DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

STUDY QUESTION: What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY: RPL is defined as loss of two or more pregnancies, affecting 1-2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION: In this case-control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION: In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS: Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Functional mechanisms of drought tolerance in subtropical maize (Zea mays L.) identified using genome-wide association mapping.

BACKGROUND: Earlier studies were focused on the genetics of temperate and tropical maize under drought. We identified genetic loci and their association with functional mechanisms in 240 accessions of subtropical maize using a high-density marker set under water stress. RESULTS: Out of 61 significant SNPs (11 were false-discovery-rate-corrected associations), identified across agronomic traits, models, and locations by subjecting the accessions to water stress at flowering stage, 48% were associated with drought-tolerant genes. Maize gene models revealed that SNPs mapped for agronomic traits were in fact associated with number of functional traits as follows: stomatal closure, 28; flowering, 15; root development, 5; detoxification, 4; and reduced water potential, 2. Interactions of these SNPS through the functional traits could lead to drought tolerance. The SNPs associated with ABA-dependent signalling pathways played a major role in the plant's response to stress by regulating a series of functions including flowering, root development, auxin metabolism, guard cell functions, and scavenging reactive oxygen species (ROS). ABA signalling genes regulate flowering through epigenetic changes in stress-responsive genes. ROS generated by ABA signalling are reduced by the interplay between ethylene, ABA, and detoxification signalling transductions. Integration of ABA-signalling genes with auxin-inducible genes regulates root development which in turn, maintains the water balance by regulating electrochemical gradient in plant. CONCLUSIONS: Several genes are directly or indirectly involved in the functioning of agronomic traits related to water stress. Genes involved in these crucial biological functions interacted significantly in order to maintain the primary as well as exclusive functions related to coping with water stress. SNPs associated with drought-tolerant genes involved in strategic biological functions will be useful to understand the mechanisms of drought tolerance in subtropical maize.

Unraveling the genetic architecture of subtropical maize (Zea mays L.) lines to assess their utility in breeding programs.

BACKGROUND: Maize is an increasingly important food crop in southeast Asia. The elucidation of its genetic architecture, accomplished by exploring quantitative trait loci and useful alleles in various lines across numerous breeding programs, is therefore of great interest. The present study aimed to characterize subtropical maize lines using high-quality SNPs distributed throughout the genome. RESULTS: We genotyped a panel of 240 subtropical elite maize inbred lines and carried out linkage disequilibrium, genetic diversity, population structure, and principal component analyses on the generated SNP data. The mean SNP distance across the genome was 70 Kb. The genome had both high and low linkage disequilibrium (LD) regions; the latter were dominant in areas near the gene-rich telomeric portions where recombination is frequent. A total of 252 haplotype blocks, ranging in size from 1 to 15.8 Mb, were identified. Slow LD decay (200-300 Kb) at r(2) ≤ 0.1 across all chromosomes explained the selection of favorable traits around low LD regions in different breeding programs. The association mapping panel was characterized by strong population substructure. Genotypes were grouped into three distinct clusters with a mean genetic dissimilarity coefficient of 0.36. CONCLUSIONS: The genotyped panel of subtropical maize lines characterized in this study should be useful for association mapping of agronomically important genes. The dissimilarity uncovered among genotypes provides an opportunity to exploit the heterotic potential of subtropical elite maize breeding lines.

DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss.

Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

Circulation of Dengue virus-1 (DENV-1) serotype in Delhi, during 2010-11 after Dengue virus-3 (DENV-3) predominance: a single centre hospital-based study.

BACKGROUND: Delhi, a city in north India, has so far witnessed several reported outbreaks of dengue. Dengue in Delhi from being epidemic is slowly changing towards being endemic and hyper-endemic. Circulating type of the virus is also changing over the years. In the absence of an effective vaccine, dengue prevention to a major extent relies on virological surveillance, and development of effective, locally adapted control programmes. In the present study, we tried to identify the between-year non-epidemic serotype of dengue virus circulating in Delhi, during 2010-11. METHODS: Acute-phase samples were collected from the patients attending the Institute of Liver & Biliary Sciences, New Delhi, India. Dengue diagnosis was done using WHO case definitions. All the samples were subjected to Dengue NS1 Ag ELISA and modified nested RT-PCR. RESULTS: A total of 75 acute-phase samples were received, of which 19 (25.3%) were positive for dengue NS1 antigen. Dengue RT-PCR was positive in 14.6% (11/75) samples. All the RT-PCR isolates were of DENV-1 serotype. No case of concomitant infection with more than one serotype was observed. Median age of involvement was 23 yr (range10-86). Maximum number of cases were seen in the age group of 21-30 yr. Male to female ratio was 1.2 : 1. Maximum number of suspected dengue cases (n=79) was seen during September and October. CONCLUSIONS: DENV-1 was circulating in Delhi in the year 2010-11 in non-epidemic period following reported predominance of DENV-3 and co-circulation of all dengue serotypes in the epidemic years 2003, 2006 and 2007.

Antidiabetic Potential of Naturally Occurring Sesquiterpenes: A Review.

Diabetes Mellitus (DM) is an endocrine disease, which is the 3rd leading cause of death in humans; additionally, it is one of the major key concerns over the globe. The high levels of glucose in the blood stream are as well characterized by hyperglycaemia leading to serious damage to the heart, blood vessels, kidney, eyes, and nerves. The best treatment of DM is still not available; many scientists worldwide are trying hard to seek out suitable treatment of DM. Though numerous synthetic drugs are developed for the treatment of diabetes but their utility has been hampered because of several side effects and poor efficacy. Among various approaches for the treatment of DM, herbal medicine, enriched extracts, and naturally derived molecules are most effective. Plant based herbal medicines contain many bioactive phytochemicals, such as terpenoids, alkaloids, flavonoids & phenolics, etc. which are used in the treatment of many diseases. The plant-derived molecules and their suitable structure modification have given many leads or drugs to the world like sesquiterpene; artemisinin and their derivatives artemether & artesunate as an antimalarial drugs. Sesquiterpenes are available in the human diet and are largely taken as components of the many folk medicines and dietary supplements. Sesquiterpenes have a wide range of biological activities, such as anti-cancer, anti-inflammatory, anti-nociceptive, immunomodulatory, antidiabetic and antimicrobial, which make them potential targets for the development of new therapeutics and their usage for medical purposes. Natural products have gained the attention of the world due to their large number of biological activities, high safety and fewer side effect. The review mainly focuses on bioactive sesquiterpenes such as ß-caryophyllene, dysidine, farnesol & eremanthin, etc., a class of terpenoids that may play an important role in the treatment or prevention of this gruesome disorder like diabetes, with their underlying mechanisms for the blood-glucose-lowering property.

Enhanced electron transfer mediated detection of hydrogen peroxide using a silver nanoparticle-reduced graphene oxide-polyaniline fabricated electrochemical sensor.

The current study aims at the development of an electrochemical sensor based on a silver nanoparticle-reduced graphene oxide-polyaniline (AgNPs-rGO-PANI) nanocomposite for the sensitive and selective detection of hydrogen peroxide (H2O2). The nanocomposite was fabricated by simple in situ synthesis of PANI at the surface of rGO sheet which was followed by stirring with AEC biosynthesized AgNPs to form a nanocomposite. The AgNPs, GO, rGO, PANI, rGO-PANI, and AgNPs-rGO-PANI nanocomposite and their interaction were studied by UV-vis, FTIR, XRD, SEM, EDX and XPS analysis. AgNPs-rGO-PANI nanocomposite was loaded (0.5 mg cm-2) on a glassy carbon electrode (GCE) where the active surface area was maintained at 0.2 cm2 for investigation of the electrochemical properties. It was found that AgNPs-rGO-PANI-GCE had high sensitivity towards the reduction of H2O2 than AgNPs-rGO which occurred at -0.4 V vs. SCE due to the presence of PANI (AgNPs have direct electronic interaction with N atom of the PANI backbone) which enhanced the rate of transfer of electron during the electrochemical reduction of H2O2. The calibration plots of H2O2 electrochemical detection was established in the range of 0.01 µM to 1000 µM (R 2 = 0.99) with a detection limit of 50 nM, the response time of about 5 s at a signal-to-noise ratio (S/N = 3). The sensitivity was calculated as 14.7 µA mM-1 cm-2 which indicated a significant potential as a non-enzymatic H2O2 sensor.

Photo-mediated optimized synthesis of silver nanoparticles for the selective detection of Iron(III), antibacterial and antioxidant activity.

The AgNPs synthesized by green method have shown great potential in several applications such as biosensing, biomedical, catalysis, electronic etc. The present study deals with the selective colorimetric detection of Fe3+ using photoinduced green synthesized AgNPs. For the synthesis purpose, an aqueous extract of Croton bonplandianum (AEC) was used as a reducing and stabilizing agent. The biosynthesis was confirmed by UV-visible spectroscopy where an SPR band at λmax 436nm after 40s and 428nm after 30min corresponded to the existence of AgNPs. The optimum conditions for biosynthesis of AgNPs were 30min sunlight exposure time, 5.0% (v/v) AEC inoculum dose and 4mM AgNO3 concentration. The stability of synthesized AgNPs was monitored up to 9months. The size and shape of AgNPs with average size 19.4nm were determined by Field Emission Scanning Electron Microscope (FE-SEM) and High-Resolution Transmission Electron Microscope (HR-TEM). The crystallinity was determined by High-Resolution X-ray Diffractometer (HR-XRD) and Selected Area Electron Diffraction (SAED) pattern. The chemical and elemental compositions were determined by Fourier Transformed Infrared Spectroscopy (FTIR) and Energy Dispersive X-ray Spectroscopy (EDX) respectively. The Atomic Force Microscopy (AFM) images represented the lateral and 3D topological characteristics of AgNPs. The XPS analysis confirmed the presence of two individual peaks which attributed to the Ag 3d3/2 and Ag 3d5/2 binding energies corresponding to the presence of metallic silver. The biosynthesized AgNPs showed potent antibacterial activity against both gram-positive and gram-negative bacterial strains as well as antioxidant activity. On the basis of results and facts, a probable mechanism was also proposed to explore the possible route of AgNPs synthesis, colorimetric detection of Fe3+, antibacterial and antioxidant activity.

Green synthesis of silver nanoparticle for the selective and sensitive colorimetric detection of mercury (II) ion.

An ecofriendly and zero cost approach has been developed for the photoinduced synthesis of more stable AgNPs using an aqueous extract of Murraya koenigii (AEM) as a reducing and stabilizing agent. The exposed reaction mixture of AEM and AgNO3 to sunlight turned dark brown which primarily confirmed the biosynthesis of AgNPs. The biosynthesis was monitored by UV-vis spectroscopy which exhibited a sharp SPR band at 430nm after 30min of sunlight exposure. The optimum conditions for biosynthesis of AgNPs were 30min of sunlight exposure, 2.0% (v/v) of AEM inoculuam dose and 4.0mM AgNO3 concentration. TEM analysis confirmed the presence of spherical AgNPs with average size 8.6nm. The crystalline nature of the AgNPs was confirmed by XRD analysis where the Bragg's diffraction pattern at (111), (200), (220) and (311) corresponded to face centered cubic crystal lattice of metallic silver. The surface texture was analyzed by AFM analysis where the average roughness of the synthesized AgNPs was found 1.8nm. FTIR analysis was recorded between 4000 and 400cm-1 which confirmed the involvement of various functional groups in the synthesis of AgNPs. On the basis of the linear relationship between SPR band intensity and different concentration of Hg2+, the synthesized AgNPs can be used for colorimetric detection of Hg2+ with a linear range from 50nm to 500µM. Based on experimental findings, an oxidation-reduction mechanism between AgNPs and Hg2+ was also proposed.

Genomewide Expression and Functional Interactions of Genes under Drought Stress in Maize.

A genomewide transcriptome assay of two subtropical genotypes of maize was used to observe the expression of genes at seedling stage of drought stress. The number of genes expressed differentially was greater in HKI1532 (a drought tolerant genotype) than in PC3 (a drought sensitive genotype), indicating primary differences at the transcriptional level in stress tolerance. The global coexpression networks of the two genotypes differed significantly with respect to the number of modules and the coexpression pattern within the modules. A total of 174 drought-responsive genes were selected from HKI1532, and their coexpression network revealed key correlations between different adaptive pathways, each cluster of the network representing a specific biological function. Transcription factors related to ABA-dependent stomatal closure, signalling, and phosphoprotein cascades work in concert to compensate for reduced photosynthesis. Under stress, water balance was maintained by coexpression of the genes involved in osmotic adjustments and transporter proteins. Metabolism was maintained by the coexpression of genes involved in cell wall modification and protein and lipid metabolism. The interaction of genes involved in crucial biological functions during stress was identified and the results will be useful in targeting important gene interactions to understand drought tolerance in greater detail.

Photo-induced biosynthesis of silver nanoparticles using aqueous extract of Erigeron bonariensis and its catalytic activity against Acridine Orange.

The green synthesis of silver nanoparticles (AgNPs) has reduced the pollution load in the environment to a greater extent by avoiding the use of hazardous chemicals. In the present work we have developed an ecofriendly and zero cost approach for the green synthesis of more stable and spherical AgNPs using aqueous extract of Erigeron bonariensis (AEE) which act as both reducing and stabilizing agent. The reaction of AEE and AgNO3 was carried out in direct sunlight for the instant biosynthesis of AgNPs within minutes. The biosynthesis was monitored by UV-vis spectroscopy which exhibited a sharp SPR band at 442 nm and 435 nm after 5 and 35 min of sunlight exposure. The optimum conditions for biosynthesis of AgNPs were found to be 2.5mM AgNO3 concentration, 1.5% (v/v) of AEE inoculum dose and 35 min of sunlight exposure. Presence of spherical AgNPs with average size 13 nm was confirmed by SEM and TEM analysis. The XRD and SAED analysis confirmed the crystalline nature of the AgNPs where the Bragg's diffraction pattern at (111), (200), (220) and (311) corresponded to face centered cubic crystal lattice of metallic silver. The average roughness of the synthesized AgNPs was 3.21 nm which was confirmed by AFM analysis. FTIR analysis was recorded between 4000 and 400 cm(-1) which confirmed the involvement of various functional groups in the synthesis of AgNPs. The AgNPs thus obtained showed catalytic activity towards degradation of Acridine Orange (AO) without involvement of any hazardous reducing agent. The concentration dependent catalytic activity of the synthesized AgNPs was also monitored using 1, 2 and 3 mL of silver colloids and was found that the degradation of AO followed pseudo first-order kinetics.

Genome-wide expression of transcriptomes and their co-expression pattern in subtropical maize (Zea mays L.) under waterlogging stress.

Waterlogging causes extensive damage to maize crops in tropical and subtropical regions. The identification of tolerance genes and their interactions at the molecular level will be helpful to engineer tolerant genotypes. A whole-genome transcriptome assay revealed the specific role of genes in response to waterlogging stress in susceptible and tolerant genotypes. Genes involved in the synthesis of ethylene and auxin, cell wall metabolism, activation of G-proteins and formation of aerenchyma and adventitious roots, were upregulated in the tolerant genotype. Many transcription factors, particularly ERFs, MYB, HSPs, MAPK, and LOB-domain protein were involved in regulation of these traits. Genes responsible for scavenging of ROS generated under stress were expressed along with those involved in carbohydrate metabolism. The physical locations of 21 genes expressed in the tolerant genotype were found to correspond with the marker intervals of known QTLs responsible for development of adaptive traits. Among the candidate genes, most showed synteny with genes of sorghum and foxtail millet. Co-expression analysis of 528 microarray samples including 16 samples from the present study generated seven functional modules each in the two genotypes, with differing characteristics. In the tolerant genotype, stress genes were co-expressed along with peroxidase and fermentation pathway genes.