Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

From top to bottom: gridded human population estimates in data-poor situations.

Where disease risks are heterogeneous across population groups or space, or dependent on transmission between individuals, spatial data on population distributions - human, livestock and wildlife - are required to estimate infectious disease risks, burdens and dynamics. As a result, large-scale, spatially explicit, high-resolution human population data are being increasingly used in a wide range of animal- and public-health planning and policy development scenarios. Official census data, aggregated by administrative unit, provide the only complete enumeration of a country's population. While census data from developed countries are generally up-to-date and of high quality, in resource-poor settings they are often incomplete, out of date, or only available at the country or province level. The challenges associated with producing accurate population estimates in regions that lack high-quality census data have led to the development of census-independent approaches to small-area population estimations. Known as bottom-up models, as opposed to the census-based top-down approaches, these methods combine microcensus survey data with ancillary data to provide spatially disaggregated population estimates in the absence of national census data. This review highlights the need for high-resolution gridded population data, discusses problems associated with using census data as top-down model inputs, and explores census-independent, or bottom-up, methods of producing spatially explicit, high-resolution gridded population data, together with their advantages.

Dans les contextes où les risques de maladie sont hétérogènes et dépendent du groupe de population ou du territoire dont il s'agit, ou des probabilités de transmission entre individus, il est nécessaire de disposer de données spatiales sur la distribution des populations (couvrant la population humaine et les populations d'animaux d'élevage et sauvages) afin d'être en mesure d'évaluer les risques de maladie infectieuse, de calculer le fardeau qu'elles représentent et de mettre en lumière les dynamiques à l'oeuvre. En conséquence, il est fait de plus en plus souvent appel à des données spatialement explicites, à grande échelle et à haute résolution pour construire les scénarios utilisés à des fins de planification et d'élaboration des politiques de santé animale et de santé publique. Les données officielles de recensement agrégées par unité administrative constituent la seule énumération complète de la population d'un pays. Si dans les pays développés ces données de recensement sont généralement actualisées et de bonne qualité, dans les configurations moins dotées en ressources elles sont souvent incomplètes, obsolètes ou n'existent qu'à l'échelle nationale ou provinciale. Les difficultés rencontrées pour produire des estimations suffisamment exactes dans les régions dépourvues de données de recensement de bonne qualité ont conduit à élaborer des méthodes visant à estimer la population de territoires limités, sans passer par le recensement. Ces modèles, qualifiés d'" ascendants " par opposition aux modèles de recensement " descendants ", associent aux données issues d'opérations de micro-recensement un certain nombre de données complémentaires afin de fournir des estimations de population ventilées par territoires, en l'absence de données nationales de recensement. Dans cet article, l'auteure souligne l'importance de disposer de données maillées de population à haute résolution ; après avoir examiné les problèmes associés à l'utilisation des résultats des modèles descendants, elle décrit les méthodes ascendantes non basées sur le recensement et leur capacité à fournir des données maillées de population spatialement explicites et à haute résolution. Elle conclut sur les avantages de ces dernières méthodes.

En circunstancias en que el riesgo de enfermedad varía según el grupo de población o el espacio de que se trate o en que dicho riesgo depende de la transmisión entre individuos, es necesario disponer de datos espaciales sobre la distribución de poblaciones (ya sean humanas, ganaderas o de animales salvajes) para calcular el riesgo y determinar la carga y la dinámica de una enfermedad infecciosa. De ahí que en muy diversas situaciones en las que se elaboran planes o políticas de sanidad animal o salud pública se vengan utilizando, cada vez más, conjuntos de datos a gran escala y alta resolución referidos expresamente a la población humana de un determinado ámbito geográfico. Los datos del censo oficial, agregados por unidad administrativa, ofrecen el único recuento completo de la población de un país. No obstante, si bien los datos censales de países desarrollados suelen estar al día y ser de buena calidad, en condiciones de escasez de recursos esos datos tienden a ser incompletos, estar obsoletos o existir únicamente a nivel de país o de provincia. La dificultad de obtener estimaciones poblacionales exactas en regiones donde no hay datos censales de buena calidad ha llevado a concebir métodos que no dependan del censo para realizar cálculos referidos a la población de pequeños territorios. Estos métodos, llamados modelos "ascendentes", por oposición a los planteamientos "descendentes" basados en el censo, permiten subsanar la falta de datos censales nacionales combinando datos de encuestas microcensales con otros datos complementarios para obtener estimaciones poblacionales desglosadas por espacio geográfico. La autora, tras subrayar la necesidad de disponer de cuadrículas de población de alta resolución, explica los problemas derivados del uso de datos censales como fuente de información en los modelos "descendentes" y expone métodos no dependientes del censo, o "ascendentes", para elaborar cuadrículas de población de alta resolución referidas expresamente a un espacio geográfico, así como las ventajas que ofrecen estos métodos.

Mitochondrial, cell cycle control and neuritogenesis alterations in an iPSC-based neurodevelopmental model for schizophrenia.

Schizophrenia is a severe psychiatric disorder of neurodevelopmental origin that affects around 1% of the world's population. Proteomic studies and other approaches have provided evidence of compromised cellular processes in the disorder, including mitochondrial function. Most of the studies so far have been conducted on postmortem brain tissue from patients, and therefore, do not allow the evaluation of the neurodevelopmental aspect of the disorder. To circumvent that, we studied the mitochondrial and nuclear proteomes of neural stem cells (NSCs) and neurons derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients versus healthy controls to assess possible alterations related to energy metabolism and mitochondrial function during neurodevelopment in the disorder. Our results revealed differentially expressed proteins in pathways related to mitochondrial function, cell cycle control, DNA repair and neuritogenesis and their possible implication in key process of neurodevelopment, such as neuronal differentiation and axonal guidance signaling. Moreover, functional analysis of NSCs revealed alterations in mitochondrial oxygen consumption in schizophrenia-derived cells and a tendency of higher levels of intracellular reactive oxygen species (ROS). Hence, this study shows evidence that alterations in important cellular processes are present during neurodevelopment and could be involved with the establishment of schizophrenia, as well as the phenotypic traits observed in adult patients. Neural stem cells (NSCs) and neurons were derived from induced pluripotent stem cells (iPSCs) from schizophrenia patients and controls. Proteomic analyses were performed on the enriched mitochondrial and nuclear fractions of NSCs and neurons. Whole-cell proteomic analysis was also performed in neurons. Our results revealed alteration in proteins related to mitochondrial function, cell cycle control, among others. We also performed energy pathway analysis and reactive oxygen species (ROS) analysis of NSCs, which revealed alterations in mitochondrial oxygen consumption and a tendency of higher levels of intracellular ROS in schizophrenia-derived cells.

Induced pluripotent stem cell-derived astrocytes from patients with schizophrenia exhibit an inflammatory phenotype that affects vascularization.

Molecular and functional abnormalities of astrocytes have been implicated in the etiology and pathogenesis of schizophrenia (SCZ). In this study, we examined the proteome, inflammatory responses, and secretome effects on vascularization of human induced pluripotent stem cell (hiPSC)-derived astrocytes from patients with SCZ. Proteomic analysis revealed alterations in proteins related to immune function and vascularization. Reduced expression of the nuclear factor kappa B (NF-κB) p65 subunit was observed in these astrocytes, with no incremental secretion of cytokines after tumor necrosis factor alpha (TNF-α) stimulation. Among inflammatory cytokines, secretion of interleukin (IL)-8 was particularly elevated in SCZ-patient-derived-astrocyte-conditioned medium (ASCZCM). In a chicken chorioallantoic membrane (CAM) assay, ASCZCM reduced the diameter of newly grown vessels. This effect could be mimicked with exogenous addition of IL-8. Taken together, our results suggest that SCZ astrocytes are immunologically dysfunctional and may consequently affect vascularization through secreted factors.

Proteomic signatures of schizophrenia-sourced iPSC-derived neural cells and brain organoids are similar to patients' postmortem brains.

BACKGROUND: Schizophrenia is a complex and severe neuropsychiatric disorder, with a wide range of debilitating symptoms. Several aspects of its multifactorial complexity are still unknown, and some are accepted to be an early developmental deficiency with a more specifically neurodevelopmental origin. Understanding the timepoints of disturbances during neural cell differentiation processes could lead to an insight into the development of the disorder. In this context, human brain organoids and neural cells differentiated from patient-derived induced pluripotent stem cells are of great interest as a model to study the developmental origins of the disease. RESULTS: Here we evaluated the differential expression of proteins of schizophrenia patient-derived neural progenitors (NPCs), early neurons, and brain organoids in comparison to healthy individuals. Using bottom-up shotgun proteomics with a label-free approach for quantitative analysis, we found multiple dysregulated proteins since NPCs, modified, and disrupted the 21DIV neuronal differentiation, and cerebral organoids. Our experimental methods have shown impairments in pathways never before found in patient-derived induced pluripotent stem cells studies, such as spliceosomes and amino acid metabolism; but also, those such as axonal guidance and synaptogenesis, in line with postmortem tissue studies of schizophrenia patients. CONCLUSION: In conclusion, here we provide comprehensive, large-scale, protein-level data of different neural cell models that may uncover early events in brain development, underlying several of the mechanisms within the origins of schizophrenia.

Age-linked suppression of lipoxin A4 associates with cognitive deficits in mice and humans.

Age increases the risk for cognitive impairment and is the single major risk factor for Alzheimer's disease (AD), the most prevalent form of dementia in the elderly. The pathophysiological processes triggered by aging that render the brain vulnerable to dementia involve, at least in part, changes in inflammatory mediators. Here we show that lipoxin A4 (LXA4), a lipid mediator of inflammation resolution known to stimulate endocannabinoid signaling in the brain, is reduced in the aging central nervous system. We demonstrate that genetic suppression of 5-lipoxygenase (5-LOX), the enzyme mediating LXA4 synthesis, promotes learning impairment in mice. Conversely, administration of exogenous LXA4 attenuated cytokine production and memory loss induced by inflammation in mice. We further show that cerebrospinal fluid LXA4 is reduced in patients with dementia and positively associated with cognitive performance, brain-derived neurotrophic factor (BDNF), and AD-linked amyloid-ß. Our findings suggest that reduced LXA4 levels may lead to vulnerability to age-related cognitive disorders and that promoting LXA4 signaling may comprise an effective strategy to prevent early cognitive decline in AD.

Altered resting-state functional connectivity in hiPSCs-derived neuronal networks from schizophrenia patients.

Schizophrenia (SZ) is a severe mental disorder that arises from abnormal neurodevelopment, caused by genetic and environmental factors. SZ often involves distortions in reality perception and it is widely associated with alterations in brain connectivity. In the present work, we used Human Induced Pluripotent Stem Cells (hiPSCs)-derived neuronal cultures to study neural communicational dynamics during early development in SZ. We conducted gene and protein expression profiling, calcium imaging recordings, and applied a mathematical model to quantify the dynamism of functional connectivity (FC) in hiPSCs-derived neuronal networks. Along the neurodifferentiation process, SZ networks displayed altered gene expression of the glutamate receptor-related proteins HOMER1 and GRIN1 compared to healthy control (HC) networks, suggesting a possible tendency to develop hyperexcitability. Resting-state FC in neuronal networks derived from HC and SZ patients emerged as a dynamic phenomenon exhibiting connectivity configurations reoccurring in time (hub states). Compared to HC, SZ networks were less thorough in exploring different FC configurations, changed configurations less often, presented a reduced repertoire of hub states and spent longer uninterrupted time intervals in this less diverse universe of hubs. Our results suggest that alterations in the communicational dynamics of SZ emerging neuronal networks might contribute to the previously described brain FC anomalies in SZ patients, by compromising the ability of their neuronal networks for rapid and efficient reorganization through different activity patterns.

DYRK1A regulates the bidirectional axonal transport of APP in human-derived neurons.

DYRK1A triplication in Down's Syndrome (DS) and its overexpression in Alzheimer's Disease (AD) suggest a role for increased DYR1A activity in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of AD, which lead to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in human neurons remains unknown. From a proteomic dataset of human cerebral organoids treated with harmine, a DYRK1A inhibitor, we found expression changes in protein clusters associated with the control of microtubule-based transport and in close interaction with the APP vesicle. Live-imaging of APP axonal transport in human-derived neurons treated with harmine or overexpressing a dominant negative DYRK1A revealed a reduction in APP vesicle density and enhanced the stochastic behavior of retrograde vesicle transport. Moreover, harmine increased the fraction of slow segmental velocities and changed speed transitions supporting a DYRK1A-mediated effect in the exchange of active motor configuration. Contrarily, the overexpression of DYRK1A in human polarized neurons increased the axonal density of APP vesicles and enhanced the processivity of retrograde APP. In addition, increased DYRK1A activity induced faster retrograde segmental velocities together with significant changes in slow to fast anterograde and retrograde speeds transitions suggesting the facilitation of the active motor configuration. Our results highlight DYRK1A as a modulator of the axonal transport machinery driving APP intracellular distribution in neurons, and stress DYRK1A inhibition as a putative therapeutic intervention to restore APP axonal transport in DS and AD.Significance StatementAxonal transport defects are early events in the progression of neurodegenerative diseases such as Alzheimer's Disease (AD). However, the molecular mechanisms underlying transport defects remain elusive. DYRK1A kinase is triplicated in Down's Syndrome and overexpressed in AD, suggesting that DYRK1A dysfunction affects molecular pathways leading to early-onset neurodegeneration. Here, we show by live imaging of human-derived neurons that DYRK1A activity differentially regulates the intracellular trafficking of the amyloid precursor protein (APP). Further, single particle analysis revealed DYRK1A as a modulator of axonal transport and the configuration of active motors within the APP vesicle. Our work highlights DYRK1A as a regulator of APP axonal transport and metabolism; supporting DYRK1A inhibition as a therapeutic strategy to restore intracellular dynamics in AD.

Nootropic effects of LSD: Behavioral, molecular and computational evidence.

The therapeutic use of classical psychedelic substances such as d-lysergic acid diethylamide (LSD) surged in recent years. Studies in rodents suggest that these effects are produced by increased neural plasticity, including stimulation of the mTOR pathway, a key regulator of metabolism, plasticity, and aging. Could psychedelic-induced neural plasticity be harnessed to enhance cognition? Here we show that LSD treatment enhanced performance in a novel object recognition task in rats, and in a visuo-spatial memory task in humans. A proteomic analysis of human brain organoids showed that LSD affected metabolic pathways associated with neural plasticity, including mTOR. To gain insight into the relation of neural plasticity, aging and LSD-induced cognitive gains, we emulated the experiments in rats and humans with a neural network model of a cortico-hippocampal circuit. Using the baseline strength of plasticity as a proxy for age and assuming an increase in plasticity strength related to LSD dose, the simulations provided a good fit for the experimental data. Altogether, the results suggest that LSD has nootropic effects.

Zika Virus Strains and Dengue Virus Induce Distinct Proteomic Changes in Neural Stem Cells and Neurospheres.

Brain abnormalities and congenital malformations have been linked to the circulating strain of Zika virus (ZIKV) in Brazil since 2016 during the microcephaly outbreak; however, the molecular mechanisms behind several of these alterations and differential viral molecular targets have not been fully elucidated. Here we explore the proteomic alterations induced by ZIKV by comparing the Brazilian (Br ZIKV) and the African (MR766) viral strains, in addition to comparing them to the molecular responses to the Dengue virus type 2 (DENV). Neural stem cells (NSCs) derived from induced pluripotent stem (iPSCs) were cultured both as monolayers and in suspension (resulting in neurospheres), which were then infected with ZIKV (Br ZIKV or ZIKV MR766) or DENV to assess alterations within neural cells. Large-scale proteomic analyses allowed the comparison not only between viral strains but also regarding the two- and three-dimensional cellular models of neural cells derived from iPSCs, and the effects on their interaction. Altered pathways and biological processes were observed related to cell death, cell cycle dysregulation, and neurogenesis. These results reinforce already published data and provide further information regarding the biological alterations induced by ZIKV and DENV in neural cells.

Schizophrenia-derived hiPSC brain microvascular endothelial-like cells show impairments in angiogenesis and blood-brain barrier function.

Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. Long-standing clinical observations and molecular data have pointed to a possible vascular deficiency that could be acting synergistically with neuronal dysfunction in SZ. As SZ is a neurodevelopmental disease, the use of human-induced pluripotent stem cells (hiPSC) allows disease biology modeling while retaining the patient's unique genetic signature. Previously, we reported a VEGFA signaling impairment in SZ-hiPSC-derived neural lineages leading to decreased angiogenesis. Here, we present a functional characterization of SZ-derived brain microvascular endothelial-like cells (BEC), the counterpart of the neurovascular crosstalk, revealing an intrinsically defective blood-brain barrier (BBB) phenotype. Transcriptomic assessment of genes related to endothelial function among three control (Ctrl BEC) and five schizophrenia patients derived BEC (SZP BEC), revealed that SZP BEC have a distinctive expression pattern of angiogenic and BBB-associated genes. Functionally, SZP BEC showed a decreased angiogenic response in vitro and higher transpermeability than Ctrl BEC. Immunofluorescence staining revealed less expression and altered distribution of tight junction proteins in SZP BEC. Moreover, SZP BEC's conditioned media reduced barrier capacities in the brain microvascular endothelial cell line HCMEC/D3 and in an in vivo permeability assay in mice. Overall, our results describe an intrinsic failure of SZP BEC for proper barrier function. These findings are consistent with the hypothesis tracing schizophrenia origins to brain development and BBB dysfunction.

How much is that doodle in the window? Exploring motivations and behaviours of UK owners acquiring designer crossbreed dogs (2019-2020).

BACKGROUND: Demand for intentional crosses of purebred dog breeds, often labelled 'designer crossbreeds' (e.g., Labrador Retriever X Poodle, the 'Labradoodle'), has recently increased in the UK. This study aimed to explore this phenomenon by comparing pre-purchase motivations, pre-purchase and purchase behaviours of UK owners of designer crossbred puppies purchased during 2019-2020 with those of owners of purebred puppies purchased during the same period. RESULTS: Data were collected in an online cross-sectional survey between November-December 2020. Responses from n = 6293 puppies (designer crossbred puppies: n = 1575; purebred puppies: n = 4718) were analysed. Perceived hypoallergenicity was cited as a motivator for breed/crossbreed choice by almost half of designer crossbreed owners (47.1%), six times more than purebred dog owners (7.86%; odds ratio [OR]: 9.12, 95% CI: 7.70-10.8). Designer crossbred puppies were more likely to have been acquired via a general selling website (e.g., Gumtree; 13.8%) compared to purebred puppies (7.67%; OR: 2.19, 95% CI: 1.77-2.71), or an animal-specific selling websites (e.g., Pets4Homes; 55.7%) compared to purebred puppies (37.4%; OR: 1.89, 95% CI: 1.65-2.17). Designer crossbreed owners were less likely to see their puppy in person prior to purchase than purebred owners (60.4% vs. 67.0%, respectively; OR: 0.74, 95% CI: 0.64-0.85), and at purchase, designer crossbred puppies were less likely to be seen with their mother (73.1% vs. 79.8%, respectively; OR: 0.82, 95% CI: 0.70-0.95), and littermates (67.7% vs. 78.1%, respectively; OR: 0.63, 95% CI: 0.55-0.73). Designer crossbreeds had a significantly higher purchase price, with 25.7% of designer crossbreed puppies costing £2000-£2999 compared to 15.1% of purebred puppies (X2 = 207.31, p <  0.001). CONCLUSIONS: The recent boom in designer crossbreeds in the UK has been fuelled by a desire for perceived hypoallergenic and generally healthy dogs that fit the lifestyles of households with children and limited experience with dogs. Some sought-after traits in designer crossbreeds are misconceptions that risk canine welfare, including relinquishment risk, if owner expectations are not met. Purchasing practices fuelling this boom support irresponsible breeding and selling practices, which combined with reduced pressure for health testing from buyers, may result in a higher disease burden and poorer future welfare for this growing designer dog population.

Dopamine signaling impairs ROS modulation by mitochondrial hexokinase in human neural progenitor cells.

Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D1R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H2O2 and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.

Microbiome associated with the tetrodotoxin-bearing anuran Brachycephalus pitanga.

The genus Brachycephalus includes small species of aposematic anurans known as microendemic, occurring in the mountains of the Atlantic Forest. Brachycephalus ephippium, B. nodoterga and B. pernix have been reported to contain the neurotoxin tetrodotoxin in skin and viscera. The biological conservation of several Brachycephalus species is currently threatened by climate change, deforestation, and the pandemic caused by the fungus Batrachochytrium dendrobatidis (Bd). Despite the well-known importance of amphibians' associated bacteria in the defensive role against pathogens, there is still a poor understanding of amphibian microbiome composition. The present study investigated the composition of B. pitanga microbial community and the presence of TTX in the host and in cultures of bacterial isolates, using a combination of metagenomics, bacterial culture isolation, mass spectrometry and metabolomic analyses. Results of culture-dependent and -independent analyses characterized the microbial communities associated with the skin and viscera of B. pitanga. Mass spectrometry analysis indicated the presence of TTX in host tissues, while bacterial production of TTX was not observed under the experimental conditions used in this investigation. This is the first report confirming the occurrence of TTX in B. pitanga.

WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that lead to damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins six, eight, 18 and tumor necrosis factor-alpha (TNF-α) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

The Age of Brain Organoids: Tailoring Cell Identity and Functionality for Normal Brain Development and Disease Modeling.

Over the past years, brain development has been investigated in rodent models, which were particularly relevant to establish the role of specific genes in this process. However, the cytoarchitectonic features, which determine neuronal network formation complexity, are unique to humans. This implies that the developmental program of the human brain and neurological disorders can only partly be reproduced in rodents. Advancement in the study of the human brain surged with cultures of human brain tissue in the lab, generated from induced pluripotent cells reprogrammed from human somatic tissue. These cultures, termed brain organoids, offer an invaluable model for the study of the human brain. Brain organoids reproduce the cytoarchitecture of the cortex and can develop multiple brain regions and cell types. Integration of functional activity of neural cells within brain organoids with genetic, cellular, and morphological data in a comprehensive model for human development and disease is key to advance in the field. Because the functional activity of neural cells within brain organoids relies on cell repertoire and time in culture, here, we review data supporting the gradual formation of complex neural networks in light of cell maturity within brain organoids. In this context, we discuss how the technology behind brain organoids brought advances in understanding neurodevelopmental, pathogen-induced, and neurodegenerative diseases.

Recurrent acute otitis media: a survey of current management in England.

OBJECTIVE: Recurrent acute otitis media is common in children. The preferred treatment measures for recurrent acute otitis media have a mixed evidence base. This study sought to assess baseline practice across ENT departments in England. METHODS: A national telephone survey of healthcare staff was conducted. Every ENT centre in England was contacted. A telephone script was used to ask about antibiotic and grommet use and duration in recurrent acute otitis media cases. RESULTS: Ninety-six centres (74 per cent) provided complete information. Recurrent acute otitis media treatment across England by ENT departments varied. The antibiotic first- and second-line prophylaxis offered varies, with trimethoprim used in 33 centres and 29 centres not offering any antibiotics. The timing or choice about when to use grommets also varies, but 87 centres (91 per cent) offer grommet surgery at one stage. CONCLUSION: The treatments received by children in England for recurrent acute otitis media vary by centre; collaborative research in this area is advised.

Spatio-temporal distribution and agroecological factors associated with canine leptospirosis in Great Britain.

Leptospirosis is an important global zoonotic disease that affects a wide range of mammalian species. Canine leptospirosis outbreaks have been reported after metereological events such as flooding (eg. in Brazil and the United States of America) suggesting an environmental association, but there has been no such study in Great Britain (GB). The distribution of cases across GB is also unreported. Objectives of this study were to: (1) assess the spatio-temporal variation of leptospirosis test submissions (2) explore associations between agroecological risk factors and distribution of different canine leptospirosis serogroups in GB, and (3) generate probability of presence maps for the different serogroups. Data analysed comprised laboratory submissions (n = 3986) to IDEXX laboratories between 1st January 2009 and 31st December 2018 for PCR or MAT leptospirosis testing. Spatial and seasonal scan statistics were used to investigate spatial and temporal clustering of positive tests, logistic regression was used to identify significant agroecological risk factors for positive tests, and the Maxent algorithm was used to model the environmental niche of four serogroups. There was an increased risk of a positive test result in the West Midlands of England (relative risk = 2.16) and between October and January (relative risk = 1.54). Logistic regression identified season and region to be significantly associated with a positive diagnosis,with higher odds of a positive test in Autumn (OR = 1.86 95 %CI 1.29-2.69) and Winter (OR = 1.51, 95 %CI 1.02-2.23) and in the East (OR = 2.20, 95 %CI = 1.31-3.71) and West Midlands (OR = 2.32, 95 %CI 1.45-3.71). The increased test-positive proportion in Autumn together with the increased odds of a positive diagnosis in Autumn suggests there may be a seasonal pattern to the canine leptospirosis in GB. The most important variable associated with higher leptospirosis presence in all ecological niche models was higher average annual temperature. The importance and retention of other variables differed between serogroups. Overall, a higher probability of leptospirosis presence was predicted in southern England and a low probability in Scotland and northern England. Although leptospirosis vaccine usage provides protection against the majority of serogroups identified here, one is not represented in the currently licensed vaccine formulations and therefore leptospirosis should remain a differential diagnosis in vaccinated dogs demonstrating consistent clinical signs of the disease.

In vitro antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19.

BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

Balance Right in Multiple Sclerosis (BRiMS): a feasibility randomised controlled trial of a falls prevention programme.

BACKGROUND: Balance, mobility impairments and falls are problematic for people with multiple sclerosis (MS). The "Balance Right in MS (BRiMS)" intervention, a 13-week home and group-based exercise and education programme, aims to improve balance and minimise falls. This study aimed to evaluate the feasibility of undertaking a multi-centre randomised controlled trial and to collect the necessary data to design a definitive trial. METHODS: This randomised controlled feasibility study recruited from four United Kingdom NHS clinical neurology services. Patients ≥ 18 years with secondary progressive MS (Expanded Disability Status Scale 4 to 7) reporting more than two falls in the preceding 6 months were recruited. Participants were block-randomised to either a manualised 13-week education and exercise programme (BRiMS) plus usual care, or usual care alone. Feasibility assessment evaluated recruitment and retention rates, adherence to group assignment and data completeness. Proposed outcomes for the definitive trial (including impact of MS, mobility, quality of life and falls) and economic data were collected at baseline, 13 and 27 weeks, and participants completed daily paper falls diaries. RESULTS: Fifty-six participants (mean age 59.7 years, 66% female, median EDSS 6.0) were recruited in 5 months; 30 randomised to the intervention group. Ten (18%) participants withdrew, 7 from the intervention group. Two additional participants were lost to follow up at the final assessment point. Completion rates were > 98% for all outcomes apart from the falls diary (return rate 62%). After adjusting for baseline score, mean intervention-usual care between-group differences for the potential primary outcomes at week 27 were MS Walking Scale-12v2: - 7.7 (95% confidence interval [CI] - 17.2 to 1.8) and MS Impact Scale-29v2: physical 0.6 (CI - 7.8 to 9), psychological - 0.4 (CI - 9.9 to 9). In total, 715 falls were reported, rate ratio (intervention:usual care) for falls 0.81 (0.41 to 2.26) and injurious falls 0.44 (0.41 to 2.23). CONCLUSIONS: Procedures were practical, and retention, programme engagement and outcome completion rates satisfied a priori progression criteria. Challenges were experienced in completion and return of daily falls diaries. Refinement of methods for reporting falls is therefore required, but we consider a full trial to be feasible. TRIAL REGISTRATION: ISRCTN13587999 Date of registration: 29 September 2016.