Primary-school-aged children inspire their peers and families to eat more vegetables in the KiiDSAY project: a qualitative descriptive study.

BACKGROUND: While vegetable intakes in Australia remain sub-optimal across all age groups, children are rarely consulted about their ideas on how to increase consumption. Qualitative research involving children provides an opportunity to consider their views. The aim of the Kids initiative inspires Dietary Success in Adults and Youth (KiiDSAY) project was to explore the views of school-aged children, who had participated in a school-based nutrition education program, about inspiring their peers and families to eat more vegetables. METHODS: A total of 26 children (15 boys) aged 10-12 years from four primary schools in New South Wales, Australia, participated in seven focus group interviews. Purposeful sampling was used to recruit participants. The study involved open-ended semi-structured questions conducted via Zoom that were audio-recorded, transcribed verbatim and analysed using thematic analysis with deductive and inductive coding in NVivo. RESULTS: Four major themes emerged: (i) taste; (ii) family environment; (iii) healthy eating; and (iv) change makers; with subthemes that were embedded within Social Cognitive Theory and Ecological Model of Health Behaviour theoretical frameworks. CONCLUSIONS: Children's inputs hold great potential for informing future interventions, particularly when designing or refining school-based nutrition programs. Children offered suggestions on how to inspire increased vegetable consumption among their peers and families that could be taken into consideration for future research and practice. These included: cooking activities in the home and school settings using recipes that creatively hide/mask/enhance the flavour of vegetables, involving positive role models and supportive school environments. Additionally, children recommended a sequential approach to the delivery of recipes starting from fruit-based and transitioning to vegetable-based recipes. Given the challenges faced in increasing children's vegetable consumption, particular focus on future research in this area is warranted. TRIAL REGISTRATION: FEAST Trial registered 14th December 2020 with the Australian and New Zealand Clinical Trials Registry (ACTRN12620001347954).

Driver mutations of cancer epigenomes

Epigenetic alterations are associated with all aspects of cancer, from tumor initiation to cancer progression and metastasis. It is now well understood that both losses and gains of DNA methylation as well as altered chromatin organization contribute significantly to cancer-associated phenotypes. More recently, new sequencing technologies have allowed the identification of driver mutations in epigenetic regulators, providing a mechanistic link between the cancer epigenome and genetic alterations. Oncogenic activating mutations are now known to occur in a number of epigenetic modifiers (i.e. IDH1/2, EZH2, DNMT3A), pinpointing epigenetic pathways that are involved in tumorigenesis. Similarly, investigations into the role of inactivating mutations in chromatin modifiers (i.e. KDM6A, CREBBP/EP300, SMARCB1) implicate many of these genes as tumor suppressors. Intriguingly, a number of neoplasms are defined by a plethora of mutations in epigenetic regulators, including renal, bladder, and adenoid cystic carcinomas. Particularly striking is the discovery of frequent histone H3.3 mutations in pediatric glioma, a particularly aggressive neoplasm that has long remained poorly understood. Cancer epigenetics is a relatively new, promising frontier with much potential for improving cancer outcomes. Already, therapies such as 5-azacytidine and decitabine have proven that targeting epigenetic alterations in cancer can lead to tangible benefits. Understanding how genetic alterations give rise to the cancer epigenome will offer new possibilities for developing better prognostic and therapeutic strategies.

Nucleotide bias of DCL and AGO in plant anti-virus gene silencing

Plant Dicer-like (DCL) and Argonaute (AGO) are the key enzymes involved in anti-virus post-transcriptional gene silencing (AV-PTGS). Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates. In comparison with genome sequences of dicot-infecting Turnip mosaic virus (TuMV) and monocot-infecting Cocksfoot streak virus (CSV), viral-derived small interfering RNAs (vsiRNAs) displayed positive correlations between AV-PTGS efficiency and G+C content (GC%). Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases. This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations, and AGO associated Arabidopsis endogenous siRNA populations, indicating that plant AGOs operated with G-preference. We further propose a hypothesis that AV-PTGS imposes selection pressure(s) on the evolution of plant viruses. This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination, suggesting that plant virus evolution to have low GC% genomes would have a unique function, which is to reduce the host AV-PTGS attack during infections.

Heat shock proteins and their role in early mammalian development

Elevated body temperature can result from many agents in the natural environment, such as fever, hot weather and heavy exercise. In our modern living conditions additional sources of induced hyperthermia including hot baths, saunas, drugs, electromagnetic radiation and ultrasound have been introduced. Hyperthermia during pregnancy has been shown to cause a wide spectrun of effects in art species studied, including humans, the outcome depending on the dose of heat absorbed by the mother and embryo and the stage of enbryonic or fetal development when exposed. The dose of heat is the product of the elevation of temperature above normal and the duration of the elevation. In relatively uncontrolled natural environmental exposures, embryonic death and resorption or abortion are probably the most common outcome. In less severe exposures (smaller doses) major or minor developmental defects can result and the central nervous system appears to be a major target for its effects. Heat damage to embryos appears to be by apoptotic and other forms of cell death in organs at critical stages of development. Over many millennia all living orgaisms appear to have developed protective mechanisms against excess heat, known collectively as the heat shock response. This response has been studied intensively over the last 20 years and its mechanisms of protection are now becoming more clearly defined. Exposures to heat (and a number of other toxic agents) trigger the heat shock response which is characterized by abrupt suspension in the normal protein synthesis and the concurrent induction of heat shock genes (hsp) and the synthesis of a set of protein families known collectively as the heat shock proteins (HSP). The hsp ape known to be involved in the response in embryos, each has at least two copies, one which appears to have functions in the normal embryonic development (cognate) and another which is induced at a certain dose of heat (induced) and which can offer some protection against damage for some time after the initiating dose. Most cognate HSP can normally be found in embryos at all stages of development. At certain critical, early stages of organ formation increased activity of one or more of the hsp families can be identified at the site of the organ analogue. The inducible HSP are usually undetectable during normal development and generally become inducible at these critical inductive stages of organ development, implying a protective function for that process. Excess heat is known to cause denaturation of proteins. Each of the known HSP families appears to protect cells through their chaperone functions in which they bind to adhesive sites on newly synthesized or heat damaged and partially unfolded structural and functional proteins. This prevents the formation of function-less aggregates. The damaged proteins are then either presented for degradation or are reconstituted by orderly disengagement from the chaperone protein. The molecular mechanisms of initiating and regulating the response are now becoming more clearly defined. Trigger mechanisms include release of prostaglandin Al which can be modulated by glucocorticoids and nonsteroidal anti-inflammatory agents. A heat shock factor (HSF) binds to the heat shock element (hse) on the gene sequence and initiates the hsp response. The signal induction pathway involves mitogen activated proteins (MAP) and stress activated proteins (SAP) which are regulated by phosphorylation. Signals are amplified by kinase cascades while they are being transmitted to the nucleus. Activated MAP and SAP kinases regulate the process by phosphorylation of proteins including transcription factors, HSP, other protein kinases and phosphorylases, growth factor receptors and cytoskeletal proteins. Although this research has defined some pathways indicating how and why heat can cause some defects, a means of preventing them has not yet emerged.

Estimating pre-symptomatic transmission of COVID-19: a secondary analysis using published data

ObjectiveTo estimate the proportion of pre-symptomatic transmission of SARS-CoV-2 infection that can occur and timing of transmission relative to symptom onset. Setting/designSecondary analysis of international published data. Data sourcesMeta-analysis of COVID-19 incubation period and a rapid systematic review of serial interval and generation time, which are published separately. ParticipantsStudies were selected for analysis if they had transparent methods and data sources and they provided enough information to simulate full distributions of serial interval or generation time. Twenty-three estimates of serial interval and five of generation time from 17 publications were included. MethodsSimulations were generated of incubation period and of serial interval or generation time. From these, transmission times relative to symptom onset were calculated and the proportion of pre-symptomatic transmission was estimated. Outcome measuresTransmission time of SARS-CoV-2 relative to symptom onset and proportion of pre-symptomatic transmission. ResultsTransmission time ranged from a mean of 2.91 (95% CI: 3.18-2.64) days before symptom onset to 1.20 (0.86-1.55) days after symptom onset. Unweighted pooling of estimates of transmission time based on serial interval resulted in a mean of 0.60 days before symptom onset (3.01 days before to 1.81 days after). Proportion of pre-symptomatic transmission ranged from 42.8% (39.8%-45.9%) to 80.6% (78.1%-83.0%). The proportion of pre-symptomatic transmission from pooled estimates was 56.4% (34.9%-78.0%). ConclusionsWhilst contact rates between symptomatic infectious and susceptible people are likely to influence the proportion of pre-symptomatic transmission, there is substantial potential for pre-symptomatic transmission of SARS-CoV-2 in a range of different contexts. Our work suggests that transmission is most likely in the day before symptom onset whereas estimates suggesting most pre-symptomatic transmission highlighted mean transmission times almost three days before symptom onset. This highlights the need for rapid case detection, contact tracing and quarantine. Strengths and weaknesses of this studyO_LIWe estimate the extent and variation of pre-symptomatic transmission of SARS-CoV-2 infection across a range of contexts. This provides important information for development and targeting of control policies and for the parameterisation of transmission models. C_LIO_LIThis is a secondary analysis using simulations based on published data, some of which is in pre-print form and not yet peer-reviewed. There is overlap in the contact tracing data that informed some of our source publications. We partially address this by summarising data at source location level as well as at study level. C_LIO_LIPopulations where symptomatic people are rapidly isolated are likely have relatively more pre-symptomatic transmission. This should be borne in mind whilst interpreting our results, but does not affect our finding that there is substantial potential for pre-symptomatic transmission of SARS-CoV-2 infection. C_LIO_LIA strength of our approach is that it builds an understanding of pre-symptomatic transmission from a range of estimates in the literature, facilitates discussion for the drivers of variation between them, and highlights the consistent message that consideration of pre-symptomatic transmission is critical for COVID-19 control policy. C_LI

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor.

Designing covalent inhibitors is a task of increasing importance in drug discovery. Efficiently designing irreversible inhibitors, though, remains challenging. Here, we present covalentizer, a computational pipeline for creating irreversible inhibitors based on complex structures of targets with known reversible binders. For each ligand, we create a custom-made focused library of covalent analogs. We use covalent docking, to dock these tailored covalent libraries and to find those that can bind covalently to a nearby cysteine while keeping some of the main interactions of the original molecule. We found ~11,000 cysteines in close proximity to a ligand across 8,386 protein-ligand complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In prospective evaluation against a panel of kinases, five out of nine predicted covalent inhibitors showed IC50 between 155 nM - 4.2 M. Application of the protocol to an existing SARS-CoV-1 Mpro reversible inhibitor led to a new acrylamide inhibitor series with low micromolar IC50 against SARS-CoV-2 Mpro. The docking prediction was validated by 11 co-crystal structures. This is a promising lead series for COVID-19 antivirals. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.