Antibiotics have transformed modern medicine in manifold ways. However, the misuse and over-consumption of antibiotics or antimicrobials have led to the rise in antimicrobial resistance (AMR). Unfortunately, robust tools or techniques for the detection of potential loci responsible for AMR before it happens are lacking. The emergence of resistance even when a strain lacks known AMR genes has puzzled researchers for a long time. Clearly, there is a critical need for the development of novel approaches for uncovering yet unknown resistance elements in pathogens and advancing our understanding of emerging resistance mechanisms. To aid in the development of new tools for deciphering AMR, here we propose a machine learning (ML) based framework that provides ML models trained and tested on (1) genotypic AMR and phenotypic antimicrobial susceptibility testing (AST) data, which can predict novel resistance factors in bacterial strains that lack already implicated resistance genes; and (2) complete gene set and AST phenotypic data, which can predict the most important genetic loci involved in resistance to specific antibiotics in bacterial strains. The validation of resistance loci prioritized by our ML pipeline was performed using homology modeling and in silico molecular docking.

Fibrinolysis pathway is an important mechanism for dissolution of fibrin clot by the action of plasmin which is formed from plasminogen, a zymogen via the action of plasminogen activators, i.e. tissue plasminogen activator and urinary plasminogen activator. The regulation of fibrinolysis system in vivo is maintained by plasminogen activators and natural inhibitors i.e. α2-antiplasmin, α2-macroglobulin, Thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor 1 and 2 (PAI-1and PAI-2). There are several fibrinolytic assays developed for human plasma but there are no reports describing fibrinolytic assay using zebrafish plasma. In this study, a fibrinolytic assay via using small amount of zebrafish plasma was developed. This assay was performed under different conditions; one by the addition of exogenous tissue plasminogen activator alone to the pooled zebrafish plasma along with calcium chloride and thromboplastin, second Dade ACTIN was used instead of tissue plasminogen activator and third Dade ACTIN along with thromboplastin was used. Epsilon amino caproic acid (EACA), a synthetic antifibrinolytic agent was used at different concentrations to inhibit fibrinolysis successfully. Similar experiments were performed on human plasma as well to check the applicability of the assay to humans and positive results were obtained. Furthermore, knockdown of tissue plasminogen activator and plasminogen genes …

Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no …

To better understand the nature and function of N-acylethanolamines (NAEs) in Camelina sativa, we used mass spectrometry analysis to identify and quantify NAE types in developing seeds, desiccated seeds and seedlings. Developing seeds showed a differential increase in individual NAE species and an overall increase in NAE content with seed development and maturation. The NAE composition in mature, desiccated seeds mostly reflected the total fatty acid composition in the seed tissues, except for a noted absence of 11-eicosenoic (20C monounsaturated) fatty acid in the NAE pool. During seed stratification and seedling growth, individual NAE species were depleted at similar rates. Simulated drought treatments during seedling development resulted in a significant rise in NAE levels for the major 18C NAE types compared with untreated seedlings. Arabidopsis and Camelina mutants with reported altered fatty acid profiles were analyzed for their NAE compositions; both Arabidopsis and Camelina had relatively similar changes between compositions of total seed fatty acids and NAEs. Furthermore, seeds were analyzed from transgenic Arabidopsis and Camelina with engineered, non-native, long-chain polyunsaturated fatty acids (18C, 20C and 22C), and the results showed the production of novel N-acylphosphatidylethanolamines (presumed precursors of NAEs) and NAEs with the same long acyl chains. These results …

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for …

The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of …

Symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. Within nodule cells, N-fixing rhizobia are surrounded by plant-derived symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. First, I investigated the impact of P deprivation on wild-type Medicago truncatula plants. My observations that plants had impaired SNF activity, reduced growth, and accumulated less phosphate in P-deficient tissues (leaves, roots and nodules) is consistent with those of similar previous studies. Galactolipids decreased with increase in phospholipids in all P-starved organs. Matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI-MSI) of phosphatidylcholine (PC) species in nodules showed that under low P environments distributions of some PC species changed, indicating that membrane lipid remodeling during P stress is not uniform across the nodule. Secondly, a metabolomics study was carried out to test the alterations in the metabolic profile of the nodules in P-stress. GC-MS based untargeted metabolomics showed increased levels of amino acids and sugars and decline in amounts of organic acids in P deprived nodules. Subsequently, …

Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant protein containing three Kunitz domains, K1, K2 and K3. K1 inhibits Factor VIIa, K2 inhibits Factor Xa, and K3 enhances the Factor Xa inhibition by its interaction with Protein S. Since zebrafish is an excellent genetic model, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of tfpia in zebrafish. Subsequently, we performed knockdown of tfpia, and knockout of tfpia in K3 domain using CRISPR/Cas9. Both the tfpia knockdown and tfpia homozygous deletion mutants showed increased coagulation activities. Our data suggest that zebrafish tfpia is an orthologue for human TFPIα, and silencing it results in a thrombotic phenotype. We then optimized the piggyback knockdown method, where we could simultaneously piggyback 3 or 6 ASOs corresponding to 3 or 6 genes, respectively, using one VMO. These multiple gene knockdowns will increase the efficiency of genome-wide knockdowns. Since there are no studies on chromatin remodeling that control TFPI expression, we hypothesized that the genome-wide knockdowns of the Chromatin Binding and Regulatory Proteins (CBRPs) in zebrafish could help identify novel tfpia gene regulators. We chose 69 CBRPs and subjected them to simultaneous gene knockdowns. Our …