Comprehensive Analyses of miRNA and DNA Methylation in 17beta-Estradiol Exposed Juvenile Rainbow Trout Skeletal Muscle

Reproductive development in female teleosts like rainbow trout starts with release of the sex steroid estrogen, necessary for synthesis of the egg yolk protein vitellogenin. Increase in estrogen secretion during these reproductive phases results in increased energy demands. Skeletal muscle serves as an immediate endogenous source to address energy demands during vitellogenesis and spawning due to which it undergoes rapid degradation resulting in water accumulation, poor fillet and low egg quality. An imbalance in protein turnover is observed not only because of rapid increase in degradative pathways but also due to decreased protein synthesis. These changes in protein metabolism in skeletal muscle were identified within 24 hours of E2 exposure and confirmed in both in vivo and in vitro models. Normally during adult myogenesis myogenic precursor cells proliferate and differentiate further to form a mature muscle fiber resulting in either hyperplasty or hypertrophy or both in fishes. Signaling pathways and MRFs need to function in coordination with epigenetic factors and non-coding RNAs to positively or negatively regulate muscle synthesis. Interestingly, estrogen also influences the expression of miRNA and in turn their target genes. However, the effects of estrogen regulating protein imbalance in rainbow trout skeletal muscle is not completely understood. Hence studies to understand the influence of estrogen were carried out using juvenile, sexually immature fish. Juvenile rainbow trout were treated with either estrogen or vehicle and their skeletal muscle collected after 24 hours and 72 hours of treatment to understand the role of miRNA and DNA methylation in regulation of myogenesis. Findings from the miRNA study revealed diluted effects of estrogen after 72 hours of treatment, hence further studies were carried out with samples from 24-hour post treatment.;miRNAs play a crucial role in regulation of gene expression along with epigenetics. These are small non-coding RNAs with length ranging from 21--23bp mostly functioning as post-transcriptional regulators of gene expression. Generally, they bind to the 3\u27 region of transcribed mRNA with a complementary region called seed sequence. These genes are called target genes of miRNA. A single miRNA can target more than one mRNA. Binding of miRNA to its target gene results in either translational repression or deadenylation or both. Deadenylation results in loss of RNA stability, which leads to its degradation. miRNAs express in tissue specific manner and those that express specifically in muscle are called myomiRNA. Various myomiRNA have been identified and studied for their functions. Estrogen influence the expression of miRNA and in turn their target genes. Therefore, rainbow trout skeletal muscle samples were sequenced for small RNAs to understand the effects of estrogen. Differentially expressed miRNA were identified in estrogen treated samples when compared to control. A total of 36 miRNAs were either upregulated or down regulated in E2 exposed skeletal muscle of which two were novel. Target genes of these differentially expressed miRNAs were identified followed by gene ontology enrichment. These target genes are involved in various biological and molecular functions including their role in signaling pathways, cell cycle, DNA methylation, signal transduction and transcription factor binding. Genes regulated by miRNAs were also involved specifically in proliferation and differentiation of myogenic precursor cells and degradative pathways. E2 induced expression of miR-17 and miR-20, which are important in regulation of cell cycle. Further gene expression analysis of myogeneic regulatory factor, Pax7 specific to MPCs confirms increase in their number with decreased expression of MyoD, specific to differentiation. Similarly, miR-23a revealed its role in maintenance of mitochondrial outer membrane permeability and post-transcriptional regulation of atrophic genes. Decreased expression of miR-23a with increase in permeability was supported by increased gene expressions including fbxo32, caspases 3a and 9 that are involved in atrophy and apoptosis respectively. Expression analysis of one of the novel genes, miR-nov-285, was performed to know its expression pattern in different tissues, highest expression was observed in testis. C-5 methylation of cytosine was one of the GO term that was enriched during the target gene analysis of this novel miRNA. Further regulation of Dnmt1 gene expression by this novel miRNA was confirmed by luciferase assay. All together these observations indicate that E2 influences differential expression of various miRNAs which in turn regulate gene expression contributing to muscle metabolism.;Reduced expression of MyoD was observed in our previous study and direct regulation of this differentiation factor was not observed by any of the differentially expressed miRNA. Since gene expression is also regulated transcriptionally by epigenetic mechanisms including DNA methylation, efforts were made to understand its role in reduced MyoD expression. Generally, DNA methylation reduces gene expression by directly blocking transcription factor binding or by recruiting other epigenetic contributors like histone deacetylases or histone methyltransferases. (Abstract shortened by ProQuest.)

STABILITY-INDICATING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF METHYLCOBALAMIN, ALPHA-LIPOIC ACID, PYRIDOXINE HCL, AND FOLIC ACID IN BULK AND COMBINED DOSAGE FORM

Objectives: The purpose of the research is to develop a simple, precise, economical, accurate, reproducible, and sensitive method for the estimation of methylcobalamin, alpha-lipoic acid, pyridoxine hydrochloride, and folic acid drug product by reversed-phase high-performance liquid chromatography (RP-HPLC) method.Methods: New analytical method was developed for the estimation of methylcobalamin, alpha-lipoic acid, pyridoxine hydrochloride, and folic acid in drug product by RP-HPLC. The chromatographic separation was achieved on the Inertsil C18, 250 mm × 4.6 mm, 5 μm at ambient temperature. The separation achieved employing a mobile phase consists of buffer (added 5.05 g hexane-1-sulfonic acid is dissolved into 1000 mL of distilled water):acetonitrile in the ratio of 10:90% v/v. The flow rate was 1 mL/min and UV-visible spectrophotometer at 285 nm. The average retention time for methylcobalamin, alpha-lipoic acid, pyridoxine hydrochloride, and folic acid was found to be 3.5, 6.7, 8.5, and 9.3, respectively.Results: The developed method was validated as per ICH guidelines. All validation parameters were within the acceptable ranges. The assay methods were found to be linear from 0 to 2130 μg/mL for methylcobalamin, 0 to 142.5 μg/mL for alpha-lipoic acid, 0–4.54 μg/mL for pyridoxine hydrochloride, and 0–2 μg/mL for folic acid. The correlation coefficient was 0.999 for all drugs, respectively. The mean percentage values for the developed method were found to be within the range of 98–100.6%. The developed method was also found to be robust.Conclusion: It is concluded that developed method was accurate, precise, linear, reproducible, robust, and sensitive

Unscarred uterine rupture: a retrospective analysis

Uterine rupture is an obstetrical emergency associated with significant maternal and fetal mortality and morbidity. Spontaneous rupture of an unscarred uterus, though rare, can occur in developing countries. Many risk factors for uterine rupture, as well as a wide range of clinical presentations, have been identified. The aim of the study was to analyze the incidence, predisposing factors and to determine the maternal and perinatal outcomes of unscarred uterine rupture. A retrospective analysis of cases of unscarred uterine rupture was conducted at the department of obstetrics and gynecology, Andhra Medical College/King George Hospital, Visakhapatnam from 01 October 2020 to 31 October 2021. Out of the 8657 deliveries in our hospital during the study period, there were 11 cases of ruptured unscarred uteri giving an incidence of 0.127%. All of these (100%) were unbooked cases. Most of them (90.9%) were multigravida. Uterine rupture occurred at term in 9 cases and 2 were pre term. Maternal mortality was 18.18% (2 cases) and perinatal mortality was 72.72% (8 cases). Sub-total hysterectomy was done in 4 and laparotomy with repair of the rent was performed in the remaining 7 cases. Unscarred uterine rupture though a rare complication of pregnancy, can occur commonly in developing countries. Obstructed labour, mismanaged labour, injudicious use of oxytocin and grand multiparity are the common risk factors associated with unscarred uterine rupture

Preparation and Evaluation of Curcumin Invasomes

Curcumin has poor aqueous solubility and has bioavailability problems. Hence in the present study the solubility of curcumin was increased by complexing with cyclodextrin (CD) and Hydroxy propyl β cyclodextrin(HPβCD). This complex was incorporated in to invasomes and then into HPMC gel to prepare as a transdermal formulation. Curcumin cyclodextrin complexes were prepared by physical mixture and co-precipitation method. Different formulations of invasomes containing 0.5, 1.0, 1.5 % of limonene, fenchone, nerolidol  were prepared using mechanical dispersion technique. Invasomes were characterized for vesicular size, surface morphology, zeta potential, entrapment efficiency and percutaneous permeation. Formulations CHL1 and CHL2 were optimized for further studies. It was found in the study that complexation with HPβCD in 1:2 proportion prepared by co-precipitation method was found to bind 90% of curcumin. Invasomal preparation with 0.5% limonene, 4% ethanol was found to enhance permeation by 8.11 times the control. In vivo diffusion studies were conducted using franz diffusion cell, ex vivo skin permeation studies of CHL1 using rat abdominal skin showed cumulative drug permeated (Q24­) of 70.32 µg/cm2, steady state transdermal flux of 3.344 µg/cm2/hr-1, permeability coefficient of 5.35 cm/hr and lag time of 1 hr when compared with control formulation. From the results it was concluded that the solubility of curcumin was increased by complexing with HPβCD and invasomal preparation with 0.5% limonene has improved the permeation through the skin

Seroprevalence of syphilis in human immunodeficiency virus patients

Background: Syphilis is a sexually transmitted infection caused by, Treponema pallidum. Syphilis facilitates the transmission and acquisition of human immunodeficiency virus (HIV) and causes transient increase in the viral load. Sexually transmitted infections (STI) are 3-5 times more likely to acquire HIV infection, if exposed to the virus through sexual contact. Aim of the study was to estimate the seroprevalence of Syphilis in HIV patients.Methods: A total of 920 blood samples were collected from HIV patients attending ART (Antiretroviral therapy) centre and were tested for Syphilis by using Rapid Plasma Reagin (RPR) and Treponema pallidum Hemagglutination Assay (TPHA). A total of 100 HIV non-reactive individuals were taken as a control group.Results: Out of 920 samples, 102 (11.1%) were positive for Syphilis. Out of 102 Syphilis seropositive patients, males (76.5%) were more commonly affected in age group of 21-40 years. Both RPR and TPHA were reactive in 46% of cases and only TPHA reactive in 53.9% of cases. Out of 100 HIV non-reactive patients, 5% of patients are reactive for Syphilis.Conclusions: In the present study, prevalence of Syphilis was more in HIV patients compared to HIV non-reactive persons. Persons with HIV infection acquired through sexual route should be screened for Syphilis by one nonspecific test along with specific test to confirm the diagnosis. This will help in proper management of the patients having Syphilis and HIV co-infection

Lhx8 interacts with a novel germ cell-specific nuclear factor containing an Nbl1 domain in rainbow trout (Oncorhynchus mykiss)

Lhx8 is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. In this study, we report the characterization of rainbow trout lhx8 genes and identification of a novel germ cell-specific nuclear factor that interacts with Lhx8. Two lhx8genes, lhx8a and lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Collectively, results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout

Effectiveness of Behavioral Skills Training (BST) on Knowledge of Sexual Abuse and Resistance Ability among Children with Intellectual Disability: a pilot study

Sexual abuse among children with Intellectual Disability is 2-4 times the rate of the general population. 39% - 83% of girls and 16% - 32% of boys with Intellectual Disability typically experience sexual abuse by the time they reach the age of 18. In order to protect themselves, children with Intellectual Disability must have the skills to independently recognize potentially abusive situations and respond appropriately. The current study focused on assessing the effectiveness of Behavioural Skills Training on knowledge of sexual abuse and resistance ability among children with Intellectual Disability. A true experimental, pre-test post-test control group design was adopted. Study was conducted among 14 children with mild or moderate disability (7 in the experimental and control groups respectively), randomly selected from a selected special school. Pre-assessment of subjects was done using the Personal Safety Questionnaire (PSQ), and video based modified What If Situation test (WIST). BST was administered for one month (12 sessions). Post-assessment was done one week after the intervention. The Experimental Group demonstrated greater knowledge regarding sexual abuse (t=3.27, p=0.008) and resistance ability (WIST female t=5.85, p< 0.001, WIST male t=4.47, p=0.001) when compared to the control group. Current study findings suggest that BST is effective for children with mild or moderate Intellectual Disability in the experimental group. Further research with the same protocol to gain more subjects, Is suggested in order to confirm the result before being applied as a primary prevention intervention for child sexual abuse in children with Intellectual Disability

Issues and challenges in sexuality and sex education in Intellectually Disabled

All human beings are sexual from birth to death, and the Intellectually Disabled (ID) are not exempt from this. The issues of sexual needs and sexual behaviour of the ID have been a sensitive and controversial topic. Perhaps because of the taboos associated with Intellectual Disability and sexuality, individually or in combination, very little is known about the sexuality of people with ID, but research studies do indicate that it is much the same as the general population, although opportunities for sexual expression are limited. This article provides an overview of sexuality issues in ID, with regard to sexual rights, sexual development and maturation, menstruation and menstrual hygiene, masturbation, petting, sexual activity, marriage, pregnancy, parenting, sexual abuse, fertility and birth control, menopause, and the progress and issues in sexuality education for ID. Prior research shows that individuals with ID show secondary sexual characteristics and development similar to that of intellectually averaged peers; those in the lower range of functioning develop those characteristics at a slower pace. ID children often neglect menstruation and are non-co operative in menstrual grooming due to poor understanding, irritability, fatigue and increased seizure. Research reports vaginal hysterectomy as management of this. They show a lower rate of offspring production than the non retarded. It is estimated that ID children are sexually victimized at 4-10 times the rate of the general population, 39-83% of girls and 16-32% of boys experience sexual abuse before they reach the age of 18. Parents and professionals are pessimistic in the sexual needs of people with IDD and consider them as asexual. There is an urgent need for development of methods to teach them about sexuality and sexual abuse prevention, since research on sex education revealed poor generalization of skills to real life situations. In situ training revealed better generalization, but has the issue of causing emotional distress and dissonance among ID