Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G.

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.

Early prediction of pre-eclampsia using circulating placental exosomes: Newer insights.

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.

Development and characterization of a new muscle cell culture system from Clarias magur (Hamilton, 1822).

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%.

Identifying novel risk conferring genes involved in glycosylation processes with familial schizophrenia in an Indian cohort: Prediction of ADAMTS9 gene variant for structural stability.

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.

Development and characterization of a new gill cell line from the striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878).

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.

Development and characterization of a new DRCF cell line from Indian wild strain zebrafish Danio rerio (Hamilton 1822).

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.

Novel Technique of Vaginoplasty Developing Normal Vagina, Role of Stemness Markers and Translational Genes.

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.

Lenz-Majewski syndrome: Report of a case with novel mutation in PTDSS1 gene.

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.

Novel mutation in forkhead box G1 (FOXG1) gene in an Indian patient with Rett syndrome.

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.

Identification of novel mutations in STAR gene in patients with lipoid congenital adrenal hyperplasia: a first report from India.

Lipoid congenital adrenal hyperplasia (LCAH), a rare disorder of steroid biosynthesis, is the most severe form of CAH. We report novel molecular findings of three unrelated infants with LCAH diagnosed at our center. A known missense mutation c.653C>T (p.A218V) and two novel mutations [premature termination c.441G>A (or p.W147X) and frameshift deletion c.del815G (or p.R272PfsX35)] were identified after complete sequencing of the STAR gene. Prenatal diagnosis was carried out for the family with mutation c.815delG by molecular testing wherein the fetus was found to be homozygous for the mutation. This is the first report of molecular diagnosis and prenatal testing for LCAH from India.

Mutational analysis of methyl-CpG binding protein 2 (MECP2) gene in Indian cases of Rett syndrome.

Rett syndrome (RTT) is an X-linked postnatal neurological disorder, primarily affecting females and characterized by regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. RTT is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 200 individual nucleotide changes in the gene, which cause pathogenic mutations, have been reported; however, eight most commonly occurring missense and nonsense mutations account for almost 70% of all mutations. RTT cases have also been reported from India. The phenotype (classical and atypical inclusive) has many differentials. However, a genetically based confirmed diagnosis would help in management and counseling. In this pilot study we have analyzed MECP2 mutations in ten Indian sporadic patients diagnosed clinically as having RTT. Two mutations and one novel variant in MECP2 have been detected. Missense mutations p.R133C and c.806delG have been detected. The missence mutation p.R133C was the part of eight hotspots reported in Rett patients. This patient met all the essential criteria except delayed onset of regression. The other c.806delG mutation positive patient also fulfilled all the obligatory criteria of classical RTT. Another clinically atypical Rett patient showed a novel mutation p.C339S in MECP2 gene. The preliminary result necessitates a large-scale study of RTT patients to determine more precisely the influence of MECP2 mutations in Indian patients and their correlation with clinical phenotypes.

Mutation analysis of mitogen activated protein kinase 1 gene in Indian cases of 46,XY disorder of sex development.

BACKGROUND: Determination of sex is the result of cascade of molecular events that cause undifferentiated bipotential gonad to develop as a testis or an ovary. A series of genes such as SRY, steroidogenic factor-1 (SF1), AR, SRD5 α, Desert hedgehog (DHH) etc., have been reported to have a significant role in development of sex in the fetus and secondary sexual characteristics at the time of puberty. Recently, mitogen activated protein kinase kinase kinase 1 (MAP3K1) gene was found to be associated with 46, XY disorders of sex development (DSD). AIM: The present study is focused to identify mutations in MAP3K1 gene in the cohort of 10 Indian patients with 46,XY DSD including one family with two affected sisters. These patients were already screened for SRY, SF1 and DHH gene, but no mutation was observed in any of these genes. MATERIALS AND METHODS: The entire coding regions of MAP3K1 were amplified and sequenced using the gene specific primers. RESULTS AND DISCUSSIONS: Sequence analysis of MAP3K1 gene has revealed four variants including one missense, two silent and one deletion mutation. The missense mutation p.D806N was observed in four patients with hypospadias. Two patients showed the presence of silent mutation p.Q1028Q present in exon 14. Another silent mutation p.T428T was observed in a patient with gonadal dysgenesis. We have also observed one deletion mutation p. 942insT present in two patients. The pathogenicity of the missense mutation p.D806N was carried out using in-silico approach. Sequence homology analysis has revealed that the aspartate at 806 was found to be well-conserved across species, indicated the importance of this residue. The score for polyphen analysis of this mutation was found to be 0.999 indicating to be pathogenic mutation. Since, p.D806N mutation was found to be important residue; it might contribute to sexual development. We have reported the presence of mutations/polymorphism in MAP3K1 gene. All the mutations were found to be polymorphism upon comparing to single nucleotide polymorphism database. However, in-silico analysis of the missense mutation revealed to be a pathogenic mutation.

Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome. A total of 62 (69%) patients remained without molecular genetics diagnosis that necessitates further search for mutations in other genes like CDKL5 and FOXG1 that are known to cause Rett phenotype. The majority of mutations are detected in exon 4 and only one mutation was present in exon 3. Therefore, our study suggests the need for screening exon 4 of MECP2 as first line of diagnosis in these patients.

Novel mutations in cyclin-dependent kinase-like 5 (CDKL5) gene in Indian cases of Rett syndrome.

Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Both the classic and atypical forms of Rett syndrome are primarily due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with atypical Rett syndrome, X-linked infantile spasms sharing common features of generally early-onset seizures and mental retardation. CDKL5 is known as serine/threonine protein kinase 9 (STK9) and is mapped to the Xp22 region. It has a conserved serine/threonine kinase domain within its amino terminus and a large C-terminal region. Disease-causing mutations are distributed in both the amino terminal domain and in the large C-terminal domain. We have screened the CDKL5 gene in 44 patients with atypical Rett syndrome who had tested negative for MECP2 gene mutations and have identified 6 sequence variants, out of which three were novel and three known mutations. Two of these novel mutations p.V966I and p.A1011V were missense and p.H589H a silent mutation. Other known mutations identified were p.V999M, p.Q791P and p.T734A. Sequence homology for all the mutations revealed that the two mutations (p.Q791P and p.T734A) were conserved across species. This indicated the importance of these residues in structure and function of the protein. The damaging effects of these mutations were analysed in silico using PolyPhen-2 online software. The PolyPhen-2 scores of p.Q791P and p.T734A were 0.998 and 0.48, revealing that these mutations could be deleterious and might have potential functional effect. All other mutations had a low score suggesting that they might not alter the activity of CDKL5. We have also analysed the position of the mutations in the CDKL5 protein and found that all the mutations were present in the C-terminal domain of the protein. The C-terminal domain is required for cellular localization through protein-protein interaction; any mutations in this domain might alter this function of the protein. This is the first report from India showing the mutation in CDKL5 gene in Indian cases of Rett syndrome. Our study emphasizes the role of CDKL5 mutation screening in cases of atypical Rett syndrome with congenital seizure variant.

Novel homozygous mutations in Desert hedgehog gene in patients with 46,XY complete gonadal dysgenesis and prediction of its structural and functional implications by computational methods.

Male to female sex reversal in patients with 46,XY karyotype results from the failure of development of testis which may be due to mutations in the SRY gene. Only 10-15% of cases of 46,XY gonadal dysgenesis are accounted for by different types of mutations in the SRY gene. Hence, majority of such patients may have mutations in other genes involved in the testicular differentiation pathway. Besides SRY, other autosomal and X-linked genes are also involved in sexual development during embryogenesis. We describe here the first report from India wherein, two cases of 46,XY complete gonadal dysgenesis that could be attributable to mutations in the Desert hedgehog (DHH) gene. The mutations found in these two patients were a homozygous deletion (c.271_273delGAG) that resulted in deletion of one amino acid (p.D90del) and a homozygous duplication (c.57-60dupAGCC) that resulted in premature termination resulting in non-functional DHH protein. The structure-function implications of the p.D90del mutation were predicted using computational tools. Structural studies on the p.D90del mutant revealed that the mutation could seriously perturb the interaction of DHH with its binding partners. This is the second report in literature showing homozygous mutation in cases with 46,XY complete gonadal dysgenesis.

Molecular cloning and characterization of beta-defensin cDNA expressed in distal ileum of buffalo (Bubalus bubalis).

Defensins play a prominent role in protection of various epithelial surfaces. In this study, we have cloned and characterized the mRNA from the distal ileum of Bubalus bubalis. Total RNA after isolation from ileal epithelium was reverse transcribed to synthesize cDNA using primers designed by taking conserved region of cattle enteric beta-defensin (EBD) mRNA, goat beta-defensin 2 (BD 2) and cattle lingual antimicrobial peptide (LAP) mRNA sequences. The PCR amplified cDNA of 254 bp was ligated to pDrive cloning vector and transformed into XL-blue strain of E coli. The sequence analysis indicated 29 nucleotide substitutions with reported cattle EBD mRNA sequence sharing 86.2% homology, 92.1% with cattle LAP, 81.6% with cattle tracheal antimicrobial peptide and 84.6% with goat BD 2. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide. Both nucleotide and amino acid sequence homology shows that the cloned sequence is closer to cattle LAP.