Single-stranded DNA binding protein hitches a ride with the Escherichia coli YoaA-χ helicase.

The Escherichia coli XPD/Rad3-like helicase, YoaA, and DNA polymerase III subunit, χ, are involved in E. coli DNA damage tolerance and repair. YoaA and χ promote tolerance to the DNA chain-terminator, 3 -azidothymidine (AZT), and together form the functional helicase complex, YoaA-χ. How YoaA-χ contributes to DNA damage tolerance is not well understood. E. coli single-stranded DNA binding protein (SSB) accumulates at stalled replication forks, and the SSB-χ interaction is required to promote AZT tolerance via an unknown mechanism. YoaA-χ and SSB interactions were investigated in vitro to better understand this DNA damage tolerance mechanism, and we discovered YoaA-χ and SSB have a functional interaction. SSB confers a substrate-specific effect on the helicase activity of YoaA-χ, barely affecting YoaA-χ on an overhang DNA substrate but inhibiting YoaA-χ on forked DNA. A paralog helicase, DinG, unwinds SSB-bound DNA in a similar manner to YoaA-χ on the substrates tested. Through use of ensemble experiments, we believe SSB binds behind YoaA-χ relative to the DNA ds/ss junction and show via single-molecule assays that SSB translocates along ssDNA with YoaA-χ. This is, to our knowledge, the first demonstration of a mechanoenzyme pulling SSB along ssDNA.

ESCRT disruption provides evidence against trans-synaptic signaling via extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types, including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating endosomal sorting complex required for transport (ESCRT) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo evenness interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell-autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease.

Diagnostic tests were heralded as crucial during the Coronavirus disease (COVID-19) pandemic with most of the key methods using bioanalytical approaches that detected larger molecules (RNA, protein antigens or antibodies) rather than conventional clinical biochemical techniques. Nucleic Acid Amplification Tests (NAATs), like the Polymerase Chain Reaction (PCR), and other molecular methods, like sequencing (that often work in combination with NAATs), were essential to the diagnosis and management during COVID-19. This was exemplified both early in the pandemic but also later on, following the emergence of new genetic SARS-CoV-2 variants. The 100 day mission to respond to future pandemic threats highlights the need for effective diagnostics, therapeutics and vaccines. Of the three, diagnostics represents the first opportunity to manage infectious diseases while also being the most poorly supported in terms of the infrastructure needed to demonstrate effectiveness. Where performance targets exist, they are not well served by consensus on how to demonstrate they are being met; this includes analytical factors such as limit of detection (LOD) false positive results as well as how to approach clinical evaluation. The selection of gold standards or use of epidemiological factors such as predictive value, reference ranges or clinical thresholds are seldom correctly considered. The attention placed on molecular diagnostic tests during COVID-19 illustrates important considerations and assumptions on the use of these methods for infectious disease diagnosis and beyond. In this manuscript, we discuss state-of-the-art approaches to diagnostic evaluation and explore how they may be better tailored to diagnostic techniques like NAATs to maximise the impact of these highly versatile bioanalytical tools, both generally and during future outbreaks.

ESCRT disruption provides evidence against transsynaptic signaling functions for extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo Evenness Interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

Identification of two novel subtypes of hepatitis C virus genotype 8 and a potential new genotype successfully treated with direct acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral (DAA) efficacy and global HCV elimination. METHODS: We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences. RESULTS: Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F). CONCLUSIONS: This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.

Still just a matter of taste? Sensorial appreciation of seafood is associated with more frequent and diverse consumption.

Improving health and sustainability outcomes in WEIRD (Western, Educated, Industrial, Rich, Democratic) nations necessitates a reduction in red meat consumption. Seafood is often overlooked in achieving this goal. However, simply consuming more of familiar fish species places high stress on production of these species. For this reason, diversification of seafood consumption is also critical. Here the motives for seafood consumption (frequency and diversity) are investigated across two studies by adapting the 4Ns survey to the seafood category. This 16-item survey measures four factors underpinning meat consumption: namely that it is 'Natural', 'Necessary', 'Normal' and 'Nice'. Swedish consumers' hedonic and sensory expectations of two herring concepts (traditional pickled contra novel minced and presented as a burger) are also evaluated in relation to the 4Ns. Study 1 (N = 304) revealed that the seafood 4Ns scale had a similar underlying structure to that of meat and had good test-retest reliability. Study 2 (N = 514) showed that consumers expected to like the pickled herring (associated with being 'seasoned', 'salty', 'sweet', 'firm', 'juicy', 'chewy', and 'slimy') more than the minced herring (associated with being 'mushy', 'fishy', 'grainy', 'dry' and having 'small bones'), and that 'Nice' scores affected expectations of both herring concepts. Food neophobia correlated inversely with seafood consumption frequency, expected liking, the 'Nice' subscale, and food agency. Critically, in both studies, enjoyment of seafood (higher 'Nice' scores) predicted more frequent and diverse seafood consumption, whilst agreeing that seafood is 'Necessary' for health predicted only consumption frequency, not diversity. Communicating the positive sensory attributes of seafood and developing novel product concepts in ways that disconfirm sceptical consumers' negative sensory expectations may increase acceptance of both familiar and unfamiliar seafood concepts.

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.