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Expression of G-protein Coupled Receptors in Young and Mature Thrombocytes and Knockdown of Gpr18 in Zebrafish
In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.
The Microbial Retting Environment of Hibiscus Cannabinus and Its Implications in Broader Applications
Fiber-yielding plants is an area of increased interest due to the potential use in a variety of green-based materials. These biocomposites can be incorporated into multiple uses; for example, to replace building materials and interior vehicular paneling. The research here aims to focus in on the crop Hibiscus cannabinus for utilization into these functions. H. cannabinus is economically attractive due to the entire process being able to be accomplished here in the United States. The plant can be grown in a relatively short growth period (120-180 days), and then processed and incorporated in a biocomposite. The plant fiber must first be broken down into a useable medium. This is accomplished by the retting process, which occurs when microbial constituents breakdown the heteropolysaccharides releasing the fiber. The research aims to bridge the gap between the primitive process of retting and current techniques in molecular and microbiology. Utilizing a classical microbiological approach, which entailed enrichment and isolation of pectinase-producing bacteria for downstream use in augmented microbial retting experiments. The tracking of the bacteria was accomplished by using the 16S rRNA which acts as “barcodes” for bacteria. Next-generation sequencing can then provide data from each environment telling the composition and microbial diversity of each tested variable. The main environments tested are: a natural environment, organisms contributed by the plant material solely, and an augmented version in which pectinase-producing bacteria are added. In addition, a time-course experiment was performed on the augmented environment providing data of the shift to an anaerobic environment. Lastly, a drop-in set was performed using each isolate separately to determine which contributes to the shift in microbial organization. This research provided a much needed modernization of the retting technique. Previous studies have been subject to simple clone libraries and growth plate assays and next-generation sequencing will bring the understanding of …
Metabolism and Action of Polyunsaturated N-acylethanolamines in Arabidopsis thaliana Seedlings
The lipoxygenase (LOX) pathway plays an important role in the oxidative metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). The LOX pathway functions in conjugation with hydrolysis by fatty acid amide hydrolase (FAAH) and to produce oxidized NAEs during seed germination and early seedling development. When Arabidopsis seedlings were grown in low micromolar concentrations of lauroylethanolamide (NAE 12:0), growth retardation and elevated endogenous PU-NAE levels were observed due to the competitive inhibition of LOX by NAE 12:0. The elevated levels of endogenous PU-NAEs were more pronounced in genotypes with reduced NAE hydrolase capacity (faah knockouts), and less evident with overexpression of FAAH. Alterations in PU-NAE metabolism were studied in seedlings of various lox and FAAH mutants. The partitioning of PU-NAEs into oxylipin metabolites was exaggerated in the presence of exogenous linolenoylethanolamide (NAE18:3) and resulted in bleaching of cotyledons. The bleaching phenotype was restricted to a narrow developmental window (3-to-5 days after sowing), and was attributed to a reversible disruption of thylakoid membranes in chloroplasts. Biochemical and genetic evidence suggested that 9-hydro(pero)xy and 13-hydro(pero)xy octadecatrienoylethanolamides (9- and 13-NAE-H(P)OT), but not their corresponding hydro(pero)xy free fatty acids, induced cotyledon bleaching. The LOX-mediated metabolites of NAE18:3 shared some overlapping effects on seedling development with those of linoleoylethanolamide (NAE18:2) such as a reduction in seedling root growth. On the other hand, NAE18:3 oxylipin metabolites also exhibited distinct effects during seedling development such as the inhibition of photomorphogenesis. Biochemical and genetic evidence indicated that a LOX-mediated metabolite of NAE18:2, 9-hydro(pero)xy octadecadienoylethanolamide (9-NAE-H(P)OD), acted as a potent negative regulator of seedling root development, and this depended on an intact abscisic acid (ABA) signaling pathway. Synergistic inhibition of root elongation between 9-NAE-H(P)OD and ABA was restricted to a narrow developmental window (3-to-5 d after sowing) of seedling development. Genetic evidence with Arabidopsis mutants in ABA synthesis (aba1, aba2), perception (pyr1, …
Identification of Genes Involved in Flocculation by Whole Genome Sequencing of Thauera aminoaromatica Strain MZ1T Floc-defective Mutants
Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated sludge wastewater treatment plant, overproduces exopolysaccharide (EPS) leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole genome phenotypic sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants were subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type genome. We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The FTIR spectra of EPS isolated from the wild-type showed reduced C=O peak of the N-acetyl group at 1665 cm-1 as compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated the deacetylase gene transcript increased before flocculation occurred. The results suggest that the deacetylation of MZ1T EPS is crucial for flocculation. The information obtained from this study will be useful for preventing viscous bulking and wastewater treatment system failure, and may have potential applications in the biotechnology sector for the controlled removal of cells.
Identification of Hox Genes Controlling Thrombopoiesis in Zebrafish
Thrombocytes are functional equivalents of mammalian platelets and also possess megakaryocyte features. It has been shown earlier that hox genes play a role in megakaryocyte development. Our earlier microarray analysis showed five hox genes, hoxa10b, hoxb2a, hoxc5a, hoxc11b and hoxd3a, were upregulated in zebrafish thrombocytes. However, there is no comprehensive study of genome wide scan of all the hox genes playing a role in megakaryopoiesis. I first measured the expression levels of each of these hox genes in young and mature thrombocytes and observed that all the above hox genes except hoxc11b were expressed equally in both populations of thrombocytes. hoxc11b was expressed only in young thrombocytes and not in mature thrombocytes. The goals of my study were to comprehensively knockdown hox genes and identify the specific hox genes involved in the development of thrombocytes in zebrafish. However, the existing vivo-morpholino knockdown technology was not capable of performing such genome-wide knockdowns. Therefore, I developed a novel cost- effective knockdown method by designing an antisense oligonucleotides against the target mRNA and piggybacking with standard control morpholino to silence the gene of interest. Also, to perform knockdowns of the hox genes and test for the number of thrombocytes, the available techniques were both cumbersome or required breeding and production of fish where thrombocytes are GFP labeled. Therefore, I established a flow cytometry based method of counting the number of thrombocytes. I used mepacrine to fluorescently label the blood cells and used the white cell fraction. Standard antisense oligonucleotide designed to the central portion of each of the target hox mRNAs, was piggybacked by a control morpholino and intravenously injected into the adult zebrafish. The thrombocyte count was measured 48 hours post injection. In this study, I found that the knockdown of hoxc11b resulted in increased number of thrombocytes and knockdown of hoxa10b, …
Phylogenetic and Functional Characterization of Cotton (Gossypium hirsutum) CENTRORADIALIS/TERMINAL FLOWER1/SELF-PRUNING Genes
Plant architecture is an important agronomic trait driven by meristematic activities. Indeterminate meristems set repeating phytomers while determinate meristems produce terminal structures. The centroradialis/terminal flower1/self pruning (CETS) gene family modulates architecture by controlling determinate and indeterminate growth. Cotton (G. hirsutum) is naturally a photoperiodic perennial cultivated as a day-neutral annual. Management of this fiber crop is complicated by continued vegetative growth and asynchronous fruit set. Here, cotton CETS genes are phylogenetically and functionally characterized. We identified eight CETS genes in diploid cotton (G. raimondii and G. arboreum) and sixteen in tetraploid G. hirsutum that grouped within the three generally accepted CETS clades: flowering locus T (FT)-like, terminal flower1/self pruning (TFL1/SP)-like, and mother of FT and TFL1 (MFT)-like. Over-expression of single flower truss (GhSFT), the ortholog to Arabidopsis FT, accelerates the onset of flowering in Arabidopsis Col-0. In mutant rescue analysis, this gene driven by its native promoter rescues the ft-10 late flowering phenotype. GhSFT upstream sequence was used to drive expression of the uidA reporter gene. As anticipated, GUS accumulated in the vasculature of Arabidopsis leaves. Cotton has five TFL1-like genes, all of which delay flowering when ectopically expressed in Arabidopsis; the strongest phenotypes fail to produce functional flowers. Three of these genes, GhSP, GhTFL1-L2, and GhBFT-L2, rescue the early flowering tfl1-14 mutant phenotype. GhSPpro:uidA promoted GUS activity specifically in plant meristems; whereas, other GhTFL1-like promoters predominately drove GUS activities in plant vascular tissues. Finally, analysis of Gossypium CETS promoter sequences predicted that GhSFT, GhSP, GhTFL1-L1, GhTFL1-L2 and GhBFT-L2 are regulated by transcription factors involved in shoot and flowering development. Analysis of cotton's two MFT homologs indicated that neither gene functions to control shoot architecture. Our results emphasize the functional conservation of members of this gene family in flowering plants and also suggest this family as targets during artificial selection …
Stability of Myosin Subfragment-2 Modulates the Force Produced by Acto-Myosin Interaction of Striated Muscle
Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 and light meromyosin (LMM). This dissertation examines whether the myosin S2 coiled coil could regulate the amount of myosin S1 heads available to bind actin thin filaments by modulating the stability of its coiled coil. A stable myosin S2 coiled coil would have less active myosin S1 heads compared to a more flexible myosin S2 coiled coil, thus causing increased force production through acto-myosin interaction. The stability of the myosin S2 coiled coil was modulated by the binding of a natural myosin S2 binding protein, myosin binding protein C (MyBPC), and synthetic myosin S2 binding proteins, stabilizer and destabilizer peptide, to myosin S2. Competitive enzyme linked immunosorbent assay (cELISA) experiments revealed the cross specificity and high binding affinity of the synthetic peptides to the myosin S2 of human cardiac and rabbit skeletal origins. Gravitational force spectroscopy (GFS) was performed to test the stability of myosin S2 coiled coil in the presence of these myosin S2 binding proteins. GFS experiments demonstrated the stabilization of the myosin S2 coiled coil by the binding of MyBPC and stabilizer peptide to myosin S2, while the binding of destabilizer peptide to the same resulted in a flexible myosin S2 coiled coil. The binding of MyBPC and stabilizer peptide respectively, resulted in 3.35 and 1.5 times increase in force required to uncoil the myosin S2, while the binding of destabilizer peptide resulted in 1.6 times decrease in force required to uncoil the myosin S2. The myofibrillar contractility assay was performed to test the effect of synthetic myosin S2 binding proteins on the sarcomere shortening in myofibrils. The stabilizer peptide resulted in decreased sarcomere shortening of myofibrils as a result of decreased acto-myosin interaction, on the other hand, the binding of destabilizer peptide caused an increase …
Rapid Metabolic Response of Plants Exposed to Light Stress
Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could be used to augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in initial stage of light stress response. Light-induced rapid systemic signaling and systemic acquired acclimation (SAA) are thought to play an important role in the response of plants to different abiotic stresses. Although molecular and metabolic responses to light stress have been extensively studied in local leaves, and to a lesser degree in systemic leaves, very …
Revisiting the Neuroprotective Role of 17B-Estradiol (E2): A Multi-Omics Based Analysis of the Rat Brain and Serum
The ovarian hormone 17β-estradiol (E2) is one of the central regulators of the female reproductive system. E2 is also a pleiotropic regulator since it can exert its non-reproductive role on other organ systems. E2 is neuroprotective, it maintains body's energy homeostasis, participates in various repair mechanism and is required for neural development. However, there is a substantial evidence suggesting that there might be a molecular reprogramming of E2's action when it is supplied exogenously after E2 deprivation. Though the length of E2 deprivation and age has been linked to this phenomenon, the molecular components and how they activate this reprogramming is still elusive. Our main goal was to perform global proteomics and metabolomics study to identify the molecular components and their interaction networks that are being altered in the brain and serum after a short-term E2 treatment following ovariectomy (OVX) in Sprague Dawley rats. One of the strength of our global study is that it gave us extensive information on the brain proteome itself by identification of a wide number of proteins in different brain sections. By analyzing the differentially expressed proteins, our proteomics study revealed 49 different networks to be altered in 7 sections of the brain. Most of the perturbed networks were involved in cell metabolism, neural development, protein synthesis, cellular trafficking and degradation, and several stress response signaling pathways. We assessed the neuroenergetic status of the brain based on E2's response to various energy generating pathways, including glycolysis, TCA cycle, and oxidative phosphorylation, and several signaling pathways. All energetics pathways were shown to be downregulated in E2 treatment, which suggests that E2 exerts its neuroprotective role by restoring energy homeostasis in OVX rat model by regulating complex signaling and metabolic networks. Our second focus was to determine the metabolite response (amino acids and lipids) after E2 treatment …
Studies in Trypsin as an Alarm Substance in Zebrafish
Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our …
Compartmentalization of Jojoba Seed Lipid Metabolites
Seeds from the desert shrub Simmondsia chinensis (jojoba) are one of the only known natural plant sources to store a majority of its oil in the form of liquid wax esters (WE) instead of triacylglycerols (TAGs) and these oils account for ~55% of the seed weight. Jojoba oil is highly valued as cosmetic additives and mechanical lubricants, yet despite its value much is still unknown about its neutral lipid biosynthetic pathways and lipid droplet packaging machinery. Here, we have used a multi-"omics" approach to study how spatial differences in lipid metabolites, gene expression, and lipid droplet proteins influence the synthesis and storage of jojoba lipids. Through these studies mass spectrometry analyses revealed that WEs are compartmentalized primarily in the cotyledonary tissues, whereas TAGs are, surprisingly, localized to the embryonic axis tissues. To study the differences in gene expression between these two tissues, a de novo transcriptome was assembled from high throughput RNAseq data. Differential gene expression analysis revealed that the Jojoba Wax Synthase, which catalyzes the formation of wax esters, and the Diacylglycerol O-Acyltransferase1, which catalyzes the final acylation of triacylglycerol synthesis, were differentially expressed in the cotyledons and embryonic axis tissues, respectively. Furthermore, through proteomic analysis of lipid droplet proteins from lipid droplets of the cotyledons and embryonic axis, it was estimated that each of these tissues contains a different proportion of the major lipid droplet proteins, oleosins, steroleosins, caleosins, and lipid droplet associated proteins. The Jojoba Olesosin1, Lipid Droplet Associated Protein 1, and Lipid Droplet Associated Protein 3, were identified as potential lipid droplet proteins that could be important for storage of wax esters. The coding sequences of these genes were transiently expressed in N. benthamiana leaves individually, and with co-expression of Mus musculus diacylglycerol acyltransferase 2, and in all cases were able to induce neutral lipid accumulation. …
Isolation and Genomic Characterization of 45 Novel Bacteriophages Infecting the Soil Bacterium Streptomyces griseus
Bacteriophages, or simply "phages," are the most abundant biological entities on the planet and are thought to be the largest untapped reservoir of available genetic information. They are also important contributors to both soil health and nutrient recycling and have significantly influenced our current understanding of molecular biology. Bacteria in the genus Streptomyces are also known to be important contributors to soil health, as well as producing a number of useful antibiotics. The genetic diversity of large (> 30) groups of other actinobacteriophages, i.e. phages infecting a few close relatives of the Streptomycetes, has been explored, but this is the first formal effort for Streptomyces-infecting phages. Described here are a group of 45 phages, isolated from soil using a single Streptomycete host, Streptomyces griseus ATCC 10137. All 45 phages are tailed phages with double-stranded DNA. Siphoviruses predominate, six of the phages are podoviruses, and no myoviruses were observed. Notably present are seven phages with prolate icosahedral capsids. Genome lengths and genome termini vary considerably, and the distributions of each are in line with findings among other groups of studied actinobacteriophages. Interestingly, the average G+C among the 45 phages is around 11% lower than that of the isolation host, a larger disparity than reported for other groups of actinobacteriophages. Eighteen of the phages carry between 17 and 45 tRNAs and 12 of those carry a single tmRNA. Forty-three phages were grouped into seven clusters and two subclusters based on dot plot analysis, average nucleotide identities, and gene content similarities. Two phages were not clustered with other phages in this dataset. A total of 5250 predicted genes were sorted into 1300 gene "phamilies," with about 8% of the total phamilies having only a single member. Analysis of gene content among the 45 phages indicates first that most clusters presented here appear to …
Lipogenic Proteins in Plants: Functional Homologues and Applications
Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes in plant vegetative tissues. Collectively, our findings in this study expand our knowledge of the broader cellular mechanisms of LD biogenesis that are partially conserved in eukaryotes and distinct in plants and suggest novel targets that can be introduced into plants to collaborate with other factors in lipid metabolism and elevate oil content in plant tissues.
Defining Components Linked to Bacterial Nutritional Utilization of Cyanide as a Sole Nitrogen Source
One of the challenges in biology is placing a function on the myriad of gene sequences having become available from rapid advances in genome sequencing. One such example is a gene cluster (Nit1C) found in bacteria that is tied to the unusual ability of certain bacteria to grow when supplied cyanide as the sole nitrogen source. The term cyanotrophs has been applied to such bacteria, for which a genetic linkage between cyanotrophy and Nit1C was demonstrated for 10 separate bacteria. In addition to growth, cyanide induced the expression of Nit1C genes in all organisms tested, and in one case, deletion of one of the Nit1C genes (nitC) caused a loss of growth. Of the ten bacteria able to grow cyanotrophically, all gave evidence of harboring Nit1C on their genome except for two (Pseudomonas fluorescens Pf11764 and P. monteilii BCN3), which were sequenced and the presence of Nit1C was also confirmed. A broader search of bacteria identified 270 separate strains with the cluster, all limited to bacteria spanning the phyla Firmicutes, Actinobacteria, Proteobacteria and Cyanobacteria. Remarkably, many examples of a single representative of a given taxon contained Nit1C, most poignantly displayed by Pf11764 and PmBCN3; the interpretation being the cluster was likely acquired by horizontal gene transfer in response to cyanide as an environmental cue. Consistent with its absence in Archaea is the time line for the emergence of cyanide producing organisms (cyanogens) on earth dating back only 400-500 million years.
A Genetic Approach to Identify Proteins that Interact with Eukaryotic Microtubule Severing Proteins via a Yeast Two Hybrid System
Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. Finally, a Biomolecular Fluorescence Complementation (BiFC) assay was initiated as a proof of concept for confirmation of in vivo protein-protein interaction.
Novel Approaches for Enhancing Resistance to Fusarium graminearum in Arabidopsis and Wheat by Targeting Defense and Pathogenicity Factors
Fusarium head blight (FHB) is an important disease of small grain cereals including wheat that affects grain quality and yield. The fungus Fusarium graminearum (Fg) is the major agent of this disease. Lack of natural resistance has limited ability to control wheat losses to this disease. Developing new approaches is critical for increasing host plant resistance to this fungus. This work has identified four processes that can be targeted for enhancing host plant resistance to FHB. The first involves targeting the pattern-triggered immunity mechanism to promote host plant resistance. Two other approaches involved reducing activity of susceptibility factors in the host to enhance plant resistance. The susceptibility factors targeted include accumulation of the phytohormone jasmonic acid and the 9-lipoxygenase pathway that oxidizes fatty acids. Besides suppressing host defenses against Fg, jasmonic acid also directly acts on the fungus to promote fungal growth. 9- lipoxygenases similarly suppress host defenses to promote fungal pathogenicity. Another approach that was developed involved having the plant express double stranded RNA to target fungal virulence genes for silencing. This host-induced gene silencing approach was employed to target two fungal virulence genes, the lipase encoding FGL1 and salicylate hydroxylase encoding FgNahG, which the fungus secretes into the host to promote turnover of the plant defense signaling metabolite salicylic acid. FGL1 in contrast acts on host lipids to release fatty acids, which suppress the deposition of callose that provides a physical barrier to limit fungal spread.
Role of Arabidopsis thaliana WRKY45 in Response to Green Peach Aphid Infestation, Drought, and Salinity Stresses
This study shows that Arabidopsis thaliana WRKY45 gene has an important role in limiting green peach aphid (GPA; Myzus persicae Sülzer) infestation. WRKY45 belongs to the WRKY family of transcription factors, which is one of the largest transcription factor family in plants. In response to GPA infestation, expression of WRKY45 was systemically upregulated in leaves and roots, with highest expression in the vascular tissues, which are the site of aphid feeding. GPA colonization was better on the wrky45 mutant compared to the wild-type (WT) plant. In contrast, GPA poorly colonized plants that were overexpressing (OE) WRKY45, thus confirming an important role for WRKY45 in plant defense to the GPA. A WRKY45-dependent process adversely impacted the reproductive rate of GPA and feeding from the sieve elements. RNA-seq experiments indicated a major impact of WRKY45 overexpression on expression of genes associated with dehydration and abscisic acid biosynthesis and signaling. In agreement with the RNA-seq data, ABA content was also higher in WRKY45-OE plants. However, genetic studies with an ABA-insensitive mutant (abi2-2) indicates that the WRKY45-OE conferred resistance to GPA is mediated through an ABA-independent mechanism. WRKY45-OE plants showed enhanced tolerance to drought and salt stresses. Genetic studies indicate that ABA signaling is critical for WRKY45's involvement in promoting plant tolerance to drought. Taken together, these results demonstrate that WRKY45 acts as a positive regulator of plant responses to GPA infestation, and drought and salt stress responses.
Impact of Anti-S2 Peptides on a Variety of Muscle Myosin S2 Isoforms and Hypertrophic Cardiomyopathy Mutants Revealed by Fluorescence Resonance Energy Transfer and Gravitational Force Spectroscopy
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 impact on MYH11, and the stabilizer peptide had dissociation binding constants of 2.12 × 10-2 μM to MYH7 isoform, 3.41 × 10-1 μM to MYH2 isoform, and no impact on MYH11 revealed by FRET. In presence of the stabilizer, FRET assay, affinity of the E930del and E924K increased by 10.23 and 0.60 fold respectively. The force required to uncoil muscle myosin S2 peptides in the presence of the stabilizer peptide was more than in its absence in muscle myosin S2 isoforms of MYH7 (1.80 fold higher), MYH2 (1.40 fold higher), and E930del (2.60 fold higher) and no change for MYH11 …
Analysis of the Cytochrome P450 and UDP-Glucuronosyltransferase Families and Vitamin D3- Supplementation in Anoxia Survival in Caenorhabditis elegans
Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia response phenotypes and the identification of human orthologs for the detoxification enzymes and provides insight into the gene expression pattern.
Identification and Characterization of a Mutation Causing Stunted Growth in Arabidopsis that is Linked to Phosphate Perception
Plant yield is an agronomic trait dependent on the transport of photosynthate from mature source leaves to sink tissues. Manipulating phloem transport may lead to increased yield, however in a previous study, Arabidopsis thaliana overexpressing sucrose transporter AtSUC2 in the phloem resulted in stunted growth and an apparent P-deficiency. In the course of further characterizing the phenotype and identifying the causative mutation, this research included 1) reverse genetics to test genes hypothesized to modulate carbon-phosphate interactions; 2) whole genome sequencing to identify all T-DNA insertions in plants displaying the phenotype; 3) genetic crosses and segregation analysis to isolate the causative mutation; and 4) transcriptomics to capture gene-expression profiles in plants displaying the phenotype. These phenotypes were traced to a T-DNA insertion located on chromosome 4. Transcriptomics by RNA-Seq and data analysis through bioinformatics pipelines suggest disruptions in metabolic and transport pathways that include phosphate, but do not support a direct role of well-established phosphate acquisition mechanisms. Gene At1G78690 is immediately downstream of the T-DNA insertion site and shows modestly increased expression relative to wild type plants. At1G78690 encodes O-acyl transferase, which is involved in processing N-acylphosphotidyl ethanolamine (NAPE) to N-acyl ethanolamine (NAE). Exogenous NAE application causes stunted growth in specific conditions. From the experiments described herein, At1G78690 emerges as the strongest candidate for causing the observed phenotypes.
Identification, Characterization and Engineering of UDP-Glucuronosyltransferases for Synthesis of Flavonoid Glucuronides
Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology modeling and site-directed mutagenesis towards increasing the repertoire of UG(A)Ts applicable for synthesis of flavonoid glucuronides with potential human health benefits in neurological protection.
Role of 5.8S rRNA in Zebrafish and Human Blood Coagulation
Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. Previously, it has been shown that the RNA released from damaged blood cells activates clotting. However, the nature of RNA released from hemolysis is still elusive. We found that after hemolysis, the red blood cells from both zebrafish and humans release 5.8S rRNA. This RNA activated coagulation in zebrafish and human plasmas. Using both natural and synthetic 5.8S rRNA and its synthetic truncated fragments, we found that the 3'-end 26 nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor blocked 3'-26 RNA-mediated coagulation activation of both zebrafish and human plasma. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activates normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via FXII-like protein. Since zebrafish has no FXII and hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. In conclusion, we identified 5.8S rRNA released in hemolysis activates clotting in human and zebrafish plasma. Only 3'-end 26 nucleotides of the 5.8S rRNA is needed for the clotting activity. Furthermore, we showed that fish Hgfac plays a role in 5.8S rRNA-mediated activation of coagulation.
Toxicological and Biochemical Changes Induced by Sub-Acute Exposure of Biological Organisms to Silver Nanoparticles Using Soft-Landing Ion Mobility Instrument
In this study, we have developed a novel way of generating and exposing biological organisms (both prokaryotic and eukaryotic) to silver nanoparticles (AgNPs) and studying the biochemical changes induced by these particles. We analyzed the various organs of Wistar rats for localization and quantification of these particles using mass spectrometric and molecular biological techniques. Highest levels of AgNP was found in the lung tissue in addition to being present in the liver and kidneys. Analysis of the of the blood plasma from AgNP exposed rats revealed elevated levels of glutathione-disulfide, which is indicative of reactive oxygen species (ROS) generation, which was further validated using ROS specific immunofluorescence staining of liver tissue. Quantification of blood lactate levels of the AgNP exposed rats showed increased lactate levels, which is indicative of anaerobic respiration and may result from AgNP-induced oxidative stress. Further analysis of bone marrow cells from AgNP exposed rats showed a higher number of micronuclei formation in developing erythrocytes and bone marrow cytotoxicity. Finally, analysis of the genes involved in the renin-angiotensin system (RAS) and inflammatory response revealed upregulation in transcript levels of many of these important genes in the liver tissue. Taken together, our study provides an initial road map for the identification of different signaling pathways that are altered by the AgNP exposure and contributes to a comprehensive understanding of the mechanism involved in silver nanoparticle-induced toxicity.
The Development of Potential Therapeutic Anti-Myosin S2 Peptides that Modulate Contraction and Append to the Heart Homing Adduct Tannic Acid without Noticeable Effect on Their Functions
This dissertation aimed to explore the S2 region with an attempt to modulate its elasticity in order to tune the contraction output. Two peptides, the stabilizer and destabilizer, showed high potential in modifying the S2 region at the cellular level, thus they were prepared for animal model testing. In this research, (i) S2 elasticity was studied, and the stabilizer and destabilizer peptides were built to tune contraction output through modulating S2 flexibility; (ii) the peptides were attached to heart homing adducts and the bond between them was confirmed; and (iii) it was shown that minor changes were imposed on the modulating peptides' functionality upon attaching to the heart homing adducts. S2 flexibility was confirmed through comparing it to other parts of myosin using simulated force spectroscopy. Modulatory peptides were built and computationally tested for their efficacy through interaction energy measurement, simulated force spectroscopy and molecular dynamics; these were attached to heart homing adducts for heart delivery. Interaction energy tests determined that tannic acid (TA) served well for this purpose. The stoichiometry of the bond between the TA and the modulating peptides was confirmed using mass spectroscopy. The functionality of the modulating peptides was shown to be unaltered through expansion microscopy where they located to the same position on the sarcomere with and without TA. They were also shown to cause the sarcomeres to contract similarly with and without the TA in contractility assay. Taken together, this work prepared the modulating peptides for animal model tests by attaching them to tannic acid.
Multi-Omics Based Investigation of Distinct Early Oxidative Stress Responses of Saccharomyces cerevisiae to Various Oxidants
The early signaling mechanism(s) that control oxidant perception and signal transduction leading to activation of the antioxidant defense response and survival mechanisms tailored toward specific oxidative insult remains unknown. Here, we identified early changes in metabolome and proteome of S. cerevisiae in response to hydrogen peroxide, menadione, cumene hydroperoxide, and diamide. Firstly, global untargeted LC–MS/MS analysis allowed us to identify 196 proteins in response to hydrogen peroxide, 569 proteins in response to cumene hydroperoxide, 369 proteins in response to menadione and 207 proteins in response to diamide that were significantly regulated at 3 min after exposure. We revealed that each oxidant triggered unique signaling mechanisms associated with survival and repair mechanisms as early as 3 minutes of post treatment with a set of proteins that uniquely responded to the particular oxidant. In addition, our comprehensive pathway analysis revealed signaling pathways and the molecular players that are regulated globally by all oxidants at early time points namely Ran, mTOR, Rho, and eIF2. Additionally, we analyzed metabolic response using targeted GC-MS/MS that allowed us to identity 35 metabolites that were consistently detected in all samples at 3 min of exposure. These metabolites showed distinct response to the four oxidants in carbohydrate metabolism, tricarboxylic acid, amino acid metabolism and glutathione cycle. Furthermore, temporal analysis showed targeted metabolites significantly regulated at different time points by different oxidants suggesting specificity in the response to oxidants having different mode of actions.
Effect of Phosphorus Starvation on Metabolism and Spatial Distribution of Phosphatidylcholine in Medicago truncatula Wild-Type and PDIL3 Genotypes
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, LC-MS/MS was used to quantify these compounds including phosphorylated metabolites in whole plant. My findings showed strong drop in levels of organic acids and phosphorylated compounds in P deprived leaves with moderate reduction in P deprived roots and nodules. Moreover, sugars and amino acids were elevated in whole plant under P deprivation. Finally, the last project of my thesis involved studying the response of PDIL3 (Phosphate Deficiency-Induced LncRNA-3) a long non-coding RNA (lncRNA) mutant under severe P stress. PDIL3 is known to regulate Pi-deficiency signaling and transport in M. truncatula (Wang et al., 2017). My results confirmed that in P …
Exploring Flavonoid Glycosylation in Kudzu (Pueraria lobata)
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 recombinant UGT71T5 with daidzein was confirmed in the present work. Following the development of a kudzu hairy root system, UGT71T5 expression was then knocked down by RNA interference (RNAi). When compared to control hairy roots there was a large reduction in puerarin content in the UGT71T5-RNAi roots, confirming the role of this enzyme in puerarin biosynthesis. Isotopic labeling of kudzu plants revealed that labeled daidzein could be directly incorporated into puerarin; however, the percent incorporation of daidzein was substantially lower than that of L-phenylalanine, a compound at the start of the pathway to isoflavone synthesis. The knockdown of 2-hydroxisoflavanone synthase …
Studies on Intrinsic Coagulation Pathway of Zebrafish
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 unknown etiologies in Type 1 VWD. Moreover, therapeutic interventions could be designed in the future by modulation of these gene products to control bleeding or thrombosis.Zebrafish has three f9 genes, f9a, f9b, and f9l and the ortholog to human F9 is unknown. RNA analysis showed an age-dependent increase in expression of all three genes from larval stages to adults, comparable to those observed in mice and humans while mass spectrometry and immunohistochemistry confirmed the presence of all three proteins in the fish. Based on coagulation assays performed after individual gene knockdown and immunodepletion, we identified that zebrafish f9a has …
Studies on Tissue Factor Pathway Inhibitor in Zebrafish
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 results have identified 5 novel regulators for tfpia. We exploited this information to discover UNC6852, a drug that enhances tfpia mRNA levels. This could be used as an antithrombotic drug. The approach developed here could be used to study the regulation of other coagulant and anticoagulant factors.
Manipulation of Lipid Droplet Biogenesis for Enhanced Lipid Storage in Arabidopsis thaliana and Nicotiana benthamiana
In this study, I examined the use of mouse (Mus musculus) Fat Specific Protein 27 (FSP27) ectopically expressed in Arabidopsis thaliana and Nicotiana benthamiana as a means to increase lipid droplet (LD) presence in plant tissues. In mammalian cells, this protein induces cytoplasmic LD clustering and fusion and helps prevent breakdown of LDs contributing to the large, single LD that dominates adipocytes. When expressed in Arabidopsis thaliana and Nicotiana benthamiana, FSP27 retained its functionality and supported the accumulation of numerous and large cytoplasmic LDs, although it failed to produce the large, single LD that typifies adipose cells. FSP27 has no obvious homologs in plants, but a search for possible distant homologs in Arabidopsis returned a Tudor/PWWP/MBT protein coded for by the gene AT1G80810 which for the purposes of this study, we have called LIPID REGULATORY TUDOR DOMAIN CONTAINING GENE 1 (LRT1). As a possible homolog of FSP27, LRT1 was expected to have a positive regulatory effect on LDs in cells. Instead, a negative regulatory effect was observed in which disruption of the gene induced an accumulation of cytoplasmic LDs in non-seed tissue. A study of lrt1 mutants demonstrated that disruption this gene is the causal factor of the cytoplasmic LD accumulation observed in the mutants, that this phenotype occurs in above ground tissues and is present throughout the early growth stages of the plant. Further examination of lrt1 mutant plants has allowed a preliminary understanding of the role LRT1 may play in LD regulation. Taken together, the results of this study point towards some promising strategies to increase LD content in plant tissues.
Role of DEFECTIVE IN SYSTEMIC DEFENSE INDUCED BY ABIETANE DITERPENOID 1 (DSA1), a Putative O-Fucosyltransferase, in Plant Systemic Acquired Resistance (SAR)
Dehydroabietinal (DA), an abietane diterpenoid, was previously demonstrated to be a potent activator of systemic acquired resistance (SAR). DA also promotes flowering time in Arabidopsis thaliana by repressing expression of the flowering repressor FLOWERING LOCUS C (FLC) while simultaneously upregulating expression of FLOWERING LOCUS D (FLD), FLOWERING LOCUS VE (FVE) and RELATIVE OF EARLY FLOWERING 6 (REF6), a set of flowering time promoters. To further understand the mechanism underlying signaling by abietane diterpenoids, Arabidopsis mutants exhibiting reduced responsiveness to abietane diterpenoids were identified. One such mutant plant, ems2/7, exhibited SAR-deficiency and delayed flowering, which were found to be associated with two independent, but linked loci. The gene responsible for the SAR defect in ems2/7 was identified as DEFECTIVE IN SYSTEMIC DEFENSE INDUCED BY ABIETANE DITERPENOID 1 (DSA1). Similar to the missense mutant dsa1-1 identified in the mutant screen, the T-DNA insertion bearing null allele dsa1-2 exhibited SAR deficiency that could be complemented by a genomic copy of DSA1. The gene responsible for the delayed flowering phenotype of ems2/7 remains to be identified. DSA1 encodes a protein that is homologous to human protein O-fucosyltransferase 2. DSA1 is required for long-distance transport of the SAR signal. It is hypothesized that DSA1 is involved in the O-fucosylation-facilitated channeling through the ER/Golgi network of a protein involved in long distance SAR signaling. In a yeast two-hybrid screen, all the DSA1-interacting proteins identified are chloroplast-localized proteins, thus raising the interesting possibility of ER interaction with chloroplast and its potential role in SAR signaling.
A Sensitive and Robust Machine Learning-Based Framework for Deciphering Antimicrobial Resistance
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.
Identification and Characterization of Two Putative Sulfate Transporters Essential for Symbiotic Nitrogen Fixation in Medicago truncatula
The process of symbiotic nitrogen fixation (SNF) in legume root nodules requires the channeling and exchange of nutrients within and between the host plant cells and between the plant cells and their resident rhizobia. Using a forward genetics approach in the Medicago truncatula Tnt1 mutant population followed by whole genome sequencing, two putative sulfate transporter genes, MtSULTR3;5 and MtSULTR3;4b, were identified. To support the hypothesis that the defective putative sulfate transporter genes were the causative mutation for the mutants' phenotypes, the M. truncatula Tnt1 population was successfully reverse screened to find other mutant alleles of the genes. The F2 progeny of mutants backcrossed with wildtype R108 demonstrated co-segregation of mutant phenotypes with the mutant alleles confirming that the mutated mtsultr3;5 and mtsultr3;4b genes were the cause of defective SNF in the mutant lines mutated in the respective genes. This finding was further established for mtsultr3;4b by successful functional complementation of a mutant line defective in the gene with the wildtype copy of MtSULTR3;4b. A MtSULTR3;4b promoter-GUS expression experiment indicated MtSULTR3;4b expression in the vasculature and infected and uninfected plant cells of root nodules. MtSULTR3;4b was found to localize to the autophagosome membrane when expressed in Nicotiana benthamiana. A transcriptomics study on the mutant nodules revealed the probable impact of mutated mtsultr3;5 and mtsultr3;4b on expression of genes involved in N fixation and on other biological processes, including possible effects of the mutated genes on the transcriptional regulation of sulfate assimilation pathway in the respective mutants' nodules. The RNAseq study also demonstrated the mis-regulation of nodule zone-specific genes in mtsultr3;5 and mtsultr3;4b mutants. A PCR-based approach was used to study the transcription of MtSULTR3;5 and MtSULTR3;4b in the respective mutant lines. The study demonstrated formation of readthrough chimeric gene-Tnt1 transcripts in mtsultr3;5 mutant alleles and truncated chimeric gene-Tnt1 transcripts and aberrantly …
Medicago truncatula NPF1.7: Structure-Function Assessment and Potential as a Phytohormone Transporter
In Medicago truncatula, the MtNPF1.7 transporter has been shown to be essential for root morphology and nodulation development. The allelic MtNPF1.7 mutants, Mtnip-1 (A497V), Mtnip-3 (E171K), and Mtlatd (W341STOP), show altered lateral root growth and compromised legume-rhizobium symbiosis. To assess the role of a series of distinct amino acids in the transporter's function, in silico structural predictions were combined with in planta complementation of the severely defective Mtnip-1 mutant plants. The findings support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. The results also question the existence of a putative TMH4-TMH10 salt bridge, which may not form in MtNPF1.7. Results reveal that a motif conserved among MFS proteins, Motif A, is essential for function. Hypothetically, the Motif A participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. The mutated valine (A497V) in Mtnip-1 may interfere with the lateral helix. Mutating a residue (L253) on the lateral helix with reduced side chain restored Mtnip-1 function. The predicted residue (Q351) for substrate binding is not essential for protein function. To probe the possibility that MtNPF1.7 transports auxin, two heterologous assay systems were attempted. The first was a Xenopus laevis oocyte assay. However, MtNPF1.7 expressed in oocytes failed to show substrate transport, which may due to low expression levels of proteins on the membrane or may be caused by other factors. Second, yeast (Saccharomyces cerevisiae) strains expressing MtNPF1.7 were constructed. They showed an increased flux of radiolabeled IAA and differential susceptibility to 5-fluoroindole-3-acetic acid (F-IAA), a toxic IAA-like compound. These results suggested that MtNPF1.7 may function as an auxin transporter in yeast. Unexpectedly, the Mtnip-1 (A497V) and Mtnip-3 (E171K) proteins when expressed in yeast also showed influx of F-IAA transport …
Role of MicroRNAs and Their Downstream Targets in Zebrafish Thrombopoiesis
Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on microRNAs' role in zebrafish thrombopoiesis. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, I identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Knockdown of three microRNAs, mir-7148, let-7b, and mir-223, by the piggyback method in zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. I then verified these findings in zebrafish larvae after the knockdown of the above microRNAs followed by an arterial laser thrombosis assay. I concluded mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. Furthermore, I explored let-7b downstream genes in thrombocytes detected by RNA-seq analysis and chose 14 targets based on their role in cell differentiation (rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b) that are transcriptional regulators. The qRT-PCR analysis of expression levels the above genes following let-7b knockdown showed significant changes in the expression of 13 targets. I then studied the effect of the 14 targets on thrombocytes production and identified 5 genes (irf5, tgif1, irf8, cebpa, and rorca) that showed thrombocytosis and one gene ikzf1 that showed thrombocytopenia. Furthermore, I tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. I also identified that tgif1, cebpa, …
Anti-S2 Peptides and Antibodies Binding Effect on Myosin S2 and Anti-S2 Peptide's Ability to Reach the Cardiomyocytes in vivo and Interfere in Muscle Contraction
The anti-S2 peptides, the stabilizer and destabilizer, were designed to target myosin sub-fragment 2 (S2) in muscle. When the peptides are coupled to a heart-targeting molecule, they can reach the cardiomyocytes and interfere with cardiac muscle contraction. Monoclonal antibodies, MF20 and MF30, are also known to interact with light meromyosin and S2 respectively. The MF30 antibody compared to anti-S2 peptides and the MF20 antibody is used as a control to test the central hypothesis that: Both the anti-S2 peptides and antibodies bind to myosin S2 with high affinity, compete with MyBPC, and possibly interact with titin, in which case the anti-S2 peptides have further impact on myosin helicity and reach the heart with the aid of tannic acid to modulate cardiomyocytes' contraction in live mice. In this research, the effects of anti-S2 peptides and antibodies on myosin S2 were studied at the molecular and tissue levels. The anti-myosin binding mechanism to whole myosin was determined based on total internal reflectance fluorescence spectroscopy (TIRFS), and a modified cuvette was utilized to accommodate this experiment. The binding graphs indicated the cooperative binding of the peptides and antibodies with high affinity to myosin. Anti-myosin peptides and antibodies competition with Myosin Binding Protein C (MyBPC) was revealed through the super-resolution expansion microscopy using wildtype skeletal and cardiac myofibrils, and MyBPC knock-out cardiac myofibril. This new emerging technique depends on using the regular confocal microscope in imaging expanded myofibril after embedding in a swellable hydrogel polymer and digestion. A decrease in the fluorescent intensity at the C-zone was observed in myofibrils labeled with fluorescently labeled anti-S2 peptides or antibodies supporting the competition with MyBPC, which further was confirmed by the absence of this reduction at the C-zone in the knockout MyBPC cardiac tissue. The anti-S2 peptide's ability to reach inside the cardiomyocytes was tested by …
Identification and Characterization of Genes Required for Symbiotic Nitrogen Fixation in Medicago truncatula Tnt1 Insertion Mutants
In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by which MtVTL8 functions.
Fatty Acid Amide Hydrolases in Upland Cotton (Gossypium hirsutum L.) and the Legume Model Medicago truncatula
Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.
Glucose-Induced Developmental Delay is Modulated by Insulin Signaling and Exacerbated in Subsequent Glucose-Fed Generations in Caenorhabditis elegans
In this study, we have used genetic, cell biological and transcriptomic methods in the nematode C. elegans as a model to examine the impact of glucose supplementation during development. We show that a glucose-supplemented diet slows the rate of developmental progression (termed "glucose-induced developmental delay" or GIDD) and induces the mitochondrial unfolded protein response (UPRmt) in wild-type animals. Mutation in the insulin receptor daf-2 confers resistance to GIDD and UPRmt in a daf-16-dependent manner. We hypothesized that daf-2(e1370) animals alter their metabolism to manage excess glucose. To test this, we used RNA-sequencing which revealed that the transcriptomic profiles of glucose-supplemented wildtype and daf-2(e1370) animals are distinct. From this, we identified a set of 27 genes which are both exclusively upregulated in daf-2(e1370) animals fed a glucose-supplemented diet and regulated by daf-16, including a fatty acid desaturase (fat-5), and two insulin-like peptides (ins-16 and ins-35). Mutation of any of these genes suppresses the resistance of daf-2(e1370) to GIDD. Additionally, double mutation of ins-16 and ins-35 in a daf-2(e1370) background results in an increase in constitutive dauer formation which is suppressed by glucose supplementation. Further investigation of the insulin-like peptides revealed that ins-16 mutation in a wild-type background results in upregulation of ins-35 and DAF-16 nuclear translocation regardless of diet; however, unlike daf-2(e1370), this translocation is not associated with resistance to GIDD. Taken together, these data suggest that glucose-supplemented daf-2(e1370) animals maintain developmental trajectory in part through upregulation of specific insulin-like peptide genes and fatty acid desaturation and contribute to a deeper understanding of the mechanisms underlying the resistance of daf-2(e1370) animals to GIDD. We also showed another fascinating aspect of GIDD: it becomes more pronounced in subsequent generations exposed to a glucose-supplemented diet, suggesting that the parental glucose diet has an impact on the developmental progression of their offspring.
Investigating Novel Streptomyces Bacteriophage Endolysins as Potential Antimicrobial Agents
As antibiotic resistance has become a major global threat, the World Health Organization has urgently called scientists for alternative strategies for control of bacterial infections. Endolysin, a protein encoded by a phage gene, can degrade bacterial peptidoglycan (PG). Currently, there are three endolysin products in the clinical phase. We, thus, are interested in exploring novel endolysins from Streptomyces phages as only a few of them have been experimentally characterized. Using bioinformatics tools, we identified nine functional domain groups from 250 Streptomyces phages putative endolysins. NootNoot gp34 (transglycosylase; Nt34lys), Nabi gp26 (amidase; Nb26lys), Tribute gp42 (PGRP; Tb42lys), and LazerLemon gp35 (CHAP; LL35lys) were selected for experimental studies. We hypothesized that (1) the proteins of interest will have the ability to degrade PG, and (2) the proteins will be potential antimicrobial agents against ESKAPE safe relatives. The results showed that LL35lys, Nb26lys and Tb42lys exhibit PG-degrading activity on zymography and hydrolysis assay. The enzymes (400 µg/mL) can reduce PG turbidity to 32-40%. The killing assay suggested that Tb42lys possess a boarder range (Escherichia coli, Pseudomonas putida, Acinetobacter baylyi and Klebsiella aerogenes). While Nb26lys can attack Gram-negative bacteria, LL35lys can only reduce the growth of the Gram-positive strains with an MIC90 of 2 µg/mL. A higher concentration (≥300 µg/mL) of Nb26lys is needed to treat P. putida and K. aerogenes. Therefore, endolysins from Streptomyces phage have potential as possible antimicrobial agents against ESKAPE bacteria.
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