Points to consider when initiating clinical investigations in autistic paediatric populations-A White Paper.

Many individuals with autism spectrum disorder (ASD) experience various degrees of impairment in social interaction and communication, restricted, repetitive behaviours, interests/activities. These impairments make a significant contribution to poorer everyday adaptive functioning. Yet, there are no pharmacological therapies to effectively treat the core symptoms of ASD. Since symptoms of ASD likely emerge from a complex interplay of vulnerabilities, environmental factors and compensatory mechanisms during the early developmental period, pharmacological interventions arguably would have the greatest impact to improve long-term outcomes when implemented at a young age. It is essential therefore, that clinical development programmes of investigational drugs in ASD include the paediatric population early on in clinical trials. Such trials need to offer the prospect of direct benefit (PDB) for participants. In most cases in drug development this prospect is supported by evidence of efficacy in adults. However, the effectiveness of treatment approaches may be age-dependent, so that clinical trials in adults may not provide sufficient evidence for a PDB in children. In this white paper, we consolidate recommendations from regulatory guidelines, as well as advice from the Food and Drug Administration, USA (FDA) and the Committee for Human Medicinal Products (CHMP) consultations on various development programmes on: 1) elements to support a PDB to participants in early paediatric clinical trials in ASD, including single-gene neurodevelopment disorders, 2) aspects of study design to allow for a PDB. This white paper is intended to be complementary to existing regulatory guidelines in guiding industry and academic sponsors in their conduct of early paediatric clinical trials in ASD.

Big data and its impact on the 3Rs: a home cage monitoring oriented review.

Undisturbed home cage recording of mouse activity and behavior has received increasing attention in recent years. In parallel, several technologies have been developed in a bid to automate data collection and interpretation. Thanks to these expanding technologies, massive datasets can be recorded and saved in the long term, providing a wealth of information concerning animal wellbeing, clinical status, baseline activity, and subsequent deviations in case of experimental interventions. Such large datasets can also serve as a long-term reservoir of scientific data that can be reanalyzed and repurposed upon need. In this review, we present how the impact of Big Data deriving from home cage monitoring (HCM) data acquisition, particularly through Digital Ventilated Cages (DVCs), can support the application of the 3Rs by enhancing Refinement, Reduction, and even Replacement of research in animals.

A minimal metadata set (MNMS) to repurpose nonclinical in vivo data for biomedical research.

Although biomedical research is experiencing a data explosion, the accumulation of vast quantities of data alone does not guarantee a primary objective for science: building upon existing knowledge. Data collected that lack appropriate metadata cannot be fully interrogated or integrated into new research projects, leading to wasted resources and missed opportunities for data repurposing. This issue is particularly acute for research using animals, where concerns regarding data reproducibility and ensuring animal welfare are paramount. Here, to address this problem, we propose a minimal metadata set (MNMS) designed to enable the repurposing of in vivo data. MNMS aligns with an existing validated guideline for reporting in vivo data (ARRIVE 2.0) and contributes to making in vivo data FAIR-compliant. Scenarios where MNMS should be implemented in diverse research environments are presented, highlighting opportunities and challenges for data repurposing at different scales. We conclude with a 'call for action' to key stakeholders in biomedical research to adopt and apply MNMS to accelerate both the advancement of knowledge and the betterment of animal welfare.

Advancements in the use of xenopus oocytes for modelling neurological disease for novel drug discovery.

INTRODUCTION: Introduced about 50 years ago, the model of Xenopus oocytes for the expression of recombinant proteins has gained a broad spectrum of applications. The authors herein review the benefits brought from using this model system, with a focus on modeling neurological disease mechanisms and application to drug discovery. AREAS COVERED: Using multiple examples spanning from ligand gated ion channels to transporters, this review presents, in the light of the latest publications, the benefits offered from using Xenopus oocytes. Studies range from the characterization of gene mutations to the discovery of novel treatments for disorders of the central nervous system (CNS). EXPERT OPINION: Development of new drugs targeting CNS disorders has been marked by failures in the translation from preclinical to clinical studies. As progress in genetics and molecular biology highlights large functional differences arising from a single to a few amino acid exchanges, the need for drug screening and functional testing against human proteins is increasing. The use of Xenopus oocytes to enable precise modeling and characterization of clinically relevant genetic variants constitutes a powerful model system that can be used to inform various aspects of CNS drug discovery and development.

Selective and brain-penetrant HCN1 inhibitors reveal links between synaptic integration, cortical function, and working memory.

Hyperpolarization-activated and cyclic-nucleotide-gated 1 (HCN1) ion channels are proposed to be critical for cognitive function through regulation of synaptic integration. However, resolving the precise role of HCN1 in neurophysiology and exploiting its therapeutic potential has been hampered by minimally selective antagonists with poor potency and limited in vivo efficiency. Using automated electrophysiology in a small-molecule library screen and chemical optimization, we identified a primary carboxamide series of potent and selective HCN1 inhibitors with a distinct mode of action. In cognition-relevant brain circuits, selective inhibition of native HCN1 produced on-target effects, including enhanced excitatory postsynaptic potential summation, while administration of a selective HCN1 inhibitor to rats recovered decrement working memory. Unlike prior non-selective HCN antagonists, selective HCN1 inhibition did not alter cardiac physiology in human atrial cardiomyocytes or in rats. Collectively, selective HCN1 inhibitors described herein unmask HCN1 as a potential target for the treatment of cognitive dysfunction in brain disorders.

Striatum-projecting prefrontal cortex neurons support working memory maintenance.

Neurons in the medial prefrontal cortex (mPFC) are functionally linked to working memory (WM) but how distinct projection pathways contribute to WM remains unclear. Based on optical recordings, optogenetic perturbations, and pharmacological interventions in male mice, we report here that dorsomedial striatum (dmStr)-projecting mPFC neurons are essential for WM maintenance, but not encoding or retrieval, in a T-maze spatial memory task. Fiber photometry of GCaMP6m-labeled mPFC→dmStr neurons revealed strongest activity during the maintenance period, and optogenetic inhibition of these neurons impaired performance only when applied during this period. Conversely, enhancing mPFC→dmStr pathway activity-via pharmacological suppression of HCN1 or by optogenetic activation during the maintenance period-alleviated WM impairment induced by NMDA receptor blockade. Moreover, cellular-resolution miniscope imaging revealed that >50% of mPFC→dmStr neurons are active during WM maintenance and that this subpopulation is distinct from neurons active during encoding and retrieval. In all task periods, neuronal sequences were evident. Striatum-projecting mPFC neurons thus critically contribute to spatial WM maintenance.

Manipulating Agaricus bisporus developmental patterns by passaging microbial communities in complex substrates.

IMPORTANCE: Agaricus bisporus is an economically important edible mushroom and manipulating its developmental patterns is crucial for maximizing yield and quality. One of the potential strategies for achieving such a goal is passaging microbial communities in compost or casing. The current study demonstrated that passaging substrates develop enriched microbial communities, and after a few passages, certain levels of changes in mushroom developmental patterns (the timing of fruiting bodies formation) were observed as well as shifts in the bacterial communities. Overall, a better understanding of the complex interactions between microorganisms present in the cultivation system may help farmers and researchers to develop more efficient and sustainable cultivation practices that can both benefit the environment and human health.

A randomized, double-blind, placebo-controlled trial of intravenous alpha-1 antitrypsin for ARDS secondary to COVID-19.

Background: Patients with severe coronavirus disease 2019 (COVID-19) develop a febrile pro-inflammatory cytokinemia with accelerated progression to acute respiratory distress syndrome (ARDS). Here we report the results of a phase 2, multicenter, randomized, double-blind, placebo-controlled trial of intravenous (IV) plasma-purified alpha-1 antitrypsin (AAT) for moderate to severe ARDS secondary to COVID-19 (EudraCT 2020-001391-15). Methods: Patients (n = 36) were randomized to receive weekly placebo, weekly AAT (Prolastin, Grifols, S.A.; 120 mg/kg), or AAT once followed by weekly placebo. The primary endpoint was the change in plasma interleukin (IL)-6 concentration at 1 week. In addition to assessing safety and tolerability, changes in plasma levels of IL-1ß, IL-8, IL-10, and soluble tumor necrosis factor receptor 1 (sTNFR1) and clinical outcomes were assessed as secondary endpoints. Findings: Treatment with IV AAT resulted in decreased inflammation and was safe and well tolerated. The study met its primary endpoint, with decreased circulating IL-6 concentrations at 1 week in the treatment group. This was in contrast to the placebo group, where IL-6 was increased. Similarly, plasma sTNFR1 was substantially decreased in the treatment group while remaining unchanged in patients receiving placebo. IV AAT did not definitively reduce levels of IL-1ß, IL-8, and IL-10. No difference in mortality or ventilator-free days was observed between groups, although a trend toward decreased time on ventilator was observed in AAT-treated patients. Conclusions: In patients with COVID-19 and moderate to severe ARDS, treatment with IV AAT was safe, feasible, and biochemically efficacious. The data support progression to a phase 3 trial and prompt further investigation of AAT as an anti-inflammatory therapeutic. Funding: ECSA-2020-009; Elaine Galwey Research Bursary.

Stochastic Collisional Quantum Thermometry.

We extend collisional quantum thermometry schemes to allow for stochasticity in the waiting time between successive collisions. We establish that introducing randomness through a suitable waiting time distribution, the Weibull distribution, allows us to significantly extend the parameter range for which an advantage over the thermal Fisher information is attained. These results are explicitly demonstrated for dephasing interactions and also hold for partial swap interactions. Furthermore, we show that the optimal measurements can be performed locally, thus implying that genuine quantum correlations do not play a role in achieving this advantage. We explicitly confirm this by examining the correlation properties for the deterministic collisional model.

Efficacy of FFP3 respirators for prevention of SARS-CoV-2 infection in healthcare workers.

Background: Respiratory protective equipment recommended in the UK for healthcare workers (HCWs) caring for patients with COVID-19 comprises a fluid-resistant surgical mask (FRSM), except in the context of aerosol generating procedures (AGPs). We previously demonstrated frequent pauci- and asymptomatic severe acute respiratory syndrome coronavirus 2 infection HCWs during the first wave of the COVID-19 pandemic in the UK, using a comprehensive PCR-based HCW screening programme (Rivett et al., 2020; Jones et al., 2020). Methods: Here, we use observational data and mathematical modelling to analyse infection rates amongst HCWs working on 'red' (coronavirus disease 2019, COVID-19) and 'green' (non-COVID-19) wards during the second wave of the pandemic, before and after the substitution of filtering face piece 3 (FFP3) respirators for FRSMs. Results: Whilst using FRSMs, HCWs working on red wards faced an approximately 31-fold (and at least fivefold) increased risk of direct, ward-based infection. Conversely, after changing to FFP3 respirators, this risk was significantly reduced (52-100% protection). Conclusions: FFP3 respirators may therefore provide more effective protection than FRSMs for HCWs caring for patients with COVID-19, whether or not AGPs are undertaken. Funding: Wellcome Trust, Medical Research Council, Addenbrooke's Charitable Trust, NIHR Cambridge Biomedical Research Centre, NHS Blood and Transfusion, UKRI.

Transmission of mushroom virus X and the impact of virus infection on the transcriptomes and proteomes of different strains of Agaricus bisporus.

Cultivation of Agaricus bisporus is a large horticultural industry for many countries worldwide, where a single variety is almost grown exclusively. Mushroom virus X (MVX), a complex of multiple positive-sense single stranded RNA (ss(+)RNA) viruses, is a major pathogen of typical A. bisporus crops. MVX can manifest a variety of symptoms in crops and is highly infective and difficult to eradicate once established in host mycelium. Currently our knowledge regarding the molecular response of A. bisporus fruit bodies to MVX infection is limited. In order to study the response of different A. bisporus strains with different susceptibilities to MVX, we designed a model system to evaluate the in-vitro transmission of viruses in A. bisporus hyphae over a time-course, at two crucial phases in the crop cycle. The symptom expression of MVX in these varieties and the transcriptomic and proteomic response of fruit bodies to MVX-infection were examined. Transmission studies revealed the high potential of MVX to spread to uninfected mycelium yet not into the fruit bodies of certain strains in a crop. MVX affected colour and quality of multiple fruit bodies. Gene expression is significantly altered in all strains and between times of inoculation in the crop. Genes related to stress responses displayed differential expression. Proteomic responses revealed restriction of cellular signalling and vesicle transport in infected fruit bodies. This in-depth analysis examining many factors relevant to MVX infection in different A. bisporus strains, will provide key insights into host responses for this commercially important food crop.

Alpha-1 antitrypsin for cystic fibrosis complicated by severe cytokinemic COVID-19.

BACKGROUND: The clinical course of severe COVID-19 in cystic fibrosis (CF) is incompletely understood. We describe the use of alpha-1 antitrypsin (AAT) as a salvage therapy in a critically unwell patient with CF (PWCF) who developed COVID-19 while awaiting lung transplantation. METHODS: IV AAT was administered at 120 mg/kg/week for 4 consecutive weeks. Levels of interleukin (IL)-1ß, IL-6, IL-8, and soluble TNF receptor 1 (sTNFR1) were assessed at regular intervals in plasma, with IL-1ß, IL-6, IL-8 and neutrophil elastase (NE) activity measured in airway secretions. Levels were compared to baseline and historic severe exacerbation measurements. RESULTS: Systemic and airway inflammatory markers were increased compared to both prior exacerbation and baseline levels, in particular IL-6, IL-1ß and NE activity. Following each AAT dose, rapid decreases in each inflammatory parameter were observed. These were matched by marked clinical and radiographic improvement. CONCLUSIONS: The results support further investigation of AAT as a COVID-19 therapeutic, and re-exploration of its use in CF.

A linear prognostic score based on the ratio of interleukin-6 to interleukin-10 predicts outcomes in COVID-19.

BACKGROUND: Prognostic tools are required to guide clinical decision-making in COVID-19. METHODS: We studied the relationship between the ratio of interleukin (IL)-6 to IL-10 and clinical outcome in 80 patients hospitalized for COVID-19, and created a simple 5-point linear score predictor of clinical outcome, the Dublin-Boston score. Clinical outcome was analysed as a three-level ordinal variable ("Improved", "Unchanged", or "Declined"). For both IL-6:IL-10 ratio and IL-6 alone, we associated clinical outcome with a) baseline biomarker levels, b) change in biomarker level from day 0 to day 2, c) change in biomarker from day 0 to day 4, and d) slope of biomarker change throughout the study. The associations between ordinal clinical outcome and each of the different predictors were performed with proportional odds logistic regression. Associations were run both "unadjusted" and adjusted for age and sex. Nested cross-validation was used to identify the model for incorporation into the Dublin-Boston score. FINDINGS: The 4-day change in IL-6:IL-10 ratio was chosen to derive the Dublin-Boston score. Each 1 point increase in the score was associated with a 5.6 times increased odds for a more severe outcome (OR 5.62, 95% CI -3.22-9.81, P = 1.2 × 10-9). Both the Dublin-Boston score and the 4-day change in IL-6:IL-10 significantly outperformed IL-6 alone in predicting clinical outcome at day 7. INTERPRETATION: The Dublin-Boston score is easily calculated and can be applied to a spectrum of hospitalized COVID-19 patients. More informed prognosis could help determine when to escalate care, institute or remove mechanical ventilation, or drive considerations for therapies. FUNDING: Funding was received from the Elaine Galwey Research Fellowship, American Thoracic Society, National Institutes of Health and the Parker B Francis Research Opportunity Award.

Characterization of the Inflammatory Response to Severe COVID-19 Illness.

Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.

Proteomic investigation of interhyphal interactions between strains of Agaricus bisporus.

Hyphae of filamentous fungi undergo polar extension, bifurcation and hyphal fusion to form reticulating networks of mycelia. Hyphal fusion or anastomosis, a ubiquitous process among filamentous fungi, is a vital strategy for how fungi expand over their substrate and interact with or recognise self- and non-self hyphae of neighbouring mycelia in their environment. Morphological and genetic characterisation of anastomosis has been studied in many model fungal species, but little is known of the direct proteomic response of two interacting fungal isolates. Agaricus bisporus, the most widely cultivated edible mushroom crop worldwide, was used as an in vitro model to profile the proteomes of interacting cultures. The globally cultivated strain (A15) was paired with two distinct strains; a commercial hybrid strain and a wild isolate strain. Each co-culture presented a different interaction ranging from complete vegetative compatibility (self), lack of interactions, and antagonistic interactions. These incompatible strains are the focus of research into disease-resistance in commercial crops as the spread of intracellular pathogens, namely mycoviruses, is limited by the lack of interhyphal anastomosis. Unique proteomic responses were detected between all co-cultures. An array of cell wall modifying enzymes, plus fungal growth and morphogenesis proteins were found in significantly (P < 0.05) altered abundances. Nitrogen metabolism dominated in the intracellular proteome, with evidence of nitrogen starvation between competing, non-compatible cultures. Changes in key enzymes of A. bisporus morphogenesis were observed, particularly via increased abundance of glucanosyltransferase in competing interactions and certain chitinases in vegetative compatible interactions only. Carbohydrate-active enzyme arsenals are expanded in antagonistic interactions in A. bisporus. Pathways involved in carbohydrate metabolism and genetic information processing were higher in interacting cultures, most notably during self-recognition. New insights into the differential response of interacting strains of A. bisporus will enhance our understanding of potential barriers to viral transmission through vegetative incompatibility. Our results suggest that a differential proteomic response occurs between A. bisporus at strain-level and findings from this work may guide future proteomic investigation of fungal anastomosis.

Depression of Accumbal to Lateral Hypothalamic Synapses Gates Overeating.

Overeating typically follows periods of energy deficit, but it is also sustained by highly palatable foods, even without metabolic demand. Dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) of the nucleus accumbens shell (NAcSh) project to the lateral hypothalamus (LH) to authorize feeding when inhibited. Whether plasticity at these synapses can affect food intake is unknown. Here, ex vivo electrophysiology recordings reveal that D1-MSN-to-LH inhibitory transmission is depressed in circumstances in which overeating is promoted. Endocannabinoid signaling is identified as the induction mechanism, since inhibitory plasticity and concomitant overeating were blocked or induced by CB1R antagonism or agonism, respectively. D1-MSN-to-LH projectors were largely non-overlapping with D1-MSNs targeting ventral pallidum or ventral midbrain, providing an anatomical basis for distinct circuit plasticity mechanisms. Our study reveals a critical role for plasticity at D1-MSN-to-LH synapses in adaptive feeding control, which may underlie persistent overeating of unhealthy foods, a major risk factor for developing obesity.

FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation.

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a 'halo-effect' of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

Whole Genome Sequence of the Commercially Relevant Mushroom Strain Agaricus bisporus var. bisporus ARP23.

Agaricus bisporus is an extensively cultivated edible mushroom. Demand for cultivation is continuously growing and difficulties associated with breeding programs now means strains are effectively considered monoculture. While commercial growing practices are highly efficient and tightly controlled, the over-use of a single strain has led to a variety of disease outbreaks from a range of pathogens including bacteria, fungi and viruses. To address this, the Agaricus Resource Program (ARP) was set up to collect wild isolates from diverse geographical locations through a bounty-driven scheme to create a repository of wild Agaricus germplasm. One of the strains collected, Agaricus bisporus var. bisporus ARP23, has been crossed extensively with white commercial varieties leading to the generation of a novel hybrid with a dark brown pileus commonly referred to as 'Heirloom'. Heirloom has been successfully implemented into commercial mushroom cultivation. In this study the whole genome of Agaricus bisporus var. bisporus ARP23 was sequenced and assembled with Illumina and PacBio sequencing technology. The final genome was found to be 33.49 Mb in length and have significant levels of synteny to other sequenced Agaricus bisporus strains. Overall, 13,030 putative protein coding genes were located and annotated. Relative to the other A. bisporus genomes that are currently available, Agaricus bisporus var. bisporus ARP23 is the largest A. bisporus strain in terms of gene number and genetic content sequenced to date. Comparative genomic analysis shows that the A. bisporus mating loci in unifactorial and unsurprisingly highly conserved between strains. The lignocellulolytic gene content of all A. bisporus strains compared is also very similar. Our results show that the pangenome structure of A. bisporus is quite diverse with between 60-70% of the total protein coding genes per strain considered as being orthologous and syntenically conserved. These analyses and the genome sequence described herein are the starting point for more detailed molecular analyses into the growth and phenotypical responses of Agaricus bisporus var. bisporus ARP23 when challenged with economically important mycoviruses.

Author Correction: Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria.

In the version of this Article originally published, it was incorrectly stated that "16,687 protein-coding genes were inferred for the most recent common ancestor (MRCA) of Armillaria"; the value was incorrect and it should have read "15,787". This has now been corrected.