Systematic strategies for developing phage resistant Escherichia coli strains.

Phages are regarded as powerful antagonists of bacteria, especially in industrial fermentation processes involving bacteria. While bacteria have developed various defense mechanisms, most of which are effective against a narrow range of phages and consequently exert limited protection from phage infection. Here, we report a strategy for developing phage-resistant Escherichia coli strains through the simultaneous genomic integration of a DNA phosphorothioation-based Ssp defense module and mutations of components essential for the phage life cycle. The engineered E. coli strains show strong resistance against diverse phages tested without affecting cell growth. Additionally, the resultant engineered phage-resistant strains maintain the capabilities of producing example recombinant proteins, D-amino acid oxidase and coronavirus-encoded nonstructural protein nsp8, even under high levels of phage cocktail challenge. The strategy reported here will be useful for developing engineered E. coli strains with improved phage resistance for various industrial fermentation processes for producing recombinant proteins and chemicals of interest.

Qualitative analysis of chemical components in Lianhua Qingwen capsule by HPLC-Q Exactive-Orbitrap-MS coupled with GC-MS.

The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases. In particular, it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, due to its complex composition, little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule. This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry (GC-MS). An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds. A total of 104 compounds, consisting of alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were successfully characterized. In addition, the fragmentation pathways and characteristic fragments of some representative compounds were elucidated. GC-MS analysis was conducted to characterize the volatile compounds present in LHQW. In total, 17 compounds were putatively characterized by comparing the acquired data with that from the NIST library. The major constituent was menthol, and all the other compounds were terpenoids. This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS, coupled with GC-MS, and the results of this study can be used for the quality control and standardization of LHQW capsules.

Rapid Profiling of Chemical Constituents in Qingfei Paidu Granules Using High Performance Liquid Chromatography Coupled with Q Exactive Mass Spectrometry.

Qingfei Paidu (QFPD) granules have played a critical role during the Coronavirus Disease 2019 (COVID-19) in China. However, worldwide acceptance has been a problem because of the complex ingredients and unique theory of treatment. In this study, high-performance liquid chromatography (HPLC)-Q Exactive Orbitrap-mass spectrometry (MS) and the Orbitrap traditional Chinese medicine library (OTCML) were used to investigate the chemical constituents of QFPD granules. By comparing retention times, masses, isotope ion patterns, and MS2 profiles, 108 compounds were putatively identified using the OTCML combined with manual verification, including 12 alkaloids, 49 flavonoids, 13 terpenoids, 14 phenylpropanoids, 4 phenolic acids, 5 phenols, and 11 other phytochemicals. Of these compounds, 17 were confirmed using reference standards. In addition, representative compounds of these different chemical types were used as examples to analyze the fragmentation pathways and characteristic product ions. Moreover, 20 herbs within the QFPD granules were also identified to establish the sources of these chemical components. This is the first rapid profiling of the chemical constituents of QFPD granules using HPLC-Q Exactive Orbitrap-MS and yields valuable information for further quality control and mechanistic studies of QFPD granules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10337-021-04085-0.

An aurora of natural products-based drug discovery is coming.

Natural products (NPs), a nature's reservoir possessing enormous structural and functional diversity far beyond the current ability of chemical synthesis, are now proving themselves as most wonderful gifts from mother nature for human beings. Many of them have been used successfully as medicines, as well as the most important sources of drug leads, food additives, and many industry relevant products for millennia. Most notably, more than half of the antibiotics and anti-cancer drugs currently in use are, or derived from, natural products. However, the speed and outputs of NP-based drug discovery has been slowing down dramatically after the fruitful harvest of the "low-hanging fruit" during the golden age of 1950s-1960s. With recent scientific advances combining metabolic sciences and technology, multi-omics, big data, combinatorial biosynthesis, synthetic biology, genome editing technology (such as CRISPR), artificial intelligence (AI), and 3D printing, the "high-hanging fruit" is becoming more and more accessible with reduced costs. We are now more and more confident that a new age of natural products discovery is dawning.

Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses.

The ongoing global novel coronavirus pneumonia COVID-19 outbreak has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false-negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long-read, real-time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS-CoV-2 reaches 100%. Parallel testing with approved real-time reverse transcription-polymerase chain reaction kits for SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS-CoV-2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.