Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein Stability and Unfolding Dynamics.

How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.

Aqueous alkaline phosphate facilitates the non-exchangeable deuteration of peptides and proteins.

The incorporation of deuterium into peptides and proteins holds broad applications across various fields, such as drug development and structural characterization. Nevertheless, current methods for peptide/protein deuteration often target exchangeable labile sites or require harsh conditions for stable modification. In this study, we present a late-stage approach utilizing an alkaline phosphate solution to achieve deuteration of non-exchangeable backbone sites of peptides and proteins. The specific deuteration regions are identified through ultraviolet photodissociation (UVPD) and mass spectrometry analysis. This deuteration strategy demonstrates site and structure selectivity, with a notable affinity for labeling the α-helix regions of myoglobin. The deuterium method is particularly suitable for peptides and proteins that remain stable under high pH conditions.

Inhibition of SARS-CoV-2 replication by a ssDNA aptamer targeting the nucleocapsid protein.

The nucleocapsid protein of SARS-CoV-2 plays significant roles in viral assembly, immune evasion, and viral stability. Due to its immunogenicity, high expression levels during COVID-19, and conservation across viral strains, it represents an attractive target for antiviral treatment. In this study, we identified and characterized a single-stranded DNA aptamer, N-Apt17, which effectively disrupts the liquid-liquid phase separation (LLPS) mediated by the N protein. To enhance the aptamer's stability, a circular bivalent form, cb-N-Apt17, was designed and evaluated. Our findings demonstrated that cb-N-Apt17 exhibited improved stability, enhanced binding affinity, and superior inhibition of N protein LLPS; thus, it has the potential inhibition ability on viral replication. These results provide valuable evidence supporting the potential of cb-N-Apt17 as a promising candidate for the development of antiviral therapies against COVID-19.IMPORTANCEVariants of SARS-CoV-2 pose a significant challenge to currently available COVID-19 vaccines and therapies due to the rapid epitope changes observed in the viral spike protein. However, the nucleocapsid (N) protein of SARS-CoV-2, a highly conserved structural protein, offers promising potential as a target for inhibiting viral replication. The N protein forms complexes with genomic RNA, interacts with other viral structural proteins during virion assembly, and plays a critical role in evading host innate immunity by impairing interferon production during viral infection. In this investigation, we discovered a single-stranded DNA aptamer, designated as N-Apt17, exhibiting remarkable affinity and specificity for the N protein. Notably, N-Apt17 disrupts the liquid-liquid phase separation (LLPS) of the N protein. To enhance the stability and molecular recognition capabilities of N-Apt17, we designed a circular bivalent DNA aptamer termed cb-N-Apt17. In both in vivo and in vitro experiments, cb-N-Apt17 exhibited increased stability, enhanced binding affinity, and superior LLPS disrupting ability. Thus, our study provides essential proof-of-principle evidence supporting the further development of cb-N-Apt17 as a therapeutic candidate for COVID-19.

The Interaction between SARS-CoV-2 Nucleocapsid Protein and UBC9 Inhibits MAVS Ubiquitination by Enhancing Its SUMOylation.

Severe COVID-19 patients exhibit impaired IFN-I response due to decreased IFN-ß production, allowing persistent viral load and exacerbated inflammation. While the SARS-CoV-2 nucleocapsid (N) protein has been implicated in inhibiting innate immunity by interfering with IFN-ß signaling, the specific underlying mechanism still needs further investigation for a comprehensive understanding. This study reveals that the SARS-CoV-2 N protein enhances interaction between the human SUMO-conjugating enzyme UBC9 and MAVS. Increased MAVS-UBC9 interaction leads to enhanced SUMOylation of MAVS, inhibiting its ubiquitination, resulting in the inhibition of phosphorylation events involving IKKα, TBK1, and IRF3, thus disrupting IFN-ß signaling. This study highlights the role of the N protein of SARS-CoV-2 in modulating the innate immune response by affecting the MAVS SUMOylation and ubiquitination processes, leading to inhibition of the IFN-ß signaling pathway. These findings shed light on the complex mechanisms utilized by SARS-CoV-2 to manipulate the host's antiviral defenses and provide potential insights for developing targeted therapeutic strategies against severe COVID-19.

Effects of Luteolin-7-O-Glucoside on Intestinal Microbiota Dysbiosis and Drug Resistance Transmission Caused by Raoultella ornithinolytica B1645-1: Modulating the Composition of Intestinal Microbiota and Promoting the Transfer of blaNDM-1 Gene from Genus Enterococcus to Lactobacillus in Mice.

Raoultella ornithinolytica is an Enterobacteriaceae bacterium that can infect both humans and animals, while luteolin-7-O-glucoside (IOG) is a flavonoid that has broad effects on the intestinal microbiota of healthy animals. However, current studies lack sufficient data on intestinal microbiota dysbiosis and drug resistance transmission caused by R. ornithinolytica and the possible role of IOG. In this study, BALB/c mice were infected with R. ornithinolytica carrying blaNDM-1 gene and treated with IOG (3 mg/kg·d and 6 mg/kg·d) to analyze the diversity of intestinal microbiota and the transfer of blaNDM-1 between bacteria. The findings indicated that R. ornithinolytica B1645-1 exhibited a significant ability to enhance the Firmicutes/Bacteroidota ratio and increase the relative abundance of Lactobacillus and Bacillus after 48 h, where as 6 mg/kg·d IOG had an opposite effect. Moreover, R. ornithinolytica B1645-1 facilitated the emergence of drug-resistant bacteria and promoted blaNDM-1 gene transfer in Enterococcus, Escherichia, Klebsiella, Acinetobacter, Bacillus, Brevibacterium, and Lactobacillus. Enterococcus was the predominant genus at 48 h. Surprisingly, 6 mg/kg·d IOG significantly inhibited the production of drug-resistant bacteria and promoted blaNDM-1 gene transfer from Enterococcus to Lactobacillus at 144 h. However, the role of Lactobacillus as a recipient for drug-resistant genes should be of more concern.

Evolution of carbapenem resistance in klebsiella pneumoniae and escherichia coli carrying blaNDM-1 gene: imipenem exposure results in sustained resistance memory of strains in vitro.

BACKGROUND: Antibiotics exert an outstanding selective pressure on bacteria, forcing their chromosomal gene mutations and drug resistance genes to spread. The objective of this study is to evaluate the expression of the New Delhi Metallo-ß-Lactamase-1 gene (blaNDM-1) in the clinical isolate (Klebsiella pneumoniae TH-P12158), transformant strains Escherichia coli BL21 (DE3)-blaNDM-1, and Escherichia coli DH5α- blaNDM-1 when exposed to imipenem. METHODS: ß-Lactamase genes (blaSHV, blaTEM-1, blaCTX-M-9, blaIMP, blaNDM-1, blaKPC, blaOXA, blaGES, and blaDHA) from randomly selected carbapenems-sensitive K.pneumoniae (n = 20) and E.coli (n = 20) strains were amplified by PCR. The recombinant plasmid of pET-28a harboring blaNDM-1 was transformed into E.coli BL21 (DE3) and E.coli DH5α by electroporation. The resistance phenotype and higher blaNDM-1 expression in K.pneumoniae TH-P12158, transformant E.coli BL21 (DE3)-blaNDM-1, and E.coli DH5α-blaNDM-1 were observed when exposed to imipenem with grade increasing, decreasing, and canceling doses, respectively. RESULTS: After being exposed to different doses of imipenem, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of antimicrobial drugs and blaNDM-1 expression of strains increased, which was positively correlated with doses of imipenem. On the contrary, with the decrease or cancellation of imipenem doses, the blaNDM-1 expression was deteriorated, while the MIC and MBC values remained relatively stable. These results demonstrated that low doses of imipenem (˂MIC) could press blaNDM-1 positive strains producing stable drug resistance memory and altered blaNDM-1 expression. CONCLUSIONS: Low doses of imipenem could press blaNDM-1 positive strains producing sustained resistance memory and altered blaNDM-1 expression. In particular, the positive correlation between the resistance genes expression and antibiotics exposure shows promising guiding significance for clinical medication.

Investigation of Gut Bacterial Communities of Asian Citrus Psyllid (Diaphorina citri) Reared on Different Host Plants.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) can cause severe damage to citrus plants, as it transmits Candidatus Liberibacter spp., a causative agent of Huanglongbing disease. Symbiotic bacteria play vital roles in the ecology and biology of herbivore hosts, thereby affecting host growth and adaptation. In our research, the effects of Rutaceous plants (i.e., Citrus reticulata cv. Shatangju, Citrus poonensis cv. Ponkan, Murraya paniculata (orange jasmine), Citrus limon (lemon), and Citrus sinensis (navel orange)) on the gut microbiota (GM) and microbial diversity of D. citri adults were investigated by 16S rRNA high-throughput sequencing. It was found that Proteobacteria dominated the GM communities. The gut microbe diversity was the highest in the ponkan-feeding population, and the lowest in the Shatangju-feeding population. The NMDS analysis revealed that there were obvious differences in the GM communities among the different hosts. PICRUSt function prediction indicated significant differences in host function, and those pathways were crucial for maintaining population reproduction, growth, development, and adaptation to environmental stress in D. citri. Our study sheds new light on the interactions between symbionts, herbivores, and host plants and expands our knowledge on host adaptation related to GM in D. citri.

Lewis-Acid-Catalyzed Selective Friedel-Crafts Reaction or Annulation between Anilines and Glyoxylates.

A Lewis-acid-catalyzed selective reaction between anilines and glyoxylates was developed for synthesis of diarylmethanes or oxoimidazolidines. Under the catalysis of AgOTf, a series of anilines-based diarylmethanes, including primary, secondary, and tertiary anilines, were obtained in moderate to good yields. Moreover, stereoselective oxoimidazolidines were performed with the catalysis of Cu(OAc)2·H2O.

SARS-CoV-2 Accessory Protein ORF7b Mediates Tumor Necrosis Factor-α-Induced Apoptosis in Cells.

The accessory proteins of coronaviruses are essential for virus-host interactions and the modulation of host immune responses. It has been reported that accessory protein ORF3a encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce apoptosis, and accessory protein ORF6 and ORF8 could be inhibitors of the type-I interferon (IFN) signaling pathway. However, the function of accessory protein ORF7b is largely unknown. We investigated the apoptosis-inducing activity of ORF7b in cells. Cytokine levels and host innate immune responses, including expression of interferon regulatory transcription factor (IRF)-3, signal transducer and activator of transcription (STAT)-1, interferon (IFN)-ß, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, were also investigated. We found that ORF7b promoted expression of IFN-ß, TNF-α, and IL-6, activated type-I IFN signaling through IRF3 phosphorylation, and activated TNFα-induced apoptosis in HEK293T cells and Vero E6 cells. These results could provide deeper understanding about the pathogenicity of SARS-CoV-2 as well as the interaction between the accessory protein ORF7b with host immune responses.

In vitro transcriptomes analysis identifies some special genes involved in pathogenicity difference of the Beauveria bassiana against different insect hosts.

Typical entomopathogenic filamentous fungi such as Beauveria bassiana infect susceptible hosts via penetration of insect cuticle. The pathogenicity of B. bassiana strain to diverse insect hosts is different. While the molecular mechanisms of B. bassiana adapt to different insects are not well clear. B. bassiana GXsk1011 is a hyper-virulent strain from silkworm, which was investigated on the metabolic responses to three cuticle extracts of Bombyx mori, Helicoverpa armigera and Clanis bilineata at 24 h by RNA-seq method. A total of 638 up- and 400 down-regulated differentially expressed genes (DEGs) were identified in B. bassiana grown on H. armigera compared with B. mori, and 910 up- and 401 down-regulated genes for C. bilineata compared with B. mori. Functional categorization showed that DEGs are mainly involved in metabolic processes, localization, catalytic activity and transporter activity. Analysis of 20 highest fold change genes in DEGs showed that when B. bassiana transferred to non-original hosts as H. armigera and C. bilineata, the adhesion (Mad1), protease (Pr2) and cell surface protein (BBA_09174), etc. were down-regulated. While the class III chitinase ChiA2 (BBA_05353, Bbchi-17), major allergen Asp f 2-like protein (BBA_05395, Bb-f2) and nonribosomal peptide synthase, etc. were up-regulated. The secretory lipase that responded to H. armigera and the phosphate permease responded to C. bilineata were also up-regulated in the Top 20 DEGs. These special expressed genes indicate when the B. bassiana transferred to non-original hosts (or called as non-natural hosts), the strain appeared the changes of metabolic response and infection strategies to adapt to new hosts, and implied the key actions of infected adaptation were to break the barrier of different cuticle chitin component and against the immune stress of hosts. This study provided an insight into the B. bassiana that with wide host ranges how to adapt to infect different insect hosts, which will help us to further understand the pathogenesis of B. bassiana infection.

Occurrence of two NDM-1-producing Raoultella ornithinolytica and Enterobacter cloacae in a single patient in China: probable a novel antimicrobial resistance plasmid transfer in vivo by conjugation.

OBJECTIVES: To identify the general features of acquisition of drug-resistance genes in two multi-drug resistant Enterobacteriaceae strains isolated from a single patient in China. METHODS: The whole-plasmid was sequenced by Illumina Hiseq 4000 and Pacbio RSII procedures. The plasmid conjugation transfer experiment were performed by the mating-out assay. Drug-resistance genes were amplified by PCR assay. RESULTS: We identified two New Delhi metallo-ß-lactamase type 1(NDM-1)-producing isolates, named Raoultella ornithinolytica B1645-1 and Enterobacter cloacae B1645-2, which shared the same sulfonamide-resistant dihydropteroate synthase sul2 gene and aminoglycoside O-phosphotransferase aph(3'')-Ib gene. A novel antimicrobial resistance plasmid pCYNDM01 was first discovered from the multi-drug resistant R. ornithinolytica B1645-1. Interestingly, plasmid pCYNDM01 carried a Gifsy-2 prophage gene. The blaNDM-1 gene was located on a novel complex class 1 integron with a structure of sul1-qacEΔ1-ΔISAba125-blaNDM-1-blaMBL-trpC-ISCR1-catb8-aacA4-IS1-IS6100-dfrA14-intI1. The carrying the blaNDM-1 gene plasmid pCYNDM01 was transferred to the E. cloacae B1645-2 recipient strain. This 149.44 kb plasmid pCYNDM01 belonged to the IncFII type. CONCLUSIONS: A novel antimicrobial resistance plasmid pCYNDM01 was first recovered from a multi-drug resistance R. ornithinolytica B1645-1 isolated from China. The novel complex sul1-type class 1 integron might play an essential role in the mobilization of the blaNDM-1 gene among different enterobacterial species. The occurrence of plasmid pCYNDM01 transfer from R. ornithinolytica to E. cloacae in vitro by conjugation showed that plasmid pCYNDM01 was a self-conjugative plasmid and might cause dissemination of drug-resistance genes within different enterobacterial species from a single patient in vivo by conjugation. The novel variant F-like T4SS of plasmid pCYNDM01 might be as a tool of R. ornithinolytica B1645-1 for resistance genes transfer. The emergence of the two NDM-1-producing Enterobacteriaceae strains should be attracted China attentions and required to prevent its future prevalence.

Epidemiological and Clinical Characteristics of Patients With Coronavirus Disease-2019 in Shiyan City, China.

To investigate the early epidemic of COVID-19, a total of 176 confirmed COVID-19 cases in Shiyan city, Hubei province, China were surveyed. Our data indicated that the rate of emergence of early confirmed COVID-19 cases in Hubei province outside Wuhan was dependent on migration population, and the second-generation of patients were family clusters originating from Wuhan travelers. Epidemiological investigation indicated that the reproductive number (R0) under containment strategies was 1.81, and asymptomatic SARS-CoV-2 carriers were contagious with a transmission rate of 10.7%. Among the 176 patients, 53 were admitted to the Renmin Hospital of Hubei University of Medicine. The clinical characteristics of these 53 patients were collected and compared based on a positive RT-PCR test and presence of pneumonia. Clinical data showed that 47.2% (25/53) of COVID-19 patients were co-infected with Mycoplasma pneumoniae, and COVID-19 patients coinfected with M. pneumoniae had a higher percentage of monocytes (P < 0.0044) and a lower neutrophils percentage (P < 0.0264). Therefore, it is important to assess the transmissibility of infected asymptomatic individuals for SARS-CoV-2 transmission; moreover, clinicians should be alert to the high incidence of co-infection with M. pneumoniae in COVID-19 patients.

Down-regulation of lincRNA-EPS regulates apoptosis and autophagy in BCG-infected RAW264.7 macrophages via JNK/MAPK signaling pathway.

Macrophages play a major role in the control and elimination of invading Mycobacterium tuberculosis (Mtb). Long intergenic noncoding RNA erythroid prosurvival (lincRNA-EPS) plays an important role in regulating various biologic processes in macrophages, including inflammatory responses, cell apoptosis, and autophagy. Whereas the effect of lincRNA-EPS in regulating the immune response of macrophages to Mtb is little studied. This study aimed to explore lincRNA-EPS expression in monocytes from patients with active pulmonary tuberculosis (PTB) and from healthy individuals. We also sought to investigate the effect of lincRNA-EPS on Bacillus Calmette-Guérin (BCG)-infected macrophages apoptosis and autophagy. Our study found that lincRNA-EPS expression was down-regulated in the monocytes from patients with active PTB compared with healthy individuals, accompanied by significant attenuated monocyte apoptosis and enhanced autophagy. In vitro, knockdown of lincRNA-EPS inhibited apoptosis and promoted autophagy in BCG-infected RAW264.7 macrophages. Moreover, we revealed that lincRNA-EPS regulated apoptosis and autophagy of BCG-infected RAW264.7 macrophages via JNK/MAPK signaling pathway. In conclusion, our findings demonstrated that knockdown of lincRNA-EPS inhibits apoptosis and enhances autophagy by activating the JNK/MAPK signaling pathway in BCG-infected RAW264.7 macrophages. Suggesting that lincRNA-EPS could serve as a new potential therapeutic target for PTB.

[Antagonism against Beauveria bassiana by lipopeptide metabolites produced by entophyte Bacillus amyloliquefaciens strain SWB16].

OBJECTIVE: We screened bacterial strains that have strong antagonism against Beauveria bassiana, an important pathogen of silkworm industry, and detected the antagonistic activity of lipopeptide metabolites. METHODS: We identified bacterium SWB16 by morphological observation, physiological and biochemical experiments, 16SrRNA, and gyrA gene sequence analysis, tested antagonistic activity of strain SWB16 against Beauveria bassiana by measuring the inhibition zone diameter using filter paper diffusion method (Kirby-Bauer method), obtained lipopeptide metabolites of the strain using methanol extraction and observed the antagonism of strain SWB16 lipopeptide extracts against the conidia and hyphae of Beauveria bassiana, detected main ingredients and genes of lipopeptide metabolites by high-performance liquid chromatography-mass spectrometry and PCR amplification. RESULTS: SWB16 isolated from tissue of plant Dioscorea zingiberensis C. H. Wright belongs to Bacillus amyloliquefaciens and showed high antagonistic activity to Beauveria bassiana, and the lipopeptide extracts of isolate SWB16 exhibited significant inhibition to conidial germination and mycelial growth of Beauveria bassiana. The result of mass spectrometric detection indicated main component of the lipopeptide metabolites were fengcin and iturin, and genes fenB, ituA involved in the synthesis of them were amplified in the genome. CONCLUSION: Bacillus amyloliquefaciens strain SWB16 could produce lipopeptide antibiotics with strong antagonism to the entomopathogenic fungus Beauveria bassiana, and the results suggested that strain SWB16 has potential application value for controlling white muscardine of economic insects including silkworm.

Antibacterial effect and cytotoxicity of beta-1,3-1, 4-glucanase from endophytic Bacillus subtilis SWB8.

OBJECTIVE: We studied the antibiotic activity and selective cytotoxicity of beta-1,3-1,4-glucanase from endophytic Bacillus subtilis SWB8. METHODS: Based on gel permeation chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry methods, protein fragments of beta-1,3-1,4-glucanase from endophytic Bacillus subtilis strain SWB8 were purified and identified. Then, beta-1,3-1,4-glucanase was used to evaluate the antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Salmonella typhi, Salmonella paratyphi A, Shigella dysenteriae, Candida albicans and Cryptococcus neoformans and cytotoxicity against human pulmonary adenocarcinoma cells (A549) and human bone marrow mesenchymal stem cells (MSCs) by using the disc diffusion, methyl thiazolyl tetrazolium and flow cytometry methods, respectively. RESULTS: Bacterial beta-1,3-1,4-glucanase showed broad antimicrobial spectrum against all nine bacterial and fungal strains. Furthermore, beta-1,3-1,4-glucanase possessed significant anticancer activity against A549 cells that the IC50 and IC90 values were 11.5 and 20.1 microg/mL, respectively. The percentage of apoptotic A549 cells treated with different concentrations of beta-1,3-1,4-glucanase was significantly increased from 4.43% of the control to 43.1% of 19.2 microg/mL glucanase in a dose dependent manner. In contrast, these changes could not be observed in human bone marrow mesenchymal stem cells. CONCLUSION: Beta-1,3-1,4-glucanase could be a potential source of desirable antimicrobial agent, or anticancer compounds with higher efficiency and lower toxicity.

Apoptosis of human lung adenocarcinoma A549 cells induced by prodigiosin analogue obtained from an entomopathogenic bacterium Serratia marcescens.

An entomopathogenic bacterial strain SCQ1 was isolated from silkworm (Bombyx mori) and identified as Serratia marcescens via 16S rRNA gene analysis. This strain produces a red pigment that causes acute septicemia of silkworm. The red pigment of strain SCQ1 was identified as prodigiosin analogue (PGA) with various reported biological activities. In this study, we found that low concentration of PGA showed significant anticancer activity in human lung adenocarcinoma A549 cells, but has little effect in human bone marrow stem cells, in vitro. By exposure to different concentrations of PGA for 24 h, morphological changes and the MTT assay showed that A549 cell line was very sensitive to PGA, with IC(50) value about 2.2 mg/L. Early stage of apoptosis was detected by flow cytometry while A549 cells were treated with PGA for 4 and 12 h, respectively. The proportion of dead cells was increased with treatment time or the concentrations of PGA, but it was inversely proportional to that of apoptotic cells. These results indicate that PGA obtained from strain SCQ1 induces apoptosis in A549 cells, but the molecular mechanisms of cell death are complicated, and the S. marcescens strain SCQ1 may serve as a source of the anticancer compound, PGA.