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.

Short and long TNF-alpha exposure recapitulates canonical astrogliosis events in human-induced pluripotent stem cells-derived astrocytes.

Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1ß, IL-6, and TNF-α in the first hours following TNF-α stimulation. After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNF-α-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover, ~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction. Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing human astrogliosis with potential applicability as a platform to uncover novel biomarkers and drug targets to prevent or mitigate astrogliosis associated with human brain disorders.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual.

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.

Liquid biopsy evaluation of circulating tumor DNA, miRNAs, and cytokines in meningioma patients.

Introduction: Liquid biopsy is a non-invasive method used to detect cancer and monitor treatment responses by analyzing blood or other bodily fluids for cancer biomarkers. Meningiomas are the most common primary central nervous system tumors, and biomarkers play a crucial role in their diagnosis, prognosis, and treatment monitoring. The World Health Organization (WHO) classifies meningiomas based on tumor grades and molecular alterations in genes such as in NF2, AKT1, TRAF7, SMO, PIK3CA, KLF4, SMARCE1, BAP1, H3K27me3, TERT promoter, and CDKN2A/B. Liquid biopsy, specifically cell-free DNA (cfDNA) analysis, has shown potential for monitoring meningiomas as it can detect ctDNA release in the blood, unaffected by the blood-brain barrier. MicroRNAs (miRNAs) have also been found to be deregulated in various cancers, including meningiomas, presenting potential as diagnostic biomarkers. Additionally, studying cytokines in the tumor microenvironment may aid in establishing prognostic or diagnostic panels for meningiomas. Methods: In the present study we analyzed the DNA coming from both the plasma and tumor samples, in addition to analyze miRNA-21 and cytokines in the plasma of 28 meningioma patients. Discussion and Conclusion: Our findings indicate that the detection of ctDNA in the plasma of meningioma patients is feasible. However, it's important to note that certain challenges persist when comparing plasma DNA analysis to that of tumor tissues. In our study, we observed a paired identification of mutations in only one patient, highlighting the complexities involved. Furthermore, we successfully identified miR-21 and cytokines in the plasma samples. Notably, our analysis of Interleukin 6 (IL-6) unveiled higher expression in the clear cell subtype compared to the other types. Despite the ongoing research, the clinical implementation of liquid biopsy in meningiomas remains somewhat limited. Nevertheless, our promising results underscore the need for further investigation.

Pathophysiology and mechanisms of hearing impairment related to neonatal infection diseases.

The inner ear, the organ of equilibrium and hearing, has an extraordinarily complex and intricate arrangement. It contains highly specialized structures meticulously tailored to permit auditory processing. However, hearing also relies on both peripheral and central pathways responsible for the neuronal transmission of auditory information from the cochlea to the corresponding cortical regions. Understanding the anatomy and physiology of all components forming the auditory system is key to better comprehending the pathophysiology of each disease that causes hearing impairment. In this narrative review, the authors focus on the pathophysiology as well as on cellular and molecular mechanisms that lead to hearing loss in different neonatal infectious diseases. To accomplish this objective, the morphology and function of the main structures responsible for auditory processing and the immune response leading to hearing loss were explored. Altogether, this information permits the proper understanding of each infectious disease discussed.

Osteosarcoma, chondrosarcoma and Ewing sarcoma: Clinical aspects, biomarker discovery and liquid biopsy.

Bone sarcomas, although rare, are associated with significant morbidity and mortality. The most frequent primary bone cancers include osteosarcoma, chondrosarcoma and Ewing sarcoma. The treatment approaches are heterogeneous and mainly chosen based on precise tumour staging. Unfortunately, clinical outcome has not changed significantly in over 30 years and tumour grade is still the best prognosticator of metastatic disease and survival. An option to improve this scenario is to identify molecular biomarkers in the early stage of the disease, or even before the disease onset. Blood-based liquid biopsies are a promising, non-invasive way to achieve this goal and there are an increasing number of studies which investigate their potential application in bone cancer diagnosis, prognosis and personalised therapy. This review summarises the interplay between clinical and molecular aspects of the three main bone sarcomas, alongside biomarker discovery and promising applications of liquid biopsy in each tumour context.

Graphene: Insights on Biological, Radiochemical and Ecotoxicological Aspects.

Graphene, including graphene quantum dots, its oxide and unoxidized forms (pure graphene) have several properties, like fluorescence, electrical conductivity, theoretical surface area, low toxicity, and high biocompatibility. In this study, we evaluated genotoxicity (in silico analysis using the functional density theory-FDT), cytotoxicity (human glioblastoma cell line), in vivo pharmacokinetics, in vivo impact on microcirculation and cell internalization assay. It was also radiolabeled with lutetium 177 (177Lu), a beta emitter radioisotope to explore its therapeutic use as nanodrug. Finally, the impact of its disposal in the environment was analyzed using ecotoxicological evaluation. FDT analysis demonstrated that graphene can construct covalent and non-covalent bonds with different nucleobases, and graphene oxide is responsible for generation of reactive oxygen species (ROS), corroborating its genotoxicity. On the other hand, non-cytotoxic effect on glioblastoma cells could be demonstrated. The pharmacokinetics analysis showed high plasmatic concentration and clearance. Topical application of 0.1 and 1 mg/kg of graphene nanoparticles on the hamster skinfold preparation did not show inflammatory effect. The cell internalization assay showed that 1-hour post contact with cells, graphene can cross the plasmatic membrane and accumulate in the cytoplasm. Radio labeling with 177Lu is possible and its use as therapeutic nanosystem is viable. Finally, the ecotoxicity analysis showed that A. silina exposed to graphene showed pronounced uptake and absorption in the nauplii gut and formation of ROS. The data obtained showed that although being formed exclusively of carbon and carbon-oxygen, graphene and graphene oxide respectively generate somewhat contradictory results and more studies should be performed to certify the safety use of this nanoplatform.

Neuromechanisms of SARS-CoV-2: A Review.

Recent studies have suggested the neuroinvasive potential of severe acute respiratory coronavirus 2 (SARS-CoV-2). Notably, neuroinvasiveness might be involved in the pathophysiology of coronavirus disease 2019 (COVID-19). Some studies have demonstrated that synapse-connected routes may enable coronaviruses to access the central nervous system (CNS). However, evidence related to the presence of SARS-CoV-2 in the CNS, its direct impact on the CNS, and the contribution to symptoms suffered, remain sparse. Here, we review the current literature that indicates that SARS-CoV-2 can invade the nervous system. We also describe the neural circuits that are potentially affected by the virus and their possible role in the progress of COVID-19. In addition, we propose several strategies to understand, diagnose, and treat the neurological symptoms of COVID-19.

Zika virus infection leads to mitochondrial failure, oxidative stress and DNA damage in human iPSC-derived astrocytes.

Zika virus (ZIKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZIKV. However, the consequences of ZIKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZIKV infection in iPSC-derived astrocytes are presented. Our results show ROS imbalance, mitochondrial defects and DNA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the Northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit.

Characterization of a human induced Pluripotent Stem (iPS) cell line (INCABRi002-A) derived from a primary myelofibrosis patient harboring the 5-bp insertion in CALR and the p.W146X mutation in TP53.

Primary myelofibrosis (PMF) is a hematological malignancy characterized by activation of the JAK/STAT pathway and risk of leukemic transformation. In this study, we generated an induced Pluripotent Stem (iPS) cell line derived from a 65-year old male PMF patient carrying the 5-pb insertion in the CALR gene (CALRins5) and the c.437 G > A mutation in the TP53 gene (p.W146X). The newly derived PMF3.17 iPS cell line harbors the original mutations and was characterized as bona fide iPS. Resource table.

Generation and characterization of a human induced pluripotent stem (iPS) cell line derived from an acute myeloid leukemia patient evolving from primary myelofibrosis carrying the CALR 52bp deletion and the ASXL1 p.R693X mutation.

Peripheral blood sample was donated by a 61years old female patient diagnosed with acute myeloid leukemia secondary to a primary myelofibrosis harboring the 52-bp deletion in the CALR gene (c.1092_1143del, p.L367fs*46) and the R693X mutation in the ASXL1 gene (c.2077C>T, p.R693X). CD34+ cells were isolated from the sample and subjected to the reprogramming procedure by using the Sendai virus carrying the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc. iPS colonies generated retained the original mutations and displayed all the features of bona fide iPS cells.

Generation of urine iPS cell lines from patients with Attention Deficit Hyperactivity Disorder (ADHD) using a non-integrative method.

Urine samples were collected from three patients (2 males, 1 female) with clinically diagnosed Attention Deficit Hyperactivity Disorder (ADHD) according to DSM-5 criteria using semi-structured interviews (K-SADS adapted for adults) by a trained psychiatrist. Urine epithelial cell lines were established and expanded for subsequent reprogramming procedure. Induced pluripotent stem cells (iPSCs) were derived using integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, which includes Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

Centrosomal localisation of the cancer/testis (CT) antigens NY-ESO-1 and MAGE-C1 is regulated by proteasome activity in tumour cells.

The Cancer/Testis (CT) antigen family of genes are transcriptionally repressed in most human tissues but are atypically re-expressed in many malignant tumour types. Their restricted expression profile makes CT antigens ideal targets for cancer immunotherapy. As little is known about whether CT antigens may be regulated by post-translational processing, we investigated the mechanisms governing degradation of NY-ESO-1 and MAGE-C1 in selected cancer cell lines. Inhibitors of proteasome-mediated degradation induced the partitioning of NY-ESO-1 and MAGE-C1 into a detergent insoluble fraction. Moreover, this treatment also resulted in increased localisation of NY-ESO-1 and MAGE-C1 at the centrosome. Despite their interaction, relocation of either NY-ESO-1 or MAGE-C1 to the centrosome could occur independently of each other. Using a series of truncated fragments, the regions corresponding to NY-ESO-1(91-150) and MAGE-C1(900-1116) were established as important for controlling both stability and localisation of these CT antigens. Our findings demonstrate that the steady state levels of NY-ESO-1 and MAGE-C1 are regulated by proteasomal degradation and that both behave as aggregation-prone proteins upon accumulation. With proteasome inhibitors being increasingly used as front-line treatment in cancer, these data raise issues about CT antigen processing for antigenic presentation and therefore immunogenicity in cancer patients.

A comprehensive promoter landscape identifies a novel promoter for CD133 in restricted tissues, cancers, and stem cells.

PROM1 is the gene encoding prominin-1 or CD133, an important cell surface marker for the isolation of both normal and cancer stem cells. PROM1 transcripts initiate at a range of transcription start sites (TSS) associated with distinct tissue and cancer expression profiles. Using high resolution Cap Analysis of Gene Expression (CAGE) sequencing we characterize TSS utilization across a broad range of normal and developmental tissues. We identify a novel proximal promoter (P6) within CD133(+) melanoma cell lines and stem cells. Additional exon array sampling finds P6 to be active in populations enriched for mesenchyme, neural stem cells and within CD133(+) enriched Ewing sarcomas. The P6 promoter is enriched with respect to previously characterized PROM1 promoters for a HMGI/Y (HMGA1) family transcription factor binding site motif and exhibits different epigenetic modifications relative to the canonical promoter region of PROM1.

Prevalence and genetic diversity of torque teno virus in patients with systemic lupus erythematosus in a reference service in Mato Grosso do Sul.

UNLABELLED: Recent studies on the torque teno virus (TTV), genus Anellovirus, have allowed formulating the hypothesis that TTV may trigger autoimmune rheumatic diseases or have some pathogenic role in them. OBJECTIVES: To determine the frequency of TTV infection in patients with systemic lupus erythematosus (SLE), the genetic diversity of TTV, the correlation between TTV infection and SLE clinical manifestations, and SLE clinical course and serological profile. PATIENTS AND METHODS: Serum samples were obtained from 46 SLE patients treated at the University-Affiliated Hospital of Campo Grande (NHU/FAMED/UFMS), Brazil. For controls, serum samples were obtained from 46 healthy volunteer blood donors. Viral DNA was extracted from samples using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and amplified using nested PCR. RESULTS: Positivity for TTV was found in 17 (37%) of SLE patients and in only seven (15.2%) of the controls (z test, P = 0.03). There was no correlation between TTV infection, SLE clinical manifestations, SLE clinical course, and the serological profile of the patients evaluated. CONCLUSION: Further studies on the presence of TTV in SLE patients are required.

Implications of aneuploidy for stem cell biology and brain therapeutics.

Understanding the cellular basis of neurological disorders have advanced at a slow pace, especially due to the extreme invasiveness of brain biopsying and limitations of cell lines and animal models that have been used. Since the derivation of pluripotent stem cells (PSCs), a novel source of cells for regenerative medicine and disease modeling has become available, holding great potential for the neurology field. However, safety for therapy and accurateness for modeling have been a matter of intense debate, considering that genomic instability, including the gain and loss of chromosomes (aneuploidy), has been repeatedly observed in those cells. Despite the fact that recent reports have described some degree of aneuploidy as being normal during neuronal differentiation and present in healthy human brains, this phenomenon is particularly controversial since it has traditionally been associated with cancer and disabling syndromes. It is therefore necessary to appreciate, to which extent, aneuploid pluripotent stem cells are suitable for regenerative medicine and neurological modeling and also the limits that separate constitutive from disease-related aneuploidy. In this review, recent findings regarding chromosomal instability in PSCs and within the brain will be discussed.

Frequent MAGE mutations in human melanoma.

BACKGROUND: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. SIGNIFICANCE: These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.

Prevalência e diversidade genética do torque teno vírus em pacientes com lúpus eritematoso sistêmico em serviço de referência no Mato Grosso do Sul / Prevalence and genetic diversity of torque teno virus in patients with systemic lupus erythematosus in a reference service in Mato Grosso do Sul

Estudos recentes sobre o torque teno vírus (TTV), gênero Anellovirus, permitiram construir a hipótese de que esse vírus pode ser um desencadeante ou tenha algum papel patogênico nas doenças reumáticas autoimunes. OBJETIVOS: Verificar a frequência da infecção pelo TTV em pacientes com lúpus eritematoso sistêmico (LES), e sua diversidade gênica, a existência de correlação entre a infecção pelo TTV e as manifestações clínicas do LES, sua evolução clínica e o perfil sorológico. PACIENTES E MÉTODOS: Foram obtidas 46 amostras de soro de pacientes com LES atendidos no Ambulatório de Reumatologia do Hospital Universitário de Campo Grande (NHU/FAMED/UFMS). Para os controles, utilizaram-se 46 amostras de soro de doadores de sangue. O DNA viral foi extraído das amostras utilizando o QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Alemanha), e amplificado utilizando a técnica de nested PCR. RESULTADOS: Foi encontrada positividade para o TTV em 17 (37 por cento) dos pacientes lúpicos, e em apenas sete (15,2 por cento) dos controles (teste z, P = 0,03). Não houve correlação entre a infecção pelo TTV, as manifestações clínicas, o perfil sorológico e a evolução clínica dos pacientes avaliados neste estudo. CONCLUSÃO: A presença do TTV nos pacientes com LES necessita ser mais bem compreendida a partir deste estudo inicial.

Recent studies on the torque teno virus (TTV), genus Anellovirus, have allowed formulating the hypothesis that TTV may trigger autoimmune rheumatic diseases or have some pathogenic role in them. OBJECTIVES: To determine the frequency of TTV infection in patients with systemic lupus erythematosus (SLE), the genetic diversity of TTV, the correlation between TTV infection and SLE clinical manifestations, and SLE clinical course and serological profile. PATIENTS AND METHODS:Serum samples were obtained from 46 SLE patients treated at the University-Affiliated Hospital of Campo Grande (NHU/FAMED/UFMS), Brazil. For controls, serum samples were obtained from 46 healthy volunteer blood donors. Viral DNA was extracted from samples using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) and amplified using nested PCR. RESULTS: Positivity for TTV was found in 17 (37 percent) of SLE patients and in only seven (15.2 percent) of the controls (z test, P = 0.03). There was no correlation between TTV infection, SLE clinical manifestations, SLE clinical course, and the serological profile of the patients evaluated. CONCLUSION: Further studies on the presence of TTV in SLE patients are required.