The role and mechanism of action of BRK in breast cancer progression

Breast cancer is unanimously considered a highly heterogeneous disease due to its diverse molecular features. Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase that is highly expressed in over 80% of breast carcinomas. The role and mechanism of action of enzymatically activated BRK in breast pathology are unclear. The objectives of this project were to reveal the effect of BRK activation on cell migration, proliferation and tumorigenesis. We also aimed to determine the mechanism of action of BRK in the promotion of cell proliferation. We used BRK-negative cells (MCF10A, MDA-MB-231 and HEK293) to generate three sets of stable cell lines that stably expressed GFP alone, GFP-BRK-WT or GFP-BRK-Y447F (constitutively active) by retroviral infections. We also stably knocked down BRK from BRK-positive cells BT20 and SKBR3 by RNA interference using shRNAs against BRK. Western blotting, immunoprecipitation and qPCR studies were conducted to evaluate protein expression, protein-protein interaction and mRNA expression, respectively. Both sets of cell lines were used to determine the effect of BRK on cell proliferation (automated cell counter), cell migration (transwell and wound healing assay), transformation (colony formation assay) and tumor formation (mouse Xenograft assay). To investigate the mechanism of action of BRK, we validated downstream of tyrosine kinases 1 (Dok1), a tumor suppressor, as a BRK substrate. Deletion or site-directed mutagenesis was performed to map BRK-targeted tyrosines in Dok1 protein. Results obtained from this research project showed that stable expression of the constitutively active mutant of BRK (BRK-Y447F) in MDA-MB-231 cells led to a significant increase in the cell proliferation, migration rate and promoted colony formation and drastically enhanced tumor formation in athymic nude mice in comparison to control cells. Additionally, depletion of BRK abrogated the migration of BT20 and SKBR3 cells. Furthermore, we showed that BRK interacts with and phosphorylates Dok1, inducing Dok1 downregulation via a ubiquitin-proteasome-mediated mechanism. Together, our results show that the activation of BRK is essential for mammary gland tumorigenesis and suggest that targeting of Dok1 for degradation is a novel mechanism of action of BRK in the promotion of cell proliferation, migration and tumor formation

Subtractive proteomic analysis for identification of potential drug targets and vaccine candidates against Burkholderia pseudomallei K96243

Burkholderia pseudomallei is regarded as a global threat because of its ability to cause melioidosis, and potential as bioweapon. Intrinsic resistance to frontline antibiotics remains a major challenge in treating melioidosis. The increasing global burden of B. pseudomallei triggers the need to develop effective drugs and vaccine for controlling this pathogen. Therefore, we analyzed the whole proteome of B. pseudomallei K96243 using subtractive proteomic and immune-informatic analysis to find out potential drug target and vaccine candidate. Our analysis revealed 45 essential and non-homologous metabolic proteins, involved in different unique metabolic pathways. 36 out of 45 proteins can serve as novel drug targets and the remaining proteins had already been targeted with experimental drugs. 2 novel targets of cytoplasmic localization, namely dihydroneopterin aldolase and phosphoribosyl transferase were subjected to molecular docking with 14 antibiotics, which revealed the strong binding affinity of cefiderocol and tetracycline to the identified targets. Moreover, 100 ns molecular dynamic simulation and MM-PBSA calculation revealed the stability of drug-protein complexes. On the contrary, highly antigenic, non-allergic and non-toxic epitopes from three essential outer membrane proteins were joined by linkers and adjuvant to construct a chimeric peptide vaccine. The vaccine construct could efficiently bind to TLR-4 and generated robust immune response. The vaccine-receptor complex showed minimum deformability and good stability. Overall, our findings will aid in designing new inhibitors that disrupts the functionality of proposed targets and in the development of subunit vaccine for the therapeutic and prophylactic management of B. pseudomallei

Molecular docking and dynamics simulation study of medicinal fungi derived secondary metabolites as potential inhibitor for COVID-19 treatment

The severity of COVID-19, lack of specific treatment, and controversies on the vaccine's efficacy demand the development of new drugs against SARS-CoV-2. Fungi produce various metabolites with diverse molecular structures that have emerged as promising antiviral drug candidates. Therefore, the present study aimed to investigate medicinal fungi derived secondary metabolites as potential inhibitors of 3 different targets associated with viral entry (human TMPRSS2) and replication (main and papain-like protease) through molecular docking and dynamic simulation studies. Based on our findings, we identified Phelligridin E, Lepiotaprocerine G, and Inoscavin A as the potential blockers of SARS-CoV-2 main protease, papain-like protease, and human TMPRSS2, respectively. These compounds strongly interacted with their corresponding target, passed Lipinski Rule's and had acceptable ADMET properties. Drug-protein complexes showed good stability during MD simulation. Estimation of binding free energy using the MM-GBSA method validated the inhibitor potential of identified compounds. Taken together, we believe that further in vitro and in vivo investigations on our proposed molecules may contribute to expanding the therapeutic arsenal in our fight against COVID-19

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size

Comparison with other devices.

<p>Comparison with other devices.</p

Cost breakdown of our prototype.

<p>Cost breakdown of our prototype.</p

Results showing (a) Normal cell; (b)-(c) Cancer cells as viewed from our prototype; (d) Same cancer cell when viewed from the commercial microscope (model Olympus IX51).

<p>Results showing (a) Normal cell; (b)-(c) Cancer cells as viewed from our prototype; (d) Same cancer cell when viewed from the commercial microscope (model Olympus IX51).</p

Fluorescence Microscope (a) Our prototype (b) Commercial microscope [12] (c) Experimental setup showing our proposed microscope connected to computer/laptop.

<p>Fluorescence Microscope (a) Our prototype (b) Commercial microscope [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167863#pone.0167863.ref012" target="_blank">12</a>] (c) Experimental setup showing our proposed microscope connected to computer/laptop.</p

(a) Block diagram of the fluorescence microscope; (b) absorbance/emission spectra of the sample under test (adapted from [10]).

<p>The excitation filter passes through lights of 400-499nm which is reflected by the dichroic mirror. The peak absorption and peak emission of the sample is 495nm and 519nm respectively. Emitted light is then passed through the dichroic mirror followed by the barrier filter whose band pass wavelength is 520±18nm. Finally a small camera microscope captures the emitted wavelength, forms an image and sends to a computer.</p