HORMONAL AND NUTRITIONAL REGULATION OF MOLTING, METAMORPHOSIS, AND REPRODUCTION IN BED BUGS, \u3cem\u3eCimex lectularius\u3c/em\u3e

The bed bug, Cimex lectularius is an obligate hematophagous ectoparasite that feeds on humans. The increase in travel and development of insecticide resistance to commercially available insecticides have enabled the bed bug population to resurge, causing economical and psychological trauma to the human population. Lack of knowledge about the basic molecular biology of bed bugs has motivated us to study the key aspects of molting, metamorphosis, and reproduction. A blood meal triggers expression of various genes that enable bed bugs to molt or undergo metamorphosis. Molting and metamorphosis in bed bugs are regulated by two key hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH induces expression of Krüppel homologue 1 (Kr-h1) gene. Higher expression of Kr-h1 in the penultimate nymphal instar represses ecdysone inducible gene E93 and the development of adult characteristics in the insect. E93 is expressed during the last instar stage in the absence of Kr-h1. E93 promotes the development of adult structures and metamorphosis to the adult stage. Studies on reproduction have also shown that blood meal and mating are essential for vitellogenin gene expression and oocyte maturation in bed bugs. JH and 20E regulate reproduction in bed bugs. Detailed studies on the involvement of juvenile hormone in reproduction using the next generation sequencing technology identified genes that regulate reproduction in bed bugs. V-Maf, avian musculoaponeurotic fibrosarcoma oncogene homolog B (MafB), forkhead box protein (Foxl2) and heparanase were found to play key roles in regulation of reproduction. The role of ABC transporters was also studied using RNA interference. ABC transporters (ATP-binding cassette) are involved in active transport of various molecules including steroid hormones, cuticle lipids, and other molecules. ABC transporters were also shown to be involved regulation of reproduction, molting and metamorphosis in bed bugs. This study lays a foundation for future research aimed at the development of novel methods for controlling bed bugs

Juvenile Hormone Regulation of Female Reproduction in the Common Bed Bug, \u3cem\u3eCimex lectularius\u3c/em\u3e

To begin studies on reproduction in common bed bug, Cimex lectularius, we identified three genes coding for vitellogenin (Vg, a protein required for the reproductive success of insects) and studied their hormonal regulation. RNA interference studied showed that expression of Vg3 gene in the adult females is a prerequisite for successful completion of embryogenesis in the eggs laid by them. Juvenile hormone (JH) receptor, Methoprene-tolerant (Met), steroid receptor coactivator (SRC) and GATAa but not ecdysone receptor (EcR) or its partner, ultraspiracle (USP) are required for expression of Vg genes. Feeding and mating working through Vg, Met, SRC, EcR, and GATAa regulate oocyte development. Knockdown of the expression of Met, SRC, EcR, USP, BR-C (Broad-Complex), TOR (target of rapamycin), and GATAa in female adults resulted in a reduction in the number eggs laid by them. Interestingly, Kruppel homolog 1 (Kr-h1) knockdown in the adult females did not reduce their fecundity but affected the development of embryos in the eggs laid by females injected with Kr-h1 double-stranded RNA. These data suggest that JH functioning through Met and SRC regulate both vitellogenesis and oogenesis in C. lectularius. However, JH does not work through Kr-h1 but may work through transcription factors not yet identified

FORMULATION AND EVALUATION OF AN ANTIMICROBIAL MUCOADHESIVE DENTAL GEL OF AZADIRACHTA INDICA AND GLYCYRRHIZA GLABRA

Objective: Objective of this study was to formulate and evaluate an antimicrobial mucoadhesive dental gel of herbal drugs for the prevention and treatment of dental plaque, dental caries, and periodontitis. Methods: Azadirachta indica leaves extract was prepared with ethanol: water (80:20 v/v) and Glycyrrhiza glabra roots extract was prepared with ethanol: water (30:70 v/v). Both the extracts were evaluated for organoleptic properties, pH, phytochemical screening and total phenolic content. Thin layer chromatography (TLC) and Reverse-phase high-performance liquid chromatography (RP-HPLC) studies were performed. Antibacterial activity of the extract was done on Mueller Hinton agar media against Streptococcus mutans using the disc diffusion method. A mucoadhesive gel was prepared using carbopol 934, polyethylene glycol (PEG) 400 as a bioadhesive polymer and penetration enhancer, respectively. Three gel formulations were prepared at various concentrations of carbopol 934. Dental gel formulations were evaluated for different parameters such as appearance, pH, viscosity, spreadabilty, syringeability. Optimised batch was used for further studies viz. stability study, drug content, diffusion study to determine percent cumulative release of drug from gel formulation and in vitro mucoadhesion study. Results: Rf value of nimbin and glycyrrhizin in TLC study, were found to be 0.08 and 0.56, respectively. RP-HPLC method was developed for estimation of active constituents present in both extracts using mobile phase acetonitrile: water [containing 0.1 % v/v glacial Acetic acid (GAA)]: methanol in the ratio of 30:60:10 at the flow rate of 0.8 ml/min. Rt of active constituents from both drug extracts was found to be 3.119 and 4.15 min, respectively. 2.5 % w/v of Azadirachta indica leaves extract showed a good zone of inhibition (10.66±0.577 mm) near to chlorhexidine (11.33±0.5773 mm). 1 % of Glycyrrhiza glabra roots extract exhibit antibacterial activity (9±1 mm) and masks the bitter taste of formulation. Batch (F2) was selected on the basis of viscosity, spreadabilty and syringeability. The optimised batch was found to be stable and has 83 % and 80.3 % of drug content. Percent cumulative releases of the drug from gel formulation during the diffusion study were found to be 87.52 % and 85.43 %. Adhesive force and adhesiveness were found to be 11.90 g and 0.92 millijoule, respectively during mucoadhesion study. Conclusion: The evaluation parameters of optimized batch indicate the prepared dental gel is mucoadhesive, stable, good delivery drug system containing antimicrobial agents for prevention of plaque formation, dental caries and periodontitis

STABILITY INDICATING RP-HPLC ASSAY METHOD FOR ESTIMATION OF MIDODRINE HYDROCHLORIDE IN BULK AND TABLETS

Objective: To develop an accurate, simple, sensitive and precise stability indicating reverse phase-high performance liquid chromatographic (RP-HPLC) assay method for estimation of Midodrine hydrochloride (MD) in bulk and tablets.Methods: The chromatographic separation was performed on enable C18, (250 mm X 4.6 mm, 5 μm) column. The mobile phase consists of triethylamine buffer 0.02%, pH-3: acetonitrile (38:62 v/v) was delivered at a flow rate of 0.6 ml/min and UV detection at 289 nm. The method was validated with forced degradation studies as per ICH guidelines.Results: The retention time of the drug was found to be 3.56 min. The developed method was found to be linear in a concentration range of 19.98-99.9μg/ml of the drug (r2= 0.9998). The low value of % RSD indicates reproducibility of the method. The low value of LOD and LOQ suggests the sensitivity of the method. The results of forced degradation studies indicated that the drug was stable in acidic condition and degraded in basic, oxidative and hydrolytic conditions.Conclusion: The present study represents first stability-indicating HPLC assay method that deals with the estimation of midodrine hydrochloride. It can be concluded from the results that the developed method is simple, rapid, accurate, specific, sensitive and precise. Thus, this method can be used for routine analysis of midodrine hydrochloride formulation and to check the stability of bulk samples.Â

Unique Features of a Global Human Ectoparasite Identified Through Sequencing of the Bed Bug Genome

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite

Krüppel homolog 1 and E93 Mediate Juvenile Hormone Regulation of Metamorphosis in the Common Bed Bug, \u3cem\u3eCimex lectularius\u3c/em\u3e

The common bed bug is an obligate hematophagous parasite of humans. We studied the regulation of molting and metamorphosis in bed bugs with a goal to identify key players involved. qRT-PCR studies on the expression of genes known to be involved in molting and metamorphosis showed high levels of Krüppel homolog 1 [Kr-h1, a transcription factor that plays key roles in juvenile hormone (JH) action] mRNA in the penultimate nymphal stage (N4). However, low levels of Kr-h1 mRNA were detected in the fifth and last nymphal stage (N5). Knockdown of Kr-h1 in N4 resulted in a precocious development of adult structures. Kr-h1 maintains the immature stage by suppressing E93 (early ecdysone response gene) in N4. E93 expression increases during the N5 in the absence of Kr-h1 and promotes the development of adult structures. Knockdown of E93 in N5 results in the formation of supernumerary nymphs. The role of JH in the suppression of adult structures through interaction with Kr-h1 and E93 was also studied by the topical application of JH analog, methoprene, to N5. Methoprene induced Kr-h1 and suppressed E93 and induced formation of the supernumerary nymph. These data show interactions between Kr-h1, E93 and JH in the regulation of metamorphosis in the bed bugs

The Roles of Human DNA Methyltransferases and Their Isoforms in Shaping the Epigenome

A DNA sequence is the hard copy of the human genome and it is a driving force in determining the physiological processes in an organism. Concurrently, the chemical modification of the genome and its related histone proteins is dynamically involved in regulating physiological processes and diseases, which overall constitutes the epigenome network. Among the various forms of epigenetic modifications, DNA methylation at the C-5 position of cytosine in the cytosine⁻guanine (CpG) dinucleotide is one of the most well studied epigenetic modifications. DNA methyltransferases (DNMTs) are a family of enzymes involved in generating and maintaining CpG methylation across the genome. In mammalian systems, DNA methylation is performed by DNMT1 and DNMT3s (DNMT3A and 3B). DNMT1 is predominantly involved in the maintenance of DNA methylation during cell division, while DNMT3s are involved in establishing de novo cytosine methylation and maintenance in both embryonic and somatic cells. In general, all DNMTs require accessory proteins, such as ubiquitin-like containing plant homeodomain (PHD) and really interesting new gene (RING) finger domain 1 (UHRF1) or DNMT3-like (DNMT3L), for their biological function. This review mainly focuses on the role of DNMT3B and its isoforms in de novo methylation and maintenance of DNA methylation, especially with respect to their role as an accessory protein

Profiling DNA methylation differences between inbred mouse strains on the Illumina Human Infinium MethylationEPIC microarray

<div><p>The Illumina Infinium MethylationEPIC provides an efficient platform for profiling DNA methylation in humans at over 850,000 CpGs. Model organisms such as mice do not currently benefit from an equivalent array. Here we used this array to measure DNA methylation in mice. We defined probes targeting conserved regions and performed differential methylation analysis and compared between the array-based assay and affinity-based DNA sequencing of methyl-CpGs (MBD-seq) and reduced representation bisulfite sequencing. Mouse samples consisted of 11 liver DNA from two strains, C57BL/6J (B6) and DBA/2J (D2), that varied widely in age. Linear regression was applied to detect differential methylation. In total, 13,665 probes (1.6% of total probes) aligned to conserved CpGs. Beta-values (β-value) for these probes showed a distribution similar to that in humans. Overall, there was high concordance in methylation signal between the EPIC array and MBD-seq (Pearson correlation r = 0.70, p-value < 0.0001). However, the EPIC probes had higher quantitative sensitivity at CpGs that are hypo- (β-value < 0.3) or hypermethylated (β-value > 0.7). In terms of differential methylation, no EPIC probe detected a significant difference between age groups at a Benjamini-Hochberg threshold of 10%, and the MBD-seq performed better at detecting age-dependent change in methylation. However, the top most significant probe for age (cg13269407; uncorrected p-value = 1.8 x 10⁻⁵) is part of the clock CpGs used to estimate the human epigenetic age. For strain, 219 EPIC probes detected significant differential methylation (FDR cutoff 10%) with ~80% CpGs associated with higher methylation in D2. This higher methylation profile in D2 compared to B6 was also replicated by the MBD-seq data. To summarize, we found only a small subset of EPIC probes that target conserved sites. However, for this small subset the array provides a reliable assay of DNA methylation and can be effectively used to measure differential methylation in mice.</p></div