Biosensing Detection of the SARS-CoV-2 D614G Mutation.

The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.

Discovery of G-quadruplex-forming sequences in SARS-CoV-2.

The outbreak caused by the novel coronavirus SARS-CoV-2 has been declared a global health emergency. G-quadruplex structures in genomes have long been considered essential for regulating a number of biological processes in a plethora of organisms. We have analyzed and identified 25 four contiguous GG runs (G2NxG2NyG2NzG2) in the SARS-CoV-2 RNA genome, suggesting putative G-quadruplex-forming sequences (PQSs). Detailed analysis of SARS-CoV-2 PQSs revealed their locations in the open reading frames of ORF1 ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes. Identical PQSs were also found in the other members of the Coronaviridae family. The top-ranked PQSs at positions 13385 and 24268 were confirmed to form RNA G-quadruplex structures in vitro by multiple spectroscopic assays. Furthermore, their direct interactions with viral helicase (nsp13) were determined by microscale thermophoresis. Molecular docking model suggests that nsp13 distorts the G-quadruplex structure by allowing the guanine bases to be flipped away from the guanine quartet planes. Targeting viral helicase and G-quadruplex structure represents an attractive approach for potentially inhibiting the SARS-CoV-2 virus.

Fluorescence detection of the human angiotensinogen protein by the G-quadruplex aptamer.

Noncanonical G-quadruplex nucleic acid structures can be used as probes in biosensors for the detection of metal ions, proteins and nucleic acids. Angiotensinogen (AGT) is a glycosylated globulin found in serum, which can regulate blood pressure and body fluid homeostasis. AGT is an important part of the renin-angiotensin system (RAS) and can be potentially used as a biomarker of diseases with RAS alterations. G-quadruplex based biosensors can detect targets with high accuracy and speed and low cost. Employing the magnetic bead enrichment method we constructed a reliable and efficient fluorescent biosensor platform for G-quadruplex based detection of the human AGT protein. The primary antibody in our biosensor is recognized by fluorescently labeled secondary antibodies, which leads to the detection of AGT captured by the G-quadruplex aptamer coupled magnetic beads. This G-quadruplex based fluorescent biosensor designed with a detection limit of 5 × 10-5 mg mL-1 was used for the successful detection of AGT at the cellular level. Our G-quadruplex based fluorescent biosensor will contribute to the more reliable and efficient detection of AGT.

KPC-Mediated Resistance to Ceftazidime-Avibactam and Collateral Effects in Klebsiella pneumoniae.

Carbapenem-resistant Enterobacterales, such as Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, represent a major threat to public health due to their rapid spread. Novel drug combinations such as ceftazidime-avibactam (CZA), combining a broad-spectrum cephalosporin along with a broad-spectrum ß-lactamase inhibitor, have recently been introduced and have been shown to exhibit excellent activity toward multidrug-resistant KPC-producing Enterobacterales strains. However, CZA-resistant K. pneumoniae isolates are now being increasingly reported, mostly corresponding to producers of KPC variants. In this study, we evaluated in vitro the nature of the mutations in the KPC-2 and KPC-3 ß-lactamase sequences (the most frequent KPC-type enzymes) that lead to CZA resistance and the subsequent effects of these mutations on susceptibility to other ß-lactam antibiotics. Single-step in vitro selection assays were conducted, resulting in the identification of a series of mutations in the KPC sequence which conferred the ability of those mutated enzymes to confer resistance to CZA. Hence, 16 KPC-2 variants and 10 KPC-3 variants were obtained. Production of the KPC variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to broad-spectrum cephalosporins and carbapenems, with the exceptions of ceftazidime and piperacillin-tazobactam, compared to wild-type KPC enzymes. Enzymatic assays showed that all of the KPC variants identified exhibited an increased affinity toward ceftazidime and a slightly decreased sensitivity to avibactam, sustaining their impact on CZA resistance. However, their respective carbapenemase activities were concurrently negatively impacted.

Genetic Features Leading to Reduced Susceptibility to Aztreonam-Avibactam among Metallo-ß-Lactamase-Producing Escherichia coli Isolates.

Metallo-ß-lactamase (MBL)-producing Escherichia coli isolates resistant to the newly developed ß-lactam/ß-lactamase inhibitor drug combination aztreonam-avibactam (ATM-AVI) have been reported. Here, we analyzed a series of 118 clinical MBL-producing E. coli isolates of various geographical origins for susceptibility to ATM-AVI. The nature of the PBP3 protein sequence and the occurrence of blaCMY genes for susceptibility to ATM-AVI were investigated. We showed here that elevated MICs of ATM-AVI among MBL-producing E. coli isolates resulted from a combination of different features, including modification of PBP3 protein sequence through specific amino acid insertions and production of CMY-type enzymes, particularly, CMY-42. We showed here that those insertions identified in the PBP3 sequence are not considered the unique basis of resistance to ATM-AVI, but they significantly contribute to it.

KPC-50 Confers Resistance to Ceftazidime-Avibactam Associated with Reduced Carbapenemase Activity.

KPC-50 is a KPC-3 variant identified from a Klebsiella pneumoniae clinical isolate recovered in Switzerland in 2019. Compared to KPC-3, KPC-50 shows (i) a three-amino-acid insertion (Glu-Ala-Val) between amino acids 276 and 277, (ii) an increased affinity to ceftazidime, (iii) a decreased sensitivity to avibactam, explaining the ceftazidime-avibactam resistance, and (iv) an association with a sharp reduction of its carbapenemase activity.

Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens.

Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4'-deoxy-4'-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or rRNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.

In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii.

OBJECTIVES: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants. METHODS: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity. RESULTS: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin. CONCLUSIONS: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

Recent Advances in Aptamer Discovery and Applications.

Aptamers are short, single-stranded DNA, RNA, or synthetic XNA molecules that can be developed with high affinity and specificity to interact with any desired targets. They have been widely used in facilitating discoveries in basic research, ensuring food safety and monitoring the environment. Furthermore, aptamers play promising roles as clinical diagnostics and therapeutic agents. This review provides update on the recent advances in this rapidly progressing field of research with particular emphasis on generation of aptamers and their applications in biosensing, biotechnology and medicine. The limitations and future directions of aptamers in target specific delivery and real-time detection are also discussed.

High molecular weight DNA assembly in vivo for synthetic biology applications.

DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

On the road to synthetic life: the minimal cell and genome-scale engineering.

Synthetic biology employs rational engineering principles to build biological systems from the libraries of standard, well characterized biological parts. Biological systems designed and built by synthetic biologists fulfill a plethora of useful purposes, ranging from better healthcare and energy production to biomanufacturing. Recent advancements in the synthesis, assembly and "booting-up" of synthetic genomes and in low and high-throughput genome engineering have paved the way for engineering on the genome-wide scale. One of the key goals of genome engineering is the construction of minimal genomes consisting solely of essential genes (genes indispensable for survival of living organisms). Besides serving as a toolbox to understand the universal principles of life, the cell encoded by minimal genome could be used to build a stringently controlled "cell factory" with a desired phenotype. This review provides an update on recent advances in the genome-scale engineering with particular emphasis on the engineering of minimal genomes. Furthermore, it presents an ongoing discussion to the scientific community for better suitability of minimal or robust cells for industrial applications.

Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome.

BACKGROUND: Escherichia coli K-12 is a frequently used host for a number of synthetic biology and biotechnology applications and chassis for the development of the minimal cell factories. Novel approaches for integrating high molecular weight DNA into the E. coli chromosome would therefore greatly facilitate engineering efforts in this bacterium. RESULTS: We developed a reliable and flexible lambda Red recombinase-based system, which utilizes overlapping DNA fragments for integration of the high molecular weight DNA into the E. coli chromosome. Our chromosomal integration strategy can be used to integrate high molecular weight DNA of variable length into any non-essential locus in the E. coli chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the fliK locus of the flagellar region 3b in the E. coli K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of Bacillus subtilis 168 DNA into two target sites in the E. coli K12 MG1655 chromosome. The chromosomal integrations into the fliK locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of E. coli. CONCLUSIONS: In addition to the rational design of synthetic biology devices, our high molecular weight DNA chromosomal integration system will facilitate metabolic and genome-scale engineering of E. coli.

Integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models reveals that pathogenicity is host specific.

Pseudomonas aeruginosa is a multi-host opportunistic pathogen causing a wide range of diseases because of the armoury of virulence factors it produces, and it is difficult to eradicate because of its intrinsic resistance to antibiotics. Using an integrated whole-genome approach, we searched for P. aeruginosa virulence genes with multi-host relevance. We constructed a random library of 57 360 Tn5 mutants in P. aeruginosa PAO1-L and screened it in vitro for those showing pleiotropic effects in virulence phenotypes (reduced swarming, exo-protease and pyocyanin production). A set of these pleiotropic mutants were assayed for reduced toxicity in Drosophila melanogaster, Caenorhabditis elegans, human cell lines and mice. Surprisingly, the screening revealed that the virulence of the majority of P. aeruginosa mutants varied between disease models, suggesting that virulence is dependent on the disease model used and hence the host environment. Genomic analysis revealed that these virulence-related genes encoded proteins from almost all functional classes, which were conserved among P. aeruginosa strains. Thus, we provide strong evidence that although P. aeruginosa is capable of infecting a wide range of hosts, many of its virulence determinants are host specific. These findings have important implication when searching for novel anti-virulence targets to develop new treatments against P. aeruginosa.

Pseudomonas aeruginosa essentials: an update on investigation of essential genes.

Pseudomonas aeruginosa is the leading cause of nosocomial infections, particularly in immunocompromised, cancer, burn and cystic fibrosis patients. Development of novel antimicrobials against P. aeruginosa is therefore of the highest importance. Although the first reports on P. aeruginosa essential genes date back to the early 2000s, a number of more sensitive genomic approaches have been used recently to better define essential genes in this organism. These analyses highlight the evolution of the definition of an 'essential' gene from the traditional to the context-dependent. Essential genes, particularly those indispensable under the clinically relevant conditions, are considered to be promising targets of novel antibiotics against P. aeruginosa. This review provides an update on the investigation of P. aeruginosa essential genes. Special focus is on recently identified P. aeruginosa essential genes and their exploitation for the development of antimicrobials.

Horizontal gene transfer in human pathogens.

Horizontal gene transfer has a tremendous impact on the genome plasticity, adaptation and evolution of bacteria. Horizontally transferred mobile genetic elements are involved in the dissemination of antibiotic resistance and virulence genes, thus contributing to the emergence of novel "superbugs". This review provides update on various mechanisms of horizontal gene transfer and examines how horizontal gene transfer contributes to the evolution of pathogenic bacteria. Special focus is paid to the role horizontal gene transfer plays in pathogenicity of the emerging human pathogens: hypervirulent Clostridium difficile and Escherichia coli (including the most recent haemolytic uraemic syndrome outbreak strain) and methicillin-resistant Staphylococcus aureus (MRSA), which have been associated with largest outbreaks of infection recently.

Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering.

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell 'chassis'. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

Identification of another module involved in the horizontal transfer of the Haemophilus genomic island ICEHin1056.

A significant part of horizontal gene transfer is facilitated by genomic islands. Haemophilus influenzae genomic island ICEHin1056 is an archetype of a genomic island that accounts for pandemic spread of antibiotics resistance. ICEHin1056 has modular structure and harbors modules involved in type IV secretion and integration. Previous studies have shown that ICEHin1056 encodes a functional type IV secretion system; however, other modules have not been characterized yet. Here we show that the module on the 5' extremity of ICEHin1056 consists of 15 genes that are well conserved in a number of related genomic islands. Furthermore by disrupting six genes of the investigated module of ICEHin1056 by site-specific mutagenesis we demonstrate that in addition to type IV secretion system module, the investigated module is also important for the successful conjugal transfer of ICEHin1056 from donor to recipient cells.

Aptamers targeting SARS-COV-2: a promising tool to fight against COVID-19.

SARS-CoV-2, the causative agent of COVID-19, remains among the main causes of global mortality. Although antigen/antibody-based immunoassays and neutralizing antibodies targeting SARS-CoV-2 have been successfully developed over the past 2 years, they are often inefficient and unreliable for emerging SARS-CoV-2 variants. Novel approaches against SARS-CoV-2 and its variants are therefore urgently needed. Aptamers have been developed for the detection and inhibition of several different viruses such as HIV, influenza viruses, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV. Aptamers targeting SARS-CoV-2 represent a promising tool in the fight against COVID-19, which is of paramount importance for the current and any future pandemics. This review presents recent advances and future trends in the development of aptamer-based approaches for SARS-CoV-2 diagnosis and treatment.

Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics.

The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹4C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface.

Correction of out-of-focus microscopic images by deep learning.

Motivation: Microscopic images are widely used in basic biomedical research, disease diagnosis and medical discovery. Obtaining high-quality in-focus microscopy images has been a cornerstone of the microscopy. However, images obtained by microscopes are often out-of-focus, resulting in poor performance in research and diagnosis. Results: To solve the out-of-focus issue in microscopy, we developed a Cycle Generative Adversarial Network (CycleGAN) based model and a multi-component weighted loss function. We train and test our network in two self-collected datasets, namely Leishmania parasite dataset captured by a bright-field microscope, and bovine pulmonary artery endothelial cells (BPAEC) captured by a confocal fluorescence microscope. In comparison to other GAN-based deblurring methods, the proposed model reached state-of-the-art performance in correction. Another publicly available dataset, human cells dataset from the Broad Bioimage Benchmark Collection is used for evaluating the generalization abilities of the model. Our model showed excellent generalization capability, which could transfer to different types of microscopic image datasets. Availability and Implementation: Code and dataset are publicly available at: https://github.com/jiangdat/COMI.