Oxidative stress and DNA damage in newborns born to mothers with hyperglycemia - a prospective cohort study.

BACKGROUND: Hyperglycemic mothers have increased oxidative stress during pregnancy, which can adversely affect the outcome in their neonates. OBJECTIVES: To measure the oxidative stress and DNA damage in newborns born to mothers with hyperglycemia and correlate their immediate outcome with the amount of oxidative stress and DNA damage. METHODS: This prospective cohort study conducted in a tertiary care teaching hospital, South India included three groups - newborns born to pregestational diabetic mothers, gestational diabetic mothers, and euglycemic mothers with 24 newborns in each. Oxidative stress parameters - malondialdehyde (MDA), total antioxidant capacity (TAC), and DNA damage (comet assay) were assessed in umbilical cord blood. Association between these parameters and immediate neonatal outcome among three groups were studied. RESULTS: Newborns born to diabetic mothers had more MDA levels and DNA damage compared to euglycemic mothers. There was a positive correlation between comet parameters and MDA levels. There was an inverse correlation between comet parameters and TAC. Newborns born to diabetic mothers were at high risk to develop respiratory distress syndrome, hypoglycemia, and sepsis. CONCLUSIONS: Newborns born to diabetic mothers had increased oxidative stress and DNA damage compared to those born to mothers with euglycemia.

Assessment of Sperm Deoxyribose Nucleic Acid Fragmentation Using Sperm Chromatin Dispersion Assay.

The integrity of sperm deoxyribose nucleic acid (DNA) is one of the determinants that ensure normal fertilization, implantation, pregnancy, and the generation of a healthy progeny. Assessment of sperm DNA fragmentation has gained importance as a tool to provide significant information regarding sperm quality, and it can independently predict sperm fertilizing potential. The sperm chromatin dispersion (SCD) assay is one of the available techniques to detect sperm DNA damage with results comparable to the gold standard - Sperm Chromatin Structure Assay. We present here a detailed methodology of an alkaline modification of SCD that can be carried out with minimal laboratory equipment. The SCD assay is a modified halo assay that utilizes chemical methods to demonstrate sperm DNA fragmentation. It involves the embedding of sperms in an agarose medium followed by exposure to alkaline denaturation and deproteinization. The results are interpreted under a light microscope. A visual scoring system is utilized to differentiate the sperms with possible DNA fragmentation from those without fragmented DNA and to determine the sperm DNA fragmentation index (SDFI) % for each semen sample. The SDFI % is directly proportional to the quality of sperm. The SCD assay is a simple, cost-effective, and reliable technique that can detect sperm DNA fragmentation, thus providing information regarding sperm functional quality and reproductive capacity. It is of significance in clinical and research areas of andrology and reproductive medicine, toxicology, and pharmacotherapeutics.

Body Donation- Consent from Non-Related Persons: Case Series, Review, and Recommendations.

Learning anatomy by dissection of cadavers is the best way to learn anatomy. Voluntary body donation is one of the sources of procuring cadavers. In the case of donations after hospital or non-institutional deaths, the family members of the deceased approach the hospital authorities regarding body donation of the deceased. There are situations, where there is no available accompanying near relatives of the deceased, which pose a challenge for personnel involved in the process of body donation. In two of the reported cases, the body donation was done by the live-in partner and a friend of the deceased. In another reported case, the son of the deceased was nominated by the donor at the time of registration. As the son of the deceased was not available at the time of death of the donor, donation was executed by another near relative. Anatomy Acts of individual States in the Union of India and Acts of other countries are examined about the consent for body donation by persons other than near relatives. None of the Anatomy Acts of various States in India provide an alternative in the absence of near relatives for claiming the body or for donating the body, except for Acts of Delhi and Kerala. There is a need to bring in a Unified Anatomy Act, common for all the States and Union territories in India and include: friend, live-in partner and a nominated person in the provisions of the Act to enable them to give consent for body donation.

Anatomic Variant of Liver, Gall Bladder and Inferior Vena Cava.

The morphology and relations of liver, gall bladder and inferior vena cava are cardinal. Their anatomical variations may be a reason for the adverse surgical outcome. During routine anatomy dissection of an abdomen, we noticed a variant liver, gall bladder and inferior vena cava in a 63-year-old male cadaver. In the specimen, a retrohepatic segment of inferior vena cava was found to be intrahepatic. On dissection, it was observed that inferior vena cava was covered entirely by a liver tissue on its dorsal aspect. In the same specimen, the gall bladder had undulated inferior surface. On dissection of the gall bladder, numerous mucosal folds were present in the interior. A band of fibrous tissue was found, which was extending from the right side of the gall bladder to the falciform ligament. Hence, preoperative scanning of congenital variations of the liver, gall bladder and inferior vena cava may be compassionate in planning safe surgeries and interventional abdominal procedures.

DNA Damage Analysis in Children with Non-syndromic Developmental Delay by Comet Assay.

INTRODUCTION: Majority of the developmental delays in children are non-syndromic and they are believed to have an underlying DNA damage, though not well substantiated. Hence the present study was carried out to find out if there is any increased DNA damage in children with non-syndromic developmental delay by using the comet assay. AIM: The present case-control study was undertaken to assess the level of DNA damage in children with non syndromic developmental delay and compare the same with that of age and sex matched controls using submarine gel electrophoresis (Comet Assay). MATERIALS AND METHODS: The blood from clinically diagnosed children with non syndromic developmental delay and controls were subjected for alkaline version of comet assay - Single cell gel electrophoresis using lymphocytes isolated from the peripheral blood. The comets were observed under a bright field microscope; photocaptured and scored using the Image J image quantification software. Comet parameters were compared between the cases and controls and statistical analysis and interpretation of results was done using the statistical software SPSS version 20. RESULTS: The mean comet tail length in cases and control was 20.77+7.659µm and 08.97+4.398µm respectively which was statistically significant (p<0.001). Other comet parameters like total comet length and % DNA in tail also showed a statistically significant difference (p < 0.001) between cases and controls. CONCLUSION: The current investigation unraveled increased levels of DNA damage in children with non syndromic developmental delay when compared to the controls.

CometQ: An automated tool for the detection and quantification of DNA damage using comet assay image analysis.

BACKGROUND AND OBJECTIVE: DNA damage analysis plays an important role in determining the approaches for treatment and prevention of various diseases like cancer, schizophrenia and other heritable diseases. Comet assay is a sensitive and versatile method for DNA damage analysis. The main objective of this work is to implement a fully automated tool for the detection and quantification of DNA damage by analysing comet assay images. METHODS: The comet assay image analysis consists of four stages: (1) classifier (2) comet segmentation (3) comet partitioning and (4) comet quantification. Main features of the proposed software are the design and development of four comet segmentation methods, and the automatic routing of the input comet assay image to the most suitable one among these methods depending on the type of the image (silver stained or fluorescent stained) as well as the level of DNA damage (heavily damaged or lightly/moderately damaged). A classifier stage, based on support vector machine (SVM) is designed and implemented at the front end, to categorise the input image into one of the above four groups to ensure proper routing. Comet segmentation is followed by comet partitioning which is implemented using a novel technique coined as modified fuzzy clustering. Comet parameters are calculated in the comet quantification stage and are saved in an excel file. RESULTS: Our dataset consists of 600 silver stained images obtained from 40 Schizophrenia patients with different levels of severity, admitted to a tertiary hospital in South India and 56 fluorescent stained images obtained from different internet sources. The performance of "CometQ", the proposed standalone application for automated analysis of comet assay images, is evaluated by a clinical expert and is also compared with that of a most recent and related software-OpenComet. CometQ gave 90.26% positive predictive value (PPV) and 93.34% sensitivity which are much higher than those of OpenComet, especially in the case of silver stained images. The results are validated using confusion matrix and Jaccard index (JI). Comet assay images obtained after DNA damage repair by incubation in the nutrient medium were also analysed, and CometQ showed a significant change in all the comet parameters in most of the cases. CONCLUSIONS: Results show that CometQ is an accurate and efficient tool with good sensitivity and PPV for DNA damage analysis using comet assay images.

Assessment of DNA damage and repair efficiency in drug naïve schizophrenia using comet assay.

The etiology of schizophrenia continues to be confounding and elusive. Some knowledge gaps exist in the neurodegenerative theory of schizophrenia. Oxidative DNA damage and repair deficits are relevant to the mechanisms of neurodegeneration but have not been studied in drug naïve schizophrenia. The present study used the comet assay technique to study the extent of DNA damage in circulating peripheral lymphocytes of patients with drug naïve schizophrenia (n = 40) along with an age and gender matched control group (n = 40). We also assessed the DNA repair efficiency in cases following incubation in a nutrient medium. All the assayed comet parameters demonstrated significantly greater baseline DNA damage in cases in comparison to the controls except for head diameter (p < 0.001 for all significant results, p = 0.32 for head diameter). Gender, age and duration of illness (p = 0.21, 0.69 and 0.12 respectively for tail length) did not influence any of the parameters significantly. Significant decrease was noted in the comet tail length and percentage of DNA in comet tail (p < 0.001 for both) in cases following incubation suggesting that the DNA repair machinery was preserved. No difference in DNA repair efficiency was noted between the genders (p = 0.23 for tail length). Our findings confirm the presence of significant baseline DNA damage in schizophrenia even prior to the initiation of anti-psychotic treatment. Additionally, intact genomic repair efficiency was noted in this group as a whole. These results provide some evidence for oxidative DNA damage as molecular link underpinning neurodegeneration in drug naïve schizophrenia.

Perception of MBBS students to "flipped class room" approach in neuroanatomy module.

A flipped classroom is a learner centered approach in which the learner is responsible to attend the class with basic understanding of the subject to fully participate and engage in discussions. The aim of this study was to evaluate students' perception of flipped classroom approach for neuroanatomy module and assess the impact on their performance and attitudes. The subject chosen to evaluate the flipped classroom model for first year medical students was clinical neuroanatomy. One hundred and thirty first year medical students participated in the study module. Students were divided into five groups and five case scenarios pertaining to various clinically relevant regions of the neuraxis, with varying anatomical complexity were generated. The pre- and post-tests were designed to specifically test the declared learning objectives of the session. The perception of the students regarding this model of teaching and learning was also evaluated. Eighty-six percent of students felt that the flipped classroom approach was better at fulfilling the stated learning objectives than the conventional didactic teaching, 92% felt that the work-sheet with questions provided prior to the class enabled a better understanding of the subject and 87% were of the opinion that the web sources with references kindled a greater interest to read as compared with didactic lectures. The paired t test showed highly significant differences between the pre and post-test scores. Student response to the flipped classroom structure was largely positive, indicating it to be an approach worth pursuing in future years.

A comprehensive review on clinical applications of comet assay.

Increased levels of DNA damage and ineffective repair mechanisms are the underlying bio-molecular events in the pathogenesis of most of the life-threatening diseases like cancer and degenerative diseases. The sources of DNA damage can be either exogenous or endogenous in origin. Imbalance between the oxidants and antioxidants resulting in increased reactive oxygen species mostly accounts for the endogenously derived attacks on DNA. Among the various methods employed in the estimation of DNA damage, alkaline comet assay is proven to be a relatively simple and versatile tool in the assessment of DNA damage and also in determining the efficacy of DNA repair mechanism. The aim of this article is to review the application of comet assay in the field of medicine towards human biomonitoring, understanding the pathogenesis of cancer and progression of chronic and degenerative diseases, prediction of tumour radio & chemosensitivity and in male infertility. A standardized protocol and analysis system of various variants of comet assay in different types of cells, across the labs will be of useful and reliable clinical tool in the field of Medicine for the estimation of levels of DNA damage and repair mechanisms.

Hypoxia induced DNA damage in children with isolated septal defect and septal defect with great vessel anomaly of heart.

BACKGROUND AND AIM: In Congenital Heart Disease (CHD), shunting of blood occurs through the anatomical defects which lead to mixing of oxygenated and deoxygenated blood. Chronic hypoxia which occurs due to the above said mechanism has the potency to cause DNA damage in children with CHD. In chronic hypoxia, there is a liberation of Reactive Oxygen Species (ROS) due to tissue injury as a result of ischemia and induction of hypoxia inducible factor - 1HIF-1 and p53 which in turn activates pro-apoptotic factors leading to alteration in the regulation of pro-apoptotic gene Blc-2 to be involved in causing the DNA damage. The extent of chronic hypoxia and the DNA damage depends on the nature of the anatomical heart defect. Hence, the present case-control study was conducted to find out the DNA damage in children with isolated septal defect and septal defect with great vessel anomaly of heart and to compare the same. MATERIALS AND METHODS: The study group was categorized into those with isolated septal defects and septal defects associated with great vessel anomaly based on echo-cardiogram. Age and sex matched healthy children were taken as controls. Single-cell gel electrophoresis - Comet Assay of Alkaline Version was performed conventionally and the comets were analyzed using comet score software. RESULTS AND CONCLUSION: The comet metrics was found to be statistically significant in children with isolated septal defect and septal defect with great vessel anomaly when compared with that of the controls. In addition, comet metrics also showed significantly increased DNA damage among children with septal defects associated with great vessel anomaly when compared to isolated septal defects. The data strongly suggests a linear correlation of severity of the anomaly involved with the degree of DNA damage as evidenced by lesser extent of DNA damage in isolated septal defect and greater in septal defect with great vessel anomaly.

Variations in the branching pattern of posterior division of mandibular nerve: a case report.

PURPOSE: Abnormal communications among the branches of mandibular nerve especially the posterior division are significant due to various procedures undertaken in this region. These variations are worth reporting as they pose serious implications in several interventions in this region, and may even lead to false diagnosis. METHODS: During routine dissection, the mandibular nerve and its branches were dissected in the infratemporal fossa. The branches from the posterior division of the mandibular nerve namely the inferior alveolar and auriculotemporal nerves were carefully dissected, and their abnormal branching pattern was noted. RESULTS: There was a communicating branch between left inferior alveolar and auriculotemporal nerve. There was also a variant recurrent branch from the left inferior alveolar nerve that supplied the lateral pterygoid muscle. CONCLUSIONS: Such variant branches and communications between the branches of mandibular nerve as seen in this case have an embryological basis and are clinically important in this region especially for dental surgeries and anesthesia.

Assessment of folic Acid and DNA damage in cleft lip and cleft palate.

Studies have identified the risk factors like folic acid deficiency during gestational period, family history for orofacial clefts, drugs like antiepileptic, vitamin A. But, the data regarding the folic acid status in children with cleft lip/palate is hardly evaluated in depth. Here, an assessment of folic acid and DNA damage were carried out in children with orofacial anomalies. Folic acid level and DNA damage were evaluated by folic acid assay (direct chemiluminescent technology) and single cell gel electrophoresis or comet assay method respectively. The mean value of plasma folic acid by direct chemiluminescent technology was 6.5±3.6 nmol/L and the normal value in children ranges from 11.3 to 47.6 nmol/L. The amount of damaged DNA, measured as the tail length of the comet in cases, was 19.4±8.9 µm and the mean percentage of DNA in tail was 16.5±3.7. Folic acid deficiency could be the reason for DNA damage.

Deoxyribonucleic acid damage study in primary amenorrhea by comet assay and karyotyping.

AIM: This study aims at evaluating the chromosomal abnormalities and deoxyribonucleic acid (DNA) damage in cases with primary amenorrhea by karyotyping and comet assay. STUDY DESIGN: A total of 30 cases of primary amenorrhea were recruited. Secondary sexual characters were assessed by Tanner staging. Chromosomal analysis was performed by conventional phytohemagglutinin stimulated lymphocyte cell culture technique. Alkaline version of comet assay was used to evaluate DNA damage. RESULTS: The chromosomal pattern of 20 subjects (66.7%) was found to be normal (46,XX). Two subjects had 46,XY pattern and eight subjects had Turner syndrome (45,X or 45,X/46,XX). The comet parameters were found to be increased among subjects with 45,X monosomy, when compared to the rest of the study group and also in subjects with Tanner stage 1 when compared to stage 2. CONCLUSION: Comet assay revealed increased DNA damage in cases with 45,X monosomy, compared with subjects with 46,XX and 46,XY karyotype, which correlated with clinical features.