Toward self-adjuvanting subunit vaccines: model peptide and protein antigens incorporating covalently bound toll-like receptor-7 agonistic imidazoquinolines

Toll-like receptor (TLR)-7 agonists show prominent Th1-biased immunostimulatory activities. A TLR7-active N¹-(4-aminomethyl)benzyl substituted imidazoquinoline 1 served as a convenient precursor for the syntheses of isothiocyanate and maleimide derivatives for covalent attachment to free amine and thiol groups of peptides and proteins. 1 was also amenable to direct reductive amination with maltoheptaose without significant loss of activity. Covalent conjugation of the isothiocyanate derivative 2 to α-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting α-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models. The premise of covalently decorating protein antigens with adjuvants offers the possibility of drastically reducing systemic exposure of the adjuvant, and yet eliciting strong, Th1-biased immune responses.     

Cystamine prevents haloperidol-induced decrease of BDNF/TrkB signaling in mouse frontal cortex

The role of brain-derived neurotrophic factor (BDNF) has been implicated in the pathophysiology as well as treatment outcome of schizophrenia. Rodent studies indicate that several antipsychotic drugs have time-dependent (and differential) effects on BDNF levels in the brain. Earlier studies from our laboratory have indicated that long-term treatment with haloperidol (HAL) decreases BDNF, reduced GSH and anti-apoptotic marker, Bcl-xl protein levels and increases the expression of pro-apoptotic proteins in rat frontal cortex. Furthermore, findings from human as well as rodent studies suggest that treatment of schizophrenia must involve the neuroprotective strategies to improve the neuropathology and thereby clinical outcome. In the present study, we investigated the potential of cystamine (CYS), an anti-oxidant and anti-apoptotic compound, to prevent HAL-induced reduction in BDNF, GSH, and Bcl-xl protein levels in mice and the signaling mechanism(s) involved in the beneficial effects of CYS. The results indicated that CYS as well as cysteamine (the FDA-approved precursor of CYS) increased BDNF protein levels in mouse frontal cortex 7 days after treatment. CYS co-treatment prevented chronic HAL treatment-induced reduction in BDNF, GSH, and Bcl-xl protein levels. CYS treatment enhanced TrkB-tyrosine phosphorylation and activated Akt and extracellular signal-regulated kinase (ERK)1/2, downstream molecules of TrkB signaling. In addition, in vitro experiments with mouse cortical neurons showed that CYS prevented the HAL-induced reduction in neuronal cell viability and BDNF protein levels, and increase in apoptosis. BDNF-neutralizing antibody as well as K252a, a selective inhibitor of neurotrophin signaling blocked the CYS-mediated neuroprotection. Moreover, CYS-mediated neuroprotection is also blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. Thus, CYS protects cortical neurons through a mechanism involving TrkB receptor activation, and a signaling pathway involving phosphatidylinositol 3-kinase and MAPK. The findings from the present study may be helpful for the development of novel neuroprotective strategies to improve the treatment outcome of schizophrenia.     

Molecular modeling studies of repandusinic acid as potent small molecule for hepatitis B virus through molecular docking and ADME analysis

Background: Hepatitis B virus (HBV) has affected over 300 million people worldwide which causes to induce mostly liver disease and liver cancer. It is a member of the family Hepadnaviridae which is a small DNA virus with unusual characters like retroviruses. Generally, hepatoprotective drugs provoke some side effects in human beings. For the reason, this study aims to identify alternative drug molecules from the natural source of medicinal plants with smaller quantity of side effects than those conventional drugs in treating HBV. Methods: We developed computational methods for calculating drug and target binding resemblance using the Maestro v10.2 of Schrodinger suite. The target and ligand molecules were obtained from recognized databases. Ligand molecules of 40 phytoconstituents were retrieved from variety of plants after we executed crucial analyses such as molecular docking and absorption, distribution, metabolism, and excretion (ADME) analysis.Results: In the docking analysis, the natural analogues repandusinic acid showed better docking scores of –14.768 with good binding contacts. The remaining bioactive molecules corilagin, furosin, nirurin, iso-quercetin and gallocatechin also showed better docking scores.Conclusion: This computational analysis reveals that repandusinic acid is a suitable drug candidate for HBV. Therefore, we recommend that this analogue is suitable in further exploration using in vitro studies.     

BEST: a novel computational approach for comparing gene expression patterns from early stages of Drosophila melanogaster development

Embryonic gene expression patterns are an indispensable part of modern developmental biology. Currently, investigators must visually inspect numerous images containing embryonic expression patterns to identify spatially similar patterns for inferring potential genetic interactions. The lack of a computational approach to identify pattern similarities is an impediment to advancement in developmental biology research because of the rapidly increasing amount of available embryonic gene expression data. Therefore, we have developed computational approaches to automate the comparison of gene expression patterns contained in images of early stage Drosophila melanogaster embryos (prior to the beginning of germ-band elongation); similarities and differences in gene expression patterns in these early stages have extensive developmental effects. Here we describe a basic expression search tool (BEST) to retrieve best matching expression patterns for a given query expression pattern and a computational device for gene interaction inference using gene expression pattern images and information on the associated genotypes and probes. Analysis of a prototype collection of Drosophila gene expression pattern images is presented to demonstrate the utility of these methods in identifying biologically meaningful matches and inferring gene interactions by direct image content analysis. In particular, the use of BEST searches for gene expression patterns is akin to that of BLAST searches for finding similar sequences. These computational developmental biology methodologies are likely to make the great wealth of embryonic gene expression pattern data easily accessible and to accelerate the discovery of developmental networks.     

Source of a micro-nutrient in a semi-synthetic basal diet as a causative factor in inducing urinary calculi in rats and its inhibition by PSC 833, a potent inhibitor of P-glycoprotein

We report a serendipitous finding of urinary calculi in rats fed a semi-synthetic basal diet. This observation was made during ongoing studies to evaluate the inhibitory effect of PSC 833, a potent inhibitor of P-glycoprotein, on development of tumors in rodent tumor model systems. A large number of specific-pathogen-free (SPF) female Sprague-Dawley and SPF male Fischer 344 rats being fed the diet were euthanized when it became evident clinically that they were uremic. At necropsy, the renal pelvis, ureters, and urinary bladder contained numerous calculi. The presence of urinary calculi was determined to be related to the source of a Food Chemical Codex grade of choline bitartrate. Rats being fed the same basal diet containing the United States Pharmacopia grade of choline bitartrate failed to develop urinary calculi. Interestingly, rats treated with the P-glycoprotein inhibitor were at significantly reduced risk of developing urinary calculi. This finding highlights how something seemingly innocuous as a minor dietary constituent can have a profound impact and, thereby, affect experimental outcome.     

Validation of Smartphone Based Retinal Photography for Diabetic Retinopathy Screening

Aim

To evaluate the sensitivity and specificity of “fundus on phone’ (FOP) camera, a smartphone based retinal imaging system, as a screening tool for diabetic retinopathy (DR) detection and DR severity in comparison with 7-standard field digital retinal photography.

Design

Single-site, prospective, comparative, instrument validation study.

Methods

301 patients (602 eyes) with type 2 diabetes underwent standard seven-field digital fundus photography with both Carl Zeiss fundus camera and indigenous FOP at a tertiary care diabetes centre in South India. Grading of DR was performed by two independent retina specialists using modified Early Treatment of Diabetic Retinopathy Study grading system. Sight threatening DR (STDR) was defined by the presence of proliferative DR(PDR) or diabetic macular edema. The sensitivity, specificity and image quality were assessed.

Results

The mean age of the participants was 53.5 ±9.6 years and mean duration of diabetes 12.5±7.3 years. The Zeiss camera showed that 43.9% had non-proliferative DR(NPDR) and 15.3% had PDR while the FOP camera showed that 40.2% had NPDR and 15.3% had PDR. The sensitivity and specificity for detecting any DR by FOP was 92.7% (95%CI 87.8–96.1) and 98.4% (95%CI 94.3–99.8) respectively and the kappa (ĸ) agreement was 0.90 (95%CI-0.85–0.95 p<0.001) while for STDR, the sensitivity was 87.9% (95%CI 83.2–92.9), specificity 94.9% (95%CI 89.7–98.2) and ĸ agreement was 0.80 (95%CI 0.71–0.89 p<0.001), compared to conventional photography.

Conclusion

Retinal photography using FOP camera is effective for screening and diagnosis of DR and STDR with high sensitivity and specificity and has substantial agreement with conventional retinal photography.     

Metabolism of 3H-testosterone in epididymis & accessory glands of reproduction in the castrate hamster Mesocricetus auratus

The metabolism of tritiated-testosterone in epididymis and accessory glands of reproduction was studied in the castrate hamster, Mesocricetus auratus. The amount of tritium accumulating in the epididymis was similar to that in the accessory glands 60 minutes postinjection but was significantly higher (p .02-p .001) at 150 minutes. In all accessory organs the rate of tritiated testosterone was slow; marked quantities of testosterone and androstanediol were present but the accumulation of dihydrotestosterone (DHT) was relatively minimal at both time intervals studied. These results may indicate a simultaneous requirement of testosterone and androstanediol to mediate the action of androgens in the hamster while DHT may be of secondary importance.