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.

Therapeutic metformin concentrations positively regulate proliferation in endometrial epithelial cells via mTOR activation and augmented mitochondrial strength.

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.

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%.

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.

Genetic heterogeneity and respiratory chain enzyme analysis in pediatric Indian patients with mitochondrial disorder: Report of novel variants in POLG1 gene and their functional implication using molecular dynamic simulation.

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.

Molecular insights into flavone-mediated quorum sensing interference: A novel strategy against Serratiamarcescens biofilm-induced antibiotic resistance".

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.

Differential proteomics of circulating extracellular vesicles of placental origin isolated from women with early-onset preeclampsia reveal aberrant innate immune and hemostasis processes.

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 ∼150 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.

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.

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.

Cytomegalovirus reactivation in a SARS-CoV-2 infected woman experiencing fetal demise in the first trimester with fetal trisomy 21: A case report.

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.

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.

Molecular cloning and evolution of the gene encoding the precursor of Nilgai (Boselaphus tragocamelus) interleukin 2.

Interleukin 2 is a key immunoregulatory cytokine that is involved in T helper differentiation and is known to induce interferon gamma (IFN-gamma) in natural killer (NK) and T-cell. The gene for interleukin-2 (IL-2) was amplified from cDNA pool prepared from ConAstimulated peripheral blood mononuclear cells (PBM cells) isolated from Nilgai reared in semicaptivity. The amplified IL-2 gene was cloned and nucleotide sequences were determined. The sequencing result showed that Nilgai IL-2 cDNA contains 468 bp long ORF encoding 155 amino acids (Genbank Accession No DQ017831). Homology comparison of nucleotide revealed that Nilgai IL-2 sequence is evolutionary more related to buffalo than cattle IL-2 sequence.

Role of progenitor cell producing normal vagina by metaplasia in laparoscopic peritoneal vaginoplasty.

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.

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.