A novel role for calpain in the endothelial dysfunction induced by activation of angiotensin II type 1 receptor signaling.

RATIONALE: The cytosolic protease calpain has been recently implicated in the vascular remodeling of angiotensin II (Ang II) type 1 receptor (AT(1)R) signaling. The role of Ang II/AT(1)R/calpain signaling on endothelial function, an important and early determinant of vascular pathology, remains though totally unknown. Accordingly, we investigated the role of calpain in the endothelial dysfunction of Ang II. OBJECTIVE: To demonstrate a mechanistic role for calpain in the endothelial dysfunction induced by Ang II/AT(1)R signaling. To establish endothelial-expressed calpains as an important target of AT(1)R signaling. METHODS AND RESULTS: Subchronic administration of nonpressor doses of Ang II to rats and mice significantly increased vascular calpain activity via AT(1)R signaling. Intravital microscopy studies revealed that activation of vascular expressed calpains causes endothelial dysfunction with increased leukocyte-endothelium interactions and albumin permeability in the microcirculation. Western blot and immunohistochemistry studies confirmed that Ang II/AT(1)R signaling preferentially activates the constitutively expressed µ-calpain isoform and demonstrated a calpain-dependent degradation of IκBα, along with upregulation of nuclear factor κB-regulated endothelial cell adhesion molecules. These physiological and biochemical parameters were nearly normalized following inhibition of AT(1)R or calpain in vivo. RNA silencing studies in microvascular endothelial cells, along with knockout and transgenic mouse studies, further confirmed the role of µ-calpain in the endothelial adhesiveness induced by Ang II. CONCLUSIONS: This study uncovers a novel role for calpain in the endothelial dysfunction of Ang II/AT(1)R signaling and establishes the calpain system as a novel molecular target of the vascular protective action of renin-angiotensin system inhibition. Our results may have significant clinical implications in vascular disease.

Tracking the COVID-19 vaccines: The global landscape.

ABBREVIATIONS: COVID-19: Coronavirus disease 2019; SARS-CoV-2: Severe acute respiratory syndrome coronavirus-2; VLPs: Virus like particles; WHO: World Health Organization; E: Envelope; M: Membrane; S: Spike; N: Nucleocapsid; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; FDA: Food and Drug Administration; LNP: lipid-nanoparticle; AZD1222: ChAdOx1 nCoV-19; BNT162b2: Pfizer-BioNTech mRNA vaccine; mRNA-1273: Moderna vaccine; Ad26.COV2.S: Johnson and Johnson - Janssen's vaccine; Gam-COVID-Vac: Sputnik Vaccine; NVX-CoV2373: Novavax vaccine with Matrix-M™ adjuvant.

Protein kinase C upregulates intercellular adhesion molecule-1 and leukocyte-endothelium interactions in hyperglycemia via activation of endothelial expressed calpain.

OBJECTIVE: We tested the hypothesis of a role for the calcium-dependent protease calpain in the endothelial dysfunction induced by hyperglycemic activation of protein kinase C (PKC). METHODS AND RESULTS: Chronic hyperglycemia with insulin deficiency (type 1 diabetes) was induced in rats by streptozotocin. Total PKC and calpain activities, along with activity and expression level of the 2 endothelial-expressed calpains isoforms, µ- and m-calpain, were measured in vascular tissue homogenates by enzymatic assays and Western blot analysis, respectively. Intravital microscopy was used to measure and correlate leukocyte-endothelium interactions with calpain activity in the microcirculation. Expression levels and endothelial localization of the inflammatory adhesion molecule intercellular adhesion molecule-1 were studied by Western blot analysis and immunofluorescence, respectively. The mechanistic role of hyperglycemia alone in the process of PKC-induced calpain activation and actions was also investigated. We found that in the type 1 diabetic vasculature, PKC selectively upregulates the activity of the µ-calpain isoform. Mechanistic studies confirmed a role for hyperglycemia and PKCß in this process. The functional implications of PKC-induced calpain activation were upregulation of endothelial expressed intercellular adhesion molecule-1 and leukocyte-endothelium interactions. CONCLUSIONS: Our results uncover the role of µ-calpain in the endothelial dysfunction of PKC. Calpain may represent a novel molecular target for the treatment of PKC-associated diabetic vascular disease.

Fabrication and Evaluation of Basil Essential Oil-Loaded Halloysite Nanotubes in Chitosan Nanocomposite Film and Its Application in Food Packaging.

Increasing health concerns regarding the use of plasticware have led to the development of ecofriendly biodegradable packaging film from natural polymer and food additives. In the present study, basil essential oil (BEO) loaded halloysite nanotubes (HNTs) composite films were synthesized using a solution casting method. The effects of BEO and nanotube concentration on the mechanical, physical, structural, barrier, and antioxidant properties of films were evaluated. Scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) demonstrated well-dispersed HNTs and BEO in tailored composite films. The addition of BEO in Chitosan (Ch) film caused darkening of the film color; furthermore, the incorporation of HNTs in varied concentrations increased opaqueness in Ch/BEO film. The Ch/BEO film, upon adding HNTs 5-30 wt%, exhibited a corresponding increase in the film thickness (0.108-0.135 mm) when compared with the Ch/BEO film alone (0.081 mm). The BEO-loaded HNTs composite films displayed reduced moisture content and characteristic barrier and UV properties. The Ch/BEO film with 15 wt% HNTs was found to have enhanced antioxidant activity. The Ch/BEO/HNTs composite also managed to prevent broccoli florets from losing weight and firmness during storage. The enhanced barrier and antioxidant qualities of the nanocomposite film suggest its potential application in the food processing and packaging sector. This is the first ever report on the fabrication of nanocomposite film using BEO and HNTs for food packaging. The low production cost and ecofriendly approach make the film acceptable for further research and commercialization thereafter.

Recombinant vaccines for COVID-19.

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation. Considering the crucial role of SARS-CoV-2 spike (S) glycoprotein in virus attachment, entry, and induction of neutralizing antibodies, S protein is being widely used as a target for vaccine development. Based on advances in techniques for vaccine design, inactivated, live-vectored, nucleic acid, and recombinant COVID-19 vaccines are being developed and tested for their efficacy. Phase3 clinical trials are underway or will soon begin for several of these vaccines. Assuming that clinical efficacy is shown for one or more vaccines, safety is a major aspect to be considered before deploying such vaccines to the public. The current review focuses on the recent advances in recombinant COVID-19 vaccine research and development and associated issues.

TiO2 decorated functionalized halloysite nanotubes (TiO2@HNTs) and photocatalytic PVC membranes synthesis, characterization and its application in water treatment.

In this study photocatalyst, TiO2@HNTs were prepared by  synthesizing TiO2 nanoparticles in situ on the  functionalized halloysite nanotubes (HNTs) surface. Photocatalytic PVC membrane TiO2@HNTs M2 (2 wt.%) and TiO2@HNTs M3 (3 wt.%) were also prepared. Photocatalyst TiO2@HNTs and photocatalytic PVC membranes were used to study the photocatalytic activity against the methylene blue (MB) and rhodamine B (RB) dyes in UV batch reactor. The structure and morphology of photocatalyst and photocatalytic PVC membrane were characterized by fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray (EDX), transmission electron microscopy (TEM), UV-Vis spectrophotometer and photoluminescence (PL). The PL study showed that the oxygen vacancies and surface hydroxyl groups present on the surface of TiO2@HNTs act as excellent traps for charge carrier, reducing the electron-hole recombination rate.TiO2@HNTs 2 (2 wt.%) and TiO2@HNTs 3 (3 wt.%) degraded MB dye up to 83.21%, 87.47% and RB dye up to 96.84% and 96.87%, respectively. TiO2@HNT photocatalyst proved to be stable during the three consecutive cycle of photocatalytic degradation of the RB dye. TiO2@HNTs M2 and TiO2@HNTs M3 degraded MB dye up to 27.19%, 42.37% and RB dye up to 30.78%, 32.76%, respectively. Photocatalytic degradation of both the dyes followed the first-order kinetic model. Degradation product analysis was done using the liquid chromatography-mass spectrometry (LC-MS) and the results showed that the dye degradation was initiated by demethylation of the molecule. MB and RB dye degradation reaction were tested by TBA and IPA as OH* and H+ scavengers respectively. Mechanism of photocatalytic activity of TiO2@HNTs and photocatalytic PVC membrane were also explained.

Serine/threonine kinase (pk-1) is a component of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) very late gene transcription complex and it phosphorylates a 102 kDa polypeptide of the complex.

The baculovirus gene, protein kinase-I (pk-1) encodes a serine/threonine kinase that is essential for very late gene expression. Late and very late genes of the baculoviruses are transcribed by an alpha-amanitin resistant RNA polymerase. The very late gene promoter transcription initiation complex was isolated from nuclei of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Sf9 cells by DNA affinity purification and found to contain 4 major polypeptides of sizes approximately 102, 38, 32, and 18 kDa. The 32 kDa polypeptide was immunoreactive to AcMNPV anti-pk-1 antibody and phosphorylated the 102 kDa polypeptide, earlier reported as late gene expression factor LEF-8. Electrophoretic mobility shift assays with anti-pk-1 antibody indicated the binding of promoter DNA with recombinant AcMNPV-pk-1 and transcription initiation complex proteins. All these results suggested AcMNPV-pk-1 to be a component of the viral very late gene transcription initiation complex.

Inhibition of Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin gene expression by DNAzyme knockout of its serine/threonine kinase (pk1) gene.

DNAzyme is known to selectively cleave RNA at predetermined site. Transfection of Autographa californica nucleopolyhedrovirus (AcNPV) infected Sf9 cells with serine/threonine kinase (pk1) mRNA specific DNAzymes, DZ1 and DZ2 to cleave the viral coded (pk1) mRNA in between 87th and 88th, and 250th and 251st nucleotide, respectively inhibited the pk1 mRNA and its protein expressions. Interestingly, polh mRNA and protein expressions were also inhibited by these DNAzymes despite their inability to cleave polh mRNA. The polyhedrin promoter driven green fluorescent protein (GFP) mRNA and protein expressions were also inhibited by these pk1 specific DZs. Surprisingly the extents of inhibition of polyhedrin and GFP at different concentrations of both DZs were higher than that of pk1 mRNA and protein expressions. These results suggested that pk1 regulates polyhedrin promoter driven transcription of AcNPV, and the effect of one gene expression on that of other can be studied by DNAzyme knockdown.

Characterization of a eukaryotic type serine/threonine kinase in Spodoptera litura nucleopolyhedrovirus-I (SpltNPV-I).

An open reading frame (ORF) of 819nt coding for a predicted protein of 272 amino acids was identified in the genome of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Sequence derived amino acid sequence analysis of this ORF suggested it to be a eukaryotic type protein kinase having conserved I-XI subdomains of Hanks kinase. In addition to kinase catalytic domains, this hypothetical protein had two bromodomains which could play regulatory roles in transcription. The ORF was expressed as approximately 31 kDa apoprotein in E. coli and approximately 33 kDa glycoprotein in Sf9 cells, and was called SpltNPV-I pk1 or pk1. The protein was localized in the nuclei of infected cells of the SpltNPV-I permissive cell line, NIV-HA-197. The recombinant protein had autophosphorylation and substrate phosphorylation activities in presence of Mn(2+) or Mg(2+), and these activities were inhibited by staurosporine. Mutation of Lys-50 to Met but not Lys-44 to Gln abolished pk1 kinase activity. Kinetics of pk1 showed that the rate of phosphorylation of SpltNPV-I pk1>MBP>histone H1, and both MBP and histone H1 had the K(m)s of 3muM. Analysis of phosphorylated protein showed the phosphorylation of serine and threonine residues, but not tyrosine. All these results suggested that identified SpltNPV-I ORF codes for a serine/threonine kinase.

MAPK-activated protein kinase 2 differentially regulates plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

Serine/Threonine kinase dependent transcription from the polyhedrin promoter of SpltNPV-I.

Polyhedrin (polh) and p10 are the two hyper-expressed very late genes of nucleopolyhedroviruses. Alpha amanitin resistant transcription from Spodoptera litura nucleopolyhedrovirus (SpltNPV-I) polyhedrin promoter was observed with virus infected nuclear extract of NIV-HA-197 cells but not with that from uninfected nuclear extract. Anti-protein kinase-1 (pk1) antibody inhibited the transcription and the inhibition reversed on addition of pk1, however, pk1 mutant protein, K50M having no phosphorylation activity did not overcome the transcription inhibition. Chromatin immuno-precipitation assays with viral anti-pk1 antibody showed the interaction of pk1 with the polh while electrophoretic mobility shift assays indicated the strong binding affinity (K(d) approximately 5.5x10(-11)) of purified pk1 with the polh promoter. These results suggested that the viral coded pk1 acts as a transcription factor in transcribing baculovirus very late genes.