Structure-based drug design approach to target toll-like receptor signaling pathways for disease treatment

Toll-like receptor (TLR) signaling pathways are the first line of defence against many microbial organisms. The question of how TLRs recognize endogenous ligands remains controversial. Several studies have shown that TLRs are implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Therefore, in structure-based drug design, TLRs are now viewed as potential therapeutic targets in the treatment of autoimmune diseases. This review shows how proteins, specifically TLRs, are used as therapeutic targets to design inhibitors (drugs) using the structure-based drug design approach for disease treatment.Keywords: Structure-based drug design, Toll-like receptors, Autoimmune diseases, Endogenous ligands, X-ray crystallography, Homology modelin

Knowledge about anesthesia and the role of anesthesiologists among Jeddah citizens

Background: The anesthesiologist has a vital role in the operating theatres. Awareness of the role of the anesthesiologist and the types of anesthesia is essential for every person. This study was made to estimate how much information the general population have about the anesthesiologist and the different types of anesthesia.Methods: This research was a cross sectional non-interventional study. The research team conducted a questionnaire in which each participant in the study was interviewed by the research team. The sample size was 159 participants.Results: From the participants,99 (62.2%) recognized the anesthesiologist as a specialized doctor who administers the anesthetics,62 (38.9%) know that the anesthesiologist has a role in resuscitating the patient with the team if crises occurred. However, 85 (53.4%) believe that the surgeon has the responsibility of postoperative pain management. Physicians were the source of knowledge for most participant’s information.Conclusions: A reasonable percentage of people appreciated the role of the anesthesiologist in administrating the anesthesia, however there is a lack of information about the role of the anesthesiologist intra and postoperatively. The need for more education for people about anesthesia is essential as the amount of information about anesthesia in general is rather low

Expression and production optimization of the cationic antimicrobial peptide : indolicidin by the recombinant E. coli C41 (DE3) clones

The cytoplasmic granules of bovine neutrophils naturally possess indolicidin - a promising cationic antimicrobial peptide as it displays inherent inhibitory activities against a broad type of microbial pathogens. In this study, a shake flask level production and expression optimizations of the indolicidin by the recombinant Escherichia coli C41 (DE3) clones (transformed with pET21a(+) plasmid carrying indolicidin gene) were carried out under standard conditions, as to determine the conditions required for maximal production. It was determined that a concentration of 1 mM of IPTG was effective, the 2×YT with salts and LB media at pH 7.5 with 3-6 h of incubation were required for maximal indolicidin expression

Crystallization and X-ray diffraction analysis of the N-terminal domain of the Toll-like receptor signalling adaptor protein TRIF/TICAM-1

As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 Å resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 Å resolution. The TRIF-NTD and TRIF-NTDA66M/L113M crystals have the symmetry of space groups P2 12121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the TIR domain from the Brucella melitensis TIR-domain-containing protein TcpB

In mammals, Toll-like receptors (TLRs) recognize conserved microbial molecular signatures and induce an early innate immune response in the host. TLR signalling is mediated by interactions between the cytosolic TIR (Toll/interleukin-1 receptor) domains of the receptor and the adaptor proteins. Increasingly, it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses. A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis. Studies have shown that TcpB interferes with the TLR2 and TLR4 signalling pathways to inhibit TLR-mediated inflammatory responses. Such interference may involve TIR-TIR-domain interactions between bacterial and mammalian proteins, but there is a lack of information about these interactions at the molecular level. In this study, the cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the protein construct corresponding to the TIR domain of TcpB (residues 120-250) are reported. The crystals diffracted to 2.6 angstrom resolution, have the symmetry of the monoclinic space group P2(1) and are most likely to contain four molecules in the asymmetric unit. The structure should help in understanding the molecular basis of how TcpB affects the innate immunity of the host

The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins

TRIF/TICAM-1 (TIR domain-containing adaptor inducing interferon-beta/TIR domain-containing adaptor molecule 1) is the adaptor protein in the Toll-like receptor (TLR) 3 and 4 signalling pathway that leads to the production of type 1 interferons and cytokines. The signalling involves TIR (Toll/interleukin-1 receptor) domain-dependent TRIF oligomerization. A protease-resistant N-terminal region is believed to be involved in self-regulation of TRIF by interacting with its TIR domain. Here, the structural and functional characterization of the N-terminal domain of TRIF (TRIF-NTD) comprising residues 1-153 is reported. The 2.22 angstrom resolution crystal structure was solved by single-wavelength anomalous diffraction (SAD) using selenomethionine-labelled crystals of TRIF-NTD containing two additional introduced Met residues (TRIF-NTDA66M/L113M). The structure consists of eight antiparallel helices that can be divided into two subdomains, and the overall fold shares similarity to the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both the recognition of viral RNA and modulation of innate immune signalling. Analysis of TRIF-NTD surface features and the mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or interaction with other signalling molecules or ligands. TRIF-NTD suppresses TRIF-mediated activation of the interferon-beta promoter, as well as NF-kappa B-dependent reporter-gene activity. These findings thus identify opportunities for the selective targeting of TLR3- and TLR4-mediated inflammation

Predictive Modeling and Validation on Growth, Production of Asexual Spores and Ochratoxin A of Aspergillus Ochraceus Group under Abiotic Climatic Variables

This study aimed to generate predictive models for growth, sporulation, and ochratoxin A (OTA) production under abiotic climatic variables, including temperatures (15–35 °C) and water activity levels (0.99–0.90 aw) by Aspergillus ochraceus group. The data were divided into three sets: one for training, one for testing, and the third one for model validation. Optimum growth occurred at 0.95 aw and 25 °C and 0.95 aw and 30 °C for A. westerdijkiae and A. steynii, respectively. Significantly improved A. westerdijkiae and A. steynii spore production occurred at 0.95 aw and 20 °C and 0.90 aw and 35 °C, respectively. A. steynii and A. westerdijkiae produced the majority of OTA at 35 °C and 0.95 aw and 25–30 °C at 0.95–0.99 aw, respectively. The accuracy of the third-order polynomial regression model reached 96% in growth cases, 94.7% in sporulation cases, and 90.9% in OTA production cases; the regression coefficients (R2) ranged from 0.8819 to 0.9978 for the Aspergillus ochraceus group. A reliable agreement was reached between the predicted and observed growth, sporulation, and OTA production. The effects of abiotic climatic variables on growth, sporulation, and OTA production of A. ochraceus group have been effectively defined, and the models generated were responsible for adequately predicted and validated models against data from other strains within A. ochraceus group that had been published in the literature under the current treatments. These models could be successfully implemented to predict fungal growth and OTA contamination on food matrices for these strains under these conditions

Multidrug-Resistant Bacteria Associated with Cell Phones of Healthcare Professionals in Selected Hospitals in Saudi Arabia

Cell phones may be an ideal habitat for colonization by bacterial pathogens, especially in hot climates, and may be a reservoir or vehicle in transmitting nosocomial infections. We investigated bacterial contamination on cell phones of healthcare workers in three hospitals in Saudi Arabia and determined antibacterial resistance of selected bacteria. A questionnaire was submitted to 285 healthcare workers in three hospitals, and information was collected on cell phone usage at the work area and in the toilet, cell phone cleaning and sharing, and awareness of cell phones being a source of infection. Screening on the Vitek 2 Compact system (bioMérieux Inc., USA) was done to characterize bacterial isolates. Of the 60 samples collected from three hospitals, 38 (63.3%) were positive with 38 bacterial isolates (4 Gram-negative and 34 Gram-positive bacteria). We found 38.3% of cell phones were contaminated with coagulase-negative staphylococci, particularly Staphylococcus epidermidis (10 isolates). Other bacterial agents identified were S. aureus, S. hominis, Alloiococcus otitis, Vibrio fluvialis, and Pseudomonas stutzeri. Antimicrobial susceptibility testing showed that most coagulase-negative staphylococci were resistant to benzylpenicillin, erythromycin, and rifampicin. Eight isolates were resistant to oxacillin, specifically S. epidermidis (3), S. hominis (2), and S. warneri (2). A. otitis, a cause of acute otitis media showed multidrug resistance. One isolate, a confirmed hetero-vancomycin intermediate-resistant S. aureus, was resistant to antibiotics, commonly used to treat skin infection. There was a significant correlation between the level of contamination and usage of cell phone at toilet and sharing. Our findings emphasize the importance of hygiene practices in cell phone usage among healthcare workers in preventing the transmission of multidrug-resistant microbes

Immunoinformatics-Based Identification of B and T Cell Epitopes in RNA-Dependent RNA Polymerase of SARS-CoV-2

Introduction: The ongoing coronavirus disease 2019 (COVID-19), which emerged in December 2019, is a serious health concern throughout the world. Despite massive COVID-19 vaccination on a global scale, there is a rising need to develop more effective vaccines and drugs to curb the spread of coronavirus. Methodology: In this study, we screened the amino acid sequence of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 (the causative agent of COVID-19) for the identification of B and T cell epitopes using various immunoinformatic tools. These identified potent B and T cell epitopes with high antigenicity scores were linked together to design the multi-epitope vaccine construct. The physicochemical properties, overall quality, and stability of the designed vaccine construct were confirmed by suitable bioinformatic tools. Results: After proper in silico prediction and screening, we identified 3 B cell, 18 CTL, and 10 HTL epitopes from the RdRp protein sequence. The screened epitopes were non-toxic, non-allergenic, and highly antigenic in nature as revealed by appropriate servers. Molecular docking revealed stable interactions of the designed multi-epitope vaccine with human TLR3. Moreover, in silico immune simulations showed a substantial immunogenic response of the designed vaccine. Conclusions: These findings suggest that our designed multi-epitope vaccine possessing intrinsic T cell and B cell epitopes with high antigenicity scores could be considered for the ongoing development of peptide-based novel vaccines against COVID-19. However, further in vitro and in vivo studies need to be performed to confirm our in silico observations