCI + ibuprofen group, and CCI + stigmasterol group. We performed behavioral tests, enzyme-linked immunosorbent assay, hematoxylin-esoin staining (H&E) staining and immunohistochemistry, immunofluorescence, and Western blotting. In cell experiments, we performed flow cytometry, immunofluorescence, Western blotting, and qRT-PCR. Stigmasterol reduced thermal and mechanical hyperalgesia and serum IL-1ß and IL-8 levels and increased serum IL-4 and TGF-ß levels in CCI rats. Stigmasterol reduced IL-1ß, COX-2, and TLR4 expression in the right sciatic nerve and IL-1ß expression in the spinal cord. Stigmasterol reduced the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, COX-2, IL-1ß, and CD32 in the spinal cord of CCI rats while increasing the expression of IL-10 and CD206. Stigmasterol decreased M1 polarization markers and increased M2 polarization markers in lipopolysaccharide (LPS)-induced microglia and decreased the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, iNOS, COX-2, and IL-1ß in LPS-treated microglia while increasing the expression of Arg-1 and IL-10. Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate NP.

Chiral Skyrmions Interacting with Chiral Flowers.

The chiral nature of active matter plays an important role in the dynamics of active matter interacting with chiral structures. Skyrmions are chiral objects, and their interactions with chiral nanostructures can lead to intriguing phenomena. Here, we explore the random-walk dynamics of a thermally activated chiral skyrmion interacting with a chiral flower-like obstacle in a ferromagnetic layer, which could create topology-dependent outcomes. It is a spontaneous mesoscopic order-from-disorder phenomenon driven by the thermal fluctuations and topological nature of skyrmions that exists only in ferromagnetic and ferrimagnetic systems. The interactions between the skyrmions and chiral flowers at finite temperatures can be utilized to control the skyrmion position and distribution without applying any external driving force or temperature gradient. The phenomenon that thermally activated skyrmions are dynamically coupled to chiral flowers may provide a new way to design topological sorting devices.

Deterministic Magnetization Switching via Tunable Noncollinear Spin Configurations in Canted Magnets.

Spin obit torque (SOT) driven magnetization switching has been used widely for encoding consumption-efficient memory and logic. However, symmetry breaking under a magnetic field is required to realize the deterministic switching in synthetic antiferromagnets with perpendicular magnetic anisotropy (PMA), which limits their potential applications. Herein, we report all electric-controlled magnetization switching in the antiferromagnetic Co/Ir/Co trilayers with vertical magnetic imbalance. Besides, the switching polarity could be reversed by optimizing the Ir thickness. By using the polarized neutron reflection (PNR) measurements, the canted noncollinear spin configuration was observed in Co/Ir/Co trilayers, which results from the competition of magnetic inhomogeneity. In addition, the asymmetric domain walls demonstrated by micromagnetic simulations result from introducing imbalance magnetism, leading to the deterministic magnetization switching in Co/Ir/Co trilayers. Our findings highlight a promising route to electric-controlled magnetism via tunable spin configuration, improve our understanding of physical mechanisms, and significantly promote industrial applications in spintronic devices.

Ginsenoside Rb1, Compound K and 20(S)-Protopanaxadiol Attenuate High-Fat Diet-Induced Hyperlipidemia in Rats via Modulation of Gut Microbiota and Bile Acid Metabolism.

Hyperlipidemia, characterized by elevated serum lipid concentrations resulting from lipid metabolism dysfunction, represents a prevalent global health concern. Ginsenoside Rb1, compound K (CK), and 20(S)-protopanaxadiol (PPD), bioactive constituents derived from Panax ginseng, have shown promise in mitigating lipid metabolism disorders. However, the comparative efficacy and underlying mechanisms of these compounds in hyperlipidemia prevention remain inadequately explored. This study investigates the impact of ginsenoside Rb1, CK, and PPD supplementation on hyperlipidemia in rats induced by a high-fat diet. Our findings demonstrate that ginsenoside Rb1 significantly decreased body weight and body weight gain, ameliorated hepatic steatosis, and improved dyslipidemia in HFD-fed rats, outperforming CK and PPD. Moreover, ginsenoside Rb1, CK, and PPD distinctly modified gut microbiota composition and function. Ginsenoside Rb1 increased the relative abundance of Blautia and Eubacterium, while PPD elevated Akkermansia levels. Both CK and PPD increased Prevotella and Bacteroides, whereas Clostridium-sensu-stricto and Lactobacillus were reduced following treatment with all three compounds. Notably, only ginsenoside Rb1 enhanced lipid metabolism by modulating the PPARγ/ACC/FAS signaling pathway and promoting fatty acid ß-oxidation. Additionally, all three ginsenosides markedly improved bile acid enterohepatic circulation via the FXR/CYP7A1 pathway, reducing hepatic and serum total bile acids and modulating bile acid pool composition by decreasing primary/unconjugated bile acids (CA, CDCA, and ß-MCA) and increasing conjugated bile acids (TCDCA, GCDCA, GDCA, and TUDCA), correlated with gut microbiota changes. In conclusion, our results suggest that ginsenoside Rb1, CK, and PPD supplementation offer promising prebiotic interventions for managing HFD-induced hyperlipidemia in rats, with ginsenoside Rb1 demonstrating superior efficacy.

Transcriptional Self-Regulation of the Master Nitrogen Regulator GlnR in Mycobacteria.

As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic pathways. To date, most studies have been focused on the GlnR regulon, while little is known about the transcriptional regulator for glnR itself. It has been observed that glnR transcription can be upregulated in Mycobacterium smegmatis under nitrogen-limited growth conditions; however, the detailed regulatory mechanism is still unclear. Here, we demonstrate that the glnR gene in M. smegmatis is transcriptionally activated by its product GlnR in response to nitrogen limitation. The precise GlnR binding site was successfully characterized in its promoter region using the electrophoretic mobility shift assay and the DNase I footprinting assay. Site mutagenesis and genetic analyses confirmed that the binding site was essential for in vivo self-activation of glnR transcription. Moreover, based on bioinformatic analyses, we discovered that most of the mycobacterial glnR promoter regions (144 out of 147) contain potential GlnR binding sites, and we subsequently proved that the purified M. smegmatis GlnR protein could specifically bind 16 promoter regions that represent 119 mycobacterial species, including Mycobacterium tuberculosis. Together, our findings not only elucidate the transcriptional self-regulation mechanism of glnR transcription in M. smegmatis but also indicate the ubiquity of the mechanism in other mycobacterial species. IMPORTANCE In actinomycetes, the nitrogen metabolism not only is essential for the construction of life macromolecules but also affects the biosynthesis of secondary metabolites and even virulence (e.g., Mycobacterium tuberculosis). The transcriptional regulation of genes involved in nitrogen metabolism has been thoroughly studied and involves the master nitrogen regulator GlnR. However, the transcriptional regulation of glnR itself remains elusive. Here, we demonstrated that GlnR functions as a transcriptional self-activator in response to nitrogen starvation in the fast-growing model Mycobacterium species Mycobacterium smegmatis. We further showed that this self-regulation mechanism could be widespread in other mycobacteria, which might be beneficial for those slow-growing mycobacteria to adapt to the nitrogen-starvation environments such as within human macrophages for M. tuberculosis.

zPoseScore model for accurate and robust protein-ligand docking pose scoring in CASP15.

We introduce a deep learning-based ligand pose scoring model called zPoseScore for predicting protein-ligand complexes in the 15th Critical Assessment of Protein Structure Prediction (CASP15). Our contributions are threefold: first, we generate six training and evaluation data sets by employing advanced data augmentation and sampling methods. Second, we redesign the "zFormer" module, inspired by AlphaFold2's Evoformer, to efficiently describe protein-ligand interactions. This module enables the extraction of protein-ligand paired features that lead to accurate predictions. Finally, we develop the zPoseScore framework with zFormer for scoring and ranking ligand poses, allowing for atomic-level protein-ligand feature encoding and fusion to output refined ligand poses and ligand per-atom deviations. Our results demonstrate excellent performance on various testing data sets, achieving Pearson's correlation R = 0.783 and 0.659 for ranking docking decoys generated based on experimental and predicted protein structures of CASF-2016 protein-ligand complexes. Additionally, we obtain an averaged local distance difference test (lDDT pli = 0.558) of AIchemy LIG2 in CASP15 for de novo protein-ligand complex structure predictions. Detailed analysis shows that accurate ligand binding site prediction and side-chain orientation are crucial for achieving better prediction performance. Our proposed model is one of the most accurate protein-ligand pose prediction models and could serve as a valuable tool in small molecule drug discovery.

Development of a mammalian cell-based ZZ display system for IgG quantification.

BACKGROUND: Biological laboratories and companies involved in antibody development need convenient and versatile methods to detect highly active antibodies. METHODS: To develop a mammalian cell-based ZZ display system for antibody quantification, the eukaryotic ZZ-displayed plasmid was constructed and transfected into CHO cells. After screening by flow cytometric sorting, the stable ZZ display cells were incubated with reference IgG and samples with unknown IgG content for 40 min at 4℃, the relative fluorescence intensity of cells was analyzed and the concentration of IgG was calculated. RESULTS: By investigating the effects of different display-associated genetic elements, a eukaryotic ZZ-displaying plasmid with the highest display efficiency were constructed. After transfection and screening, almost 100% of the cells were able to display the ZZ peptide (designated CHO-ZZ cells). These stable CHO-ZZ cells were able to capture a variety of IgG, including human, rabbit, donkey and even mouse and goat. CHO-ZZ cells could be used to quantify human IgG in the range of approximately 12.5-1000 ng/mL, and to identify high-yielding engineered monoclonal cell lines. CONCLUSIONS: We have established a highly efficient CHO-ZZ display system in this study, which enables the quantification of IgG from various species under physiological conditions. This system offers the advantage of eliminating the need for antibody purification and will contribute to antibody development.

Exploiting spatial dimensions to enable parallelized continuous directed evolution.

Current strategies to improve the throughput of continuous directed evolution technologies often involve complex mechanical fluid-controlling system or robotic platforms, which limits their popularization and application in general laboratories. Inspired by our previous study on bacterial range expansion, in this study, we report a system termed SPACE for rapid and extensively parallelizable evolution of biomolecules by introducing spatial dimensions into the landmark phage-assisted continuous evolution system. Specifically, M13 phages and chemotactic Escherichia coli cells were closely inoculated onto a semisolid agar. The phages came into contact with the expanding front of the bacterial range, and then comigrated with the bacteria. This system leverages competition over space, wherein evolutionary progress is closely associated with the production of spatial patterns, allowing the emergence of improved or new protein functions. In a prototypical problem, SPACE remarkably simplified the process and evolved the promoter recognition of T7 RNA polymerase (RNAP) to a library of 96 random sequences in parallel. These results establish SPACE as a simple, easy to implement, and massively parallelizable platform for continuous directed evolution in general laboratories.