A mRNA Vaccine Encoding for a RBD 60-mer Nanoparticle Elicits Neutralizing Antibodies and Protective Immunity Against the SARS-CoV-2 Delta Variant in Transgenic K18-hACE2 Mice.

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the Delta variant elicits robust neutralizing antibody response, and also provides protective immunity against the Delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern

Inhibition of influenza H7 hemagglutinin-mediated entry.

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50 ~ 6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound

TBHQ inhibits H3 and H7 HA-mediated entry.

<p>Each concentration point was performed in triplicate.</p

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in Cyprinidae fish: Towards hints of their arrangements using advanced classification methods

To tackle the ever-present global concern regarding human exposure to persistent organic pollutants (POPs) via food products, this study strived to indicate associations between organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in lake-fish tissue depending on the species and sampling season. Apart from the monitoring initiatives recommended in the Global Monitoring Plan for POPs, the study discussed 7 OCPs and 18 PCB congeners determined in three Cyprinidae species (rudd, carp, and Prussian carp) from Vransko Lake (Croatia), which are widely domesticated and reared as food fish across Europe and Asia. We exploit advanced classification algorithms, the Kohonen self-organizing maps (SOM) and Decision Trees (DT), to search for POP patterns typical for the investigated species. As indicated by SOM, some of the dioxin-like and non-dioxin-like PCBs (PCB-28, PCB-74, PCB-52, PCB-101, PCB-105, PCB-114, PCB-118, PCB-156 and PCB-157), alpha-HCH and beta-HCH caused dissimilarities among fish species, but regardless of their weight and length. To support these suggestions, DT analysis sequenced the fish species and seasons based on the concentration of heavier congeners. The presented assumptions indicated that the supplemental application of SOM and DT offers advantageous features over the usually rough interpretation of POPs pattern and over the single use of the methods

TBHQ stabilizes the neutral pH conformation of H7 HA.

<p>Limited proteolysis experiment for H7 HA in the presence (open squares, dotted line) and absence (filled circles, solid line) of TBHQ at different pH. The curves correspond to fits using [H⁺]_mp  = 1.30×10⁻⁵ and n = 1.6 in the absence of TBHQ and [H+]_mp  = 2.83×10⁻⁵ and n = 1.8 in the presence of TBHQ (c.f. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076363#s2" target="_blank">Experimental Procedures</a>).</p

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in Cyprinidae fish: Towards hints of their arrangements using advanced classification methods

To tackle the ever-present global concern regarding human exposure to persistent organic pollutants (POPs) via food products, this study strived to indicate associations between organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in lake-fish tissue depending on the species and sampling season. Apart from the monitoring initiatives recommended in the Global Monitoring Plan for POPs, the study discussed 7 OCPs and 18 PCB congeners determined in three Cyprinidae species (rudd, carp, and Prussian carp) from Vransko Lake (Croatia), which are widely domesticated and reared as food fish across Europe and Asia. We exploit advanced classification algorithms, the Kohonen self-organizing maps (SOM) and Decision Trees (DT), to search for POP patterns typical for the investigated species. As indicated by SOM, some of the dioxin-like and non-dioxin-like PCBs (PCB-28, PCB-74, PCB-52, PCB-101, PCB-105, PCB-114, PCB-118, PCB-156 and PCB-157), alpha-HCH and beta-HCH caused dissimilarities among fish species, but regardless of their weight and length. To support these suggestions, DT analysis sequenced the fish species and seasons based on the concentration of heavier congeners. The presented assumptions indicated that the supplemental application of SOM and DT offers advantageous features over the usually rough interpretation of POPs pattern and over the single use of the methods

Discovery of Selective Inhibitors of the <i>Clostridium difficile</i> Dehydroquinate Dehydratase

<div><p>A vibrant and healthy gut flora is essential for preventing the proliferation of <i>Clostridium difficile</i>, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most <i>C. difficile</i> infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a <i>C. difficile</i> specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the <i>C. difficile</i> enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3^rd of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I <i>C. difficile</i> DHQD but not the type II DHQD from <i>Bacteroides thetaiotaomicron</i>, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the <i>C. difficile</i> enzyme with K_i values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the <i>C. difficile</i> but not the highly homologous <i>Salmonella enterica</i> DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of <i>C. difficile</i> specific antibiotics.</p></div

Protocol for analyzing antibody responses to glycoprotein antigens using electron-microscopy-based polyclonal epitope mapping

Summary: Electron microscopy-based polyclonal epitope mapping (EMPEM) can delineate epitope specificities of serum antibodies to a given antigen following vaccination or infection. Here, we present a protocol for the EMPEM method for rapid high-throughput assessment of antibody responses to glycoprotein antigens in vaccination and infection studies. We describe steps for antibody isolation and digestion, antigen complex and purification, and electron microscope imaging. We then detail procedures for processing and analysis of EMPEM data.For complete details on the use and execution of this protocol, please refer to Bianchi et al. (2018).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

NMR demonstrates that Compounds 1–3 bind selectively to <i>cd</i>DHQD.

<p>NMR titration of (A) compound <b>1,</b> (B) compound <b>2</b>, (C) compound <b>3</b> to <i>cd</i>DHQD. All three compounds demonstrate binding to <i>cd</i>DHQD with K_d values of ∼25 µM for compounds <b>1</b> & <b>2</b>, and ∼65 µM for compound <b>3</b>. (D) NMR titration of compound <b>3</b> to <i>se</i>DHQD reveals much reduced affinity, with a K_d of ∼400 µM binding. In (A) and (B), the red and black lines represent the results from two different resonances.</p

STD NMR experiments map the binding of compounds 2 and 3 on <i>cd</i>DHQD.

<p>The relative proximity of compounds’ carbons to <i>cd</i>DHQD atoms based on STD NMR data is shown.</p