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2021 ◽  
Author(s):  
Eman Salem Algariri ◽  
Rabiatul Basria S. M. N. Mydin ◽  
Emmanuel Jairaj Moses ◽  
Simon Imakwu Okekpa ◽  
Nur Arzuar Abdul Rahim ◽  
...  

Abstract Stromal interaction molecule 1 (STIM1) is a critical regulator of calcium homeostasis through store-operated calcium entry (SOCE) and recently considered a potential therapeutic target for cancer. However, the role of STIM1 in acute myeloid leukemia (AML) remains unclear. The present study investigates the role of STIM1 in AML cell line (THP-1) proliferation and survival and its effect on reactive oxygen species (ROS) activities. Dicer-substrate siRNA (dsiRNA) - mediated STIM1 knockdown inhibited the THP-1 cells proliferation and colony formation ability. Further observation on ROS profile showed a significant reduction in the ROS level, which was associated with a significant down-regulation of NOX2 and protein kinase C (PKC). Furthermore, STIM1 knockdown exhibited significant down-regulation of Akt, KRAS, and MAPK which are critical proliferative and survival pathway-related genes. This study unveiled the importance of STIM1 in the regulation of AML cells proliferation and survival which could be through maintaining ROS at level keeping the proliferative and survival pathways at an active state. These findings represent STIM1 as a potential therapeutic target for AML treatment.


Author(s):  
Shari Wiegreffe ◽  
Daniel Löhrer ◽  
Monika Wirtz ◽  
Dominik Wiemuth

AbstractThe bile acid-sensitive ion channel (BASIC) is a member of the Deg/ENaC family of ion channels that is activated by bile acids. Despite the identification of cholangiocytes in the liver and unipolar brush cells in the cerebellum as sites of expression, the physiological function of BASIC in these cell types is not yet understood. Here we used a cholangiocyte cell line, normal rat cholangiocytes (NRCs), which expresses BASIC to study the role of the channel in epithelial transport using Ussing chamber experiments. Apical application of bile acids induced robust and transient increases in transepithelial currents that were carried by Na+ and partly blocked by the BASIC inhibitor diminazene. Genetic ablation of the BASIC gene in NRC using a CRISPR-cas9 approach resulted in a decrease of the bile acid-mediated response that matched the diminazene-sensitive current in NRC WT cells, suggesting that cholangiocytes respond to bile acids with a BASIC-mediated Na+ influx. Taken together, we have identified BASIC as a component of the cholangiocyte transport machinery, which might mediate a bile acid-dependent modification of the bile and thus control bile flux and composition.


2021 ◽  
Vol 3 (Supplement_4) ◽  
pp. iv8-iv8
Author(s):  
Kathryn Blethen ◽  
Samuel Sprowls ◽  
Tasneem Arsiwala ◽  
Ross Fladeland ◽  
Dhruvi Panchal ◽  
...  

Abstract Lung cancer is the most prevalent malignancy to affect both men and women. Around 80% of all lung cancers are classified as non-small cell lung cancer (NSCLC). This subtype of lung cancer is also the most likely to metastasize to the brain. Clinically, the common treatment for NSCLC is epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), due to the high occurrence of EGFR mutations. However, the cancer cells quickly develop resistance to the EGFR TKIs. This resistance and the added difficulty of delivering drugs across the blood-tumor barrier in efficacious concentrations to treat brain lesions are important to consider when developing treatment strategies for lung cancer brain metastases. Our study utilizes a NSCLC cell line, PC-9-Br6, which was developed in our laboratory to preferentially metastasize to the brain. This cell line was demonstrated by our collaborator to express higher levels of Bcl-2 in comparison to the parental PC-9-P cell line. We hypothesized combining novel Bcl-2 inhibitors (ABT-199/ABT-263) with an EGFR inhibitor (gefitinib) would increase survival and decrease tumor burden in our clinically relevant mouse model of lung cancer brain metastases.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bjoern Meyer ◽  
Jeanne Chiaravalli ◽  
Stacy Gellenoncourt ◽  
Philip Brownridge ◽  
Dominic P. Bryne ◽  
...  

AbstractSARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.


Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1388
Author(s):  
Paulina Sobierajska ◽  
Blazej Pozniak ◽  
Marta Tikhomirov ◽  
Julia Miller ◽  
Lucyna Mrowczynska ◽  
...  

Li+/Eu3+ dual-doped calcium apatite analogues were fabricated using a microwave stimulated hydrothermal technique. XRPD, FT-IR, micro-Raman spectroscopy, TEM and SAED measurements indicated that obtained apatites are single-phased, crystallize with a hexagonal structure, have similar morphology and nanometric size as well as show red luminescence. Lithium effectively modifies the local symmetry of optical active sites and, thus, affects the emission efficiency. Moreover, the hydrodynamic size and surface charge of the nanoparticles have been extensively studied. The protein adsorption (lysozyme, LSZ; bovine serum albumin, BSA) on the nanoparticle surface depended on the type of cationic dopant (Li+, Eu3+) and anionic group (OH−, Cl−, F−) of the apatite matrix. Interaction with LSZ resulted in a positive zeta potential, and the nanoparticles had the lowest hydrodynamic size in this protein medium. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS), murine macrophages (J774.E), as well as human red blood cells (RBCs). The studied apatites were not cytotoxic to RBCs and J774.E cells; however, at higher concentrations of nanoparticles, cytotoxicity was observed against the U2OS cell line. No antimicrobial activity was detected against Gram-negative bacteria with one exception for P. aeruginosa treated with Li+-doped fluorapatite.


Author(s):  
Farahnaz Zare ◽  
Sedigheh Sharifzadeh ◽  
Abbas Behzad-Behbahani ◽  
Gholamreza Rafiei Dehbidi ◽  
Zahra Yousefi ◽  
...  

Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.


2021 ◽  
Author(s):  
Canran Feng ◽  
Kyosuke Torimaru ◽  
Mandy Yu Theng Lim ◽  
Li-Ling Chak ◽  
Kosuke Tsuji ◽  
...  

Small regulatory RNAs (sRNAs) are involved in anti-viral defense and gene regulation. Although RNA-dependent RNA Polymerases (RdRPs) are known to produce sRNA in nematodes, plants and fungi, whether they play roles in sRNA biogenesis in other animals remains controversial. In this study, we study sRNAs in the ISE6 cell line, which is derived from the black-legged tick, an important vector of human and animal pathogens. We identify abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs). RdRP-dependent sRNAs are mainly derived from sense and antisense strands of RNA polymerase III-transcribed genes and repetitive elements. Unlike C. elegans sRNA pathways, 5′-tri-phosphorylated sRNAs are not detected, suggesting that the tick pathways are distinct from the pathways known in worms. Knockdown of one of the RdRPs unexpectedly results in downregulation of a subset of viral transcripts, in contrast to their upregulation by AGO knockdown. Furthermore, we show that knockdown of AGO/RdRP causes misregulation of protein-coding genes including RNAi-related genes, suggesting feedback regulation. Luciferase assays demonstrate that one of the RdRP-regulated genes, the MEK1 ortholog IscDsor1 is regulated through its 3′UTR, where a putative sRNA target site resides. These results provide evidence that arachnid RdRPs are important sRNA biogenesis factors, and the discovery of novel pathways underscores the importance of characterizing sRNA biogenesis in various organisms to understand virus-vector interactions and to exploit RNAi for pest control.


2021 ◽  
Author(s):  
Azam Moradi ◽  
Mohammad Yaghoubi-Avini ◽  
Joachim Wink

Abstract Several different techniques were employed for the isolation of Nannocystis from various sources. A polyphasic approach was used for identification. Twelve strains of N. pusilla, N. exedens, and N. konarekensis with distinctive distribution between climates were identified. The bioactivity was examined against a panel of eight bacteria, two yeasts, and one fungus; cytotoxicity was tested on the L929 fibroblast cell line. Eleven strains mainly inhibit Gram-positive bacteria, and only one isolate was cytotoxic. The extracts analysis by HPLC and LC-MS were compared to Myxobase, and eight different compounds were detected; a correlation was observed between compounds and producing species. 70% of strains had the potential to produce structurally diverse compounds. Nannochelins and althiomycin were the most abundant metabolites. The discovery of a new species of Nannocystis and the high potentiality of strains to produce secondary metabolites encourage further sampling and in-depth analysis of extracts to find new active metabolites.


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