A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Protein-inorganic calcium-phosphate supraparticles as a robust platform for enzyme co-immobilization.

Immobilization of enzymes provides many benefits, including facile separation and recovery of enzymes from reaction mixtures, enhanced stability, and co-localization of multiple enzymes. Calcium-phosphate-protein supraparticles imbued with a leucine zipper binding domain (ZR ) serve as a modular immobilization platform for enzymes fused to the complementary leucine zipper domain (ZE ). The zippers provide high-affinity, specific binding, separating enzymatic activity from the binding event. Using fluorescent model proteins (mCherryZE and eGFPZE ), an amine dehydrogenase (AmDHZE ), and a formate dehydrogenase (FDHZE ), the efficacy of supraparticles as a biocatalytic solid support was assessed. Supraparticles demonstrated several benefits as an immobilization support, including predictable loading of multiple proteins, structural integrity in a panel of solvents, and the ability to elute and reload proteins without damaging the support. The dual-enzyme reaction successfully converted ketone to amine on supraparticles, highlighting the efficacy of this system.

Formate Oxidase (FOx) from Aspergillus oryzae: One Catalyst Enables Diverse H2 O2 -Dependent Biocatalytic Oxidation Reactions.

An increasing number of biocatalytic oxidation reactions rely on H2 O2 as a clean oxidant. The poor robustness of most enzymes towards H2 O2 , however, necessitates more efficient systems for in situ H2 O2 generation. In analogy to the well-known formate dehydrogenase to promote NADH-dependent reactions, we here propose employing formate oxidase (FOx) to promote H2 O2 -dependent enzymatic oxidation reactions. Even under non-optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.

The Short-chain Dehydrogenase/Reductase Engineering Database (SDRED): A classification and analysis system for a highly diverse enzyme family.

The Short-chain Dehydrogenases/Reductases Engineering Database (SDRED) covers one of the largest known protein families (168 150 proteins). Assignment to the superfamilies of Classical and Extended SDRs was achieved by global sequence similarity and by identification of family-specific sequence motifs. Two standard numbering schemes were established for Classical and Extended SDRs that allow for the determination of conserved amino acid residues, such as cofactor specificity determining positions or superfamily specific sequence motifs. The comprehensive sequence dataset of the SDRED facilitates the refinement of family-specific sequence motifs. The glycine-rich motifs for Classical and Extended SDRs were refined to improve the precision of superfamily classification. In each superfamily, the majority of sequences formed a tightly connected sequence network and belonged to a large homologous family. Despite their different sequence motifs and their different sequence length, the two sequence networks of Classical and Extended SDRs are not separate, but connected by edges at a threshold of 40% sequence similarity, indicating that all SDRs belong to a large, connected network. The SDRED is accessible at https://sdred.biocatnet.de/.

Monocarbonyl analogs of curcumin inhibit growth of antibiotic sensitive and resistant strains of Mycobacterium tuberculosis.

Tuberculosis (TB) is a major public health concern worldwide with over 2 billion people currently infected. The rise of strains of Mycobacterium tuberculosis (Mtb) that are resistant to some or all first and second line antibiotics, including multidrug-resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) strains, is of particular concern and new anti-TB drugs are urgently needed. Curcumin, a natural product used in traditional medicine in India, exhibits anti-microbial activity that includes Mtb, however it is relatively unstable and suffers from poor bioavailability. To improve activity and bioavailability, mono-carbonyl analogs of curcumin were synthesized and screened for their capacity to inhibit the growth of Mtb and the related Mycobacterium marinum (Mm). Using disk diffusion and liquid culture assays, we found several analogs that inhibit in vitro growth of Mm and Mtb, including rifampicin-resistant strains. Structure activity analysis of the analogs indicated that Michael acceptor properties are critical for inhibitory activity. However, no synergistic effects were evident between the monocarbonyl analogs and rifampicin on inhibiting growth. Together, these data provide a structural basis for the development of analogs of curcumin with pronounced anti-mycobacterial activity and provide a roadmap to develop additional structural analogs that exhibit more favorable interactions with other anti-TB drugs.

A novel chimeric amine dehydrogenase shows altered substrate specificity compared to its parent enzymes.

We created a novel chimeric amine dehydrogenase (AmDH) via domain shuffling of two parent AmDHs ('L- and F-AmDH'), which in turn had been generated from leucine and phenylalanine DH, respectively. Unlike the parent proteins, the chimeric AmDH ('cFL-AmDH') catalyzes the amination of acetophenone to (R)-methylbenzylamine and adamantylmethylketone to adamantylethylamine.

Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

A family of indoles regulate virulence and Shiga toxin production in pathogenic E. coli.

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause "attaching and effacing" (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.

Antimicrobial and host defense peptides for therapeutic use against multidrug-resistant pathogens: new hope on the horizon.

The concept of using antimicrobial peptides (AMPs) and host defense peptides (HDPs) as therapeutics was first introduced in the late 1990s. However, an AMP drug has yet to reach the market. AMPs and HDPs have intriguing potential as therapeutics: the peptides are evolutionary conserved, and are critical components of the innate immune system of all eukaryotes; their evolution pre-dates the appearance of the adaptive immune system; and they do not readily engender bacterial resistance. Nevertheless, there are significant obstacles to the use of AMPs and HDPs in humans, including the need to conduct clinical trials to demonstrate efficacy, and the capacity to manufacture AMPs and HDPs in a cost-effective manner. Progress in both of these areas would support the exciting possibility that AMPs and HDPs could be developed as therapeutics that kill pathogens and facilitate the immune response.

TLR signaling mediated by MyD88 is required for a protective innate immune response by neutrophils to Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.

Enteropathogenic Escherichia coli Tir is an SH2/3 ligand that recruits and activates tyrosine kinases required for pedestal formation.

Enteropathogenic Escherichia coli (EPEC) cause intestinal inflammation, severe diarrhoea and mortality, particularly among children in developing nations. Upon attachment to intestinal epithelial cells, EPEC induces actin-filled membrane protrusions called 'pedestals' and disrupts microvilli to form attaching and effacing (A/E) lesions. EPEC also disrupts epithelial barrier function and causes colitis. Here we have investigated how virulence factors which orchestrate formation of actin pedestals interface with host tyrosine kinases. We show that Tec-family tyrosine kinases localize beneath EPEC and, with Abl-family kinases, comprise a set of redundant host kinases utilized by EPEC to form actin pedestals. We also show that Tir, a virulence factor required for pathogenesis, contains a polyproline region (PPR) that interacts with SH3 domains of redundant kinases, and a phosphorylation site (Y474) that interacts with kinase SH2 domains. These interactions are essential for pedestal formation, and mimic activation of kinases by cellular ligands. Our results suggest that a positive feedback loop exists in which initial phosphorylation of Tir on Y474 by tyrosine kinases causes recruitment of additional redundant kinases via PPR-SH3 interactions and PO(3)-Y474-SH2 interactions, which in turn phosphorylate other Tir molecules as well as proteins that catalyse formation of actin pedestals.

Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases.

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.

Enteropathogenic Escherichia coli use redundant tyrosine kinases to form actin pedestals.

Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food and induce protrusion of actin-rich membrane pedestals beneath themselves upon attachment to intestinal epithelia. EPEC then causes intestinal inflammation, diarrhea, and, among children, death. Here, we show that EPEC uses multiple tyrosine kinases for formation of pedestals, each of which is sufficient but not necessary. In particular, we show that Abl and Arg, members of the Abl family of tyrosine kinases, localize and are activated in pedestals. We also show that pyrido[2,3-d]pyrimidine (PD) compounds, which inhibit Abl, Arg, and related kinases, block pedestal formation. Finally, we show that Abl and Arg are sufficient for pedestal formation in the absence of other tyrosine kinase activity, but they are not necessary. Our results suggest that additional kinases that are sensitive to inhibition by PD also can suffice. Together, these results suggest that EPEC has evolved a mechanism to use any of several functionally redundant tyrosine kinases during pathogenesis, perhaps facilitating its capacity to infect different cell types. Moreover, PD compounds are being developed to treat cancers caused by dysregulated Abl. Our results raise the possibility that PD may be useful in treating EPEC infections, and because PD affects host and not bacterium, selecting resistant strains may be far less likely than with conventional antibiotics.

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells.

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.