Evaluations of the peroxidative susceptibilities of cod liver oils by a 1H NMR analysis strategy: peroxidative resistivity of a natural collagenous and biogenic amine-rich fermented product

High-resolution 1H nuclear magnetic resonance (NMR) analysis was employed to molecularly screen the lipid, lipid oxidation product (LOP), and antioxidant compositions of four natural (unrefined) cod liver oil (CLO) products. Products 1–3 were non-fermented CLOs, whilst Product 4 was isolated from pre-fermented cod livers. Supporting analytical data that were acquired included biogenic amine, flavanone, tannin, phenolic antioxidant, α-tocopherol, and oxygen radical absorbance capacity (ORAC) determinations by recommended HPLC, LC/MS/MS, or spectrophotometric methods. SDS-PAGE, HPLC, and 1H NMR analyses investigated and determined collagenous antioxidants and their molecular mass ranges. 1H NMR analysis of aldehydic LOPs was employed to explore the susceptibilities/resistivities of each CLO product to peroxidation that is induced by thermal stressing episodes (TSEs) at 180°C, or following prolonged (42 day) storage episodes at 4 and 23 °C. Product 4 displayed extremely high ORAC values, which were much greater than those of Products 1–3, and that were predominantly ascribable to significant levels of peroxidation-blocking and/or aldehyde-consuming collagenous polypeptides/peptides and ammoniacal agents therein. Significantly lower levels of toxic aldehydes were generated in the pre-fermented Product 4 during exposure to TSEs, or the above long-term storage episodes. These results confirmed the enhanced peroxidative resistivity of a fermented, antioxidant-fortified natural CLO product over those of non-fermented unrefined products. Product 4: Green Pasture Blue Ice™ Fermented Cod Liver Oil

Determination of metabolic activity in planktonic and biofilm cells of Mycoplasma fermentans and Mycoplasma pneumoniae by nuclear magnetic resonance

Mycoplasmas are fastidious microorganisms, typically characterised by their restricted metabolism and minimalist genome. Although there is reported evidence that some mycoplasmas can develop biofilms little is known about any differences in metabolism in these organisms between the growth states. A systematic metabolomics approach may help clarify differences associated between planktonic and biofilm associated mycoplasmas. In the current study, the metabolomics of two different mycoplasmas of clinical importance (Mycoplasma pneumoniae and Mycoplasma fermentans) were examined using a novel approach involving nuclear magnetic resonance spectroscopy and principle component analysis. Characterisation of metabolic changes was facilitated through the generation of high-density metabolite data and diffusion-ordered spectroscopy that provided the size and structural information of the molecules under examination. This enabled the discrimination between biofilms and planktonic states for the metabolomic profiles of both organisms. This work identified clear biofilm/planktonic differences in metabolite composition for both clinical mycoplasmas and the outcomes serve to establish a baseline understanding of the changes in metabolism observed in these pathogens in their different growth states. This may offer insight into how these organisms are capable of exploiting and persisting in different niches and so facilitate their survival in the clinical setting

Synthesis, analysis and biological evaluation of novel indolquinonecryptolepine analogues as potential anti-tumour agents.

A small library of cryptolepine analogues were synthesised incorporating halogens and/or nitrogen containing side chains to optimise their interaction with the sugar-phosphate backbone of DNA to give improved binding, interfering with topoisomerase II hence enhancing cytotoxicity. Cell viability, DNA binding and Topoisomerase II inhibition is discussed for these compounds. Fluorescence microscopy was used to investigate the uptake of the synthesised cryptolepines into the nucleus. We report the synthesis and anti-cancer biological evaluation of nine novel cryptolepine analogues, which have greater cytotoxicity than the parent compound and are important lead compounds in the development of novel potent and selective indoloquinone anti-neoplastic agents

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2

Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs

Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%–50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8–92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron’s evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis