A Novel Quinazoline Inhibits Hsp90 Protein, EGFR and Induces Apoptosis in Leukemia Cells

The objective of the first part of this study was to investigate the Hsp90 protein possible activ ity of a novel quinazoline Her2/ EGFR inhibitor (Co mpound No. 1: 4-(2-(4-Oxo-2-thio xo-1,4-d ihydroquinazolin-3(2H)yl)ethyl)benzenesulfonamide) p reviously synthesized by a collaborating group. Heat shock protein 90 (Hsp90) has a central ro le in regulation of several client proteins involved in cancers [1,2]. Several Hsp90 inhibitors of the natural or synthetic origin d isplayed potent anticancer activity [3,4]. Accordingly, Hsp90 emerged as an attractive target in the design of anticancer agents. To evaluate the binding mode of compound No. 1 into the ATPase site of Hsp90, a co mparative mo lecular docking study was performed using AutoDock 4.2. The results of this studywas compared with that of the co-crystallized ligand (ATI-13387X, Onalespib). The energy minimization process of the chemical structures of No. 1 was done following our previous report [5]. The results of the docking study revealed that No. 1 fit n icely into the ATPase site, and it displayed a binding free energy (Gb) of-7.21 kcal/ mo l and inhibition constant (Ki) of 5.19 µM to Hsp90, co mpared to Gb of-7.90 kcal/ mol and Ki of 1.62 µM for ATI-13387X. Furthermore, to confirm this result, the surface plasmon resonance (SPR) was devised to test the Hsp90 inhibition activity of No.1, wh ich was 51 nM co mpared to Rad icico l and 17AA G (1.8 nM, and 360 nM; respectively). Overall, co mpound No. 1 exh ibited pro mising Hsp90 inhib iting activity. The second part of the study focused on the effect of No. 1, Dinaciclib and their co mbinationsin HL-60 leukemia cells. The comb ination showed synergistic EGFR inhib ition effect in HL-60 cells. Moreover, No. 1, Dinaciclib and their combination caused a significant increase in the Sub-G1 co mpared to control and doxorubicin (24h), at the expense of S and G2/M cell cycle phases. Cyclin D3, was consequently inhibited by each of the two drugs, and synergistically by their comb ination in HL-60 cells. Furthermore, each of the two drugs downregulated Survivin, wh ich was synergistically inhib ited by the co mbination. In conclusion, co mpound No.1, Dinaciclib and their comb inations showed synergestic EGFR inhibit ion; and pro-apoptoticeffect in HL-60 cells.This project was funded by the deanship of scientific research, Umm Alqura University, KSA (DSR: 15-M ED-3-1-0060). Keywords: Novel quinazoline EGFR inhi bi tor, Hs p90 protein, Leukemi a cells

Effects of β-blockers on the sympathetic and cytokines storms in Covid-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative virus in the development of coronavirus disease 2019 (Covid-19) pandemic. Respiratory manifestations of SARS-CoV-2 infection such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) leads to hypoxia, oxidative stress, and sympatho-activation and in severe cases leads to sympathetic storm (SS). On the other hand, an exaggerated immune response to the SARS-CoV-2 invasion may lead to uncontrolled release of pro-inflammatory cytokine development of cytokine storm (CS). In Covid-19, there are interactive interactions between CS and SS in the development of multi-organ failure (MOF). Interestingly, cutting the bridge between CS and SS by anti-inflammatory and anti-adrenergic agents may mitigate complications that are induced by SARS-CoV-2 infection in severely affected Covid-19 patients. The potential mechanisms of SS in Covid-19 are through different pathways such as hypoxia, which activate the central sympathetic center through carotid bodies chemosensory input and induced pro-inflammatory cytokines, which cross the blood-brain barrier and activation of the sympathetic center. β2-receptors signaling pathway play a crucial role in the production of pro-inflammatory cytokines, macrophage activation, and B-cells for the production of antibodies with inflammation exacerbation. β-blockers have anti-inflammatory effects through reduction release of pro-inflammatory cytokines with inhibition of NF-κB. In conclusion, β-blockers interrupt this interaction through inhibition of several mediators of CS and SS with prevention development of neural-cytokine loop in SARS-CoV-2 infection. Evidence from this study triggers an idea for future prospective studies to confirm the potential role of β-blockers in the management of Covid-19

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection

COVID-19-related mental health burdens: Impact of educational level and relationship status Among low-Income earners of Western Uganda

Objective: The study aimed to investigate the relationship between mental health with the level of education, relationship status, and awareness on mental health among low-income earners in Western Uganda. Methods: This was a cross-sectional descriptive study carried out among 253 participants. Anxiety, anger, and depression were assessed using a modified generalized anxiety disorder (GAD-7), Spielberger\u27s State-Trait Anger Expression Inventory-2, and Beck Depression Inventory item tools, respectively. Results: The majority of our respondents were male (n = 150/253, 59.3), had a secondary level of education (104/253, 41.1), and were single (137/253, 54.2). No formal education and primary education (r2 = 47.4% and 6.4%, respectively) had a negative correlation with awareness of mental health care. In addition, no formal education had a positive correlation with anger and depression (r2 = 1.9% and 0.3%, respectively). Singleness in this study had a negative correlation with awareness of mental health care, anger, and depression (r2 = 1.9, 0.8, and 0.3%, respectively), and a positive correlation with anxiety (r2 = 3.9%). Conclusion: It is evident that education and relationship status influenced awareness on mental health care and mental health state among low-income earners in Western Uganda during the first COVID-19 lockdown. Therefore, policymakers should strengthen social transformation through the proper engagement of low-income earners in this COVID-19 era

Profiling estrogen, progesterone, and androgen receptors in colorectal cancer in relation to gender, menopausal status, clinical stage, and tumour sidedness

BackgroundAlthough estrogen (ERα/ERβ), progesterone (PGR), and androgen (AR) receptors are pathologically altered in colorectal cancer (CRC), their simultaneous expression within the same cohort of patients was not previously measured.MethodsERα/ERβ/PGR/AR proteins were measured in archived paired normal and malignant colon specimens (n =120 patients) by immunohistochemistry, and results were analyzed by gender, age (≤50 vs. ≥60 years), clinical stages (early-stage I/II vs. late-stage III/IV), and anatomical location (right; RSCs vs. left; LSCs). Effects of 17β-estradiol (E2), progesterone (P4), and testosterone alone or combined with the specific blockers of ERα (MPP dihydrochloride), ERβ (PHTPP), PGR (mifepristone), and AR (bicalutamide) on cell cycle and apoptosis were also measured in the SW480 male and HT29 female CRC cell lines. ResultsERα and AR proteins increased, whilst ERβ and PGR declined markedly in malignant specimens. Moreover, male neoplastic tissues showed highest AR expression, whilst ERβ and PGR weakest alongside ERα strongest expression was seen in cancerous tissues from women aged ≥60 years. Late-stage neoplasms also revealed maximal alterations in the expression of sex steroid receptors. By tumor location, LSCs disclosed significant elevations in ERα with marked declines in PGR compared with RSCs, and ERα strongest alongside PGR weakest expression was detected in advanced LSCs from women aged ≥60 years. Late-stage LSCs from females aged ≥60 years also showed weakest ERβ and strongest AR expression. In contrast, male RSC and LSC tissues exhibited equal ERβ and AR expression in all clinical stages. ERα and AR proteins also correlated positively, whereas ERβ and PGR inversely, with tumor characteristics. Concomitantly, E2 and P4 monotherapies triggered cell cycle arrest and apoptosis in the SW480 and HT29 cells, and while pre-treatment with ERα-blocker enhanced the effects of E2, ERβ-blocker and PGR-blocker suppressed the E2 and P4 anti-cancer actions, respectively. In contrast, treatment with the AR-blocker induced apoptosis, whilst co-treatment with testosterone hindered the effects. ConclusionsThis study advocates that protein expression of sex steroid receptors in malignant tissues could represent prognostic markers, as well as hormonal therapy could provide an alternative strategy against CRC, and their efficacies could be dependent on gender, clinical stage, and tumor location

The role of tryptase and activation of protease activated receptor-2 (PAR-2) in human airways disease

Levels of expression of PAR-1 in bronchial and nasal tissue from subjects with asthma and allergic rhinitis respectively were studied.   Bronchial biopsies embedded in GMA resin from eight mild asthmatic subjects, from eight severe asthmatic subjects and from 15 non-asthmatic subjects were used.  In addition, bronchial biopsies were also taken from six mild asthmatic subjects six hours following exposure to saline or house dust mite allergen.  For a study of rhinitis nasal biopsies from eight perennial rhinitic subjects and six non-rhinitic subjects were used.  Nasal biopsies were also collected from six patients with seasonal allergic rhinitis (out-of-season) and six hours following saline or grass pollen allergen treatment.  Two micron sections were strained with P2A or an antibody raised against a peptide fragment of rat PAR-2 (B5 antiserum), and PAR-2 staining intensity quantified using computerised image analysis.  PAR-2 expression was detect mainly on epithelial cells and found to be significantly higher in the bronchial epithelium of severe asthmatics compared to non-asthmatic subjects (Mann Whitney U test, P &lt; 0.05).  Although PAR-2 expression in the bronchial epithelium of mild asthmatics did not differ from that in subjects with asthma, expression was significantly increased following allergen provocation (Wilcoxon’s test, P &lt; 0.05).  There was no change in the expression of PAR-2 in the nasal epithelium of perennial rhinitic subjects compared to those without rhinitis.  However, expression was significantly reduced following allergen challenge (Mann Whitney U test, P &lt; 0.05).  The difference in expression of PAR-2 in the upper and lower airways suggest that its expression may be regulated by separate elements in the nose and the bronchi in rhinitis and asthma. The expression of PAR-2 on eosinophils and the activation by tryptase on the function of these cells were investigated, using eosinophils purified from human peripheral blood.  PAR-2 expression was assessed by flow cytometry using P2A and the B5 antiserum.  PAR-2 was detected both at the surface and intracellularly on eosinophils using P2A. These studies demonstrate that tryptase activities eosinophils by a PAR-2 independent mechanism, and could involve activation of an as yet unidentified PAR.  We have also shown that PAR-2 expression may be up-regulated in asthma.  Thus, tryptase and PAR-2 could play key roles in allergic airways diseases, and as such can be considered as potential targets for therapeutic intervention.</p

The role of tryptase and activation of protease activated receptor-2 (PAR2) in human airways disease

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The anti-proliferative activity of BTG/TOB proteins is mediated via the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits of the Ccr4-not complex.

The human BTG/TOB protein family comprises six members (BTG1, BTG2/PC3/Tis21, BTG3/Ana, BTG4/PC3B, TOB1/Tob, and TOB2) that are characterised by a conserved BTG domain. This domain mediates interactions with the highly similar Caf1a (CNOT7) and Caf1b (CNOT8) catalytic subunits of the Ccr4-Not deadenylase complex. BTG/TOB proteins have anti-proliferative activity: knockdown of BTG/TOB can result in increased cell proliferation, whereas over-expression of BTG/TOB leads to inhibition of cell cycle progression. It was unclear whether the interaction between BTG/TOB proteins and the Caf1a/Caf1b deadenylases is necessary for the anti-proliferative activity of BTG/TOB. To address this question, we further characterised surface-exposed amino acid residues of BTG2 and TOB1 that mediate the interaction with the Caf1a and Caf1b deadenylase enzymes. We then analysed the role of BTG2 and TOB1 in the regulation of cell proliferation, translation and mRNA abundance using a mutant that is no longer able to interact with the Caf1a/Caf1b deadenylases. We conclude that the anti-proliferative activity of BTG/TOB proteins is mediated through interactions with the Caf1a and Caf1b deadenylase enzymes. Furthermore, we show that the activity of BTG/TOB proteins in the regulation of mRNA abundance and translation is dependent on Caf1a/Caf1b, and does not appear to require other Ccr4-Not components, including the Ccr4a (CNOT6)/Ccr4b (CNOT6L) deadenylases, or the non-catalytic subunits CNOT1 or CNOT3

Up-regulation of protease-activated receptor-2 by bFGF in cultured human synovial fibroblasts

Protease-activated receptors (PARs) have been implicated in the development of acute and chronic inflammatory responses. We have examined the expression of mRNA for PARs and their regulation by growth factors and cytokines in synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Messenger RNA for PAR-1, -2 and -3 was detected in these cells, but not that for PAR-4. Expression of mRNA for PAR-2 was up-regulated by bFGF in a concentration-dependent manner, whereas expression of mRNA for PAR-1 and PAR-3 was not affected. Levels of mRNA encoding PAR-1, PAR-2 and PAR-3 did not increase in response to IL-1beta and TNF-alpha. Expression of mRNA for PAR-2 was maximal 12 h after addition of bFGF, and maximal levels of immunoreactive PAR-2 were reached after 24 h. Furthermore, PAR-2 agonist peptide (SLIGKV-NH(2)), but not the inactive reverse peptide (VKGILS-NH(2)), induced transitory cytosolic Ca(2+) mobilization in cells, and its response was increased by pretreatment with bFGF. An important role could be played by bFGF in the regulation of functional PAR-2 expression in cultured RA synovial fibroblasts

Knockdown of BTG2 and TOB1 results in increased proliferation of MCF-7 cells.

<p>(A) Overview of the human BTG/TOB family. The BTG domain is highlighted in grey, and PAM2 motifs present in TOB1 and TOB2 are highlighted in light grey. (B) Expression of BTG/TOB family members in MCF-7 cells. Data was obtained by genome-wide expression data using microarrays and confirmed by RT-qPCR. (C) Knockdown of BTG2 and TOB1 results in increased proliferation of MCF7 cells. Cells in S-phase were detected by labelling using the nucleoside analogue EdU 48 h after siRNA transfection. No effect was observed upon knockdown of BTG1 and TOB2. * p<0.05 (compared to treatment with non-targeting control siRNA). (D) Combined knockdown of BTG2 and TOB1 results in further increased cell proliferation of MCF7 cells. Cells in S-phase were detected by labelling using the nucleoside analogue EdU 48 h after siRNA transfection. * p<0.05 (compared to non-targeting control siRNA). ** p<0.05 (compared to BTG2 or TOB1 knockdown). All experiments were carried out in triplicate. Error bars represent the standard error of the mean.</p