Electrostatic microfiltration (EM) enriches and recovers viable microorganisms at low-abundance in large-volume samples and enhances downstream detection.

Rapid and sensitive detection of pathogens in various samples is crucial for disease diagnosis, environmental surveillance, as well as food and water safety monitoring. However, the low abundance of pathogens (<10 CFU) in large volume (1 mL-1 L) samples containing vast backgrounds critically limits the sensitivity of even the most advanced techniques, such as digital PCR. Therefore, there is a critical need for sample preparation that can enrich low-abundance pathogens from complex and large-volume samples.

Facile metabolic reprogramming distinguishes mycobacterial adaptation to hypoxia and starvation: ketosis drives starvation-induced persistence in M. bovis BCG.

Mycobacteria adapt to infection stresses by entering a reversible non-replicating persistence (NRP) with slow or no cell growth and broad antimicrobial tolerance. Hypoxia and nutrient deprivation are two well-studied stresses commonly used to model the NRP, yet little is known about the molecular differences in mycobacterial adaptation to these distinct stresses that lead to a comparable NRP phenotype. Here we performed a multisystem interrogation of the Mycobacterium bovis BCG (BCG) starvation response, which revealed a coordinated metabolic shift away from the glycolysis of nutrient-replete growth to depletion of lipid stores, lipolysis, and fatty acid ß-oxidation in NRP.

Development of a cellulose-based 96-well plate vertical flow pull-down assay.

The abundance and low production cost of biomaterial cellulose paper have attracted attention for many applications. Point-of-care (PoC) diagnostic tests have been successfully developed using patterned cellulose paper. Although PoC diagnostic tests are rapid and simple to perform, their sample processing throughput is limited, allowing for only one sample to be evaluated at a time, which restricts potential applications.

Rapid Evaluation of Vaccine Booster Effectiveness against SARS-CoV-2 Variants.

As the COVID-19 pandemic continues, countries around the world are switching toward vaccinations and boosters to combat the pandemic. However, waning immunity against SARS-CoV-2 wild-type (WT) and variants have been widely reported. Booster vaccinations have shown to be able to increase immunological protection against new variants; however, the protection observed appears to decrease quickly over time suggesting a second booster shot may be appropriate.

A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies.

Background: Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive.

Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min.

Development and translation of a paper-based top readout vertical flow assay for SARS-CoV-2 surveillance.

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage.

Vertical Flow Cellulose-Based Assays for SARS-CoV-2 Antibody Detection in Human Serum.

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format.

Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism.

Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(NG37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from and , we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding.

Crystal structure and catalytic mechanism of the essential mG37 tRNA methyltransferase TrmD from .

The tRNA (mG37) methyltransferase TrmD catalyzes mG formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in (TrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of TrmD.

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(NG37) Methyltransferase (TrmD) Inhibitors.

Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(NG37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD).

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope.

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer.

Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes.

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence.

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria-which models tuberculous granulomas-are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes.

Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa.

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am.

The role of sequence context, nucleotide pool balance and stress in 2'-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA.

The misincorporation of 2'-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2'-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS).

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA: Application to Stress-Induced Reprogramming of tRNA Modifications.

Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. This chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: (1) RNA isolation, (2) RNA purification, (3) RNA hydrolysis to individual ribonucleosides, (4) chromatographic resolution of ribonucleosides, (5) identification of the full set of modified ribonucleosides, (6) mass spectrometric quantification of ribonucleosides, (6) interrogation of ribonucleoside datasets, and (7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision, and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response.

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species.

Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry.

Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA.

A multidimensional platform for the purification of non-coding RNA species.

A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species.

A novel murine infection model for Shiga toxin-producing Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin-producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage.

Alkyl hydroperoxide reductase is required for Helicobacter cinaedi intestinal colonization and survival under oxidative stress in BALB/c and BALB/c interleukin-10-/- mice.

Helicobacter cinaedi, a common human intestinal bacterium, has been implicated in various enteric and systemic diseases in normal and immunocompromised patients. Protection against oxidative stress is a crucial component of bacterium-host interactions. Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of peroxides and is important in protection from peroxide-induced stress.

The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation.

Multivariate modeling identifies neutrophil- and Th17-related factors as differential serum biomarkers of chronic murine colitis.

Background: Diagnosis of chronic intestinal inflammation, which characterizes inflammatory bowel disease (IBD), along with prediction of disease state is hindered by the availability of predictive serum biomarker. Serum biomarkers predictive of disease state will improve trials for therapeutic intervention, and disease monitoring, particularly in genetically susceptible individuals. Chronic inflammation during IBD is considered distinct from infectious intestinal inflammation thereby requiring biomarkers to provide differential diagnosis.

Toll-like receptor 4-mediated regulation of spontaneous Helicobacter-dependent colitis in IL-10-deficient mice.

Background & Aims: The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis.