A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

UnlabelledPairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.ImportanceOur insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale

DNA Checkpoint and Repair Factors Are Nuclear Sensors for Intracellular Organelle Stresses-Inflammations and Cancers Can Have High Genomic Risks.

Under inflammatory conditions, inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) which cause DNA damage. If not appropriately repaired, DNA damage leads to gene mutations and genomic instability. DNA damage checkpoint factors (DDCF) and DNA damage repair factors (DDRF) play a vital role in maintaining genomic integrity. However, how DDCFs and DDRFs are modulated under physiological and pathological conditions are not fully known. We took an experimental database analysis to determine the expression of 26 DNA D

In vivo Emergence of Colistin Resistance in Carbapenem-Resistant Klebsiella pneumoniae Mediated by Premature Termination of the mgrB Gene Regulator

Multidrug-resistant (MDR) Klebsiella pneumoniae is a severe threat to public health worldwide. Worryingly, colistin resistance, one of the last-line antibiotics for the treatment of MDR K. pneumoniae infection, has been increasingly reported. This study aims to investigate the emergence of evolved colistin resistance in a carbapenem-resistant K. pneumoniae isolate during colistin treatment. In this study, a pair of sequential carbapenem-resistant K. pneumoniae isolates were recovered from the same patient before and after colistin treatment, named KP1-1 and KP1-2, respectively. Antibiotic susceptibility testing was performed by the microdilution broth method. Whole genome sequencing was performed, and putative gene variations were analyzed in comparison of the genome sequence of both isolates. The bacterial whole genome sequence typing and source tracking analysis were performed by BacWGSTdb 2.0 server. Validation of the role of these variations in colistin resistance was examined by complementation experiments. The association between colistin resistance and the expression level of PhoP/PhoQ signaling system and its regulated genes was evaluated by quantitative real-time PCR (qRT-PCR) assay. Our study indicated that KP1-1 displayed extensively antibiotic resistant trait, but only susceptible to colistin. KP1-2 showed additional resistance to colistin. Both isolates belonged to Sequence Type 11 (ST11). The whole genome sequence analysis uncovered multiple resistance genes and virulence genes in both isolates. No plasmid-mediated mcr genes were found, but genetic variations in five chromosomal genes, especially the Gln30∗ alteration in MgrB, were detected in colistin-resistant isolate KP1-2. Moreover, only complementation with wild-type mgrB gene restored colistin susceptibility, with colistin MIC decreased from 32 to 1 mg/L. Expression assays revealed an overexpression of the phoP, phoQ, and pmrD genes in the mgrB-mutated isolate KP1-2 compared to the wild-type isolate KP1-1, confirming the MgrB alterations was responsible for increased expression levels of those genes. This study provides direct in vivo evidence that Gln30∗ alteration of MgrB is a critical region responsible for colistin resistance in K. pneumoniae clinical strains

Atmospheric new particle formation from sulfuric acid and amines in a Chinese megacity

Atmospheric new particle formation (NPF) is an important global phenomenon that is nevertheless sensitive to ambient conditions. According to both observation and theoretical arguments, NPF usually requires a relatively high sulfuric acid (H2SO4) concentration to promote the formation of new particles and a low preexisting aerosol loading to minimize the sink of new particles. We investigated NPF in Shanghai and were able to observe both precursor vapors (H2SO4) and initial clusters at a molecular level in a megacity. High NPF rates were observed to coincide with several familiar markers suggestive of H2SO4-dimethylamine (DMA)water (H2O) nucleation, including sulfuric acid dimers and H2SO4-DMA clusters. In a cluster kinetics simulation, the observed concentration of sulfuric acid was high enough to explain the particle growth to similar to 3 nanometers under the very high condensation sink, whereas the subsequent higher growth rate beyond this size is believed to result fromthe added contribution of condensing organic species. These findings will help in understanding urban NPF and its air quality and climate effects, as well as in formulating policies to mitigate secondary particle formation in China.Peer reviewe

Digital transformation and environmental information disclosure in China: the moderating role of top management team’s ability

Companies rely on advanced digital technologies to empower their operations and sustainable development. Environmental information disclosure by companies is critical in achieving net zero goals and sustainability. This study empirically investigates the impact of digital transformation on environmental information disclosure and how the ability of the top management team affects the relationship. We build a model based on the information processing theory and test it using panel data collected from 929 listed companies in China. The findings show an inverted U-shaped relationship between digital transformation and environmental information disclosure, and the top management team’s ability weakens the relationship. The study provides empirical evidence that digital transformation is a double-edged sword for environmental information disclosure. Also, the results suggest that the top management team may substitute or complement digital technologies in environmental information disclosure during the different stages of digital transformation.<br/

Uptake of Gaseous Alkylamides by Suspended Sulfuric Acid Particles: Formation of Ammonium/Aminium Salts

Amides represent an important class of nitrogen-containing compounds in the atmosphere that can in theory interact with atmospheric acidic particles and contribute to secondary aerosol formation. In this study, uptake coefficients (γ) of six alkylamides (C₁ to C₃) by suspended sulfuric acid particles were measured using an aerosol flow tube coupled to a high resolution time-of-flight chemical ionization mass spectrometer (HRToF-CIMS). At 293 K and < 3% relative humidity (RH), the measured uptake coefficients for six alkylamides were in the range of (4.8–23) × 10^–2. A negative dependence upon RH was observed for both <i>N</i>-methylformamide and <i>N</i>,<i>N</i>-dimethylformamide, likely due to decreased mass accommodation coefficients (α) at lower acidities. A negative temperature dependence was observed for <i>N</i>,<i>N</i>-dimethylformamide under < 3% RH, also consistent with the mass accommodation-controlled uptake processes. Chemical analysis of reacted sulfuric acid particles indicates that alkylamides hydrolyzed in the presence of water molecules to form ammonium or aminium. Our results suggest that multiphase uptake of amides will contribute to growth of atmospheric acidic particles and alter their chemical composition

Zoonotic fungus Arthroderma multifidum causing chronic pulmonary infection

A rare case of fungus Arthroderma multifidum infection occurred in a 63-year-old man. The patient had some risk factors, including occupational exposure, immunosuppressive state, and structural basis following pulmonary tuberculosis and pneumothorax surgery. The pathogen was repeatedly isolated from bronchoalveolar lavage fluid and identified by gene sequencing. It is the first report of human infection caused by A. multifidum. Whole genome sequencing and analysis of its genomic characterization are completed. The findings provide us with a key clinical insight that the combination of immune suppression and environmental exposure could create an ideal condition for zoonotic fungal infections

ACT-Toxin, the Key Effector for the Virulence of <i>Alternaria alternata</i> Tangerine Pathotype to Specific Citrus Species

Alternaria brown spot disease is caused by the Alternaria alternata tangerine pathotype, which relies on ACT-toxin for infection. At present, all identified ACT-toxin biosynthesis-related genes are multi-copy genes. In this study, we summarized the advances in important host-specific toxins (HSTs), and listed key genes required for the pathogenicity of the A. alternata tangerine pathotype. Toxin virulence test results revealed that different citrus species displayed distinctly different tolerances to ACT-toxin. The extraction method of ACT-toxin crude extract was described in schematic form to make the method easier to understand. In addition, target gene disruption of two copies of ACTT5 (∆∆ACTT5) displayed significantly reduced virulence, indicating that ACTT5 is essential for the pathogenicity of the A. alternata tangerine pathotype

Quantifying the Evolutionary Constraints and Potential of Hepatitis C Virus NS5A Protein.

RNA viruses, such as hepatitis C virus (HCV), influenza virus, and SARS-CoV-2, are notorious for their ability to evolve rapidly under selection in novel environments. It is known that the high mutation rate of RNA viruses can generate huge genetic diversity to facilitate viral adaptation. However, less attention has been paid to the underlying fitness landscape that represents the selection forces on viral genomes, especially under different selection conditions. Here, we systematically quantified the distribution of fitness effects of about 1,600 single amino acid substitutions in the drug-targeted region of NS5A protein of HCV. We found that the majority of nonsynonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized. The replication fitness of viruses is correlated with the pattern of sequence conservation in nature, and viral evolution is constrained by the need to maintain protein stability. We characterized the adaptive potential of HCV by subjecting the mutant viruses to selection by the antiviral drug daclatasvir at multiple concentrations. Both the relative fitness values and the number of beneficial mutations were found to increase with the increasing concentrations of daclatasvir. The changes in the spectrum of beneficial mutations in NS5A protein can be explained by a pharmacodynamics model describing viral fitness as a function of drug concentration. Overall, our results show that the distribution of fitness effects of mutations is modulated by both the constraints on the biophysical properties of proteins (i.e., selection pressure for protein stability) and the level of environmental stress (i.e., selection pressure for drug resistance).IMPORTANCE Many viruses adapt rapidly to novel selection pressures, such as antiviral drugs. Understanding how pathogens evolve under drug selection is critical for the success of antiviral therapy against human pathogens. By combining deep sequencing with selection experiments in cell culture, we have quantified the distribution of fitness effects of mutations in hepatitis C virus (HCV) NS5A protein. Our results indicate that the majority of single amino acid substitutions in NS5A protein incur large fitness costs. Simulation of protein stability suggests viral evolution is constrained by the need to maintain protein stability. By subjecting the mutant viruses to selection under an antiviral drug, we find that the adaptive potential of viral proteins in a novel environment is modulated by the level of environmental stress, which can be explained by a pharmacodynamics model. Our comprehensive characterization of the fitness landscapes of NS5A can potentially guide the design of effective strategies to limit viral evolution