High-risk genes involved in common septal defects of congenital heart disease.

The septation defect is one of the main categories of congenital heart disease (CHD). They can affect the septation of the atria leading to atrial septal defect (ASD), septation of ventricles leading to ventricular septal defect (VSD), and formation of the central part of the heart leading to atrioventricular septal defect (AVSD). Disruption of critical genetic factors involved in the proper development of the heart structure leads to CHD manifestation. Because of this, to identify the high-risk genes involved in common septal defects, a comprehensive search of the literature with the help of databases and the WebGestalt analysis tool was performed. The high-risk genes identified in the analysis were checked in 16 Indian whole-exome sequenced samples, including 13 VSD and three Tetralogy of Fallot for in silico validation. This data revealed three variations in GATA4, i.e., c.C1223A at exon 6: c.C602A and c.C1220A at exon 7; and one variation in MYH6, i.e., c.G3883C at exon 28 in two VSD cases. This study supports previously published studies that suggested GATA4 and MYH6 as the high-risk genes responsible for septal defects. Thus, this study contributes to a better understanding of the genes involved in heart development by identifying the high-risk genes and interacting proteins in the pathway.

The Evolution of Computer-Assisted Total Hip Arthroplasty and Relevant Applications.

In total hip arthroplasty (THA), the accurate positioning of implants is the key to achieve a good clinical outcome. Computer-assisted orthopaedic surgery (CAOS) has been developed for more accurate positioning of implants during the THA. There are passive, semi-active, and active systems in CAOS for THA. Navigation is a passive system that only provides information and guidance to the surgeon. There are 3 types of navigation: imageless navigation, computed tomography (CT)-based navigation, and fluoroscopy-based navigation. In imageless navigation system, a new method of registration without the need to register the anterior pelvic plane was introduced. CT-based navigation can be efficiently used for pelvic plane reference, the functional pelvic plane in supine which adjusts anterior pelvic plane sagittal tilt for targeting the cup orientation. Robot-assisted system can be either active or semi-active. The active robotic system performs the preparation for implant positioning as programmed preoperatively. It has been used for only femoral implant cavity preparation. Recently, program for cup positioning was additionally developed. Alternatively, for ease of surgeon acceptance, semi-active robot systems are developed. It was initially applied only for cup positioning. However, with the development of enhanced femoral workflows, this system can now be used to position both cup and stem. Though there have been substantial advancements in computer-assisted THA, its use can still be controversial at present due to the steep learning curve, intraoperative technical issues, high cost and etc. However, in the future, CAOS will certainly enable the surgeon to operate more accurately and lead to improved outcomes in THA as the technology continues to evolve rapidly.

The Evolution of Computer-Assisted Total Hip Arthroplasty and Relevant Applications / 대한고관절학회지

In total hip arthroplasty (THA), the accurate positioning of implants is the key to achieve a good clinical outcome. Computer-assisted orthopaedic surgery (CAOS) has been developed for more accurate positioning of implants during the THA. There are passive, semi-active, and active systems in CAOS for THA. Navigation is a passive system that only provides information and guidance to the surgeon. There are 3 types of navigation: imageless navigation, computed tomography (CT)-based navigation, and fluoroscopy-based navigation. In imageless navigation system, a new method of registration without the need to register the anterior pelvic plane was introduced. CT-based navigation can be efficiently used for pelvic plane reference, the functional pelvic plane in supine which adjusts anterior pelvic plane sagittal tilt for targeting the cup orientation. Robot-assisted system can be either active or semi-active. The active robotic system performs the preparation for implant positioning as programmed preoperatively. It has been used for only femoral implant cavity preparation. Recently, program for cup positioning was additionally developed. Alternatively, for ease of surgeon acceptance, semi-active robot systems are developed. It was initially applied only for cup positioning. However, with the development of enhanced femoral workflows, this system can now be used to position both cup and stem. Though there have been substantial advancements in computer-assisted THA, its use can still be controversial at present due to the steep learning curve, intraoperative technical issues, high cost and etc. However, in the future, CAOS will certainly enable the surgeon to operate more accurately and lead to improved outcomes in THA as the technology continues to evolve rapidly.

Effect of Structural Changes in Proteins Derived from GATA4 Nonsynonymous Single Nucleotide Polymorphisms in Congenital Heart Disease.

Congenital heart disease is the most common type of birth defect. The single nucleotide polymorphism in GATA4 is associated with various congenital heart disease phenotypes. In the present study, we analysed the nonsynonymous single nucleotide polymorphism of GATA4, which are involved in congenital heart disease by predicting the changes in protein structures. Total of 49 nonsynonymous single nucleotide polymorphisms of GATA4 was screened from congenital heart disease patients of Mysore and also globally reported nonsynonymous single nucleotide polymorphisms. To understand the role of nonsynonymous single nucleotide polymorphisms, we mutated the sequence and translated into amino acids. Further the mutated protein secondary structure is predicted and tertiary structure is predicted using homology modeling. The quantitative evaluation of protein structure quality was verified with Volume Area Dihedral Angle Reporter server. Results revealed the secondary, tertiary structural changes along with changes in free energy of folding, volume and accessible surface area. Thus, the structural changes in the mutated proteins impaired the normal function of GATA4.

Divergence of the gene aly in experimentally evolved cytoraces, the members of the nasuta-albomicans complex of Drosophila.

We generated cytoraces by crossing the chromosomal races (Drosophila nasuta nasuta and Drosophila nasuta albomicans) of the nasuta subgroup of Drosophila and maintained the offspring over many generations through sibling mating. These cytoraces, along with their parents, are members of the nasuta-albomicans complex of Drosophila. The gene always early (aly) is one of the rapidly evolving genes in the genus Drosophila and plays a central role in regulating meiosis. Here we examined the rate of molecular evolution of aly in cytoraces of Drosophila and demonstrated that the rate of substitutions amongst cytoraces is around eight times greater than their parents and even amongst species of subgenera. Thus, the presence of positive selection in the laboratory-derived cytoraces based on the analysis of the synonymous and nonsynonymous substitution rates of aly suggests the rapid evolution in cytoraces.

Association of methylenetetrahydrofolate reductase gene polymorphisms & colorectal cancer in India.

BACKGROUND & OBJECTIVES: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and involved in DNA synthesis, DNA repair and DNA methylation. The two common functional polymorphisms of MTHFR, 677 C-->T and 1298 A-->C have shown to impact several diseases including cancer. This case-control study was undertaken to analyse the association of the MTHFR gene polymorphisms 677 C-->T and 1298 A-->C and risk of colorectal cancer (CRC). METHODS: One hundred patients with a confirmed histopathologic diagnosis of CRC and 86 age and gender matched controls with no history of cancer were taken for this study. DNA was isolated from peripheral blood samples and the genotypes were determined by PCR-RFLP. The risk association was estimated by compounding odds ratio (OR) with 95 per cent confidence interval (CI). RESULTS: Genotype frequency of MTHFR 677 CC, CT and TT were 76.7, 22.1 and 1.16 per cent in controls, and 74, 25 and 1.0 per cent among patients. The 'T' allele frequency was 12.21 and 13.5 per cent in controls and patients respectively. The genotype frequency of MTHFR 1298 AA, AC, and CC were 25.6, 58.1 and 16.3 per cent for controls and 22, 70 and 8 per cent for patents respectively. The 'C' allele frequency for 1298 A-->C was 43.0 and 45.3 per cent respectively for controls and patients. The OR for 677 CT was 1.18 (95% CI 0.59-2.32, P = 0.642), OR for 1298 AC was 1.68 (95% CI 0.92-3.08, P = 0.092) and OR for 1298 CC was 0.45 (95% CI 0.18-1.12, P = 0.081). The OR for the combined heterozygous state (677 CT and 1298 AC) was 1.18 (95% CI 0.52-2.64, P =0.697). INTERPRETATION & CONCLUSION: The frequency of the MTHFR 677 TT genotype is rare as compared to 1298 CC genotype in the population studied. There was no association between 677 C-->T and 1298 A-->C polymorphisms and risk of CRC either individually or in combination. The homozygous state for 1298 A-->C polymorphism appears to slightly lower risk of CRC. This needs to be confirmed with a larger sample size.

Methylenetetrahydrofolate reductase gene polymorphisms and risk of acute lymphoblastic leukemia in children.

INTRODUCTION: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation. Genetic polymorphisms of this enzyme have been shown to impact several diseases, including cancer. Leukemias are malignancies arising from rapidly proliferating hematopoietic cells having great requirement of DNA synthesis. This case-control study was undertaken to analyze the association of the MTHFR gene polymorphisms 677 C"T and 1298 A"C and the risk of acute lymphoblastic leukemia in children. MATERIALS AND METHODS: Eighty-six patients aged below 15 years with a confirmed diagnosis of acute lymphoblastic leukemia (ALL) and 99 matched controls were taken for this study. Analysis of the polymorphisms was done using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Frequency of MTHFR 677 CC and CT were 85.9% and 14.1% in the controls, and 84.9% and 15.1% in the cases. The 'T' allele frequency was 7% and 7.5% in cases and controls respectively. The frequency of MTHFR 1298 AA, AC, and CC were 28.3%, 55.6% and 16.1% for controls and 23.3%, 59.3% and 17.4% for cases respectively. The 'C' allele frequency for 1298 A-->C was 43.9% and 47% respectively for controls and cases. The odds ratio (OR) for C677T was 1.08 (95% CI 0.48-2.45, p = 0.851) and OR for A1298C was 1.29 (95% CI 0.65-2.29, p = 0.46) and OR for 1298 CC was 1.31 (95% CI 0.53-3.26, p = 0.56). The OR for the combined heterozygous status (677 CT and 1298 AC) was 1.94 (95% CI 0.58-6.52, p = 0.286). CONCLUSION: The prevalence of 'T' allele for 677 MTHFR polymorphism was low in the population studied. There was no association between MTHFR 677 C-->T and 1298 A-->C gene polymorphisms and risk of ALL, which may be due to the small sample size.

INN-toxin, a highly lethal peptide from the venom of Indian cobra (Naja naja) venom-Isolation, characterization and pharmacological actions.

A novel toxic polypeptide, INN-toxin, is purified from the venom of Naja naja using combination of gel-permeation and ion-exchange chromatography. It has a molecular mass of 6951.6Da as determined by MALDI-TOF/MS and the N-terminal sequence of LKXNKLVPLF. It showed both neurotoxic as well as cytotoxic activities. INN-toxin is lethal to mice with a LD(50) of 1.2mg/kg body weight. IgY raised in chicks against basic peptide pool neutralized the toxicity of INN-toxin. INN-toxin did not inhibit cholinesterase activity. It is toxic to Ehrlich ascites tumor (EAT) cells, but it is not toxic to leukocyte culture. The toxin appears to be specific in its mode of action. Interaction of N-bromosuccinamide (NBS) with the peptide resulted in the modification of tryptophan residues and loss of lethal toxicity of INN-toxin.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the biosynthetic N-acetylornithine aminotransferases from Salmonella typhimurium and Escherichia coli.

Acetylornithine aminotransferase (AcOAT) is a type I pyridoxal 5'-phosphate-dependent enzyme catalyzing the conversion of N-acetylglutamic semialdehyde to N-acetylornithine in the presence of alpha-ketoglutarate, a step involved in arginine metabolism. In Escherichia coli, the biosynthetic AcOAT also catalyzes the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate, one of the steps in lysine biosynthesis. It is closely related to ornithine aminotransferase. AcOAT was cloned from Salmonella typhimurium and E. coli, overexpressed in E. coli and purified using Ni-NTA affinity column chromatography. The enzymes crystallized in the presence of gabaculine. Crystals of E. coli AcOAT (eAcOAT) only diffracted X-rays to 3.5 A and were twinned. The crystals of S. typhimurium AcOAT (sAcOAT) diffracted to 1.9 A and had a dimer in the asymmetric unit. The structure of sAcOAT was solved by the molecular-replacement method.

Hybridization and introgression of the genomes of Drosophila nasuta and Drosophila albomicans: evolution of new karyotypes.

Drosophila nasuta (2n = 8) and Drosophila albomicans (2n = 6) are cross-fertile allopatric sibling chromosomal races of the nasuta subgroup of Drosophila. Hybrids of these races can be maintained for any number of generations. Some of the introgressed hybrid lineages of D. nasuta and D. albomicans, after passing through a transient phase of karyotypic polymorphism, ended up with a stable karyotype whose composition is different from those of the parental races. Such hybrid populations were called cytoraces, in which the chromosomes of D. nasuta and D. albomicans are represented in different combinations. The karyotypic composition of 16 such cytoraces have been presented and discussed with reference to evolutionary strategies such as balancing selection, directional selection, and sex-specific effect on different components of the evolving karyotypes.

Incipient sexual isolation in the nasuta-albomicans complex of Drosophila: mating preference in male-, female- and multiple-choice mating experiments.

Interracial divergence is an important facet of speciation. The nasuta-albomicans complex of Drosophila with sixteen morphologically identical, karyotypically different but cross-fertile races is an excellent system to study a few dimensions of raciation. Drosophila nasuta nasuta, Drosophila nasuta albomicans, Cytorace 1, Cytorace 2, Cytorace 3 and Cytorace 4 of this subgroup have been subjected to male-, female- and multiple-choice mating experiments. Out of 8456 crosses conducted, 7185 had successful matings. The overall impression is that mating is far from random amongst these six closely related races of the nasuta-albomicans complex. The males of D. n. albomicans, Cytorace 1 and Cytorace 4 in male-choice, the females of Cytorace 1 and Cytorace 2 in female-choice, and the males and females of D. n. nasuta, D. n. albomicans, Cytorace 1 and Cytorace 4 against the males and females of Cytorace 2 in multiple-choice experiments, had significantly more homogamic matings than expected. Thus in this study of evolutionary experimentation on raciation under laboratory conditions, we have documented the initiation of preference for conspecific matings among closely related and independently evolving members of the nasuta-albomicans complex of Drosophila.

Autoradiographic study of transcription and dosage compensation in the sex and neo-sex chromosome of Drosophila nasuta nasuta and Drosophila nasuta albomicans.

Cellular autoradiography is used to study the transcription patterns of the polytene X chromosomes in Drosophila nasuta nasuta and D. n. albomicans. D. n. nasuta, with 2n = 8, includes a pair of complete heteromorphic sex chromosomes, whereas D. n. albomicans, with 2n = 6, has a pair of metacentric neo-sex chromosomes representing incomplete heteromorphic sex chromosomes. The neo-X chromosome has two euchromatic arms, one representing the ancestral X while the other represents the ancestral autosome 3 chromosomes. The metacentric neo-Y chromosome has one arm with a complete heterochromatic ancestral Y and the other arm with a euchromatic ancestral autosome 3. The transcription study has revealed that the X chromosome in D. n. nasuta is hyperactive, suggesting complete dosage compensation, while in the neo-X chromosome of D. n. albomicans the ancestral X chromosome is hyperactive and the ancestral autosome 3, which is part of the neo-sex chromosome, is similar to any other autosomes. This finding shows dosage compensation on one arm (XLx/-) of the neo-X chromosome, while the other arm (XR3/YR3) is not dosage compensated and has yet to acquire the dosage compensatory mechanism.

Incipient sexual isolation in the nasuta-albomicans complex of Drosophila: no-choice experiments.

Drosophila nasuta nasuta and Drosophila nasuta albomicans are cross-fertile races of Drosophila. Hybridization between these races in the laboratory has given rise to new races (Cytoraces), among which karyotypic composition differs from one another and also from those of the parental races. In this study, we search for the evidence of incipient reproductive isolation among the parental races and four Cytoraces by assessing the fraction of no-matings, mating latency and copulation duration in all possible types of homo- and heterogamic crosses (N = 4184). In no-choice conditions, the latency time (time to initiation of copulation) is lower in homogamic crosses than in heterogamic crosses for both parental races and Cytoraces. Latency time and copulation duration are negatively correlated, whereas fraction of no matings is positively correlated with latency time. Thus these six closely related races of the nasuta-albomicans complex show the initiation of the earliest stages of pre-zygotic isolation, manifested as a tendency for matings to be initiated earlier and more often, and for a longer duration, among homogamic rather than heterogamic individuals

Patterns of replication in the neo-sex chromosomes of Drosophila nasuta albomicans.

Drosophila nasuta albomicans (with 2n = 6), contains a pair of metacentric neo-sex chromosomes. Phylogenetically these are products of centric fusion between ancestral sex (X, Y) chromosomes and an autosome (chromosome 3). The polytene chromosome complement of males with a neo-X- and neo-Y-chromosomes has revealed asynchrony in replication between the two arms of the neo-sex chromosomes. The arm which represents the ancestral X-chromosome is faster replicating than the arm which represents ancestral autosome. The latter arm of the neo-sex chromosome is synchronous with other autosomes of the complement. We conclude that one arm of the neo-X/Y is still mimicking the features of an autosome while the other arm has the features of a classical X/Y-chromosome. This X-autosome translocation differs from the other evolutionary X-autosome translocations known in certain species of Drosophila.

Direct detection of Borrelia burgdorferi in Ixodes scapularis (Acari: Ixodidae) nymphs by hybridization to ribosomal RNA.

A method for direct detection of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner has been developed. Cells are lysed to facilitate release of ribosomal RNA. Lysates are filtered onto nylon membranes that are hybridized with probes specific for sequences in B. burgdorferi 23S rRNA. The technique is rapid and does not require any enzymatic amplification steps. With the use of a cocktail containing five different probes, approximately 1,000 organisms could be detected. The assay was successfully applied to direct detection of B. burgdorferi in Ixodes scapularis Say nymphs.

Racial divergence in abdominal bristles among parental races and newly evolved cytoraces of nasuta-albomicans complex of Drosophila.

Cytoraces are the products of interracial hybridization between Drosophila nasuta nasuta and D. nasuta albomicans. These races differ from their parents in the chromosome composition, mating preference, certain fitness phenotypes and also a few morphophenotypic traits. Now, these cytoraces are passing through 330 generations. Racial divergence in the 4th and 5th abdominal bristles among the parental races and the newly evolved cytorace 1 and 2 is reported. The results revealed that the parental races have more number of bristles than newly evolved cytoraces. Thus, these cytoraces are evolved/evolving with reduced abdominal bristle number and better fitness.

Does evolution reduce the body size? A study of the four members of newly evolved nasuta-albomicans complex of Drosophila.

Our long range interracial hybridization experiments between a pair of cross fertile races, Drosophila nasuta (2n = 8) and D. albomicans (2n = 6) have resulted in the evolution of two new karyotypic strains under laboratory conditions, which are named as Cytorace 1 and Cytorace 2. These Cytoraces harbor chromosomes from both parents. Here, we compare the body size of the parental races and newly evolved Cytoraces and the relationship between the body size and fitness. Analysis reveals that the parental races have reduced fertility and are larger in body size than newly evolved Cytoraces. Thus, the newly evolved Cytoraces show reduced body size and better fitness in the course of their evolution.

A direct screen identifies new flight muscle mutants on the Drosophila second chromosome.

An ethyl methanesulfonate mutagenesis of Drosophila melanogaster was undertaken, and >3000 mutagenized second chromosomes were generated. More than 800 homozygous viable lines were established, and adults were screened directly under polarized light for muscle defects. A total of 16 mutant strains in which the indirect flight muscles were reduced in volume or disorganized or were otherwise abnormal were identified. These fell into seven recessive and one semidominant complementation groups. Five of these eight complementation groups, including the semidominant mutation, have been mapped using chromosomal deficiencies and meiotic recombination. Two complementation groups mapped close to the Myosin heavy chain gene, but they are shown to be in different loci. Developmental analysis of three mutations showed that two of these are involved in the early stages of adult myogenesis while the other showed late defects. This is the first report of results from a systematic and direct screen for recessive flight muscle defects. This mutant screen identifies genes affecting the flight muscles, which are distinct from those identified when screening for flightlessness.

Molecular cloning and characterization of CFT1, a developmentally regulated avian alpha(1,3)-fucosyltransferase gene.

Although coordinate expression of carbohydrate epitopes during development is well described, mechanisms which regulate this expression remain largely unknown. In this study we demonstrate that developing chicken B cells express the LewisX terminal oligosaccharide structure in a stage-specific manner. To examine regulation of this expression, we have cloned and expressed the chicken alpha(1,3)-fucosyltransferase gene involved in LewisX biosynthesis, naming it chicken fucosyltransferase 1 (CFT1). CFT1 is characterized by a single long open reading frame of 356 amino acids encoding a type II transmembrane glycoprotein. The domain structure and predicted amino acid sequence are highly conserved between CFT1 and mammalian FucTIV genes (52.8% and 46.3% identity to mouse and human respectively). In vitro CFT1 fucosyltransferase activity utilizes LacNAc > 3'sialyl-LacNAc acceptors with almost no utilization of other neutral type II (lactose, 2-fucosyllactose), or type I (lacto-N-biose I) acceptors. CFT1-transfected cells make cell surface LewisX (COS-7) and LewisX + VIM-2 structures (Chinese hamster ovary). CFT1 gene expression is tissue-specific and includes embryonic thymus and bursa. Furthermore, expression of the CFT1 gene and cell surface LewisX structures are closely linked during B cell development. These findings reveal the evolutionary conservation between nonmammalian and mammalian alpha(1,3)-fucosyltransferase genes and demonstrate a role for fucosyltransferase gene regulation in the developmental expression of oligosaccharide structures.