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.

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.

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.

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.

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.

Inhibition of SARS-CoV-2 infection in human iPSC-derived cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoarchitecture and beating.

SARS-CoV-2 infects cardiac cells and causes heart dysfunction. Conditions such as myocarditis and arrhythmia have been reported in COVID-19 patients. The Sigma-1 receptor (S1R) is a ubiquitously expressed chaperone that plays a central role in cardiomyocyte function. S1R has been proposed as a therapeutic target because it may affect SARS-CoV-2 replication; however, the impact of the inhibition of S1R in human cardiomyocytes remains to be described. In this study, we investigated the consequences of S1R inhibition in iPSC-derived human cardiomyocytes (hiPSC-CM). SARS-CoV-2 infection in hiPSC-CM was productive and reduced cell survival. S1R inhibition decreased both the number of infected cells and viral particles after 48 hours. S1R inhibition also prevented the release of pro-inflammatory cytokines and cell death. Although the S1R antagonist NE-100 triggered those protective effects, it compromised cytoskeleton integrity by downregulating the expression of structural-related genes and reducing beating frequency. Our findings suggest that the detrimental effects of S1R inhibition in human cardiomyocytes' integrity may abrogate its therapeutic potential against COVID and should be carefully considered.

Genetic switches designed for eukaryotic cells and controlled by serine integrases.

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

The cyanobacterial saxitoxin exacerbates neural cell death and brain malformations induced by Zika virus.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.

TET2 haploinsufficiency alters reprogramming into induced pluripotent stem cells.

The discovery of the Ten-Eleven Translocation (TET) protein family was initiated by the identification of the MLL partner TET1, and of mutations in the TET2 gene in hematological malignancies including myeloproliferative neoplasms (MPN). TET1, 2 and 3 proteins hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Previous studies highlight the involvement of TET proteins in somatic cells reprogramming into induced pluripotent stem cells (iPSC), particularly Tet1 and 2 in mouse and TET1 in human. Here, we asked whether endogenous TET2 knockdown also displays this function. Using different shRNA against TET2, we provide evidence that TET2 strongly decreases the reprogramming of human hematopoietic progenitor cells into iPSC. Importantly, using 2 MPN patients, we observed that TET2 mutations affecting catalytic domain allowed iPSC generation. Instead, using another TET2 and TET3-mutated patient, we could only reprogram IPSC with TET3 mutation alone, suggesting that the type of TET2 mutation and/or the cooperation with TET3 mutations may alter the reprogramming activity. Altogether, this work highlights the importance of endogenous TET in the reprogramming process of human hematopoietic progenitors.

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.

Human Cerebral Organoids and Fetal Brain Tissue Share Proteomic Similarities.

The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases.