PARTIAL CHARACTERIZATION AND CLONING OF PROTEASE FROM BACILLUS

  Objective: The present research focused on amplification of protease gene from Bacillus strain which was then assessed for maximal enzyme activity.Methods: A putative Bacillus strain was isolated from soil, inoculated into protease production media, and optimized with appropriate pH and temperature conditions for maximal enzyme activity. Genomic DNA was isolated from the strain and amplified the fragment by polymerase chain reaction (PCR) using gene-specific primers for protease. The fragment is then ligated into a T/A cloning vector and transformed into calcium chloride-treated competent Escherichia coli DH5α cells. The plasmids were then isolated and confirmed the presence of the gene.Results: A specific amplification of 1.1 kb was observed following PCR. The amplified product includes the coding sequence and a signal peptide sequence of the protease gene. After cloning with T/A cloning vector pTZ57R/T and transformed into E. coli DH5α competent cells, the recombinant plasmid was selected using blue-white selection. Plasmid DNA isolated from the recombinant strains and confirmed the presence of a gene of interest using PCR and quantified by an assay for maximal protease activity. The optimum pH was found to be 10.1 and giving an activity of 21.566 international unit (IU)/ml, and the optimum temperature was found to be on 60°C giving an activity of 38.708 IU/ml.Conclusion: Amplification of protease gene by PCR isolated from Bacillus strain and optimization of pH and temperature conditions for the assessment of subtilisin Carlsberg produced by it. Subtilisin which is protein engineered can be used in commercial products such as stain cutter, dishwashing detergents, cosmetics and food processing, and contact lens cleaner

Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF)

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs 150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations

miR-21 in the Extracellular Vesicles (EVs) of Cerebrospinal Fluid (CSF): A Platform for Glioblastoma Biomarker Development

Glioblastoma cells secrete extra-cellular vesicles (EVs) containing microRNAs (miRNAs). Analysis of these EV miRNAs in the bio-fluids of afflicted patients represents a potential platform for biomarker development. However, the analytic algorithm for quantitative assessment of EV miRNA remains under-developed. Here, we demonstrate that the reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. In this context, we quantitated EV miRNA in absolute terms and normalized this value to the input EV number. Using this method, we examined the abundance of miR-21, a highly over-expressed miRNA in glioblastomas, in EVs. In a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21. Consistent with this hypothesis, the CSF EV miR-21 levels of glioblastoma patients (n=13) were, on average, ten-fold higher than levels in EVs isolated from the CSF of non-oncologic patients (n=13, p<0.001). Notably, none of the glioblastoma CSF harbored EV miR-21 level below 0.25 copies per EV in this cohort. Using this cut-off value, we were able to prospectively distinguish CSF derived from glioblastoma and non-oncologic patients in an independent cohort of twenty-nine patients (Sensitivity=87%; Specificity=93%; AUC=0.91, p<0.01). Our results suggest that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development

miR-181d: a predictive glioblastoma biomarker that downregulates MGMT expression

Genome-wide microRNA (miRNA) profiling of 82 glioblastomas demonstrated that miR-181d was inversely associated with patient overall survival after correcting for age, Karnofsky performance status, extent of resection, and temozolomide (TMZ) treatment. This association was validated using the Cancer Genome Atlas (TCGA) dataset (n= 424) and an independent cohort (n= 35). In these independent cohorts, an association of miR-181d with survival was evident in patients who underwent TMZ treatment but was not observed in patients without TMZ therapy. Bioinformatic analysis of potential genes regulated by miR-181d revealed methyl-guanine-methyl-transferase (MGMT) as a downstream target. Indeed, transfection of miR-181d downregulated MGMT mRNA and protein expression. Furthermore, luciferase reporter assays and coprecipitation studies showed a direct interaction between miR-181d and MGMT 3′UTR. The suppressive effect of miR-181d on MGMT expression was rescued by the introduction of an MGMT cDNA. Finally, MGMT expression inversely correlated with miR-181d expression in independent glioblastoma cohorts. Together, these results suggest that miR-181d is a predictive biomarker for TMZ response and that its role is mediated, in part, by posttranscriptional regulation of MGMT

Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid

BackgroundTumor specific genetic material can be detected in extracellular vesicles (EVs) isolated from blood, cerebrospinal fluid (CSF), and other biofluids of glioblastoma patients. As such, EVs have emerged as a promising platform for biomarker discovery. However, the optimal procedure to transport clinical EV samples remains poorly characterized.MethodsWe examined the stability of EVs isolated from CSF of glioblastoma patients that were stored under different conditions. EV recovery was determined by Nanoparticle tracking analysis, and qRT-PCR was performed to determine the levels of miRNAs.ResultsCSF EVs that were lyophilized and stored at room temperature (RT) for seven days exhibited a 37-43% reduction in EV number. This reduction was further associated with decreased abundance of representative miRNAs. In contrast, the EV number and morphology remained largely unchanged if CSF were stored at RT. Total RNA and representative miRNA levels were well-preserved under this condition for up to seven days. A single cycle of freezing and thawing did not significantly alter EV number, morphology, RNA content, or miRNA levels. However, incremental decreases in these parameters were observed after two cycles of freezing and thawing.ConclusionsThese results suggest that EVs in CSF are stable at RT for at least seven days. Repeated cycles of freezing/thawing should be avoided to minimize experimental artifacts

Differential localization of glioblastoma subtype: implications on glioblastoma pathogenesis.

IntroductionThe subventricular zone (SVZ) has been implicated in the pathogenesis of glioblastoma. Whether molecular subtypes of glioblastoma arise from unique niches of the brain relative to the SVZ remains largely unknown. Here, we tested whether these subtypes of glioblastoma occupy distinct regions of the cerebrum and examined glioblastoma localization in relation to the SVZ.MethodsPre-operative MR images from 217 glioblastoma patients from The Cancer Imaging Archive were segmented automatically into contrast enhancing (CE) tumor volumes using Iterative Probabilistic Voxel Labeling (IPVL). Probabilistic maps of tumor location were generated for each subtype and distances were calculated from the centroid of CE tumor volumes to the SVZ. Glioblastomas that arose in a Genetically Modified Murine Model (GEMM) model were also analyzed with regard to SVZ distance and molecular subtype.ResultsClassical and mesenchymal glioblastomas were more diffusely distributed and located farther from the SVZ. In contrast, proneural and neural glioblastomas were more likely to be located in closer proximity to the SVZ. Moreover, in a GFAP-CreER; PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/- GEMM model of glioblastoma where tumor can spontaneously arise in different regions of the cerebrum, tumors that arose near the SVZ were more likely to be of proneural subtype (p &lt; 0.0001).ConclusionsGlioblastoma subtypes occupy different regions of the brain and vary in proximity to the SVZ. These findings harbor implications pertaining to the pathogenesis of glioblastoma subtypes

Total EV yield as determined by NTA and TRPS.

<p>Total EV yield as determined by NTA and TRPS.</p

Comparison of EV quantification by NTA and TEM.

<p>CSF EVs were fractionated into microvesicles (10,000×g) and exosomes (120,000×g) by differential ultracentrifugation and then analyzed by NTA and TEM. <b>(A)</b> Representative TEM images, scale bar = 200nm. <b>(B)</b> Total EV count as determined by NTA and TEM. Fold difference in particle detected between NTA and TEM is denoted.</p

EV quantitative analysis.

<p><b>(A)</b> Schematic representation of protocol used for the isolation of CSF microvesicles and exosomes. <b>(B)</b> In nanoparticles tracking analysis, light scattered by EVs is captured by digital camera over a series of frames. The rate of the particle movement is then used to calculate particle size using the Stokes—Einstein equation. <b>(C)</b> In tunable resistive pulse sensing, EVs change the electrical resistance as they pass through a pore-based sensor resulting in a resistive pulse signal. Signals obtained from the measurements can then be used to calculate the size, concentration and charge of each particle by correlating the signal back to a set of known standards. <b>(D)</b> In Vesicle flow cytometry, EVs were stained with an optimized concentration of a fluorogenic lipophilic probe, di-8-ANEPPS, and detected on a custom high sensitivity flow cytometer. Vesicle diameter was estimated by comparison to di-8-stained liposomes.</p