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Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-γ levels 1 d following secondary immunization. Notably, we found that natural killer cells and CD8+ T cells in the draining lymph nodes are the major producers of this circulating IFN-γ. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8+ T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.How mRNA-based coronavirus disease 2019 vaccines drive immune responses is not clear. Here the authors characterize immune responses to the BNT162b2 vaccine in mice, and show how it stimulates innate immunity, with antigen-specific CD8+ T cell responses dependent on the RNA sensor MDA5.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions 1 . TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A 2 – 6 . We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed 7 , we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology. Antisense oligonucleotides effectively decrease the inclusion of exon  8A of CACNA1C in human cells both in vitro and in rodents transplanted with human brain organoids, and a single intrathecal administration rescued both calcium changes and in vivo dendrite morphology of patient neurons.

The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain 1 , 2 – 3 , which has been challenging to study at inaccessible stages of late gestation and early postnatal human development 4 . Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development 5 , but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids 6 . Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4 . We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK . We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms. Assembloids are integrated with CRISPR screening to investigate the involvement of 425 neurodevelopmental disorder genes in human interneuron development, showing endoplasmic reticulum displacement before nuclear translocation and interference from LNPK deletion, resulting in abnormal migration.

The development of the highly efficacious mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology. However, reports of waning vaccine efficacy, coupled with the emergence of variants of concern that are resistant to antibody neutralization, have raised concerns about the potential lack of durability of immunity to vaccination. We recently reported findings from a comprehensive analysis of innate and adaptive immune responses in 56 healthy volunteers who received two doses of the BNT162b2 vaccination. Here, we analyzed antibody responses to the homologous Wu strain as well as several variants of concern, including the emerging Mu (B.1.621) variant, and T cell responses in a subset of these volunteers at six months (day 210 post-primary vaccination) after the second dose. Our data demonstrate a substantial waning of antibody responses and T cell immunity to SARS-CoV-2 and its variants, at 6 months following the second immunization with the BNT162b2 vaccine. Notably, a significant proportion of vaccinees have neutralizing titers below the detection limit, and suggest a 3rd booster immunization might be warranted to enhance the antibody titers and T cell responses. Competing Interest Statement The authors have declared no competing interest.

Neuromodulators influence critical functions of the developing human brain and regulate behavioral states. Dysfunction of neuromodulatory systems is often involved in neuropsychiatric disease and many drugs for these conditions act on these signaling pathways. Recent advances in stem cell biology have made it possible to derive a wide range of cells across the developing human nervous system in regionalized organoids and to functionally integrate them into assembloids, however they currently do not systematically incorporate neuromodulation. Here, we generated human midbrain-hindbrain organoids (hMHO) from human induced pluripotent stem (hiPS) cells and fused them with human cortical organoids (hCO) to form neuromodulatory assembloids (hNMA). We focus on serotonin (5-hydroxytryptamine, 5-HT) as one key neuromodulator and found characteristic gene expression patterns and electrophysiological properties of serotonergic neurons (5-HT neurons) in the hMHO. In hNMA, 5-HT neurons projected into hCO, released 5-HT and modulated cortical network activity. To explore the applicability of this system in human disease, we studied 22q11.2 deletion syndrome (22q11.2DS), a common microdeletion associated with high risk for neuropsychiatric disease and defects in 5-HT signaling. We found aberrant 5-HT dynamics in hNMA from patient hiPS cell lines that were rescued by administration of a selective serotonin reuptake inhibitor (SSRI). Taken together, hNMA can be used to study human 5-HT dynamics and uncover disease phenotypes which could facilitate therapeutic development.

Organizers are specialized cell populations that orchestrate cell patterning and axon guidance in the developing nervous system. Although non-human models have led to fundamental discoveries about the organization of the nervous system midline by the floor plate, an experimental model of human floor plate would enable broader insights into regulation of human neurodevelopment and midline connectivity. Here, we have developed stem cell-derived organoids resembling human floor plate (hFpO) and assembled them with spinal cord organoids (hSpO) to generate midline assembloids (hMA). We demonstrate that hFpO promote Sonic hedgehog-dependent ventral patterning of human spinal progenitors and Netrin-dependent guidance of human commissural axons, paralleling non-human models. To investigate evolutionary-divergent midline regulators, we profiled the hFpO secretome and identified 27 evolutionarily divergent genes between human and mouse. Utilizing the hMA platform, we targeted these candidates in an arrayed CRISPR knockout screen and reveal that , a gene involved in O-linked glycosylation, impairs floor plate-mediated guidance of commissural axons in humans. This novel platform extends prior axon guidance discoveries into human-specific neurobiology with implications for mechanisms of nervous system evolution and neurodevelopmental disorders.

A medicina, outrora estável e com vertentes mais curativas, passou a incidir sobre a vertente preventiva e do bem-estar físico e mental. Sendo o Imposto sobre o Valor Acrescentado (IVA) uma importante fonte de receita do Estado Português, torna-se crucial entender o seu comportamento e aplicabilidade ao ramo emergente.Neste estudo, pretende-se determinar em que medida o reconhecimento de benefícios psicológicos pode afetar a aplicação do IVA na prestação dos serviços de cirurgia plástica, reconstrutiva e medicina estética e qual tem sido o entendimento dos tribunais portugueses e do TJUEPara isso, adotou-se uma abordagem de investigação de natureza qualitativa, hermenêutica e jurídico-tributária, com análise aprofundada de normas, diretivas e jurisprudências pertinentes.As conclusões da investigação revelam que tanto a jurisprudência nacional como a comunitária estão inclinadas a reconhecer benefícios psicológicos como um fator que pode requalificar um procedimento estético como terapêutico, levando à possível isenção de IVA. No entanto, diversas áreas cinzentas permanecem, notadamente quanto à definição de \"finalidade terapêutica\" e e aos profissionais habilitados para tal avaliação. Este estudo salienta a necessidade urgente de atualizar e esclarecer as diretrizes e legislações, alinhando-as com os avanços na compreensão médica e social da terapia, e contribuindo para políticas fiscais e de saúde mais eficazes e justas.

A medicina, outrora estável e com vertentes mais curativas, passou a incidir sobre a vertente preventiva e do bem-estar físico e mental. Sendo o Imposto sobre o Valor Acrescentado (IVA) uma importante fonte de receita do Estado Português, torna-se crucial entender o seu comportamento e aplicabilidade ao ramo emergente.Neste estudo, pretende-se determinar em que medida o reconhecimento de benefícios psicológicos pode afetar a aplicação do IVA na prestação dos serviços de cirurgia plástica, reconstrutiva e medicina estética e qual tem sido o entendimento dos tribunais portugueses e do TJUEPara isso, adotou-se uma abordagem de investigação de natureza qualitativa, hermenêutica e jurídico-tributária, com análise aprofundada de normas, diretivas e jurisprudências pertinentes.As conclusões da investigação revelam que tanto a jurisprudência nacional como a comunitária estão inclinadas a reconhecer benefícios psicológicos como um fator que pode requalificar um procedimento estético como terapêutico, levando à possível isenção de IVA. No entanto, diversas áreas cinzentas permanecem, notadamente quanto à definição de \"finalidade terapêutica\" e e aos profissionais habilitados para tal avaliação. Este estudo salienta a necessidade urgente de atualizar e esclarecer as diretrizes e legislações, alinhando-as com os avanços na compreensão médica e social da terapia, e contribuindo para políticas fiscais e de saúde mais eficazes e justas.

The assembly of cortical circuits involves the generation and migration of cortical interneurons from the ventral to the dorsal forebrain, which has been challenging to study in humans as these processes take place at inaccessible stages of late gestation and early postnatal development. Autism spectrum disorder (ASD) and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development, but which of the hundreds of NDD genes impact interneuron generation and migration into circuits and how they mediate these effects remain unknown. We previously developed a stem cell-based platform to study human cortical interneurons in self-organizing organoids resembling the ventral forebrain and their migration using forebrain assembloids. Here, we integrate assembloid technology with CRISPR screening to systematically investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including the RNA-binding protein CSDE1 and the canonical TGFβ signaling activator SMAD4. Then, we ran an interneuron migration screen in ∼1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and, notably, the endoplasmic reticulum (ER)-related gene LNPK. Interestingly, we discovered that, during interneuron migration, the ER is displaced along the leading neuronal branch prior to nuclear translocation. Deletion of LNPK interfered with this ER displacement and resulted in reduced interneuron saltation length, indicating a critical role for the ER in this migratory process. Taken together, these results highlight how this versatile CRISPR-assembloid platform can be used to systematically map disease genes onto early stages of human neural development and to reveal novel mechanisms regulating interneuron development.

The COVID pandemic initially led to the unprecedented suspension of large numbers of clinical research studies and trials that required in-person study visits, highlighting the need to reevaluate how human participant research projects are conducted. During this time the University of Pittsburgh Clinical and Translational Science Institute (Pitt CTSI) embarked on a multi-year initiative to envision the Research Visit of the Future, which ultimately led to an academia–industry partnership that enables the scalable self-service model using a virtual clinical research platform for clinical studies at a major academic research institution. This model enables the flexible deployment of eConsent, Telehealth (audio/video) calls, synchronous and asynchronous data capture, integrated sensors and wearables, and patient engagement tools, all within a single participant-focused research platform. Academic investigators can configure their studies without custom coding across a wide range of study designs, including hybrid- and fully decentralized clinical trials. Here we present the journey to identifying the technology requirements to enrich data collection, structure of our partnership model, and considerations for crossing the digital divide to enable broader access to clinical research among diverse and under-represented communities.