Generation of a SARS-CoV-2 Reverse Genetics System and Novel Human Lung Cell Lines That Exhibit High Virus-Induced Cytopathology.

The global COVID-19 pandemic continues with continued cases worldwide and the emergence of new SARS-CoV-2 variants. In our study, we have developed novel tools with applications for screening antivirals, identifying virus-host dependencies, and characterizing viral variants. Using reverse genetics, we rescued SARS-CoV-2 Wuhan1 (D614G variant) wild type (WTFL) and reporter virus (NLucFL) using molecular BAC clones. The replication kinetics, plaque morphology, and titers were comparable between viruses rescued from molecular clones and a clinical isolate (VIDO-01 strain). Furthermore, the reporter SARS-CoV-2 NLucFL virus exhibited robust luciferase values over the time course of infection and was used to develop a rapid antiviral assay using remdesivir as proof-of-principle. In addition, as a tool to study lung-relevant virus-host interactions, we established novel human lung cell lines that support SARS-CoV-2 infection with high virus-induced cytopathology. Six lung cell lines (NCI-H23, A549, NCI-H1703, NCI-H520, NCI-H226, and HCC827) and HEK293T cells were transduced to stably express ACE2 and tested for their ability to support virus infection. A549ACE2 B1 and HEK293TACE2 A2 cell lines exhibited more than 70% virus-induced cell death, and a novel lung cell line, NCI-H23ACE2 A3, showed about ~99% cell death post-infection. These cell lines are ideal for assays relying on live-dead selection, such as CRISPR knockout and activation screens.

Highly Specific Sigma Receptor Ligands Exhibit Anti-Viral Properties in SARS-CoV-2 Infected Cells.

(1) Background: There is a strong need for prevention and treatment strategies for COVID-19 that are not impacted by SARS-CoV-2 mutations emerging in variants of concern. After virus infection, host ER resident sigma receptors form direct interactions with non-structural SARS-CoV-2 proteins present in the replication complex. (2) Methods: In this work, highly specific sigma receptor ligands were investigated for their ability to inhibit both SARS-CoV-2 genome replication and virus induced cellular toxicity. This study found antiviral activity associated with agonism of the sigma-1 receptor (e.g., SA4503), ligation of the sigma-2 receptor (e.g., CM398), and a combination of the two pathways (e.g., AZ66). (3) Results: Intermolecular contacts between these ligands and sigma receptors were identified by structural modeling. (4) Conclusions: Sigma receptor ligands and drugs with off-target sigma receptor binding characteristics were effective at inhibiting SARS-CoV-2 infection in primate and human cells, representing a potential therapeutic avenue for COVID-19 prevention and treatment.

Pairwise correlation of genes involved in glucose metabolism: a potential diagnostic marker of cancer?

Cancer is a highly malignant disease, killing approximately 10 million people worldwide in 2020. Cancer patient survival substantially relies on early diagnosis. In this study, we evaluated whether genes involved in glucose metabolism could be used as potential diagnostic markers for cancer. In total, 127 genes were examined for their gene expression levels and pairwise gene correlations. Genes ADH1B and PDHA2 were differentially expressed in most of the 12 types of cancer and five pairs of genes exhibited consistent correlation changes (from strong correlations in normal controls to weak correlations in cancer patients) across all types of cancer. Thus, the two differentially expressed genes and five gene pairs could be potential diagnostic markers for cancer. Further preclinical and clinical studies are warranted to prove whether these genes and/or gene pairs would indeed aid in early diagnosis of cancer.

Identifying kinase targets of PPARγ in human breast cancer.

Breast cancer is the most common cancer in women. Despite advances in screening women for genetic predisposition to breast cancer and risk stratification, a majority of women carriers remain undetected until they become affected. Thus, there is a need to develop a cost-effective, rapid, sensitive and non-invasive early-stage diagnostic method. Kinases are involved in all fundamental cellular processes and mutations in kinases have been reported as drivers of cancer. PPARγ is a ligand-activated transcription factor that plays important roles in cell proliferation and metabolism. However, the complete set of kinases modulated by PPARγ is still unknown. In this study, we identified human kinases that are potential PPARγ targets and evaluated their differential expression and gene pair correlations in human breast cancer patient dataset TCGA-BRCA. We further confirmed the findings in human breast cancer cell lines MCF7 and SK-BR-3 using a kinome array. We observed that gene pair correlations are lost in tumours as compared to healthy controls and could be used as a supplement strategy for diagnosis and prognosis of breast cancer.

Alteration in Gene Pair Correlations in Tryptophan Metabolism as a Hallmark in Cancer Diagnosis.

Tryptophan metabolism plays essential roles in both immunomodulation and cancer development. Indoleamine 2,3-dioxygenase, a rate-limiting enzyme in the metabolic pathway, is overexpressed in different types of cancer. To get a better understanding of the involvement of tryptophan metabolism in cancer development, we evaluated the expression and pairwise correlation of 62 genes in the metabolic pathway across 12 types of cancer. Only gene AOX1, encoding aldehyde oxidase 1, was ubiquitously downregulated, Furthermore, we observed that the 62 genes were widely and strongly correlated in normal controls, however, the gene pair correlations were significantly lost in tumor patients for all 12 types of cancer. This implicated that gene pair correlation coefficients of the tryptophan metabolic pathway could be applied as a prognostic and/or diagnostic biomarker for cancer.

Enhancing Drug Efficacy against Mastitis Pathogens-An In Vitro Pilot Study in Staphylococcus aureus and Staphylococcus epidermidis.

BACKGROUND: Bovine mastitis is one of the major infectious diseases in dairy cattle, resulting in large economic loss due to decreased milk production and increased production cost to the dairy industry. Antibiotics are commonly used to prevent/treat bovine mastitis infections. However, increased antibiotic resistance and consumers' concern regarding antibiotic overuse make it prudent and urgent to develop novel therapeutic protocols for this disease. MATERIALS AND METHODS: Potential druggable targets were found in 20 mastitis-causing pathogens and conserved and unique targets were identified. Bacterial strains Staphylococcus aureus (ATCC 29213, and two clinical isolates CI 1 and CI 2) and Staphylococcus epidermidis (ATCC 12228, and two clinical isolates CI 1 and CI 2) were used in the present study for validation of an effective drug combination. RESULTS: In the current study, we identified the common and the unique druggable targets for twenty mastitis-causing pathogens using an integrative approach. Furthermore, we showed that phosphorylcholine, a drug for a unique target gamma-hemolysin component B in Staphylococcus aureus, and ceftiofur, the mostly used veterinary antibiotic that is FDA approved for treating mastitis infections, exhibit a synergistic effect against S. aureus and a strong additive effect against Staphylococcus epidermidis in vitro. CONCLUSION: Based on the data generated in this study, we propose that combination therapy with drugs that work synergistically against conserved and unique targets can help increase efficacy and lower the usage of antibiotics for treating bacterial infections. However, these data need further validations in animal models of infection.

Ibrutinib as a potential therapeutic option for HER2 overexpressing breast cancer - the role of STAT3 and p21.

Treatment response rates to current anticancer therapies for HER2 overexpressing breast cancer are limited and are associated with severe adverse drug reactions. Tyrosine kinases perform crucial roles in cellular processes by mediating cell signalling cascades. Ibrutinib is a recently approved Tyrosine Kinase Inhibitor (TKI) that has been shown be an effective therapeutic option for HER2 overexpressing breast cancer. The molecular mechanisms, pathways, or genes that are modulated by ibrutinib and the mechanism of action of ibrutinib in HER2 overexpressing breast cancer remain obscure. In this study, we have performed a kinome array analysis of ibrutinib treatment in two HER2 overexpressing breast cancer cell lines. Our analysis shows that ibrutinib induces changes in nuclear morphology and causes apoptosis via caspase-dependent extrinsic apoptosis pathway with the activation of caspases-8, caspase-3, and cleavage of PARP1. We further show that phosphorylated STAT3Y705 is upregulated and phosphorylated p21T145 is downregulated upon ibrutinib treatment. We propose that STAT3 upregulation is a passive response as a result of induction of DNA damage and downregulation of phosphorylated p21 is promoting cell cycle arrest and apoptosis in the two HER2 overexpressing cell lines. These results suggest that inhibitors of STAT3 phosphorylation may be potential options for combination therapy to help increase the efficacy of ibrutinib against HER2-overexpressing tumors.

A Novel Antimicrobial-Phytochemical Conjugate With Antimicrobial Activity Against Streptococcus uberis, Enterococcus faecium, and Enterococcus faecalis.

Antimicrobial resistance is one of the major threats to human and animal health. An effective strategy to reduce and/or delay antimicrobial resistance is to use combination therapies. Research in our laboratory has been focused on combination therapies of antimicrobials and phytochemicals and development of antimicrobial-phytochemical conjugates. In this study, we report the synthesis and antimicrobial activity of a novel sulfamethoxazole-gallic acid conjugate compound (Hybrid 1). Hybrid 1 not only showed much stronger activity than sulfamethoxazole towards Streptococcus uberis 19436, Enterococcus faecium 700221, and Enterococcus faecalis 29212, which were purchased from American Type Culture Collection (ATCC), but also exhibited a promising antimicrobial effect against two E. faecalis clinical isolates, one of which was multidrug-resistant. Further studies are warranted to establish the in vivo antimicrobial activity for Hybrid 1 and develop more potent sulfamethoxazole-gallic acid-based antimicrobial conjugates using hybrid 1 as a lead compound.

A systems biology approach towards the identification of candidate therapeutic genes and potential biomarkers for Parkinson's disease.

Parkinson's disease (PD) is an irreversible and incurable multigenic neurodegenerative disorder. It involves progressive loss of mid brain dopaminergic neurons in the substantia nigra pars compacta (SN). We compared brain gene expression profiles with those from the peripheral blood cells of a separate sample of PD patients to identify disease-associated genes. Here, we demonstrate the use of gene expression profiling of brain and blood for detecting valid targets and identifying early PD biomarkers. Implementing this systematic approach, we discovered putative PD risk genes in brain, delineated biological processes and molecular functions that may be particularly disrupted in PD and also identified several putative PD biomarkers in blood. 20 of the differentially expressed genes in SN were also found to be differentially expressed in the blood. Further application of this methodology to other brain regions and neurological disorders should facilitate the discovery of highly reliable and reproducible candidate risk genes and biomarkers for PD. The identification of valid peripheral biomarkers for PD may ultimately facilitate early identification, intervention, and prevention efforts as well.

Phytochemicals as alternatives to antibiotics against major pathogens involved in bovine respiratory disease(BRD)and bovine mastitis(BM).

Bovine respiratory disease (BRD) and bovine mastitis (BM) are the most common and costly infectious diseases in beef cattle and dairy cattle, respectively. In the current study, we evaluated the antimicrobial activity of seven phytochemicals against twelve BRD- and/or BMcausing bacterial pathogens. Our results show that allyl isothiocyanate, benzyl isothiocynate, cinnamaldehyde and eugenol are effective against most of the BRD- and/or BM-causing bacterial pathogens and could be repurposed as alternatives to antibiotics for the prevention/elimination of BRD and BM in feedlots.

Identification of novel drug targets in bovine respiratory disease: an essential step in applying biotechnologic techniques to develop more effective therapeutic treatments.

BACKGROUND: Bovine Respiratory Disease (BRD) is a major problem in cattle production which causes substantial economic loss. BRD has multifactorial aetiologies, is multi-microbial, and several of the causative pathogens are unknown. Consequently, primary management practices such as metaphylactic antimicrobial injections for BRD prevention are used to reduce the incidence of BRD in feedlot cattle. However, this poses a serious threat in the form of development of antimicrobial resistance and demands an urgent need to find novel interventions that could reduce the effects of BRD drastically and also delay/prevent bacterial resistance. MATERIALS AND METHODS: We have employed a subtractive genomics approach that helps delineate essential, host-specific, and druggable targets in pathogens responsible for BRD. We also proposed antimicrobials from FDA green and orange book that could be repositioned for BRD. RESULTS: We have identified 107 putative targets that are essential, selective and druggable. We have also confirmed the susceptibility of two BRD pathogens to one of the proposed antimicrobials - oxytetracycline. CONCLUSION: This approach allows for repositioning drugs known for other infections to BRD, predicting novel druggable targets for BRD infection, and providing a new direction in developing more effective therapeutic treatments for BRD.

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes.

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, oriλ, located within gene O. Questions have persisted for years about whether O-defects can indeed be complemented in trans. We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP.

The temperate bacteriophage lambda (λ) CII protein is a positive regulator of transcription from promoter pE, a component of the lysogenic response. The expression of cII was examined in vectors devoid of phage transcription-modulating elements. Their removal enabled evaluating if the expression of the small RNA OOP, on its own, could suppress CII activities, including complementing for a lysogenic response, cell toxicity and causing rapid cellular loss of ColE1 plasmids. The results confirm that OOP RNA expression from the genetic element pO-oop-to can prevent the ability of plasmid-encoded CII to complement for a lysogenic response, suggesting that it serves as a powerful regulatory pivot in λ development. Plasmids with a pO promoter sequence of 45 nucleotides (pO45), containing the -10 and -35 regions for oop, were non-functional; whereas, plasmids with pO94 prevented CII complementation, CII-dependent plasmid loss and suppressed CII toxicity, suggesting the pO promoter has an extended DNA sequence. All three CII activities were eliminated by the deletion of the COOH-terminal 20 amino acids of CII. Host mutations in the hflA locus, in pcnB and in rpoB influenced CII activities. These studies suggest that the COOH-terminal end of CII likely interacts with the ß-subunit of RNA polymerase.

Gallic Acid Potentiates the Antimicrobial Activity of Tulathromycin Against Two Key Bovine Respiratory Disease (BRD) Causing-Pathogens.

Bovine respiratory disease (BRD) is the most common infectious disease in dairy and beef cattle. It is associated with significant morbidity and mortality and causes a huge economic loss each year. In western Canada, a one-time injection of tulathromycin is commonly used as a metaphylactic procedure to reduce BRD incidence and eliminate potential BRD outbreak. With increased global concern on antimicrobial usage in dairy and beef products and bacterial resistance to antimicrobials, it is important to develop a novel strategy to eliminate the usage or decrease the dosage of antimicrobials. In this study, we showed that gallic acid was active against both Mannheimia haemolytica and Pasteurella multocida, two key BRD associated-pathogens, with the minimum inhibitory concentration (MIC) measured at 250 and 500 µg/mL, respectively. Co-administration of tulathromycin and gallic acid exhibited a strong additive or weak synergistic effect toward both M. haemolytic and P. multocida. Tulathromycin, gallic acid and their combination were also effective against the mixed culture of M. haemolytic and P. multocida. Furthermore, we showed that pre-exposure to tulathromycin generated bacterial resistance to the antimicrobial in M. haemolytica but not in P. multocida.

Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for Rif(R), Toxicity, Mutagenicity, Plasmid Curing.

The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (Rif(R)) cells acquiring mutations within the rpoB gene encoding the ß-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the Rif(R) mutants remained P(S) and localized to the Rif binding pocket in RNAP, but a subset acquired a P(R) phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One P(R) mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP.

Microalgae mediated synthesis of silver nanoparticles and their antibacterial activity against pathogenic bacteria.

Silver nanoparticles is known to have antimicrobial affects. Cyanobacteria isolates from muthupet mangrove includes Aphanothece sp, Oscillatoria sp, Microcoleus sp, Aphanocapsa sp, Phormidium sp, Lyngbya sp, Gleocapsa sp, Synechococcus sp, Spirulina sp with were set in compliance with their cellular mechanism of nano silver creation, and were investigated by UV-VIS spectrophotometer, Energy-dispersive X-ray (EDX) and scanning electron microscopy (SEM). Silver nanoparticles were spherical shaped well distributed without aggregation in solution with an average size of about 40- 80 nm. Synthesised nano silver had antibacterial production on various organisms that provoked various diseases in humans. The cellular metabolites of Microcoleus sp. only created nano silver and it enhanced the antibacterial activity against test pathogenic bacteria from MTCC (Proteus vulgaris, Salmonella typhi, Vibrio cholera, Streptococcus sp., Bacillus subtilis, Staphylococcus aureus, Escherichia coli.) The antimicrobial assay was performed using 0.001 M concentration of nano silver in well diffusion method with positive control of appropriate standard antibiotic discs Cephotaxime, Ampicillin, Tetracyclin, Cephalexin etc. Synthesised silver nanoparticles acted as an effective antimicrobial agent and proved as an alternative for the development of new antimicrobial agents to combat the problem of resistance.

Microalgae associated Brevundimonas sp. MSK 4 as the nano particle synthesizing unit to produce antimicrobial silver nanoparticles.

The biosynthesis of silver nanoparticles and its antimicrobial property was studied using bacteria isolated from Spirulina products. Isolated bacteria were identified as Bacillus sp. MSK 1 (JX495945), Staphylococcus sp. MSK 2 (JX495946), Bacillus sp. MSK 3 (JX495947) and Brevundimonas sp. MSK 4 (JX495948). Silver nanoparticles (AgNPs) were synthesized using bacterial culture filtrate with AgNO3. The initial syntheses of Ag nanoparticles were characterized by UV-vis spectrophotometer (by measuring the color change to intense brown). Fourier Transform Infrared Spectroscopy (FTIR) study showed evidence that proteins are possible reducing agents and Energy-dispersive X-ray (EDX) study showing the metal silver as major signal. The structure of AgNPs was determined by Scanning electron microscopy (SEM) and X-ray diffraction (XRD). Synthesized Ag nanoparticles with an average size of 40-65 nm have antimicrobial property against human pathogens like Proteus vulgaris, Salmonella typhi, Vibrio cholera, Streptococcus sp., Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. Among the isolates Brevundimonas sp. MSK 4 alone showed good activity in both synthesis of AgNPs and antimicrobial activity. This work demonstrates the possible use of biological synthesized silver nanoparticles to combat the drug resistant problem.