In silico approach to inhibition of signaling pathways of Toll-like receptors 2 and 4 by ST2L

Toll-like receptors (TLRs) activate a potent immunostimulatory response. There is clear evidence that overactivation of TLRs leads to infectious and inflammatory diseases. Recent biochemical studies have shown that the membrane-bound form of ST2 (ST2L), a member of the Toll-like/IL-1 receptor superfamily, negatively regulates MyD88-dependent TLR signaling pathways by sequestrating the adapters MyD88 and Mal (TIRAP). Specifically, ST2L attenuates the recruitment of Mal and MyD88 adapters to their receptors through its intracellular TIR domain. Thus, ST2L is a potent molecule that acts as a key regulator of endotoxin tolerance and modulates innate immunity. So far, the inhibitory mechanism of ST2L at the molecular level remains elusive. To develop a working hypothesis for the interactions between ST2L, TLRs (TLR1, 2, 4, and 6), and adapter molecules (MyD88 and Mal), we constructed three-dimensional models of the TIR domains of TLR4, 6, Mal, and ST2L based on homology modeling. Since the crystal structures of the TIR domains of TLR1, 2 as well as the NMR solution structure of MyD88 are known, we utilized these structures in our analysis. The TIR domains of TLR1, 2, 4, 6, MyD88, Mal and ST2L were subjected to molecular dynamics (MD) simulations in an explicit solvent environment. The refined structures obtained from the MD simulations were subsequently used in molecular docking studies to probe for potential sites of interactions. Through protein-protein docking analysis, models of the essential complexes involved in TLR2 and 4 signaling and ST2L inhibiting processes were developed. Our results suggest that ST2L may exert its inhibitory effect by blocking the molecular interface of Mal and MyD88 adapters mainly through its BB-loop region. Our predicted oligomeric signaling models may provide a basis for the understanding of the assembly process of TIR domain interactions, which has thus far proven to be difficult via in vivo studies.     

Comparative analysis of species-specific ligand recognition in Toll-like receptor 8 signaling: a hypothesis

Toll-like receptors (TLRs) play a central role in the innate immune response by recognizing conserved structural patterns in a variety of microbes. TLRs are classified into six families, of which TLR7 family members include TLR7, 8, and 9, which are localized to endolysosomal compartments recognizing viral infection in the form of foreign nucleic acids. In our current study, we focused on TLR8, which has been shown to recognize different types of ligands such as viral or bacterial ssRNA as well as small synthetic molecules. The primary sequences of rodent and non-rodent TLR8s are similar, but the antiviral compound (R848) that activates the TLR8 pathway is species-specific. Moreover, the factors underlying the receptor's species-specificity remain unknown. To this end, comparative homology modeling, molecular dynamics simulations refinement, automated docking and computational mutagenesis studies were employed to probe the intermolecular interactions between this anti-viral compound and TLR8. Furthermore, comparative analyses of modeled TLR8 (rodent and non-rodent) structures have shown that the variation mainly occurs at LRR14-15 (undefined region); hence, we hypothesized that this variation may be the primary reason for the exhibited species-specificity. Our hypothesis was further bolstered by our docking studies, which clearly showed that this undefined region was in close proximity to the ligand-binding site and thus may play a key role in ligand recognition. In addition, the interface between the ligand and TLR8s varied depending upon the amino acid charges, free energy of binding, and interaction surface. Therefore, our current work provides a hypothesis for previous in vivo studies in the context of TLR signaling.     

Molecular Evolution and Structural Features of IRAK Family Members

The interleukin-1 receptor-associated kinase (IRAK) family comprises critical signaling mediators of the TLR/IL-1R signaling pathways. IRAKs are Ser/Thr kinases. There are 4 members in the vertebrate genome (IRAK1, IRAK2, IRAKM, and IRAK4) and an IRAK homolog, Pelle, in insects. IRAK family members are highly conserved in vertebrates, but the evolutionary relationship between IRAKs in vertebrates and insects is not clear. To investigate the evolutionary history and functional divergence of IRAK members, we performed extensive bioinformatics analysis. The phylogenetic relationship between IRAK sequences suggests that gene duplication events occurred in the evolutionary lineage, leading to early vertebrates. A comparative phylogenetic analysis with insect homologs of IRAKs suggests that the Tube protein is a homolog of IRAK4, unlike the anticipated protein, Pelle. Furthermore, the analysis supports that an IRAK4-like kinase is an ancestral protein in the metazoan lineage of the IRAK family. Through functional analysis, several potentially diverged sites were identified in the common death domain and kinase domain. These sites have been constrained during evolution by strong purifying selection, suggesting their functional importance within IRAKs. In summary, our study highlighted the molecular evolution of the IRAK family, predicted the amino acids that contributed to functional divergence, and identified structural variations among the IRAK paralogs that may provide a starting point for further experimental investigations.     

Time motion study to evaluate the impact of flattening filter free beam on overall treatment time for frameless intracranial radiosurgery using Varian TrueBeam® linear accelerator

BackgroundThe aim was to study the impact of the flattening filter free (FFF) beam on overall treatment time for frameless intracranial radiosurgery using TrueBeam^® LINAC.The development of frameless stereotactic radiosurgery (SRS) is possible due to the incorporation of image guidance in the delivery of treatment. It is important to analyze the cost and benefits of FFF beams for treating SRS by understanding the impact of FFF beams in reducing the treatment time.Materials and methodsDynamic conformal arc (DCA ) and volumetric arc therapy (VMAT) plans were generated using 6 MV with a flattening filter (FF) and FFF beams. Overall treatment time was divided into beam on time (BOT) and beam off time (BFT). Percentage beam on time reduction (PBOTR) and Percentage total time reduction (PTTR) factors were defined for the comparison.ResultsBOT reduction was observed to be significant for higher dose per fraction but subjected to the treatment technique and modulation differences. PBOTR values are much higher than PTTR values. The 39.9% of PBOTR resulted in only 8% PTTR for DCA and 65.3% resulted in 15.9% PTTR for VMAT.ConclusionMajor BFT was utilized for imaging and verification. FFF beam significantly reduced the beam on time and was found to be most effective if the fractional dose was as high as that for SRS. Newly defined PBOTR and PTTR factors are very useful indicators to evaluate the efficacy of FFF beams in terms of time reduction.     

Lint,a transmembrane serine protease,regulates growth and metabolism in Drosophila

Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signaling, we discovered lumens interrupted (lint), a gene that has previously been shown to act in tracheal development. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, our analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. We found various tracheal terminal branch defects, including reduction in the length as well as number of branches in the lint knockdown background. Our study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation.