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 …

Since phages use the host resources to replicate themselves after infection, the different sizes of the phage genome should influence the replication rate. We, therefore, hypothesized that the smaller genomes should burst the cell faster than the larger ones. As well, the shorter genomes would have greater burst sizes because they should replicate faster. Here, we obtained 16 phages of various genome length. All phages were isolated on Streptomyces griseus and available in our phage bank at the University of North Texas. We performed one-step growth studies for the 16 phages, as well as determined the host doubling time from its growth curve. The results show that S. griseus grown in nutrient broth has a doubling time of 5 hours and 22 minutes. This doubling time is used as a guideline for the phage growth studies. Because the filamentous nature of the host caused several difficulties during the experiment, we isolated single cells by sonication and centrifugation. After the cell number was determined by viable cell count, the cells were infected with each type of phage using a multiplicity of infection (MOI) of 0.5. The results show that phages' burst times range between 45 (±0, standard error) and 420 (±30) …

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 …

Bacteriophages are viruses that specifically infect bacteria. When a phage infects a bacterium, it attaches itself to the surface of the bacteria and injects its DNA into the intracellular space. The phage DNA hijacks the cellular machinery of the bacteria and forces it to produce phage proteins. Eventually, the bacteria cell bursts or lyses, releasing new phage. The bacteria act as a host for phage reproduction. The ability for a phage to infect multiple bacterial species is known as host range. In siphoviridae bacteriophages, host range is thought to primarily be determined by proteins at the tip of their tail fibers. These proteins act as anti-receptors to specific receptors on the surface of bacteria. In siphoviridae Gram-positive infecting phages, the genes that code these proteins are typically located between the tape measure protein gene and the endolysin gene. It is hypothesized that phages that have similar anti-receptor proteins will have similar host range. In this study, the host ranges of 12 BD1 bacteriophages were tested on 9 different Streptomyces species. In these 12 phages, the genes between the tape measure protein gene and endolysin gene were compared. The 12 phages had high levels of variability in these genes. Five genes …

Isolating novel phages using Streptomyces azureus, which produces antibiotic thiostrepton, as a host, and characterizing the genomes may help us to find new tools that could be used to develop antibiotics in addition to contribute to the databases of phages and specifically, Streptomyces phages. Streptomyces phages Alsaber, Omar, Attoomi, Rowa, and ZamZam were isolated using during this study. They were isolated from enriched soil and sequenced by Illumina sequencing method. They were isolated from three different geographical regions. They are siphoviridae phages that create small clear plaques with a diameter of approximately 0.5-1 mm, except for Rowa which has cloudy plaques, and they have varied sizes of their heads and tails. ZamZam was not characterized at this time. The sequencing shows that they are circular genome with 3' sticky overhang and various genomes' sizes with high percentage of GC content with the average of 66%. Alsaber was classified under sub-cluster BD3, while Omar was categorized under sub-cluster BD2. They share the same cluster of Cluster BD. Rowa was placed in Cluster BL and Attoomi is currently a singleton that does not fit into an established cluster. Alsaber yields 76 putative genes with no tRNA, Omar 81 putative genes with 1 …

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 …

Legumes are unique plants because they form special structures “nodules”, via symbiotic relationships with rhizobial bacteria present in the soil. Once rhizobia mature inside nodules, they fix atmospheric nitrogen providing a source of bioavailable nitrogen to the plant. To discover novel genetic components involved in the legume-rhizobia symbiosis by using forward genetic screening, we have isolated Medicago truncatula Tnt1 insertion mutants in the R108 ecotype, which are defective in nodule development and symbiotic nitrogen fixation in response to Sinorhizobium meliloti. Out of three mutants NF11044, NF11217 and NF8324, one of the mutants showed brown nodules and Fix- phenotype that is defective in symbiotic nitrogen fixation. The other two mutants showed white nodules and Fix- phenotype, also indicator of defects in symbiotic nitrogen fixation. To identify the underlying mutation causing the phenotype, we have developed molecular genetic markers by obtaining genomic sequences flanking the Tnt1 insertions by TAIL-PCR and Illumina sequencing. To carry out co-segregation analysis, back-crossed BC1F2 segregating populations were obtained. These are being phenotyped, genotyped and analyzed for co-segregation of the phenotype with the Tnt1 genetic markers. Back-crossing also has the effect of reducing the Tnt1 insertions, which are not linked to the nodulation defective phenotypes. Out of the …

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 …

N-Acylethanolamines (NAEs) are a class of bioactive lipids, and FAAH is one of the enzymes responsible for degrading NAEs in both plants and animals. in plants, FAAH appears to be closely associated with ABA, a phytohormone which has long been associated with plant stress responses, since the overexpression of FAAH in Arabidopsis results in ABA hypersensitivity. Therefore, it is reasonable to speculate that alterations in FAAH transcript levels will result in altered stress responses in plants. to investigate this hypothesis experiments were carried out in which wild type (WT), FAAH-overexpressing (OE), and T-DNA insertional FAAH knockouts of Arabidopsis (faah) were grown in MS media under stress conditions. the stress conditions tested included chilling stress, heavy metal stress induced by cadmium or copper, nutrient limitations induced by low phosphorus or low nitrogen, salt stress induced with NaCl, and osmotic stress induced with mannitol. the OE plants were consistently hypersensitive to all stress conditions in relation to wild type plants. Inactive FAAH overexpressors did not have the hypersensitivity to the salt and osmotic stress of the active OE plants and were instead tolerant to these stresses. FAAH2 (faah2) knockouts and FAAH 1 and 2 double knockouts (faah 1+2) were based on some …

von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.