Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.     

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.     

Association of White Blood Cell Count and Differential with the Incidence of Atrial Fibrillation: The Atherosclerosis Risk in Communities (ARIC) Study

Background

Although inflammation is involved in the development of atrial fibrillation (AF), the association of white blood cell (WBC) count and differential with AF has not been thoroughly examined in large cohorts with extended follow-up.

Methods

We studied 14,500 men and women (25% blacks, 55% women, mean age 54) free of AF at baseline (1987–89) from the Atherosclerosis Risk in Communities (ARIC) study, a community-based cohort in the United States. Incident AF cases through 2010 were identified from study electrocardiograms, hospital discharge records and death certificates. Multivariable Cox proportional hazards regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for AF associated with WBC count and differential.

Results

Over a median follow-up time of 21.5 years for the entire cohort, 1928 participants had incident AF. Higher total WBC count was associated with higher AF risk independent of AF risk factors and potential confounders (HR 1.09, 95% CI 1.04–1.15 per 1-standard deviation [SD] increase). Higher neutrophil and monocyte counts were positively associated with AF risk, while an inverse association was identified between lymphocyte count and AF (multivariable adjusted HRs 1.16, 95% CI 1.09–1.23; 1.05, 95% CI 1.00–1.11; 0.91, 95% CI 0.86–0.97 per 1-SD, respectively). No significant association was identified between eosinophils or basophils and AF.

Conclusions

High total WBC, neutrophil, and monocyte counts were each associated with higher AF risk while lymphocyte count was inversely associated with AF risk. Systemic inflammation may underlie this association and requires further investigation for strategies to prevent AF.     

Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive <Emphasis Type="Italic">GH3</Emphasis> genes during development and response to different stimuli in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

In plants, auxin-mediated responses are regulated by diverse proteins. One such class of proteins, i.e. GH3, is involved in the conjugation of IAA to amino acids and provides a negative feedback loop to control auxin homoeostasis. In order to have a better understanding of the mechanism of the auxin action, 15 genes encoding GH3 members were identified using existing EST databases of tomato. Their orthologs were identified from tobacco, potato, N. benthemiana, pepper, and petunia. Phylogenetic analysis of AtGH3, SlGH3, and their Solanaceae orthologs provided insights into various orthologous relationships among these proteins. These genes were found to be responsive to a variety of signals including, phytohormones and environmental stresses. Analysis of AuxRE elements in their promoters showed variability in the sequence as well as number of this element. Up-regulation of only 11 SlGH3 genes, in response to exogenous auxin, suggested possible relationship between the diversity in the sequence and number of AuxRE element with the auxin inducibility. Expression analysis of SlGH3 genes in different vegetative and reproductive tissues/stages suggested limited or no role for most of the SlGH3 genes at the initiation of fruit ripening. However, up-regulation of SlGH3-1 and -2 at the onset of fruit ripening indicates that these genes could have a role in fruit ripening. The present study characterizes GH3 gene family of tomato and its evolutionary relationship with members of this family from other Solanaceae species and Arabidopsis. It could help in the identification of GH3 genes and revelation of their function during vegetative/reproductive development stages from other Solanaceae members.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural,conformational and thermodynamic aspects of groove-directed-intercalation of flavopiridol into DNA

Certain plant-derived alkaloids and flavonoids have shown propitious cytotoxic acitvity against different types of cancer, having deoxyribose nucleic acid (DNA) as their main cellular target. Flavopiridol, a semi-synthetic derivative of rohitukine (a natural compound isolated from Dysoxylum binectariferum plant), has attained much attention owing to its anticancer potential against various haematological malignancies and solid tumours. This work focuses on investigating interaction between flavopiridol and DNA at molecular level in order to decipher its underlying mechanism of action, which is not well understood. To define direct influence of flavopiridol on the structural, conformational and thermodynamic aspects of DNA, various spectroscopic and calorimetric techniques have been used. ATR-FTIR and SERS spectral outcomes indicate a novel insight into groove-directed-intercalation of flavopiridol into DNA via direct binding with nitrogenous bases guanine (C6=O6) and thymine (C2=O2) in DNA groove together with slight external binding to its sugar–phosphate backbone. Circular dichroism spectral analysis of flavopiridol–DNA complexes suggests perturbation in native B-conformation of DNA and its transition into C-form, which may be localized up to a few base pairs of DNA. UV–visible spectroscopic results illustrate dual binding mode of flavopiridol when interacts with DNA having association constant, K_a = 1.18 × 10⁴ M^?1. This suggests moderate type of interaction between flavopiridol and DNA. Further, UV melting analysis also supports spectroscopic outcomes. Thermodynamically, flavopiridol–DNA complexation is an enthalpy-driven exothermic process. These conclusions drawn from this study could be helpful in unveiling mechanism of cytoxicity induced by flavopiridol that can be further applied in the development of flavonoid-based new chemotherapeutics with more specificity and better efficacy.     

Acute exposure of rhesus monkeys to DDT: Effect on carbohydrate metabolism

Four furocoumarins, two having a linear molecule, psoralen and 8-methylpsoralen and two having an angular molecule, angelicin and 4,5′-dimethylangelicin were tested for mutagenesis in Escherichia coli B wild type and in various strains deficient in known repair systems. The results indicate that both monoadducts and crosslinks are mutagenic. The mutagenic efficiency of the furocourmarins ranks in the following order 8-methylpsoralen > psoralen > angelicin > 4,5′-dimethylangelicin.     

A Quadrupole Dalton-based multi-attribute method for product characterization,process development,and quality control of therapeutic proteins

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.