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PROBLEM: Early-onset preeclampsia (EOPE) is a severe gestational hypertensive disorder with significant feto-maternal morbidity and mortality due to uteroplacental insufficiency. Circulating extracellular vesicles of placental origin (EV-P) are known to be involved in the pathophysiology of EOPE and might serve as an ideal reservoir for its specific biomarkers. Therefore, we aimed to characterize and perform comparative proteomics of circulating EV-P from healthy pregnant and EOPE women before delivery. METHOD OF STUDY: The EV-P from both groups were isolated using immunoaffinity and were characterized using transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis, and immunoblotting. Following IgG albumin depletion, the pooled proteins that were isolated from EV-P of both groups were subjected to quantitative TMT proteomics. RESULTS: Circulating term EV-P isolated from both groups revealed â¼150 nm spherical vesicles containing CD9 and CD63 along with placental PLAP and HLA-G proteins. Additionally, the concentration of EOPE-derived EV-P was significantly increased. A total of 208 proteins were identified, with 26 among them being differentially abundant in EV-P of EOPE women. This study linked the pathophysiology of EOPE to 19 known and seven novel proteins associated with innate immune responses such as complement and TLR signaling along with hemostasis and oxygen homeostasis. CONCLUSION: The theory suggesting circulating EVs of placental origin could mimic molecular information from the parent organ-"the placenta"-is strengthened by this study. The findings pave the way for possible discovery of novel prognostic and predictive biomarkers as well as provide insight into the mechanisms driving the pathogenesis of EOPE.
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Vesículas Extracelulares , Hemostasis , Inmunidad Innata , Placenta , Preeclampsia , Proteómica , Humanos , Femenino , Embarazo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/inmunología , Preeclampsia/inmunología , Preeclampsia/metabolismo , Adulto , Placenta/metabolismo , Placenta/inmunología , Biomarcadores/metabolismoRESUMEN
Antibiotic resistance poses a significant challenge in modern medicine, urging the exploration of innovative approaches to combat bacterial infections. Biofilms, complex bacterial communities encased in a protective matrix, contribute to resistance by impeding antibiotic efficacy and promoting genetic exchange. Understanding biofilm dynamics is crucial for developing effective antimicrobial therapies against antibiotic resistance. This study explores the potential of flavone to combat biofilm-induced antibiotic resistance by employing in-vitro biochemical, cell biology, and Insilico (MD simulation), approaches. Flavone exhibited potent antibacterial effects with a low minimum inhibitory concentration by inducing intracellular reactive oxygen species. Flavones further inhibited the formation of biofilms by 50-60 % and disrupted the pre-formed biofilms by reducing the extracellular polysaccharide substance protective layer formed on the biofilm by 80 %. Quorum sensing (QS) plays a crucial role in bacterial pathogenicity and flavone significantly attenuated the production of QS-induced virulence factors like urease, protease, lipase, hemolysin and prodigiosin pigment in a dose-dependent manner. Further Insilico molecular docking studies along with molecular dynamic simulations run for 100 ns proved the stable binding affinity of flavone with QS-specific proteins which are crucial for biofilm formation. This study demonstrates the therapeutic potential of flavone to target QS-signaling pathway to combat S.marcescens biofilms.
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Antibacterianos , Biopelículas , Flavonas , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Percepción de Quorum , Biopelículas/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Flavonas/farmacología , Flavonas/química , Antibacterianos/farmacología , Antibacterianos/química , Simulación de Dinámica Molecular , Especies Reactivas de Oxígeno/metabolismo , Farmacorresistencia Microbiana/efectos de los fármacos , Factores de Virulencia/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
In brief: Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. Abstract: Extracellular vesicles (EVs) are membrane-bound nanovesicles secreted from the cells into extracellular space and body fluids. They are considered 'fingerprints of parent cells', which can reflect their physiological and functional states. During pregnancy, EVs are produced by the syncytiotrophoblasts and extravillous trophoblasts and are released into the maternal bloodstream. In the present study, placental alkaline phosphatase (PLAP)-specific extracellular vesicles were isolated from maternal serum-derived EVs (SDE) across pregnancy. Transmission electron microscopy and dynamic light scattering analysis showed that the isolated EVs exhibited a spherical morphology with ~30-150 nm size range. Nanoparticle tracking analysis indicated that the concentration of PLAP+ serum-derived EVs (PLAP+-SDE) increased across the gestation. PLAP+-SDE contained DNA with LINE1 promoter methylation pattern. C19 miRNA cluster miRNAs (miR 515-5p, 519e and 520f) were present in PLAP+-SDE along with other miRNAs (miR-133-3p, miR210-3p and miR-223-3p). PLAP+-SDE confirmed the presence of EV markers (CD63 and CD9), along with placental proteins (PLAP and cullin 7). A modified novel strategy to extract an enriched population of circulating placental/amniochorionic EVs was devised employing an additional marker of extravillous trophoblasts, human leukocyte antigen G (HLA-G), along with PLAP. The isolated pooled placental/amniochorionic (PLAP+&HLA-G+) serum-derived EVs (PP-SDE) showed ~two-fold increased protein levels of HLA-G in the third-trimester pregnant women compared to the non-pregnant controls. Future studies will be focused on validation of this novel strategy to isolate an enriched population of placental/amniochorionic EVs to facilitate a better understanding of placental physiology and pathophysiology.
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Vesículas Extracelulares , MicroARNs , Proteínas Gestacionales , Embarazo , Femenino , Humanos , Placenta/metabolismo , Antígenos HLA-G/metabolismo , Vesículas Extracelulares/metabolismo , Trofoblastos/metabolismo , MicroARNs/metabolismo , Proteínas Gestacionales/metabolismoRESUMEN
Mitochondrial disorders are a heterogeneous group of disorders caused by mutations in the mitochondrial DNA or in nuclear genes encoding the mitochondrial proteins and subunits. Polymerase Gamma (POLG) is a nuclear gene and mutation in the POLG gene are one of the major causes of inherited mitochondrial disorders. In this study, 15 pediatric patients, with a wide spectrum of clinical phenotypes were screened using blood samples (n = 15) and muscle samples (n = 4). Respiratory chain enzyme analysis in the muscle samples revealed multi-complex deficiencies with Complex I deficiency present in (1/4) patients, Complex II (2/4), Complex III (3/4) and Complex IV (2/4) patients. Multiple large deletions were observed in 4/15 patients using LR-PCR. Whole exome sequencing (WES) revealed a compound heterozygous mutation consisting of a POLG1 novel variant (NP_002684.1:p.Trp261X) and a missense variant (NP_002684.1:p. Leu304Arg) in one patient and another patient harboring a novel homozygous POLG1 variant (NP_002684.1:p. Phe750Val). These variants (NP_002684.1:p. Leu304Arg) and (NP_002684.1:p. Phe750Val) and their interactions with DNA were modelled using molecular docking and molecular dynamics (MD) simulation studies. The protein conformation was analyzed as root mean square deviation (RMSD), root mean square fluctuation (RMSF) which showed local fluctuations in the mutants compared to the wildtype. However, Solvent Accessible Surface Area (SASA) significantly increased for NP_002684.1:p.Leu304Arg and decreased in NP_002684.1:p.Phe750Val mutants. Further, Contact Order analysis indicated that the Aromatic-sulfur interactions were destabilizing in the mutants. Overall, these in-silico analysis has revealed a destabilizing mutations suggesting pathogenic variants in POLG1 gene.
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ADN Polimerasa gamma , Enfermedades Mitocondriales , Simulación de Dinámica Molecular , Humanos , ADN Polimerasa gamma/genética , Enfermedades Mitocondriales/genética , Niño , Masculino , Preescolar , Femenino , India , Lactante , Heterogeneidad Genética , Transporte de Electrón/genética , Adolescente , Mutación , Secuenciación del ExomaRESUMEN
Pre-eclampsia (PE), a multifactorial de novo hypertensive pregnancy disorder, is one of the leading causes of foeto-maternal morbidity and mortality. Currently, antihypertensive drugs are the first-line therapy for PE and evidence suggests that low-dose aspirin initiated early in high risk pregnancies may reduce the risk of development or severity of PE. However, an early prediction of this disorder remains an unmet clinical challenge. Several potential serum biomarkers associated with maternal immunoregulation and placental angiogenesis have been evaluated but are ineffective and inconsistent for early prediction. Although placental biomarkers would be more specific and sensitive in predicting the risk of PE, accessing the placenta during pregnancy is not feasible. Circulating placental exosomes (pEXO), originating from foeto-maternal interface, are being evaluated as the placenta's surrogate and the best source of non-invasive placental biomarkers. pEXO appear in the maternal circulation starting from six weeks of gestation and its dynamic biological cargo across pregnancy is associated with successful pregnancy outcomes. Therefore, monitoring changes in pEXO expression profiles could provide new insights into the prediction, diagnosis and treatment of PE. This narrative review comprehensively summarizes the available literature on the candidate predictive circulating biomarkers evaluated for PE to date. In particular, the review elucidates the current knowledge of distinct molecular signatures emanating from pEXO in pre-eclamptic women to support the discovery of novel early predictive biomarkers for effective intervention and management of the disease.
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Exosomas , Hipertensión , Preeclampsia , Embarazo , Femenino , Humanos , Placenta/metabolismo , Preeclampsia/diagnóstico , Exosomas/metabolismo , Resultado del Embarazo , BiomarcadoresRESUMEN
The cell line has been used as a novel in vitro tool for executing several studies in life sciences. The current study aimed to develop and characterize a muscle cell culture system derived from Clarias magur. The primary muscle cell cultures derived from the caudal peduncle muscle have been successfully sub cultured up to 13 passages to establish a new muscle cell culture system known as CMM. At a temperature of 28 °C, L-15 medium supplemented with 20% FBS produced the maximum growth of muscle cells. However, muscle cells were optimized to grow at 10% FBS. To enhance the proliferation capacity of the CMM cells, a growth-promoting factor bFGF (10 ng/ml) was added, thereby reducing the time interval of passages for the subsequent cultures. DNA barcoding of the CMM cell culture system authenticated the species of origin. The cell culture system was successfully cryopreserved by a slow freezing procedure at - 80 °C with a revival efficiency of 60%.
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Bagres , Animales , Bagres/metabolismo , Músculos , Línea Celular , Criopreservación/veterinaria , Técnicas de Cultivo de CélulaRESUMEN
Schizophrenia is a complex neuropsychiatric disorder and heritability is as high as 80 % making it the most heritable mental disorder. Although GWAS has identified numerous variants, the pathophysiology is still elusive. Here, an attempt was made to identify genetic risk factors in familial cases of schizophrenia that are associated with a common causative pathway. To achieve this objective, exome sequencing was done in 4 familial cases and identified six unique coding variants in five genes. Among these genes, PIGQ gene has two pathogenic variants, one nonsense and in-frame deletion. One missense variant in GALNT16 and one in GALNT5 have variable damaging score, however, the other variants, in ADAMTS9 and in LTBP4 have the highest damaging score. Further analysis showed that the variant of LTBP4 was not present in the functional domain. The other missense variant in the ADAMTS9 gene was found to be significant and was present in the thrombospondin repeat motif, one of the important motifs. Detailed molecular dynamics simulation study on this variant showed a damaging effect on structural stability. Since, all these genes culminated into the glycosylation process, it was evident that an aberrant glycosylation process may be one of the risk factors. Although, extracellular matrix formation through glycosylation have been shown to be associated, the involvement of ADAMTS9 and PIGQ gene mediated glycosylation has not been reported. In this paper, a novel link between ADAMTS9 and PIGQ gene with schizophrenia have been reported. Therefore, this novel observation has contributed immensely to the existing knowledge on risk factor of Schizophrenia.
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Trastornos Psicóticos , Esquizofrenia , Humanos , Esquizofrenia/genética , Glicosilación , Predisposición Genética a la Enfermedad , Mutación Missense , Proteína ADAMTS9/genética , Proteínas de la Membrana/genéticaRESUMEN
Cytomegalovirus (CMV) is the most common cause of congenital viral infections. Women seropositive for CMV prior to pregnancy can develop a non-primary CMV infection. Here, we present a case of first trimester pregnancy loss during active SARS-CoV-2 infection. There was no evidence of SARS-CoV-2 RNA in placenta and fetal tissue, but there was presence of congenital cytomegalovirus infection by nested PCR. To the best of our knowledge, this is the first report demonstrating association of early congenital CMV infection due to reactivation and fetal demise in a SARS-CoV-2 positive woman with fetal trisomy 21.
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COVID-19 , Infecciones por Citomegalovirus , Síndrome de Down , Embarazo , Femenino , Humanos , SARS-CoV-2 , Citomegalovirus , Primer Trimestre del Embarazo , ARN Viral , Feto , Muerte FetalRESUMEN
Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders, including reproductive disorders such as polycystic ovarian syndrome, where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mmol/L) of metformin have reported antiproliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20 and 70 µmol/L. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found to be mammalian target of rapamycin (mTOR) dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMP-activated protein kinase (AMPK) signaling. On the contrary, supratherapeutic metformin concentration (10 mmol/L) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation, and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitoTracker™ Red staining and enhanced succinate dehydrogenase expression; however, higher concentration (10 mmol/L) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR-dependent endometrial cell proliferation.
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Neoplasias Endometriales , Metformina , Animales , Femenino , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Proliferación Celular , Células Epiteliales/metabolismo , Metformina/farmacología , Metformina/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Cell lines as an in vitro model developed from different target organs of fish find their use in virus susceptibility, cytotoxicity, gene expression studies. The striped catfish, Pangasianodon hypophthalmus, is one of the main species in aquaculture, especially in Southeast Asian countries like Thailand, Indonesia, China, India, Bangladesh, and Vietnam. The present study reports the development of a new permanent cell line from the gills of P. hypophthalmus designated as PHG and its application in toxicological research. Leibovitz's L-15 cell culture medium supplemented with 15% fetal bovine serum (FBS) was used to maintain cell line PHG. The morphology of the PHG cell line was observed fibroblastic-like. PHG cells grew well at varying temperatures ranging from 24 to 30 °C with an optimum temperature of 28 °C. The PHG cell line was characterized using a sequence of mitochondrial cytochrome C oxidase subunit I, which authenticated the species of origin of the cell line. The cell line was transfected with a pEGFP-C1 plasmid, and the transfection reporter gene was successfully expressed 48 h post-transfection with 9% transfection efficiency. The toxicity assessment of two organophosphate pesticides, chlorpyrifos, and malathion using the PHG cell line revealed that the two organophosphate pesticides were cytotoxic to the cell line at varying concentrations.
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Bagres , Insecticidas , Animales , Bagres/genética , Línea Celular , Branquias , OrganofosfatosRESUMEN
Danio rerio, zebrafish, has been widely used as a non-mammalian vertebrate model organism in various studies. The present research describes to develop and characterize a new cell line from a wild strain Indian zebrafish native to Brahmaputra River, Assam, India. The new cell line designated as DRCF was developed from the caudal fin of D. rerio. The cell line was successfully subcultured up to 31 passages. Growth studies revealed that cell growth of DRCF was optimal at 28 °C in L-15 medium supplemented with 20% FBS. Molecular characterization of the DRCF cell line using mitochondrial genes namely cytochrome oxidase subunit I gene (COI) and 16S rRNA authenticated the true origin of the cell line. The chromosome analysis of the DRCF cell line expressed its 50 diploid chromosome number of D. rerio. The immunocytochemical characterization of the cell line exhibited its fibroblastic morphology. The expression of the green fluorescent protein (GFP) following transfection revealed the suitability of the cell line for transfection studies.
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Aletas de Animales/citología , Pez Cebra/anatomía & histología , Animales , Línea Celular , Proliferación Celular , Cromosomas , Complejo IV de Transporte de Electrones/genética , Genes Mitocondriales , Inmunohistoquímica , India , Microscopía de Contraste de Fase , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Ríos , Estaciones del Año , Células Madre/citología , Transfección , Pez Cebra/genéticaRESUMEN
AIMS AND OBJECTIVES: To study development of neo-vagina by metaplastic conversion of peritoneum, To identify translational Stemness markers using NANOG/OCT4/SOX2 from serial neo-vaginal mRNA, cDNA and to study role of WNT and HOXA genes in patients undergoing vaginoplasty. MATERIAL AND METHODS: 75 MRKH Syndrome women underwent laparoscopic peritoneal vaginoplasty (LPV). Two patients underwent serial neo-vaginal biopsies on day 0, 7-9, 12-14, 21 and 33. Fifteen MRKHS and twelve controls were subjected for neo-vaginal biopsy to detect genes upregulation. Remaining patients were evaluated anatomically and functionally. RESULTS: The translational stemness markers NANOG, OCT4 and SOX2 responsible for neo-vaginal formation were identified. Their appearance, concentration at different stages of conversion were demonstrated. The neo-vagina has shown up-regulation of these translational stemness markers. The study demonstrates expression of the specific genes (WNT4, WNT5A and WNT7A) and their role in formation of the neo-vagina. In the subjects stemness markers (NANOG, OCT4 and SOX2) appeared from day 9 to 14 of the neo-vaginal biopsies and after achieving the peak declined later. Genetic analysis showed low values in HOXA 9,10,11,13 and up-regulation of WNT 4A,5A,7 genes in neo-vagina. CONCLUSIONS: Study shows peritoneal metaplastic conversion to normal vagina, identified the translational stemness markers and genes responsible. The neo-vagina has shown up-regulation of these genes. The study demonstrates expression of the specific genes (WNT4, WNT5A and WNT7A) and their role in formation of the neo-vagina. Furthering this research, activating these genes may lead to treatment of developmental defects of Mullerian duct, obviating the need of transplant.
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CONTEXT: Host of vaginoplasty techniques have been described. None has been successful in developing normal vagina. Laparoscopic peritoneal vaginoplasty (LPV) is performed in Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) culminating in normal vagina. AIMS: This study aims to confirm normal development of neovagina by anatomical and functional parameters of histology, cytology, and ultrasonography (USG) in LPV. To identify peritoneal progenitor cell by OCT4/SOX2 markers. To demonstrate the metaplastic conversion of peritoneum to neovagina and the progenitor cell concentration, distribution pattern. SETTINGS AND DESIGN: This is prospective experimental study, conducted at teaching hospital and private hospital. SUBJECTS AND METHODS: Fifteen women of MRKHS underwent LPV followed by histology, cytology, two-/three-dimensional USG of neovagina. Four women underwent peritoneal biopsy for identification of progenitor cells with OCT4/SOX2 markers. One patient underwent serial biopsies for 4 weeks for histology and progenitor cell immunohistochemistry. RESULTS: Normal vaginal histology and cytology were apparent. USG of neovagina showed normal appearance and blood flow. Two peritoneal samples confirmed the presence of progenitor cells. Serial biopsies demonstrated the epithelial change from single to multilayer with stromal compaction and neoangiogenesis. The progenitor cells concentration and different distribution patterns were described using SOX2/OCT4 markers. CONCLUSIONS: We have shown successful peritoneal metaplastic conversion to normal vagina in LPV. The progenitor cell was identified in normal peritoneum using SOX2/OCT4 markers. The progenitor cell concentration and pattern were demonstrated at various stages of neovaginal development.
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Lenz-Majewski syndrome (LMS) is an extremely rare syndrome characterized by osteosclerosis, intellectual disability, characteristic facies and distinct craniofacial, dental, cutaneous and distal - limb anomalies. Recently, mutations in PTDSS1 gene have been identified as causative in six unrelated individuals. We report the seventh mutation proven case of LMS and provide a concise review of all known patients till date.
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Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/diagnóstico , Enfermedades del Desarrollo Óseo/genética , Facies , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Mutación , Transferasas de Grupos Nitrogenados/genética , Anomalías Múltiples/diagnóstico por imagen , Anomalías Múltiples/patología , Secuencia de Bases , Enfermedades del Desarrollo Óseo/diagnóstico por imagen , Enfermedades del Desarrollo Óseo/patología , Preescolar , Exones , Expresión Génica , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/patología , Masculino , Datos de Secuencia Molecular , RadiografíaRESUMEN
BACKGROUND: Congenital lipoid adrenal hyperplasia presents with adrenal insufficiency and sex reversal in 46XY genetic males. CASE CHARACTERISTICS: Two patients (46 XY karyotype), one having ambiguous genitalia and other having female external genitalia, presented with adrenal crisis at 6 months and 4 weeks of age, respectively. OBSERVATION: Steroidogenic Acute Regulatory Protein gene sequencing revealed homozygous mutations in both patients. OUTCOME: Treatment with hydrocortisone and fludrocortisone resulted in marked improvement. MESSAGE: Congenital lipoid adrenal hyperplasia should be considered in infants having female or ambiguous genitalia, and presenting with adrenal insufficiency.
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Hiperplasia Suprarrenal Congénita , Trastorno del Desarrollo Sexual 46,XY , Femenino , Humanos , Lactante , Masculino , Fenotipo , Fosfoproteínas/genéticaRESUMEN
Rett syndrome (RTT) is a severe neurodevelopmental disorder characterized by the progressive loss of intellectual functioning, fine and gross motor skills and communicative abilities, deceleration of head growth, and the development of stereotypic hand movements, occurring after a period of normal development. The classic form of RTT involves mutation in MECP2 while the involvement of CDKL5 and FOXG1 genes has been identified in atypical RTT phenotype. FOXG1 gene encodes for a fork-head box protein G1, a transcription factor acting primarily as transcriptional repressor through DNA binding in the embryonic telencephalon as well as a number of other neurodevelopmental processes. In this report we have described the molecular analysis of FOXG1 gene in Indian patients with Rett syndrome. FOXG1 gene mutation analysis was done in a cohort of 34 MECP2/CDKL5 mutation negative RTT patients. We have identified a novel mutation (p. D263VfsX190) in FOXG1 gene in a patient with congenital variant of Rett syndrome. This mutation resulted into a frameshift, thereby causing an alteration in the reading frames of the entire coding sequence downstream of the mutation. The start position of the frameshift (Asp263) and amino acid towards the carboxyl terminal end of the protein was found to be well conserved across species using multiple sequence alignment. Since the mutation is located at forkhead binding domain, the resultant mutation disrupts the secondary structure of the protein making it non-functional. This is the first report from India showing mutation in FOXG1 gene in Rett syndrome.
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Factores de Transcripción Forkhead/genética , Mutación del Sistema de Lectura , Proteínas del Tejido Nervioso/genética , Síndrome de Rett/genética , Secuencia de Aminoácidos , Animales , Niño , Femenino , Factores de Transcripción Forkhead/química , Humanos , India , Proteína 2 de Unión a Metil-CpG/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas Serina-Treonina Quinasas/genética , Síndrome de Rett/diagnóstico , Homología de Secuencia de AminoácidoRESUMEN
Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.