Exploratory algorithm to devise multi-epitope subunit vaccine by investigating Leishmania donovani membrane proteins

Visceral leishmaniasis (VL) is a deadly parasitic infection which affects poorest to poor population living in the endemic countries. Increasing resistant to existing drugs, disease burden and a significant number of deaths, necessitates the need for an effective vaccine to prevent the VL infection. This study employed a combinatorial approach to develop a multi-epitope subunit vaccine by exploiting Leishmania donovani membrane proteins. Cytotoxic T- and helper T-lymphocyte binding epitopes along with suitable adjuvant and linkers were joined together in a sequential manner to design the subunit vaccine. The occurrence of B-cell and IFN-γ inducing epitopes approves the ability of subunit vaccine to develop humoral and cell-mediated immune response. Physiochemical parameters of vaccine protein were also assessed followed by homology modeling, model refinement and validation. Moreover, disulfide engineering was performed for the increasing stability of the designed vaccine and molecular dynamics simulation was performed for the comparative stability purposes and to conform the geometric conformations. Further, molecular docking and molecular dynamics simulation study of a mutated and non-mutated subunit vaccine against TLR-4 immune receptor were performed and respective complex stability was determined. In silico cloning ensures the expression of designed vaccine in pET28a(+) expression vector. This study offers a cost-effective and time-saving way to design a novel immunogenic vaccine that could be used to prevent VL infection.

Communicated by Ramaswamy H. Sarma     

Signatures of miR-181a on the Renal Transcriptome and Blood Pressure

MicroRNA-181a binds to the 3′ untranslated region of messenger RNA (mRNA) for renin, a rate-limiting enzyme of the renin-angiotensin system. Our objective was to determine whether this molecular interaction translates into a clinically meaningful effect on blood pressure and whether circulating miR-181a is a measurable proxy of blood pressure. In 200 human kidneys from the TRANScriptome of renaL humAn TissuE (TRANSLATE) study, renal miR-181a was the sole negative predictor of renin mRNA and a strong correlate of circulating miR-181a. Elevated miR-181a levels correlated positively with systolic and diastolic blood pressure in TRANSLATE, and this association was independent of circulating renin. The association between serum miR-181a and systolic blood pressure was replicated in 199 subjects from the Genetic Regulation of Arterial Pressure of Humans In the Community (GRAPHIC) study. Renal immunohistochemistry and in situ hybridization showed that colocalization of miR-181a and renin was most prominent in collecting ducts where renin is not released into the systemic circulation. Analysis of 69 human kidneys characterized by RNA sequencing revealed that miR-181a was associated with downregulation of four mitochondrial pathways and upregulation of 41 signaling cascades of adaptive immunity and inflammation. We conclude that renal miR-181a has pleiotropic effects on pathways relevant to blood pressure regulation and that circulating levels of miR-181a are both a measurable proxy of renal miR-181a expression and a novel biochemical correlate of blood pressure.     

Gene transfer is a major factor in bacterial evolution

Lateral gene transfer in four strains of Salmonella enterica has been assessed using genomic subtraction. Strain LT2 (subspecies I serovar Typhimurium) chromosomal DNA was used as target and subtracted by three subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi (M229), and a subspecies V strain (M321). Data from probing random cosmids of LT2 DNA with preparations of the residual LT2 DNA after subtraction were used to estimate the amounts of LT2 DNA not able to hybridize to strains S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629, and 778-1,286 kb, respectively. Several lines of evidence indicate that most of this DNA is from genes not present in strain M321 and not from genes that have diverged in sequence. The amounts correlate with the divergence of the four strains as revealed by multilocus enzyme electrophoresis and sequence variation of housekeeping genes. Sequence of 39 of the fragments from the M321 subtracted residual LT2 DNA revealed only six inserts of known gene function with evidence of both gain and loss of genes during the development of S. enterica clones. Sixteen of the 39 segments have 45% or lower G+C content, below the species average, but over half are within the normal range for the species. We conclude that even within a species, clones may differ by up to 20% of chromosomal DNA, indicating a major role for lateral transfer, and that on the basis of G+C content, a significant proportion of the DNA is from distantly related species.      

Designing B‐ and T‐cell multi‐epitope based subunit vaccine using immunoinformatics approach to control Zika virus infection

The Zika virus is a rapidly spreading Aedes mosquito‐borne sickness, which creates an unanticipated linkage birth deformity and neurological turmoil. This study represents the use of the combinatorial immunoinformatics approach to develop a multiepitope subunit vaccine using the structural and nonstructural proteins of the Zika virus. The designed subunit vaccine consists of cytotoxic T‐lymphocyte and helper T‐lymphocyte epitopes accompanied by suitable adjuvant and linkers. The presence of humoral immune response specific B‐cell epitopes was also confirmed by B‐cell epitope mapping among vaccine protein. Further, the vaccine protein was characterized for its allergenicity, antigenicity, and physiochemical parameters and found to be safe and immunogenic. Molecular docking and molecular dynamics studies of the vaccine protein with the toll‐like receptor‐3 were performed to ensure the binding affinity and stability of their complex. Finally, in silico cloning was performed for the effective expression of vaccine construct in the microbial system (Escherichia coli K12 strain). Aforementioned approaches result in the multiepitope subunit vaccine which may have the ability to induce cellular as well as humoral immune response. Moreover, this study needs the experimental validation to prove the immunogenic and protective behavior of the developed subunit vaccine.     

Mesodermal Guidance of Pioneer Axon Growth

Pioneer axons in insect legs are experimentally accessible model systems for the molecular identification and cellular localization of guidance cues regulating the path of axon growth. A detailed study of the Fe2 pioneer axons in the legs of the cockroach was performed to examine the diversity of guidance mechanisms. A detailed microscopic analysis of the axons at various points in their trajectory indicates that the Fe2 axons grow on a mesodermal substratum which contains the cues guiding their growth along a stereotyped path. An identified pair of muscle pioneer cells (MPC) are likely to play an important role in enabling the Fe2 growth cones to respond to mesodermal guidance cues. The addition of heparan sulfate, heparitinase, and phosphatidylinositol-specific phospholipase C to the medium perturbs thein situpath of growth of the Fe2 axons and the location of the MPC in cultured embryos. This indicates a role for heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored proteins in axon guidance. When these results are compared to those of similar experiments performed on the well-characterized Ti1 axons, they indicate significant differences in the mechanisms that are used for axon guidance. The Fe2 neurons are a good model for elucidating the mechanisms used to guide axon growth on nonmuscle mesodermal substrates often encountered in the periphery of vertebrate embryos.     

Cardioprotective effect of lycopene in the experimental model of myocardial ischemia-reperfusion injury

The continuous advancements in cancer research have contributed to the overwhelming evidence of the presence of telomerase in primary and secondary tumours together with hsp90 and c-Myc. This review will discuss the important role of telomerase together with hsp90 and c-Myc within the initiation and progression of gliomas. Also it will review the differential expression of these genes in the different grades of gliomas and the possibility of new treatments targeting these specific genes.     

Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K_d of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.