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Biogeography of macro- and micro-organisms in the deep sea is, in part, shaped by naturally occurring heterogeneous habitat features of geological and biological origin such as seeps, vents, seamounts, whale and wood-falls. Artificial features including shipwrecks and energy infrastructure shape the biogeographic patterns of macro-organisms; how they influence microorganisms is unclear. Shipwrecks may function as islands of biodiversity for microbiomes, creating a patchwork of habitats with influence radiating out into the seabed. Here we show microbiome richness and diversity increase as a function of proximity to the historic deep-sea shipwreck Anona in the Gulf of Mexico. Diversity and richness extinction plots provide evidence of an island effect on microbiomes. A halo of core taxa on the seabed was observed up to 200 m away from the wreck indicative of the transition zone from shipwreck habitat to the surrounding environment. Transition zones around natural habitat features are often small in area compared to what was observed at Anona indicating shipwrecks may exert a large sphere of influence on seabed microbiomes. Historic shipwrecks are abundant, isolated habitats with global distribution, providing a means to explore contemporary processes shaping biogeography on the seafloor. This work is a case study for how built environments impact microbial biodiversity and provides new information on how arrival of material to the seafloor shapes benthic microbiomes.

Maternal risk factors and their interactions with each other that associate chromosome 21 nondisjunction are intriguing and need incisive study to be resolved. We determined recombination profile of nondisjoined chromosome 21 and maternal genotypes for four selected polymorphic variants from the folate regulators genes stratifying the women according to the origin of segregation error and age at conception. We conducted association study for genotype and maternal addiction to smokeless chewing tobacco, usually chopped tobacco leaves or paste of tobacco leaves with the incidence of Down syndrome birth. Additionally, we designed various logistic regression models to explore the effects of maternal genotype, maternal habit of smokeless chewing tobacco, maternal age at conception and all possible interactions among them on chromosome 21 nondisjunction. We found folate regulator gene mutations are associated with maternal meiosis II error. Regression models revealed smokeless chewing tobacco and folate polymorphic/mutant risk genotype interact with each other to increase the risk of reduced and single peri-centromeric recombination events on chromosome 21 that nondisjoined at meiosis II in the oocytes and the effect is maternal age independent. We inferred maternal folate polymorphic/mutant risk genotypes and habit of smokeless chewing tobacco interact with each other and increase the risk of meiosis II error in oocytes in maternal age-independent manner.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

In this paper we propose a system to analyze bacteria from a given Scanning Electron Microscope (SEM) image of the bacterial sample. Thousands of bacteria lives in the human gut and recent studies have shown that the quantitative features of the microbiome, such as co-existence ratio of different bacteria, can be indicative of the health condition in humans. Conventional bacteria analysis methods using microscopic images, can only be used to examine a single bacteria colony. In contrast, we propose a novel system to morphologically analyze the bacteria from SEM images. By this, we expect to enable a rapid analysis of the human gut bacteria ratio, in which various type of bacteria are mixed. However, to achieve an automatic and accurate count of the bacteria in the SEM images, it is important to accurately identify the bacteria regions, separate the connected bacteria regions and classify them. To address this, we propose a system that includes a segmentation, separation and a classification module. Our system achieves more than 90% recall for all of original three datasets that we have created. Subsequently, we show the comparison results between another state-of-the-art segmentation method and our system, and we empirically report that our system has a better performance.

Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans., Graphical Abstract

Consanguineous marriage was examined as a risk factor for Down syndrome birth. We genotyped Down syndrome family trios using short tandem repeat markers on 21q-to interpret the parental and meiotic stage of origin of errors as well as to record recombination profile along long arm of chromosome 21. We then compared nonconsanguineous (N = 811) group with-the consanguineous (N =157) marriages. We report for the first time that consanguineous marriage is associated with an increased risk for nondisjunction of chromosome 21 in oocytes-during the second meiotic division. We observed the absence of recombination more frequently in younger mothers in nonconsanguineous meiosis I cases. This was in contrast to an equal distribution of nonrecombinant cases across the age categories in the meiosis I consanguineous group. Moreover, the non-consanguineous group exhibited preferential telomeric recombination in meiosis I error among younger women and centromeric recombination in meiosis II errors in older women. In contrast, the consanguineous group exhibited medially placed recombination events in both meiosis I and meiosis II nondisjunction errors. Additionally, we recorded reduced maternal age at conception in the-consanguineous group. These findings suggest novel risk factors associated that increase the risk of chromosome 21 nondisjunction in the families with consanguinity.

An etiologically complex disorder, Attention Deficit Hyperactivity Disorder (ADHD), is often associated with various levels of cognitive deficit. Folate/vitamin B9 is crucial for numerous biochemical pathways including neural stem cell proliferation and differentiation, regulation of gene expression, neurotransmitter synthesis, myelin synthesis and repair, etc. and a scarcity has often been linked to cognitive deficit. Our pilot study in the field revealed significant association of few genetic variants with ADHD. Mild hyperhomocysteinemia and vitamin B12 deficiency was also noticed in the probands. In the present study additional genetic variants, folate and vitamin B6, which may affect folate-homocysteine metabolic pathway, were investigated in 866 individuals including nuclear families with ADHD probands (N=221) and ethnically matched controls (N=286) to find out whether ADHD associated traits are affected by these factors. Population based analysis revealed significant over representation of MTRR rs1801394 "G" allele and "GG" genotype in all as well as male probands. Stratified analysis showed significantly higher frequency of RFC1 rs1051266 and BHMT rs3733890 "AG" genotypes in full term and prematurely delivered ADHD probands respectively. Probands with rs1801394 "GG" genotype and BHMT rs3733890 "G" allele showed association with hyperhomocysteinemia. MTHFR rs1801131, MTR rs1805087 and BHMT rs3733890 also showed association with ADHD index. While rs1051266, rs1801131, and rs1805087 showed association with behavioral problems, rs3733890 was associated with ODD score. Conduct problem exhibited association with RFC1 rs1051266, MTHFR rs1801133 and MTRR rs1801394. Gene-gene interaction analysis revealed positive synergistic interactions between rs1051266, rs1801131 and rs1801394 in the probands as compared to the controls. It can be inferred from the data obtained that folate system genetic variants and mild hyperhomocysteimenia may affect ADHD associated traits by attenuating folate metabolism.

First time chemometric analyses have been carried out with the data obtained from ATR-FTIR spectroscopy of freeze-dried Aloe vera gel to study the distribution of functional groups of aloe hydro-gel at different growth periods; phenolic –OH, =CO,–CH3, COO– and –COC were detected as the predominant functional groups. The presence of conjugated double bonds of component phenolics attributes promising UV radiation absorption potential to aloe gel. The concentration of aloin as estimated with RP-HPLC/DAD ranged from 1.48 ± 0.26 to 3.51 ± 0.12 µg g-1freeze-dried gel; the upper values being observed in 3-year-aloe of winter followed by summer season

Altered patterns of recombination on 21q have long been associated with the nondisjunction chromosome 21 within oocytes and the increased risk of having a child with Down syndrome. Unfortunately the genetic etiology of these altered patterns of recombination have yet to be elucidated. We for the first time genotyped the gene MCM9, a candidate gene for recombination regulation and DNA repair in mothers with or without children with Down syndrome. In our approach, we identified the location of recombination on the maternal chromosome 21 using short tandem repeat markers, then stratified our population by the origin of meiotic error and age at conception. We observed that twenty-five out of forty-one single nucleotide polymorphic sites within MCM9 exhibited an association with meiosis I error (N = 700), but not with meiosis II error (N = 125). This association was maternal age-independent. Several variants exhibited aprotective association with MI error, some were neutral. Maternal age stratified characterization of cases revealed that MCM9 risk variants were associated with an increased chance of reduced recombination on 21q within oocytes. The spatial distribution of single observed recombination events revealed no significant change in the location of recombination among women harbouring MCM9 risk, protective, or neutral variant. Additionally, we identified a total of six novel polymorphic variants and two novel alleles that were either risk imparting or protective against meiosis I nondisjunction. In silico analyses using five different programs suggest the risk variants either cause a change in protein function or may alter the splicing pattern of transcripts and disrupt the proportion of different isoforms of MCM9 products within oocytes. These observations bring us a significant step closer to understanding the molecular basis of recombination errors in chromosome 21 nondisjunction within oocytes that leads to birth of child with Down syndrome., Author summary We studied MCM9 variations in the genome of women with a Down syndrome child by stratifying the women based on MCM9 genotypes, meiotic error group, and their age of conception. We identified polymorphisms are associated with reduced recombination and nondisjunction of chromosome 21 at the meiosis I stage of oogenesis in a maternal age-independent manner. But these variants do not affect the position of chiasma formation. In Silico analyses revealed the presence of MCM9 variants that may cause alteration in protein function due to amino acid substitution. We also identified splice variants in MCM9. We hypothesize that the polymorphisms in MCM9 predispose women to experience reduced recombination on chromosome 21 in oocytes at meiosis I, which ultimately leads to the birth of a child with Down syndrome.

Oligotrophic Lake Superior and mesotrophic Lake Erie are trophic end members of the hydrologically connected Laurentian Great Lakes system, and as such exhibit different profiles of dissolved nitrogen species. Nitrification in Lake Superior has led to increasing nitrate concentrations over the past century, as opposed to Erie, where nitrate inventories have declined due to denitrification. In this study, we examined the abundance and diversity of nitrifying microbes involved in the oxidation of ammonia to nitrite, and nitrite to nitrate. By in situ hybridization methods, we enumerated the major planktonic ammonia oxidizing bacteria (AOB) and archaea (AOA) during a July 2011 cruise from Lake Superior to Lake Erie. In Lake Superior, AOA dominated compared to AOB, typically exceeding 5 × 103 mL− 1, whereas in Erie, AOB were more abundant than AOA. These data parallel prior work on Lake Superior and Lake Erie sediments, in which AOA are far more abundant in Superior, but AOB dominate in Erie. The lakes were sampled during stratification, and AOA and AOB were largely restricted to the hypolimnion, consistent with the observation that ammonia oxidizers are photoinhibited in surface waters. In Lakes Superior and Erie, we also detected nitrite oxidizing bacteria (NOB) in a pattern paralleling AOA/AOB abundance. Phylogenetic analysis of archaeal 16S rRNA revealed that the planktonic archaea of Lake Superior are members of the ammonia oxidizing Group I.1a Thaumarchaeota most closely related to Nitrosoarchaeum limnia. These AOA are distinct from the Group I.1a AOA in Lake Superior sediments. The major AOB of Lake Erie form a subcluster within the genus Nitrosospira.

Physical and biochemical characterization of freeze-dried Aloe vera L. gel (AG) has been carried out from different age groups and harvesting seasons to get a clear scenario of AG properties at different growth periods. The stability of amorphous AG under thermal perturbation was analyzed with reference to the respective glass transition temperature by differential scanning calorimetry. The functional properties of gel in the form of water holding and fat adsorption capacity were more pronounced at 3-year-old plants in summer. A higher glass transition temperature (135 ± 4.80 °C) of gel from 3-year-old aloe accounts for higher concentration and better cross-linking of the component polysaccharides. The chemometric studies encompassing principal component and partial least square analysis depict an intrinsic structure-function relation with reference to different properties considered, which has been demonstrated to be influenced by growth periods of plants.

Regulated turnover of miRNAs—tiny posttranscriptional regulators of gene expression in metazoan cells—is important to optimize cellular response to external stimuli. Cell–cell contact alters miRNA stability by restricting its exosomal export., MicroRNAs (miRNAs) are tiny posttranscriptional regulators of gene expression in metazoan cells, where activity and abundance of miRNAs are tightly controlled. Regulated turnover of these regulatory RNAs is important to optimize cellular response to external stimuli. We report that the stability of mature miRNAs increases inversely with cell proliferation, and the increased number of microribonucleoproteins (miRNPs) in growth-restricted mammalian cells are in turn associated with polysomes. This heightened association of miRNA with polysomes also elicits reduced degradation of target mRNAs and impaired extracellular export of miRNA via exosomes. Overall polysome sequestration contributes to an increase of cellular miRNA levels but without an increase in miRNA activity. Therefore miRNA activity and turnover can be controlled by subcellular distribution of miRNPs that may get differentially regulated as a function of cell growth in mammalian cells.

Background Attention deficit hyperactivity disorder (ADHD) is an etiologically complex childhood onset neurobehavioral disorder characterized by age-inappropriate inattention, hyperactivity, and impulsivity. Symptom severity varies widely and boys are diagnosed more frequently than girls. ADHD probands were reported to have abnormal transmissions of dopamine, serotonin, and/or noradrenaline. Monoamine oxidase A (MAOA) and B (MAOB), mitochondrial outer membrane bound two isoenzymes, mediate degradation of these neurotransmitters and thus regulating their circulating levels. Case-control analyses in different populations, including Indians, suggested involvement of MAOA and MAOB genes in the etiology of ADHD. Due to high heritability rate of ADHD, we tested familial transmission of MAOA and MAOB variants to ADHD probands in 190 nuclear families having ADHD probands from Indo-Caucasoid ethnicity. Methods Subjects were recruited following the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV). Appropriate scales were used for measuring the behavioral traits in probands. Genotyping was performed through PCR-based amplification of target sites followed by DNA-sequencing and/or gel-electrophoresis. Data obtained were analyzed by family based statistical methods. Results Out of 58 variants present in the analyzed sites only 15 were found to be polymorphic (30 bp-uVNTR, rs5906883, rs1465107, rs1465108, rs5905809, rs5906957, rs6323, rs1137070 from MAOA and rs4824562, rs56220155, rs2283728, rs2283727, rs3027441, rs6324, rs3027440 from MAOB). Statistically significant maternal transmission of alleles to male probands was observed for MAOA rs5905809 ‘G’ (p = 0.04), rs5906957 ‘A’ (p = 0.04), rs6323 ‘G’ (p = 0.0001) and MAOB rs56220155 ‘A’ (p = 0.002), rs2283728 ‘C’ (p = 0.0008), rs2283727 ‘C’ (p = 0.0008), rs3027441 ‘T’ (p = 0.003), rs6324 ‘C’ (p = 0.003), rs3027440 ‘T’ (p = 0.0002). Significantly preferential maternal transmissions of different haplotype combinations to male probands were also noticed (p

We have tested maternal polymorphic variants MTR A2756G (rs1805087), MTRR A66G (rs1801394), MTHFR C677T (rs1801133) and MTHFR A1298C (rs1801131) for their association with meiotic errors in oocyte and Down syndrome birth among 730 controls and 1019 case mothers having Down syndrome child from Bengali speaking population of India. We demonstrated significant association of MTR A2756G (rs1805087), MTRR A66G (rs1801394), MTHFR A1298C (rs1801131), but not MTHFR C677T (rs1801133) variants with maternal meiosis II nondisjunction in maternal age independent manner. Moreover, the mutant genotypes of two tested loci when co-occurred exhibited synergistic effect and increase the risk of Down syndrome birth many folds. In silico analyses predicted the probable damages of the transcripts or proteins from respective genes owing to said polymorphisms among the women. These findings from the largest sample population tested ever bring us a significant step closer towards understanding the relation between folate metabolism, meiotic error and Down syndrome birth.

This paper presents an analysis of elemental distribution, microstructure at pre- and post-rehydration, and swelling of freeze-dried Aloe vera L. gel, obtained from two, three and four-year-old plant in summer. To explore the seasonal influence, three-year-old plants from rainy and winter season are also considered for analysis. Mineral distribution is studied following the estimation of Ca, K, Mg, P, Na, Mn, Se, Al, Fe, Zn, Cu and Cd. A consolidated effort has also been made to study the concentration of total C and N content of gel at different growth periods. Re-hydration mediated continuous topography in poly-dispersed, unorganized and amorphic freeze-dried gel accounts for the post-rehydration cross-linking among the component polysaccharides. Chemometric analysis (principal component and cluster analysis) suggests that the mineral content of gel change as a function of growth periods of plants. High content of organic C and Se with maximum water absorbing potential (in the form of swelling activity) are mainly associated with three-year-old aloe of summer; the contents of N, Na, Ca, K, Mg, P and Cu are observed to be high in two-year plants. To the best of our knowledge and belief, this is the first time, Se (a bioactive antioxidant activity promoting element) is found to be present in aloe gel.

Various free radicals (DPPH, super-oxide and hydroxyl) scavenging, transition metal chelating and reducing power assays were conducted to study the antioxidant potential of freeze-dried foliar gel obtained from 2, 3 and 4-year-old Aloe vera L. To explore the influence of season, apart from summer, gels from rainy and winter season of 3-year-old aloe were also considered. Gels obtained from different growth periods showed antioxidant potential with varying free radical quenching, transition metal chelating and reducing power activity. The antioxidant activity of aloe gel is changed as a factor of the age of the plants and harvesting seasons, and found to be more pronounced in 3-year-old aloe during summer. Degree of acetylation and functional group distribution studied by 1H NMR and FTIR spectroscopy respectively, and the distribution of the component polysaccharides and phenolics at different harvesting regimens are found to be critical in the regulation of bioactive potential of aloe gel in terms of pertained antioxidant activity.

Antioxidant assay, ATR-FTIR spectroscopy mediated predominant functional group detection and UV-absorption spectroscopy have been carried out with methanolic extracts of Aloe vera L. gel (gel-extract) from two, three and four-year-old plants in summer season. To explore the seasonal influence gel-extract has also been prepared from three-year-old aloe in rainy and winter season. Gel-extracts from three-year-old aloe in winter exhibited high response at IR and UV absorption spectrum. Maximum transmittance in IR spectrum has been assigned to phenolic OH stretching followed by C H, CO and COC. Total phenols, flavonoids and flavonols content range from 30.11 ± 1.89 to 35.77 ± 1.07 μg GAE/mg, 11.00 ± 0.88 to 29.75 ± 0.88 μg RE/mg and 7.47 ± 0.31 to 13.87 ± 0.08 μg RE/mg, respectively. The contents of total phenolics, flavonoids, flavonols, aloin, and different free radical scavenging, transition metal chelating and Fe3+ reducing are more pronounced in the gel-extracts from three-year-old aloe at winter and summer season. Phenolics concentration and the distribution of aloin in the gel-extract are significantly (p ≤ 0.05) influenced by the growth periods of A. vera. The presence of conjugated double bonds and high abundance of the integral phenolic OH attribute to the UV absorption and antioxidant potential of the gel-extract, respectively. The present work analysis shows that growth periods of A. vera plays a decisive factor in the regulation of antioxidant activity and UV absorption property of the gel-extracts.

Congo-red-acemannan (CR-acemannan) binding assay has been followed to compare the acemannan distribution in freeze-dried gel at two, three and four-year-old Aloe vera L. To explore the seasonal influence gels are also analyzed at rainy and winter season from three-year-old plants. Detection of predominant functional groups and elemental analysis have been carried out by ATR-FTIR and SEM-EDS, respectively. SEM and L * a * b * color space analysis have been employed to explore the microstructure and appearance of freeze-dried gel, respectively. Acidity and electrical-conductivity have also been measured as the physical properties of gel. Electrical-conductivity was significantly high ( p ≤ 0.05) at two-year-old plants and all of the gels are acidic in range (4.63 ± 0.02 to 4.93 ± 0.01). From 1 H NMR spectroscopy, no sign of retrogradation was noticed in dried gel. It has been observed that growth periods play the decisive factor influencing the acemannan and mineral concentration, and the color components of freeze-dried gel as well. Of the tested freeze-dried gels, the absorbance specific to CR-acemannan conjugate formation is maximum with three-year-old plants in summer, and minimum with two-year-old plants. Phenolic OH, CO, CH 3 , COO and C O C are the predominant functional groups present in gel. Collectively, sodium concentration was high in gel followed by magnesium, potassium and calcium. Among the considered minerals, the relative abundance is more with Mg, K and P at two-year-old Aloe . Sublimation at freeze-drying renders an unorganized dispersed structure to aloe gel by breaking down the compact parenchymatous cellular structure. Our experiments suggest that, the lightness of gel is high in winter, followed by summer, for a three-year-old plant.

The methanolic extract of Aloe vera L. gel was subjected to antioxidant guided fractionation with silica gel column chromatography to screen the potent fraction. The antioxidant capacities of different fractions were evaluated using DPPH radical scavenging assay and correlated with total phenol content. Total phenolic contents of different fractions were determined according to the Folin-Ciocalteu spectrophotometric method. The positive correlation was observed between DPPH radical scavenging assay and total phenolic contents indicating that phenolics in Aloe vera L. gel were fundamental contributor of antioxidant activity. Third pooled fraction was identified as potential fraction with highest antioxidant potential. This fraction indicated the presence of well resolved fluorescent components. Characteristic UV–vis absorption and HPLC analysis indicates that aloin take part in antioxidant potential attributed to aloe gel.

Synaptosomal-associated protein 25 (SNAP25) is an essential component for synaptic vesicle mediated release of neurotransmitters. Deficiencies or abnormal structure or function of SNAP25 protein, possibly arising through genetic variations in the relevant DNA code, has been suggested to play role in the pathology of several neurobehavioural disorders including Attention deficit Hyperactivity Disorder (ADHD) and a number of polymorphisms in the SNAP25 gene has been studied for association with the disorder. In the present investigation, for the first time association between ADHD and six SNAP25 polymorphisms, rs1889189, rs362569, rs362988, rs3746544, rs1051312, and rs8636 was explored in eastern Indian population. Subjects were recruited following the Diagnostic and Statistical Manual for Mental Disorders-IV. Genomic DNA isolated from peripheral blood leukocytes of ADHD probands (n = 150), their parents (n = 272) and ethnically matched controls (n = 100) was used for amplifying target sites. Data obtained were subjected to population- as well as family-based analyses. While case-control analysis revealed lack of any significant difference for alleles, family-based studies revealed a mild over transmission rs3746544 'T' and rs8636 'C' alleles (P = 0.05 and 0.03 respectively). Haplotypes formed between rs362569 "T", 362988 "G", rs3746544 "T", rs1051312 "T" and rs8636 "C" in different combinations showed statistically significant transmission to ADHD probands. Excepting rs3746544 and rs8636, all the tested sites showed very low linkage disequilibrium between them. Data obtained in this preliminary study indicates that rs3746544 'T' allele may have some role in the disease etiology in the studied Indian population.

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

The fission yeast Schizosaccharomyces pombe homologue of the p40/eIF3h subunit of mammalian translation initiation factor eIF3 has been characterized in this study. We show that this protein physically associates with the 40S ribosomal particles as a constituent of the multimeric eIF3 protein complex, which consists of all five known eIF3 core subunits (eIF3a, eIF3b, eIF3c, eIF3g and eIF3i) as well as the five non-core subunits (eIF3d, eIF3e, eIF3f, eIF3h and eIF3m) that constitute an eIF3 holocomplex in fission yeast. However, affinity purification of eIF3 from fission yeast cells expressing TAP-tagged eIF3h suggests the presence of distinct forms of eIF3 that differ in their composition of the non-core subunits. Further characterization of eIF3h shows that strains lacking eif3h+ (eif3hΔ) are viable and show no gross defects, either in vegetative growth or in the rate of in vivo protein synthesis. Polysome profile analysis shows no apparent defects in translation initiation. Furthermore, deletion of eif3h+ does not affect the ability of the other eIF3 subunits to remain associated with one another in a tight protein complex similar to the situation in wild-type cells. Additionally, we show that human eIF3h can functionally substitute fission yeast eIF3h in complementing in vivo a genetic deletion of eif3h+. Interestingly, mutant eif3hΔ cells show several prominent phenotypic properties. They are hypersensitive to caffeine and highly defective in meiosis, producing either no spores or incomplete tetrads with a very high frequency. The implications of these results in relation to the functions of eIF3h in Sz. pombe are discussed. Copyright © 2008 John Wiley & Sons, Ltd.

The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and 32P-labeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells.

Patients with temporal lobe epilepsy (TLE) are prescribed first-line antiepileptic drugs and surgery to the management of this disorder. Unfortunately, the surgical treatment has been shown to be beneficial for the selected patients but fails to provide a seizure-free outcome in 20-30% of TLE patients. In our present study, we investigate the possibilities of marketed antiepileptic drugs in a different manner to improve the present situation in TLE. Molecular docking simulation study and various open source computational tools were used to perform the study. AutoDock 4.2 MGL tools, Pymol visualize tools, Patch dock server, and Swarm Dock servers (protein-protein docking) were used to perform the molecular modeling. FTsite and computed atlas of surface topography of protein open source server were used to understand the pocket and ligand binding information respectively. Toxtree application was used to determine the toxicity profile of the drug by Cramers rule. The obtained molecular docking models (Caspase 3, Procaspase 8, and Fas-associated death domain [FADD]) with selected compounds (Clonazepam, Clobazepam, and Retigabine) showed promising trio blocking event of FADD, Caspase 3, and Procaspase 8 (−6.66 kcal, −8.1 kcal, 6.46 kcal) by Clonazepam respectively. Protein-protein interaction study (Swarm Dock, Patch Dock server) indicated promising results that helped to establish our hypothesis. Toxtree showed a quantitative structure toxicity relationship report that helps to clarify the toxicity of the selected compounds. Clonazepam showed a trio inhibition property that may lead to develop a new era of the new generation benzodiazepine prototype drugs in the future. Filtered compounds will further process for higher in vitro, in vivo models for better understanding of the mechanism.

Background: We have previously demonstrated a relationship between children born with Down syndrome and maternal telomere length. Similarly, exposure to tobacco and oral contraceptives has been explored in one of our earlier studies as a risk factor for Down syndrome. Objective: In the present study, we consider the interactions among these risk factors associated with Down syndrome in a population from Kolkata, India, using analyses stratified by maternal age. Methods: We estimated the telomere length of women with children with Down syndrome by restriction enzyme/Southern blot methods. Linear regression was employed to estimate telomere shortening as an indicator of the maternal age of conception. Interactions among the various factors were analyzed by logistic regression. Result: We found an association between the use of smokeless chewing tobacco and shorter telomere length among women who experienced meiosis I nondisjunction at gametogenesis; the effect is seen across all maternal age groups. In contrast, oral contraceptive use alone did not exhibit a statistically significant association with maternal telomere length, but there was an interaction with the use of smokeless chewing tobacco in the older mothers who experienced meiotic II nondisjunction. Conclusion: Environmental/habitual factors interact with molecular components of the oocyte, which ultimately increases the risk of chromosome 21 nondisjunction and subsequently of giving birth to a child with Down syndrome.

Cigarette smoke has long been recognized as a major environmental pollutant that can cause significant damage to the cellular macromolecules. Although much is known about the types of damage, little is known about the cellular responses to the stress caused by cigarette smoke. We have used the fission yeast Schizosaccharomyces pombe to elucidate the overall cellular responses towards cigarette smoke. Here, we demonstrate that fission yeast cells exposed to aqueous extract of cigarette smoke exhibit cell cycle arrest and cell death in a dose-dependent manner. Cigarette smoke treatment also results in accumulation of reactive oxygen species, unusual nuclear morphology and altered cellular structure. Our data further establish activation of the S phase checkpoint in cigarette smoke-exposed Sz. pombe cells. The checkpoint proteins Rad3, Rad26, Rad17, Rad1, Hus1 and Cds1 play key roles in this process, as evidenced by cell survival and biochemical analysis, although another checkpoint protein, Rad9, seems to be less required. Our results also suggest involvement of the stress-activated protein kinase Spc1/Sty1 and the bZIP transcription factors Atf1 and Pap1 in the cellular response towards cigarette smoke extract. These findings indicate activation of the critical S phase checkpoint and cell cycle arrest in Sz. pombe following CSE assault.

Associations between attention deficit hyperactivity disorder (ADHD) and genetic polymorphisms in the dopamine receptors, transporter and metabolizing enzymes have been reported in different ethnic groups. Gene variants may affect disease outcome by acting synergistically or antagonistically and thus their combined effect becomes an important aspect to study in the disease etiology. In the present investigation, interaction between ten functional polymorphisms in DRD4, DAT1, MAOA, COMT, and DBH genes were explored in the Indo-Caucasoid population. ADHD cases were recruited based on DSM-IV criteria. Peripheral blood samples were collected from ADHD probands (N=126), their parents (N=233) and controls (N=96) after obtaining informed written consent for participation. Genomic DNA was subjected to PCR based analysis of single nucleotide polymorphisms and variable number of tandem repeats (VNTRs). Data obtained was examined for population as well as family-based association analyses. While case-control analysis revealed higher occurrence of DAT1 intron 8 VNTR 5R allele (P=0.02) in cases, significant preferential transmission of the 7R-T (DRD4 exon3 VNTR-rs1800955) and 3R-T (MAOA-u VNTR-rs6323) haplotypes were noticed from parents to probands (P=0.02 and 0.002 respectively). Gene-gene interaction analysis revealed significant additive effect of DBH rs1108580 and DRD4 rs1800955 with significant main effects of DRD4 exon3 VNTR, DAT1 3'UTR and intron 8 VNTR, MAOA u-VNTR, rs6323, COMT rs4680, rs362204, DBH rs1611115 and rs1108580 thereby pointing towards a strong association of these markers with ADHD. Correlation between gene variants, high ADHD score and low DBH enzymatic activity was also noticed, especially in male probands. From these observations, an impact of the studied sites on the disease etiology could be speculated in this ethnic group.