Tetramerizing tGCN4 domain facilitates production of Influenza A H1N1 M2e higher order soluble oligomers that show enhanced immunogenicity in vivo.

One strategy for the development of a next generation influenza vaccine centers upon using conserved domains of the virus to induce broader and long-lasting immune responses. The production of artificial proteins by mimicking native-like structures has shown to be a promising approach for vaccine design against diverse enveloped viruses. The amino terminus of influenza A virus matrix 2 ectodomain (M2e) is highly conserved among influenza subtypes, and previous studies have shown M2e-based vaccines are strongly immunogenic, making it an attractive target for further exploration. We hypothesized that stabilizing M2e protein in the mammalian system might influence the immunogenicity of M2e with the added advantage to robustly produce the large scale of proteins with native-like fold and hence can act as an efficient vaccine candidate. In this study, we created an engineered construct in which the amino terminus of M2e is linked to the tetramerizing domain tGCN4, expressed the construct in a mammalian system, and tested for immunogenicity in BALB/c mice. We have also constructed a stand-alone M2e construct (without tGCN4) and compared the protein expressed in mammalian cells and in Escherichia coli using in vitro and in vivo methods. The mammalian-expressed protein was found to be more stable, more antigenic than the E. coli protein, and form higher-order oligomers. In an intramuscular protein priming and boosting regimen in mice, these proteins induced high titers of antibodies and elicited a mixed Th1/Th2 response. These results highlight the mammalian-expressed M2e soluble proteins as a promising vaccine development platform.

Biogenic selenium nanoparticles induce ROS-mediated necroptosis in PC-3 cancer cells through TNF activation.

BACKGROUND: Selenium is well documented to inhibit cancer at higher doses; however, the mechanism behind this inhibition varies widely depending on the cell type and selenium species. Previously, we have demonstrated that Bacillus licheniformis JS2 derived biogenic selenium nanoparticles (SeNPs) induce non-apoptotic cell death in prostate adenocarcinoma cell line, PC-3, at a minimal concentration of 2 µg Se/ml, without causing toxicity to the primary cells. However, the mechanism behind its anticancer activity was elusive. RESULTS: Our results have shown that these SeNPs at a concentration of 2 µg Se/ml were able to induce reactive oxygen species (ROS) mediated necroptosis in PC-3 cells by gaining cellular internalization. Real-time qPCR analysis showed increased expression of necroptosis associated tumor necrotic factor (TNF) and interferon regulatory factor 1 (IRF1). An increased expression of RIP1 protein was also observed at the translational level upon SeNP treatment. Moreover, the cell viability was significantly increased in the presence of necroptosis inhibitor, Necrostatin-1. CONCLUSION: Data suggest that our biogenic SeNPs induce cell death in PC-3 cells by the ROS-mediated activation of necroptosis, independent to RIP3 and MLKL, regulated by a RIP1 kinase.

Development of novel membrane active lipidated peptidomimetics active against drug resistant clinical isolates.

A new series of small cationic lipidated peptidomimetics have been synthesized and found to be highly active against several susceptible as well as drug resistant clinical isolates of bacteria and fungi. All lipidated peptidomimetics do not cause significant lysis of human erythrocytes (HC50>200µg/mL). Calcein dye leakage experiment revealed membranolytic effect of LPEP08 which was further confirmed by scanning electron microscopy (SEM). The involvement of intracellular targets as an alternate mode of action was precluded by DNA retardation assay. Additionally, LPEP08 exhibit high proteolytic stability and dose not elicit resistance against drug resistant clinical isolate of Staphylococcusaureus, even after 16 rounds of passaging. These results demonstrate the potential of lipidated peptidomimetics as biocompatible anti-infective therapeutics.

The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated ß-catenin degradation and through a mechanism independent of ß-catenin stability.

WNT/ß-catenin signaling is mediated by the transcriptional coactivator ß-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases CSNK1A1 and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigate the mechanism underlying this requirement. In the absence of CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was disproportionately smaller than the reduction in WNT target gene transcription. To test if the reduction in WNT signaling resulted from reduced CTNNB1 abundance alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells reduced WNT signaling with no change in CTNNB1 abundance. Genetic interaction and overexpression analyses revealed that the effects of HUWE1 on WNT signaling were not only mediated by GSK3A/GSK3B, but also by APC and AXIN1. Regulation of WNT signaling by HUWE1 required its ubiquitin ligase activity. These results suggest that in cells lacking CSNK1A1, a destruction complex containing APC, AXIN1 and GSK3A/GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation. HUWE1 enhances WNT signaling by antagonizing this activity. Therefore, HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that regulates CTNNB1 abundance and another that is independent of CTNNB1 stability. Coordinated regulation of CTNNB1 abundance and an independent signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.

Specificity of Biogenic Selenium Nanoparticles for Prostate Cancer Therapy With Reduced Risk of Toxicity: An in vitro and in vivo Study.

Selenium deficiency is associated with many physiological disorders including the high risk of cancer. The rehabilitation of selenium with different selenium supplements, however, fails due to their low therapeutic index. Therefore, it is advantageous to have a less toxic form of selenium for supplementation with potentially high anticancer activity. Here we show Bacillus licheniformis derived biogenic selenium nanoparticles at a minimal concentration of 2 µg Se/ml induce necroptosis in LNCaP-FGC cells, without affecting the RBC integrity. Real-time gene expression analysis indicated the overexpression of tumor necrotic factor (TNF) and interferon regulatory factor (IRF1) and decreased expression of androgen receptor (AR) and prostate-specific antigen (PSA). Furthermore, histopathological analysis showed the subsequent oral administrations of 10 times higher concentration of these endotoxin free selenium nanoparticles in C3H/HeJ mice (50 mg Se/kg of body weight), induce significantly lower toxicity compared to the L-selenomethionine (5 mg Se/kg). Our study suggested that the biogenic SeNP could emerge as the safest form of selenium supplementation with potent anticancer activity.

Trace detection of some nitro-explosives using thermal mediated immunochemical defragmented method.

A new immunoassay format using thermally induced defragmentation of some nitro-explosives with a high degree of selectivity is reported. Specific antibodies against three widely used explosives, 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN) were generated by designing suitable haptens using geometry optimization modules. These in-house generated antibodies were used in a newly developed thermal mediated immunochemical biosensing technique which involves the binding of specific antibodies to respective nitro-explosives on a microtiter strip, resulting in the formation of specific immunocomplex. Heating the specific immuno-complex formed on microtiter wells resulted in thermal lysis of nitro-explosives to generate nitrite ions. These ions react with Griess reagent to form a colored chromophore which correlates the concentration of individual explosive in the sample. The present work fulfills the need for an improved explosive detecting system that is highly specific and capable of quickly determining the presence of nitrate containing explosives from a mixture pool.

UV-FIA: UV-induced fluoro-immunochemical assay for ultra-trace detection of PETN, RDX, and TNT.

Fluorescence quenching based immunoassay format for the detection of a trace amount of some nitro-explosives with a high degree of selectivity is reported in this study. The immunoassay comprises anti-explosive antibodies functionalized microtitre strips specific to the targeted explosives, pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and 2,4,6-trinitrotoluene (TNT). UV induced photolysis of nitro-explosive bound to targeted antibodies generates primarily nitrite ions which after the quick reaction with the detector molecule, 2,3-diaminonaphthalene (DAN), a fluorophore, quenches its fluorescence intensity, however, proportionately undergo cyclization to produce a highly fluorescent product, 2,3-naphthotriazole (NAT). The synthesized product, NAT, was verified using various chromatographic and spectrophotometric techniques. This newly developed antibody-based detection method, utilizing DAN dye, demonstrated a high selectivity towards PETN, RDX, and TNT. This method can be used as an economical testing kit for direct quantification of explosives, implying the great potential for quick, low-cost trace detection of explosives.

Biogenic selenium nanoparticles inhibit Staphylococcus aureus adherence on different surfaces.

The global issue of nosocomial infection is owing to bacterial colonization and biofilm formation on medical devices which primarily affects critically ill and/or immuno-compromised patients and also leads to malfunctioning of the devices. Therefore, it is desirable to prevent bacterial colonization on these devices by coating with a non toxic antimicrobial agent or bacterial adherence inhibitor. Here we have shown Bacillus licheniformis JS2 derived selenium nanoparticles (SeNPs) inhibit Staphylococcus aureus adherence and micro-colony formation on polystyrene, glass, and catheter surface. Results indicated that, the coating of these non toxic biogenic SeNPs, at a concentration of 0.5 mgSe/ml, prohibits bacterial load to more than 60% on glass and catheter surface, when incubated at 4 °C for 24h in phosphate buffered saline. Furthermore, confocal and electron microscopic observations strongly suggested the inhibition of biofilm and micro-colony formation on SeNP coated glass and catheter surfaces when cultured at 37 °C for 72 h in a nutrient rich medium. The study suggests that coating of biogenic SeNPs on medical devices could be an alternative approach for prevention of biofilm related infections.