Abstract  Locked nucleic acid (LNA) is a modified RNA nucleotide that can be incorporated at specific positions to generate probes with the desired length, melting temperature (TM), and specificity. Here, we describe a method of multiplex genotyping based on dramatic shifts in the TM of a single dual-labeled LNA probe. Using this method, two varieties of the hairtail fish Trichiurus lepturus can be distinguished from each other, as well as from Trichiurus japonicus, based on a 1- to 2-bp difference in a fragment of mitochondrial cytochrome oxidase subunit 1. The shift in TM was 15 °C for a 1-bp mismatch and 27 °C for a 2-bp mismatch, indicating remarkable specificity. We anticipate that the method will be widely useful in applications such as species identification that require accurate, multiplex, and efficient detection of DNA polymorphisms.

Abstract  BACKGROUND: Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains.

RESULTS: These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences.

CONCLUSIONS: This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

Abstract  KEY MESSAGE: Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2-12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1-10 bp, but also vector DNA inserts of 34-236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

Abstract  Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the T0 transformants and their stable inheritance in homozygous T1∶2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i) pretreatment of immature spikes with CuSO4 solution (500 mg/L) at 4°C for 10 days; (ii) electroporation of plasmid DNA in enlarged microspores by a single pulse of ∼375 V; (iii) induction of microspores after transfection at 28°C in NPB-99 medium and regeneration at 26°C in MMS5 medium; (iv) co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v) elimination of AGL-1 cells after co-cultivation with timentin (200-400 mg/L).

Abstract  We present a study of five children from three unrelated Irish Traveller families presenting with primary ciliary dyskinesia (PCD). As previously characterized disorders in the Irish Traveller population are caused by common homozygous mutations, we hypothesised that all three PCD families shared the same recessive mutation. However, exome sequencing showed that there was no pathogenic homozygous mutation common to all families. This finding was supported by histology, which showed that each family has a different type of ciliary defect; transposition defect (family A), nude epithelium (family B) and absence of inner and outer dynein arms (family C). Therefore, each family was analysed independently using homozygosity mapping and exome sequencing. The affected siblings in family A share a novel 1 bp duplication in RSPH4A (NM_001161664.1:c.166dup; p.Arg56Profs*11), a radial-spoke head protein involved in ciliary movement. In family B, we identified three candidate genes (CCNO, KCNN3 and CDKN1C), with a 5-bp duplication in CCNO (NM_021147.3:c.258_262dup; p.Gln88Argfs*8) being the most likely cause of ciliary aplasia. This is the first study to implicate CCNO, a DNA repair gene reported to be involved in multiciliogenesis, in PCD. In family C, we identified a ∼3.5-kb deletion in DYX1C1, a neuronal migration gene previously associated with PCD. This is the first report of a disorder in the relatively small Irish Traveller population to be caused by >1 disease gene. Our study identified at least three different PCD genes in the Irish Traveller population, highlighting that one cannot always assume genetic homogeneity, even in small consanguineous populations.

Abstract  Mutation and selection are both thought to impact significantly the nucleotide composition of bacterial genomes. Earlier studies have compared closely related strains to obtain mutation patterns based on the hypothesis that these bacterial strains had diverged so recently that selection will not have had enough time to play its role. In this study, we used a SOLiD autosequencer that was based on a dual-base encoding scheme to sequence the genome of Staphylococcus aureus with a mapping coverage of over 5,000×. By directly counting the variation obtained from these ultradeep sequencing reads, we found that A → G was the predominant single-base substitution and 1 bp deletions were the major small indel. These patterns are completely different from those obtained by comparison of closely related S. aureus strains, where C → T accounted for a larger proportion of mutations and deletions were shown to occur at an almost equal frequency to insertion. These findings suggest that the genomic differences between closely related bacterial strains have already undergone selection and are therefore not representative of spontaneous mutation.

Abstract  INTRODUCTION: Experimental studies indicate that some chemicals with UV blocking properties (known as UV filters) can act as endocrine disruptors. UV filters are used in sunscreens and other cosmetic- and personal care products, as well as in other consumer products such as food packaging, clothing and furniture textiles to protect the products against UV radiation. Here we present the urinary excretion of suspected endocrine active UV filters in Danish children and adolescents recruited from the general population.

METHODS: The content of benzophenone (BP), benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), 5-chloro-2- hydroxybenzophenone (BP-7), 4-hydroxybenzophenone (4-HBP), 4-methyl-benzophenone (4-MBP), 3-(4- methylbenzylidene)-camphor (4-MBC) and 3-benzylidene camphor (3-BC) were monitored in 24h urine and two consecutive first morning samples from 129 healthy Danish children and adolescents (6-21 yrs). All 387 samples were collected during the autumn (Nov. 2007) and were analyzed by a new on-line TurboFlow-LC-MS/MS method developed for simultaneous biomonitoring of these nine UV filters in urine.

RESULTS: BP-3 and BP-1 were detected in more than 80% of the 24h samples and were significantly correlated (R(2)=0.815). BP, 4-HBP and BP-2 were found in 43, 15 and 5% of the samples, respectively. The median (range) concentrations of the UV-filters in 24-h urine were as follows: BP-3, 0.92 (LOD-115); BP-1, 0.54 (LOD-44.6); BP,<LOD (LOD-48.5); 4-HBP,<LOD (LOD-10.5); and BP-2,<LOD (LOD-8.43) ng/mL. In general BP-1 and BP-3 were higher in girls compared to boys and were also higher in the group of adolescent girls (16-21 yrs) compared to the younger age groups of girls. None of the other UV filters; BP-7, 4-MBP, 4-MBC or 3-BC were detected in urine. A highly significant correlation between first morning and 24h urine levels of BP-3, BP-1 and 4-HBP from the same day was observed.

CONCLUSION: Our project on UV filters analyzed by a new robust and sensitive LC-MS/MS method in Danish children and adolescents showed that almost all individuals were exposed to UV filters. Sun protection products have been claimed to be a major source of exposure to sunscreens. However since all children in the present study were exposed during autumn where sunscreens are not needed in Denmark our study also indicates that other sources and routes of exposure might be of relevance.

Abstract  The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.

Abstract  The nucleolus is the site of ribosome biogenesis and forms around the actively transcribed ribosomal RNA (rRNA) genes. However, the nucleolus is also implicated in cell cycle regulation, tumour suppression and chromosome segregation and nucleolar disfunction is linked to a wide range of human diseases. Interestingly, the nucleolus is also required for genome reprogramming and the establishment of heterochromatin in the mammalian embryo. Mammalian oocytes contain a subnuclear structure that is believed to be the precursor of the functional nucleolus, the Nucleolar Precursor Body (NPB). But the NPB is also required for the organisation of the zygotic heterochromatin and the establishment of pluripotency. We found that disruption of the mouse Upstream Binding Factor (UBF (UBTF)) gene caused disassembly of somatic nucleoli and the accumulation of the key rRNA gene transcription factors into dense subnuclear foci resembling NPBs. Here we show that UBF deletion causes the rRNA genes to collapse onto their centromere-proximal chromosomal sites spatially distinct from NPB-like structures, and that these structures contain rRNA gene transcription factors but not all nucleolar proteins. We further find that embryonic NPBs and their surrounding heterochromatin are both disrupted in UBF-null mouse embryos. These embryos also display subnuclear foci containing the rRNA gene transcription factors and arrest development before completing the forth cleavage division. The data suggest that the rRNA gene transcription factors have an intrinsic ability to interact and form a discrete nuclear compartment even in the absence of any rRNA gene activity and that the formation or maintenance of the zygotic NPB and surrounding heterochromatin requires UBF.

Abstract  Mitochondrial DNA depletion syndromes (MDS) are often serious autosomal recessively inherited disorders characterized by tissue-specific mtDNA copy number reduction. Many genes, including MPV17, are associated with the hepatocerebral form of MDS. MPV17 encodes for a mitochondrial inner membrane protein with a poorly characterized function. Several MPV17 mutations have been reported in association with a heterogeneous group of early-onset manifestations, including liver disease and neurological problems. Mpv17-deficient mice present renal and hearing defects. We describe here a MPV17 truncation mutation in dogs. We found a 1-bp insertion in exon 4 of the MPV17 gene, resulting in a frameshift and early truncation of the encoded protein. The mutation halves MPV17 expression in the lymphocytes of the homozygous dogs and the truncated protein is not translated in transfected cells. The insertion mutation is recurrent and exists in many unrelated breeds, although is highly enriched in the Boxer breed. Unexpectedly, despite the truncation of MPV17, we could not find any common phenotypes in the genetically affected dogs. The lack of observable phenotype could be due to a late onset, mild symptoms or potential tissue-specific compensatory mechanisms. This study suggests species-specific differences in the manifestation of the MPV17 defects and establishes a novel large animal model to further study MPV17 function and role in mitochondrial biology.

Abstract  Some studies based on ambulatory blood pressure (BP) monitoring (ABPM) have reported a reduction in sleep-time relative BP decline towards a more non-dipping pattern in the elderly, but rarely have past studies included a proper comparison with younger subjects, and no previous report has evaluated the potential role of hypertension treatment time on nighttime BP regulation in the elderly. Accordingly, we evaluated the influence of age and time-of-day of hypertension treatment on the circadian BP pattern assessed by 48-h ABPM. This cross-sectional study involved 6147 hypertensive patients (3108 men/3039 women), 54.0 ± 13.7 (mean ± SD) yrs of age, with 2137 (978 men/1159 women) being ≥60 yrs of age. At the time of study, 1809 patients were newly diagnosed and untreated, and 4338 were treated with hypertension medications. Among the later, 2641 ingested all their prescribed BP-lowering medications upon awakening, whereas 1697 ingested the full daily dose of ≥1 hypertension medications at bedtime. Diagnosis of hypertension in untreated patients was based on ABPM criteria, specifically an awake systolic (SBP)/diastolic (DBP) BP mean ≥135/85 mm Hg and/or an asleep SBP/DBP mean ≥120/70 mm Hg. Collectively, older in comparison with younger patients were more likely to have diagnoses of microalbuminuria, chronic kidney disease, obstructive sleep apnea, metabolic syndrome, anemia, and/or obesity. In addition, the group of older vs. younger patients had higher glucose, creatinine, uric acid, triglycerides, and fibrinogen, but lower cholesterol, hemoglobin, and estimated glomerular filtration rate. In older compared with younger patients, ambulatory SBP was significantly higher and DBP significantly lower (p < .001), mainly during the hours of nighttime sleep and initial hours after morning awakening. The prevalence of non-dipping was significantly higher in older than younger patients (63.1% vs. 41.1%; p < .001). The largest difference between the age groups was in the prevalence of a riser BP pattern, i.e., asleep SBP mean greater than awake SBP mean (19.9% vs. 4.9% in older vs. younger patients, respectively; p < .001). The sleep-time relative SBP decline was mainly unchanged until ~40 yrs of age, and then significantly and progressively decreasing with increasing age at a rate of .28%/yr (p < .001), reaching a minimum value of 4.38% ± .47% for patients ≥75 yrs of age. Treated compared with untreated patients showed lower awake and asleep SBP means, although the predictable changes of SBP and DBP with age were equivalent in both groups. As a consequence, there were no significant differences between untreated and treated patients in the changes of the sleep-time relative SBP and DBP declines with age. Additionally, the asleep SBP and DBP means were significantly lower and the sleep-time relative SBP and DBP declines significantly higher at all ages in patients ingesting ≥1 BP-lowering medications at bedtime as compared with those ingesting all medications upon awakening. Our findings document a significantly elevated prevalence of a blunted nighttime BP decline with increasing age ≥40 yrs. The prevalence of a riser BP pattern, associated with highest cardiovascular risk among all possible BP patterns, was 4 times more prevalent in patients ≥60 yrs of age than those <60 yr of age. Most important, there was an attenuated prevalence of a blunted nighttime BP decline at all ages when ≥1 hypertension medications were ingested at bedtime as compared with when all of them were ingested upon awakening. These findings indicate that older age should be included among the conditions for which ABPM is recommended for proper cardiovascular risk assessment.

Abstract  MYB transcription factors exist in a large copy number and control various plant phenotypes. We cloned R2R3 MYB-type transcription factors that determine the coloration of basal sheaths in barley and wheat coleoptiles. These genes are highly homologous to maize C1 and rice OsC1, regulators for anthocyanin biosynthesis, but they control seed pigmentation in maize and rice. On the basis of high homology, barley and wheat counterparts are designated HvC1 and TaC1, respectively. HvC1 gene is located on the short arm of chromosome 7H, and TaC1 genes are located on the short arms of chromosomes 7A, 7B, and 7D (TaC1-A1, B1, and D1, respectively). HvC1 is a strong candidate for Ant1 because of (1) complete co-segregation of anthocyanin pigmentation phenotype of the basal sheath with the HvC1 genotype in genetic mapping, and (2) complete deletion of the HvCl gene in two anthocyanin-decreased allelic mutants (ant1.1 and ant1.2) that were induced by irradiation. In contrast, colorless coleoptile wheat lines had lesions in all three genomes consisting of a single-nucleotide substitution or a 1-bp deletion of TaC1-A1, a 1.7-kb insertion of TaC1-B1, and a 2.0-kb insertion of TaC1-D1. At least one normal TaC1 gene appears to be sufficient to produce anthocyanin pigments in wheat coleoptiles. Previous crossing experiments localized Rc (red coleoptile) genes to homoeologous group 7 chromosomes and deduced Rc genotypes of several wheat lines. Their TaC1 gene sequence variation coincided with deduced Rc genotypes; therefore, the present molecular genetic study demonstrates that TaC1 is a strong candidate for Rc in wheat.

Abstract  We identified the minimal locus of 163-kb plasmid pSV1 of Streptomyces violaceoruber for the replication in S. lividans. This locus comprised a repA gene and an upstream 407-bp sequence containing two inverted repeats (IR-III and IR-IV) within an iteron, an AT-rich region and a 300-bp noncoding sequence (NCS). RepA protein bound specifically to a 94-bp sequence covering the intact IR-III and IR-IV to form multimers of DNA/protein complexes, but was unable to bind specifically to the NCS and the promoter of repA gene. Interestingly, this 'bound' region also leaves eight 1-bp 'unbound' spacers at 7-11-9-11-9-11-9-11-8-bp intervals. RepA protein-protein interaction could form dimers or trimers in vitro. These results suggest that a higher-order complex between pSV1 RepA protein and the long inverted repeats may be formed during the initiation of plasmid replication.

Abstract  Autosomal recessive Adams-Oliver syndrome was diagnosed in three remotely related Bedouin consanguineous families. Genome-wide linkage analysis ruled out association with known Adams-Oliver syndrome genes, identifying a single-homozygosity ∼1.8-Mb novel locus common to affected individuals (LOD score 3.37). Whole-exome sequencing followed by Sanger sequencing identified only a single mutation within this locus, shared by all affected individuals and found in patients from five additional apparently unrelated Bedouin families: a 1-bp deletion mutation in a predicted alternative splice variant of EOGT, leading to a putative truncated protein. RT-PCR demonstrated that the EOGT-predicted alternative splice variant is ubiquitously expressed. EOGT encodes EGF-domain-specific O-linked N-acetylglucosamine transferase, responsible for extracellular O-GlcNAcylation of epidermal growth factor-like domain-containing proteins, and is essential for epithelial cell-matrix interactions. F-actin staining in diseased fibroblasts showed apparently intact cell cytoskeleton and morphology, suggesting the EOGT mutation acts not through perturbation of cytoskeleton but through other mechanisms yet to be elucidated.

Abstract  BACKGROUND: The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.

METHODS AND FINDINGS: We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937-1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974-1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988-1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe.

CONCLUSIONS: In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.

Abstract  This present work describes the production and biochemical characterization of lipase by Candida rugosa and Geotrichum candidum in a culture supplemented with soybean molasses. After optimizing the fermentation times for both microorganisms, the effects of changing the soybean molasses concentration, the fermentative medium pH and the fermentation temperature were evaluated using the Central Composite Planning. When soybean molasses was used at a concentration of 200 g/L at 27 ± 1 °C and pH 3.5, the lipolytic activity measured in the broth was 12.3 U/mL after 12 h for C. rugosa and 11.48 U/mL after 24 h for G. candidum. The molecular masses were 38.3 kDa to G. candidum lipase and 59.7 kDa to C. rugosa lipase, determined by SDS-PAGE. The lipase from both microorganisms exhibited maximal hydrolytic activity at a temperature of 40 °C and were inhibited at pH 10.0. Using different concentration of p-nitrophenylbutyrate (p-NPB), the kinetic parameters were calculated, as follows: the Km of lipase from G. candidum was 465.44 μM and the Vmax 0.384 μmol/min; the Km and Vmax of lipase from C. rugosa were 129.21 μM and 0.034 μmol/min, respectively. Lipases activity were increased by metallic ions Mg(2+) and Na(+) and inhibited by metallic ion Cu(3+).

Abstract  Over-weight and obesity are serious problems that increase the risk not only for chronic diseases like diabetes and heart disease but also of various types of cancer. This study was conducted to evaluate cytokinesis-block micronucleus cytome (CBMN-cyt) assay parameters and plasma concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), and their relationship with age, body-mass index (BMI) and waist-to-hip ratio (WHR) in 83 obese, 21 over-weight and 21 normal-weight subjects. Frequencies of micronuclei (MN), nucleoplasmic bridges (NPB), nuclear buds (NBUD), and apoptotic and necrotic cells in lymphocytes of obese subjects were found to be significantly higher than those found in normal-weight and over-weight subjects (p<0.01 and p<0.05), whereas plasma concentrations of 8-OHdG in obese subjects were lower than those observed in normal-weight and over-weight subjects (p<0.05 and p<0.01, respectively). There was a negative correlation between age and frequency of necrotic cells and NDI (p<0.05), whereas there was no correlation between BMI, WHR, CBMN cyt assay parameters and plasma 8-OHdG in normal-weight subjects. In over-weight subjects, a negative correlation was observed between age and NDI (p<0.01) and a positive correlation between age and frequency of NPB (p<0.01) and between BMI and frequency of NBUD (p<0.05). In obese subjects, a negative correlation was observed between age and NDI (p<0.01) and between BMI and NDI (p<0.05), whereas no correlation was observed between WHR and CBMN-cyt assay parameters and plasma 8-OHdG. However, frequencies of MN, NPB, NBUD, apoptotic and necrotic cells in total over-weight/obese (p<0.01/p<0.05) and all subjects (p<0.01) increased with increasing BMI. The increase in genomic damage (MN, NPB and NBUD) in obese subjects and the positive correlation between genomic damage and BMI in total over-weight/obese subjects indicate that obesity increases genomic damage and may be associated with an increased risk of cancer, because an increase in MN frequency is a predictor of cancer risk.

Abstract  Osteogenesis imperfecta (OI) is a genetically heterogeneous group of disorders, characterized by abnormal bone fragility, blue sclera, deafness, joint laxity, and soft-tissue dysplasia. The purpose of this study was to elucidate the genetic or molecular basis for OI type IA in a Chinese family. We evaluated the members of a family, in which six individuals are affected with increased bone fragility and blue sclera. Results of exome sequencing revealed a novel 1-bp deletion (c.2329delG, p.A777fs) in exon 33 of the COL1A1 gene in two affected individuals, but not in a control family member without OI. The variation co-segregated with the disease in all the OI patients but not in the unaffected family members. The mutation caused a frameshift alteration after codon 777, leading to premature termination of the COL1A1 protein. Thus, our findings identified a novel frameshift deletion c.2329delG (p.A777fs) in the COL1A1 gene, which is associated with OI type IA in a Chinese family.

Abstract  BACKGROUND: Lead is a heavy toxic metal element in biological systems and is one of the major pollutants as a result of its widespread use in industries. In spite of its negative roles the coordination chemistry of Pb(II) complexes is a matter of interest. The N,N'-bidentate aromatic bases such as BPY,4-BPY and PHEN (BPY = 2,2'bipyridine, 4-BPY = 4,4'-dimethyl-2,2'-bipyridine, PHEN = 1,10-Phenanthroline) are widely used to build supramolecular architectures because of their excellent coordinating ability and large conjugated system that can easily form π-π interactions among their aromatic moieties. A series of novel Pb(II) complexes in concert with 5-CTPC, 5-BTPC (5-CTPC = 5-chlorothiophen-2-carboxylate, 5-BTPC = 5-bromothiophen-2-carboxylate) and corresponding bidentate chelating N.N' ligands have been synthesized and characterized.

RESULTS: Five new Pb (II) complexes [Pb(BPY)(5-CTPC)2] (1), [Pb(4-BPY)(5-CTPC)2] (2), [Pb2(PHEN)2(5-CTPC)4] (3), [Pb(4-BPY)(5-BTPC)2] (4) and [Pb2(PHEN)2(5-BTPC)2(ACE)2] (5) have been synthesized. Even though in all these complexes the molar ratio of Pb, carboxylate, N,N-chelating ligand are the same (1:2:1), there is a significant structural diversity. These complexes have been characterised and investigated by elemental analysis, IR, 1H-NMR,13C-NMR, TGA, and photoluminescence studies. Single crystal X-ray diffraction studies reveal that complexes (1, 2) and (4) are mononuclear while (3 and 5) are dinuclear in nature which may result from the chelating nature of the ligands, various coordination modes of the carboxylates, and the coordination geometry of the Pb(II) ions.

CONCLUSIONS: The observation of structures 2,4 and 3,5 show the structural changes made just chloro/bromo substituent of the thiophene ring. A detailed packing analysis has been undertaken to delineate the role of valuable non covalent interactions like X…π, H…X, (X = Cl/Br). A quadruple hydrogen bond linking the monomeric units and generating a supramolecular architecture is observed in (1). The metal bite unit comprised of PbN2C2 (i.e. Pb-N-C-C-N-Pb) is the repeating unit in all the five complexes and they have almost same geometrical parameters. This metal bite has been identified as the self assembly unit in complexes.