Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch.

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology. In comparison to plasmid-based multi-copy expression, the utilization of chromosomal multi-copy genes offers increased stability of expression level, diminishes the metabolic burden on host cells, and enhances overall genetic stability. In this study, we developed the "BacAmp", a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis, which was achieved by employing a combination of repressor and non-natural amino acids (ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination. When the reversible switch is turned on, genome editing and gene amplification can be achieved. Subsequently, the reversible switch was turned off therefore stabilizing the gene copy number. The stabilized gene amplification system marked by green fluorescent protein, achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations. When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid (NeuAc) synthesis, the copy number of the critical gene increased to an average of 7.7, which yielded a 1.3-fold NeuAc titer. Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B. subtilis.

Analysis of functional connectivity changes in attention networks and default mode networks in patients with depression and insomnia.

PURPOSE: Major Depressive Disorder (MDD) and Insomnia Disorder (ID) are prevalent psychiatric conditions often occurring concurrently, leading to substantial impairment in daily functioning. Understanding the neurobiological underpinnings of these disorders and their comorbidity is crucial for developing effective interventions. This study aims to analyze changes in functional connectivity within attention networks and default mode networks in patients with depression and insomnia. METHODS: The functional connectivity alterations in individuals with MDD, ID, comorbid MDD and insomnia (iMDD), and healthy controls (HC) were assessed from a cohort of 174 participants. They underwent rs-fMRI scans, demographic assessments, and scale evaluations for depression and sleep quality. Functional connectivity analysis was conducted using region-of-interest (ROI) and whole-brain methods. RESULTS: The MDD and iMDD groups exhibited higher Hamilton Depression Scale (HAMD) scores compared to HC and ID groups (P < 0.001). Both ID and MDD groups displayed enhanced connectivity between the left and right orbital frontal cortex compared to HC (P < 0.05), while the iMDD group showed reduced connectivity compared to HC and ID groups (P < 0.05). In the left insula, reduced connectivity with the right medial superior frontal gyrus was observed across patient groups compared to HC (P < 0.05), with the iMDD group showing increased connectivity compared to MDD (P < 0.05). Moreover, alterations in functional connectivity between the left thalamus and left temporal pole were found in iMDD compared to HC and MDD (P < 0.05). Correlation analyses revealed associations between abnormal connectivity and symptom severity in MDD and ID groups. CONCLUSIONS: Our findings demonstrate distinct patterns of altered functional connectivity in individuals with MDD, ID, and iMDD compared to healthy controls. These findings contribute to a better understanding of the pathophysiology of depression and insomnia, which could be used as a reference for the diagnosis and treatments of these patients.

Multiscale 3D genome organization underlies duck fatty liver with no adipose inflammation or serious injury.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Little is known about how gene expression and chromatin structure are regulated in NAFLD due to lack of suitable model. Ducks naturally develop fatty liver similar to serious human non-alcoholic fatty liver (NAFL) without adipose inflammation and liver fibrosis, thus serves as a good model for investigating molecular mechanisms of adipose metabolism and anti-inflammation. Here, we constructed a NAFLD model without adipose inflammation and liver fibrosis in ducks. By performing dynamic pathological and transcriptomic analyses, we identified critical genes involving in regulation of the NF-κB and MHCII signaling, which usually lead to adipose inflammation and liver fibrosis. We further generated dynamic three-dimensional chromatin maps during liver fatty formation and recovery. This showed that ducks enlarged hepatocyte cell nuclei to reduce inter-chromosomal interaction, decompress chromatin structure, and alter strength of intra-TAD and loop interactions during fatty liver formation. These changes partially contributed to the tight control the NF-κB and the MHCII signaling. Our analysis uncovers duck chromatin reorganization might be advantageous to maintain liver regenerative capacity and reduce adipose inflammation. These findings shed light on new strategies for NAFLD control.

Pd-Catalyzed Decarbonylative sp2 C-H Arylation: Construction of Five- and Six-Membered (Hetero)Cyclic Compounds.

The cyclic compounds have wide applications in the design and synthesis of drugs and materials; thus, their efficient construction attracts much attention from the synthetic community. In this letter, we report an efficient method for preparing cyclic compounds starting from the readily available carboxylic acids. This reaction takes place through intramolecular decarbonylative sp2 C-H arylation, enabling efficient synthesis of a wide range of five- and six-membered cyclic compounds. Both carbo- and heterocycles can be produced under the reaction conditions. Moreover, this reaction features a wide substrate scope with high functional group tolerance. The scale-up experiments also show its practicality in organic synthesis. Those experimental results indicate that this reaction would find wide applications in the synthetic community.

CuH-Catalyzed Reductive Coupling of Nitroarenes with Phosphine Oxides for the Direct Synthesis of Phosphamides.

A CuH-catalyzed reductive coupling of nitroarenes with phosphine oxides is developed, which produces a series of phosphamides in moderate to excellent yields with good functional group tolerance. Gram-scale synthesis and late-stage modification of nitro-aromatic functional molecule niclosamide are also successfully conducted. The mechanism study shows that the nitro group is transformed after being reduced to nitroso and a nucleophilic addition procedure is involved during the reaction.

Effects of micro- and nano-plastics on growth, antioxidant system, DMS, and DMSP production in Emiliania huxleyi.

Due to the potential impacts of microplastics (MPs) and nanoplastics (NPs) on algal growth and thereby affect the climate-relevant substances, dimethylsulfoniopropionate (DMSP) and dimethyl sulfide (DMS), we studied the polystyrene (PS) MPs and NPs of 1 µm and 80 nm impacts on the growth, chlorophyll content, reactive oxygen species (ROS), antioxidant enzyme activity, and DMS/DMSP production in Emiliania huxleyi. E. huxleyi is a prominent oceanic alga that plays a key role in DMS and DMSP production. The results revealed that high concentrations of MPs and NPs inhibited the growth, carotenoid (Car), and Chl a concentrations of E. huxleyi. However, short-time exposure to low concentrations of PS MPs and NPs stimulated the growth of E. huxleyi. Furthermore, high concentrations of MPs and NPs resulted in an increase in the superoxide anion radical (O2.-) production rate and a decrease in the malondialdehyde (MDA) content compared with the low concentrations. Exposure to MPs and NPs at 5 mg L-1 induced superoxide dismutase (SOD) activity as a response to scavenging ROS. High concentrations of MPs and NPs significantly inhibited the production of DMSP and DMS. The findings of this study support the potential ecotoxicological impacts of MPs and NPs on algal growth, antioxidant system, and dimethylated sulfur compounds production, which maybe potentially impact the global climate.

Domain wall magnetic tunnel junction-based artificial synapses and neurons for all-spin neuromorphic hardware.

We report a breakthrough in the hardware implementation of energy-efficient all-spin synapse and neuron devices for highly scalable integrated neuromorphic circuits. Our work demonstrates the successful execution of all-spin synapse and activation function generator using domain wall-magnetic tunnel junctions. By harnessing the synergistic effects of spin-orbit torque and interfacial Dzyaloshinskii-Moriya interaction in selectively etched spin-orbit coupling layers, we achieve a programmable multi-state synaptic device with high reliability. Our first-principles calculations confirm that the reduced atomic distance between 5d and 3d atoms enhances Dzyaloshinskii-Moriya interaction, leading to stable domain wall pinning. Our experimental results, supported by visualizing energy landscapes and theoretical simulations, validate the proposed mechanism. Furthermore, we demonstrate a spin-neuron with a sigmoidal activation function, enabling high operation frequency up to 20 MHz and low energy consumption of 508 fJ/operation. A neuron circuit design with a compact sigmoidal cell area and low power consumption is also presented, along with corroborated experimental implementation. Our findings highlight the great potential of domain wall-magnetic tunnel junctions in the development of all-spin neuromorphic computing hardware, offering exciting possibilities for energy-efficient and scalable neural network architectures.

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

Active Dynamic Weighting for multi-domain adaptation.

Multi-source unsupervised domain adaptation aims to transfer knowledge from multiple labeled source domains to an unlabeled target domain. Existing methods either seek a mixture of distributions across various domains or combine multiple single-source models for weighted fusion in the decision process, with little insight into the distributional discrepancy between different source domains and the target domain. Considering the discrepancies in global and local feature distributions between different domains and the complexity of obtaining category boundaries across domains, this paper proposes a novel Active Dynamic Weighting (ADW) for multi-source domain adaptation. Specifically, to effectively utilize the locally advantageous features in the source domains, ADW designs a multi-source dynamic adjustment mechanism during the training process to dynamically control the degree of feature alignment between each source and target domain in the training batch. In addition, to ensure the cross-domain categories can be distinguished, ADW devises a dynamic boundary loss to guide the model to focus on the hard samples near the decision boundary, which enhances the clarity of the decision boundary and improves the model's classification ability. Meanwhile, ADW applies active learning to multi-source unsupervised domain adaptation for the first time, guided by dynamic boundary loss, proposes an efficient importance sampling strategy to select target domain hard samples to annotate at a minimal annotation budget, integrates it into the training process, and further refines the domain alignment at the category level. Experiments on various benchmark datasets consistently demonstrate the superiority of our method.

Engineering the passivation routes of perovskite films towards high performance solar cells.

Passivation treatment is an effective method to suppress various defects in perovskite solar cells (PSCs), such as cation vacancies, under-coordinated Pb2+ or I-, and Pb-I antisite defects. A thorough understanding of the diversified impacts of different defect passivation methods (DPMs) on the device performance will be beneficial for making wise DPM choices. Herein, we choose a hydrophobic Lewis acid tris(pentafluorophenyl)borane (BCF), which can dissolve in both the perovskite precursor and anti-solvent, as the passivation additive. BCF treatment can immobilize organic cations via forming hydrogen bonds. Three kinds of DPMs based on BCF are applied to modify perovskite films in this work. It is found that the best DPM with BCF dissolved in anti-solvent can not only passivate multiple defects in perovskite, but also inhibit δ phase perovskite and improve the stability of devices. Meanwhile, DPM with BCF dissolved in both the perovskite precursor and anti-solvent can cause cracks and voids in perovskite films and deteriorate device performance, which should be avoided in practical applications. As a result, PSCs based on optimal DPMs of BCF present an increased efficiency of 22.86% with negligible hysteresis as well as improved overall stability. This work indicates that the selection and optimization of DPMs have an equally important influence on the photovoltaic performance of PSCs as the selection of passivation additives.

Asymmetric magnetization switching and programmable complete Boolean logic enabled by long-range intralayer Dzyaloshinskii-Moriya interaction.

After decades of efforts, some fundamental physics for electrical switching of magnetization is still missing. Here, we report the discovery of the long-range intralayer Dzyaloshinskii-Moriya interaction (DMI) effect, which is the chiral coupling of orthogonal magnetic domains within the same magnetic layer via the mediation of an adjacent heavy metal layer. The effective magnetic field of the long-range intralayer DMI on the perpendicular magnetization is out-of-plane and varies with the interfacial DMI constant, the applied in-plane magnetic fields, and the magnetic anisotropy distribution. Striking consequences of the effect include asymmetric current/field switching of perpendicular magnetization, hysteresis loop shift of perpendicular magnetization in the absence of in-plane direct current, and sharp in-plane magnetic field switching of perpendicular magnetization. Utilizing the intralayer DMI, we demonstrate programable, complete Boolean logic operations within a single spin-orbit torque device. These results will stimulate investigation of the long-range intralayer DMI effect in a variety of spintronic devices.

Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Machine learning-driven diagnostic signature provides new insights in clinical management of hypertrophic cardiomyopathy.

AIMS: In an era of evolving diagnostic possibilities, existing diagnostic systems are not fully sufficient to promptly recognize patients with early-stage hypertrophic cardiomyopathy (HCM) without symptomatic and instrumental features. Considering the sudden death of HCM, developing a novel diagnostic model to clarify the patients with early-stage HCM and the immunological characteristics can avoid misdiagnosis and attenuate disease progression. METHODS AND RESULTS: Three hundred eighty-five samples from four independent cohorts were systematically retrieved. The weighted gene co-expression network analysis, differential expression analysis (|log2(foldchange)| > 0.5 and adjusted P < 0.05), and protein-protein interaction network were sequentially performed to identify HCM-related hub genes. With a machine learning algorithm, the least absolute shrinkage and selection operator regression algorithm, a stable diagnostic model was developed. The immune-cell infiltration and biological functions of HCM were also explored to characterize its underlying pathogenic mechanisms and the immune signature. Two key modules were screened based on weighted gene co-expression network analysis. Pathogenic mechanisms relevant to extracellular matrix and immune pathways have been discovered. Twenty-seven co-regulated genes were recognized as HCM-related hub genes. Based on the least absolute shrinkage and selection operator algorithm, a stable HCM diagnostic model was constructed, which was further validated in the remaining three cohorts (n = 385). Considering the tight association between HCM and immune-related functions, we assessed the infiltrating abundance of various immune cells and stromal cells based on the xCell algorithm, and certain immune cells were significantly different between high-risk and low-risk groups. CONCLUSIONS: Our study revealed a number of hub genes and novel pathways to provide potential targets for the treatment of HCM. A stable model was developed, providing an efficient tool for the diagnosis of HCM.

Influence of the community assemblage on sulfur distributions in the South China sea.

Marine distribution of dimethylsulfoniopropionate (DMSP) and its cleavage product dimethyl sulfide (DMS) is greatly affected by the community structures of bacteria, phytoplankton, and zooplankton. Spatial distributions of dissolved and particulate DMSP (DMSPd,p), and DMS were measured and their relationships with DMSP lyase activity (DLA), abundance of DMSP-consuming bacteria (DCB), and the community structures of phytoplankton, zooplankton, and bacteria were determined during summer in the South China Sea (SCS). The depth distributions of DMSPd,p exhibited a similar trend with Chl a, reaching their maxima in the mixing layer. The DMS concentration was positively correlated with DCB abundance and DLA, indicating that DCB and DMSP lyase had a significant effect on DMS production. High DMS concentrations in the horizontal distribution coincided with high DCB abundance and DLA and may be due to the rapid growth of phytoplankton resulting from the high dissolved inorganic nitrogen concentration brought by the cold vortices. Moreover, the highest copepod abundance at station G3 coincided with the highest DMS concentrations there among stations B4, F2, and G3. These results suggest that copepod may play an important role in DMS production. The bacterial SAR11 clade was positively correlated with DLA, indicating its significant contribution to DMSP degradation in the SCS. These findings contribute to the understanding of the effect of the community assemblage on DMSP/DMS distributions in the SCS dominated by mesoscale vortices.

Factors affecting long-term myopic regression after corneal refractive surgery for civilian pilots in southwest China.

BACKGROUND: The purpose of this study was to analyze myopic regression after corneal refractive surgery (CRS) in civilian pilots and to explore the factors that may cause long-term myopic regression. METHODS: We included civilian pilots who had undergone CRS to correct their myopia and who had at least 5 years of follow-up. We collected retrospective data and completed eye examinations and a questionnaire to assess their eye habits. RESULTS: A total of 236 eyes were evaluated in this study. 211 eyes had Intrastromal ablations (167 eyes had laser in situ keratomileusis, LASIK, 44 eyes had small incision lenticule extraction, SMILE) and 25 eyes had subepithelial ablations (15 eyes had laser epithelial keratomileusis, LASEK and 10 eyes had photorefractive keratectomy, PRK). The mean preoperative spherical equivalent (SE) was - 2.92 ± 1.11 D (range from - 1.00 to -5.00 D). A total of 56 eyes (23.6%) suffered from myopic regression after CRS. Comparisons of individual and eye characteristics between the regression and non-regression groups revealed statistically significant differences in age, cumulative flight time, postoperative SE (at 6 months and current), uncorrected visual acuity (UCVA), accommodative amplitude (AA), positive relative accommodation (PRA), postoperative period, types of CRS and eye habits. Generalized propensity score weighting (GPSW) was used to balance the distribution of covariates among different age levels, types of CRS, cumulative flying time, postoperative period and continuous near-work time. The results of GPS weighted logistic regression demonstrated that the associations between age and myopic regression, types of CRS and myopic regression, continuous near-work time and myopic regression were significant. Cumulative flying time and myopic regression, postoperative period and myopic regression were no significant. Specifically, the odds ratio (OR) for age was 1.151 (P = 0.022), and the OR for type of CRS was 2.769 (P < 0.001). The OR for continuous near-work time was 0.635 with a P value of 0.038. CONCLUSIONS: This is the first report to analyze myopic regression after CRS in civilian pilots. Our study found that for each year increase in age, the risk of civilian pilots experiencing myopic regression was increased. Intrastromal ablations had a lower risk of long-term myopia regression than subepithelial ablations. There is a higher risk of myopic progression with continuous near-work time > 45 min and poor accommodative function may be related factors in this specific population.

Deficiency of betaine-homocysteine methyltransferase activates glucose-6-phosphate dehydrogenase (G6PD) by decreasing arginine methylation of G6PD in hepatocellular carcinogenesis.

Betaine-homocysteine methyltransferase (BHMT) regulates protein methylation and is correlated with tumorigenesis; however, the effects and regulation of BHMT in hepatocarcinogenesis remain largely unexplored. Here, we determined the clinical significance of BHMT in the occurrence and progression of hepatocellular carcinoma (HCC) using tissue samples from 198 patients. BHMT was to be frequently found (86.6%) expressed at relatively low levels in HCC tissues and was positively correlated with the overall survival of patients with HCC. Bhmt overexpression effectively suppressed several malignant phenotypes in hepatoma cells in vitro and in vivo, whereas complete knockout of Bhmt (Bhmt-/-) produced the opposite effect. We combined proteomics, metabolomics, and molecular biological strategies and detected that Bhmt-/- promoted hepatocarcinogenesis and tumor progression by enhancing the activity of glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolism in DEN-induced HCC mouse and subcutaneous tumor-bearing models. In contrast, restoration of Bhmt with an AAV8-Bhmt injection or pharmacological inhibition of G6PD attenuated hepatocarcinogenesis. Additionally, coimmunoprecipitation identified monomethylated modifications of the G6PD, and BHMT regulated the methylation of G6PD. Protein sequence analysis, generation and application of specific antibodies, and site-directed mutagenesis indicated G6PD methylation at the arginine residue 246. Furthermore, we established bidirectionally regulated BHMT cellular models combined with methylation-deficient G6PD mutants to demonstrate that BHMT potentiated arginine methylation of G6PD, thereby inhibiting G6PD activity, which in turn suppressed hepatocarcinogenesis. Taken together, this study reveals a new methylation-regulatory mechanism in hepatocarcinogenesis owing to BHMT deficiency, suggesting a potential therapeutic strategy for HCC treatment.

Integration of Whole-Genome Resequencing and Transcriptome Sequencing Reveals Candidate Genes in High Glossiness of Eggshell.

Eggshell gloss is an important characteristic for the manifestation of eggshell appearance. However, no study has yet identified potential candidate genes for eggshell gloss between high-gloss (HG) and low-gloss (LG) chickens. The aim of this study was to perform a preliminary investigation into the formation mechanism of eggshell gloss and to identify potential genes. The eggshell gloss of 300-day-old Rhode Island Red hens was measured from three aspects. Uterine tissues of the selected HG and LG (n = 5) hens were collected for RNA-seq. Blood samples were also collected for whole-genome resequencing (WGRS). RNA-seq analysis showed that 150 differentially expressed genes (DEGs) were identified in the uterine tissues of HG and LG hens. These DEGs were mainly enriched in the calcium signaling pathway and the neuroactive ligand-receptor interaction pathway. Importantly, these two pathways were also significantly enriched in the WGRS analysis results. Further joint analysis of WGRS and RNA-seq data revealed that 5-hydroxytryptamine receptor 1F (HTR1F), zinc finger protein 536 (ZNF536), NEDD8 ubiquitin-like modifier (NEDD8), nerve growth factor (NGF) and calmodulin 1 (CALM1) are potential candidate genes for eggshell gloss. In summary, our research provides a reference for the study of eggshell gloss and lays a foundation for improving egg glossiness in layer breeding.

Inhibition of the RLR signaling pathway by SARS-CoV-2 ORF7b is mediated by MAVS and abrogated by ORF7b-homologous interfering peptide.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and characterized by dysregulated immune response. Studies have shown that the SARS-CoV-2 accessory protein ORF7b induces host cell apoptosis through the tumor necrosis factor alpha (TNF-α) pathway and blocks the production of interferon beta (IFN-ß). The underlying mechanism remains to be investigated. In this study, we found that ORF7b facilitated viral infection and production, and inhibited the RIG-I-like receptor (RLR) signaling pathway through selectively interacting with mitochondrial antiviral-signaling protein (MAVS). MAVS439-466 region and MAVS Lys461 were essential for the physical association between MAVS and ORF7b, and the inhibition of the RLR signaling pathway by ORF7b. MAVSK461/K63 ubiquitination was essential for the RLR signaling regulated by the MAVS-ORF7b complex. ORF7b interfered with the recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) and the activation of the RLR signaling pathway by MAVS. Furthermore, interfering peptides targeting the ORF7b complex reversed the ORF7b-suppressed MAVS-RLR signaling pathway. The most potent interfering peptide V disrupts the formation of ORF7b tetramers, reverses the levels of the ORF7b-inhibited physical association between MAVS and TRAF6, leading to the suppression of viral growth and infection. Overall, this study provides a mechanism for the suppression of innate immunity by SARS-CoV-2 infection and the mechanism-based approach via interfering peptides to potentially prevent SARS-CoV-2 infection.IMPORTANCEThe pandemic coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and continues to be a threat to public health. It is imperative to understand the biology of SARS-CoV-2 infection and find approaches to prevent SARS-CoV-2 infection and ameliorate COVID-19. Multiple SARS-CoV-2 proteins are known to function on the innate immune response, but the underlying mechanism remains unknown. This study shows that ORF7b inhibits the RIG-I-like receptor (RLR) signaling pathway through the physical association between ORF7b and mitochondrial antiviral-signaling protein (MAVS), impairing the K63-linked MAVS polyubiquitination and its recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) to MAVS. The most potent interfering peptide V targeting the ORF7b-MAVS complex may reverse the suppression of the MAVS-mediated RLR signaling pathway by ORF7b and prevent viral infection and production. This study may provide new insights into the pathogenic mechanism of SARS-CoV-2 and a strategy to develop new drugs to prevent SARS-CoV-2 infection.

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.