Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2′-endo (“south”) conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

     

Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice

Breast tumor suppressor gene 1 (BRCA1) plays an essential role in maintaining genomic integrity. Here we show that mouse Brca1 is required for DNA-damage repair and crossing-over during spermatogenesis. Male Brca1(Delta11/Delta11)p53(+/-) mice that carried a homozygous deletion of Brca1 exon 11 and a p53 heterozygous mutation had significantly reduced testicular size and no spermatozoa in their seminiferous tubules. During spermatogenesis, homologous chromosomes from the mutant mice synapsed and advanced to the pachytene stage but failed to progress to the diplotene stage. Our analyses revealed that the Brca1 mutation affected cellular localization of several DNA damage-repair proteins. This included prolonged association of gammaH2AX with sites of DNA damage, reduced sex body formation, diminished Rad51 foci and absence of Mlh1 foci in the pachytene stage. Consequently, chromosomes from mutant mice did not form chiasmata, a point that connects exchanging homologous chromosomes. Brca1-mutant spermatocytes also exhibited decreased RNA expression levels of several genes that are involved in DNA-damage repair, including RuvB-like DNA helicase, XPB, p62 and TFIID. Of note, the premature termination of spermatogenesis at the pachytene stage was accompanied by increased apoptosis by both p53-dependent and p53-independent mechanisms. Thus, our study revealed an essential role of Brca1 in DNA-damage repair and crossing-over of homologous chromosomes during spermatogenesis.     

Plant communities on anthropogenically disturbed sites on the Chukotka Peninsula,Russia

Abstract. The vegetation of anthropogenically disturbed sites on the Chukotka Peninsula was investigated in 1986 and 1990. 102 sites had been bulldozed free of vegetation and soil between 2 and 20 yr ago and revegetated. Anthropogenic vegetation differs considerably from natural tundra. The total plant cover decreases, lichens are absent, moss composition changes, and graminoids predominate. A floristic classification was developed in order to describe the diversity of anthropogenic communities formed on these sites. Four associations were distinguished, all belonging to the alliance Poion glaucomalacanthae. An additional alliance characterized by Alopecurus alpinus is suggested for colder sites, but it has not yet been described. There is a correspondence of associations to substrate types. The association Phippsietum algidae was found only on loam substrate. The Artemisietum glomeratae and Saxifragetum punctatae are connected with rubble or gravel. The graminoid association Arctagrostietum arundinaceae is generally widespread in disturbed habitats and occurs on a variety of substrate types.     

Evaluation of changes in the element content and biomass of invaded with Meloidogyne arenaria Tiny Tim tomato plants under NH4VO3 treatment

The parasite-host system Meloidogyne arenaria--Tiny Tim tomato plants has been studied in order to investigate the influence of the process of invasion on the chemical composition and biomass of plants. The concentrations of seven chemical elements Cu, Zn, Mg, K, Na, Mn and Fe have been determined using AAS in controls and invaded plants, and their changes have been evaluated under treatment with NH4VO3 in three different concentrations 0.01, 0.1 and 0.13 mg/100 ml H2O. The process of treatment with NH4VO3 disbalances significantly the trace element content of plants. The lowest concentration (0.01 mg NH4VO3) causes bigger changes in the concentrations of Mn, Fe and Na in non-invaded plants. The highest concentration (0.13 mg NH4VO3) balances the content of the elements back to their levels in the control plants for the elements Zn, Fe and Na. The pure effect of the process of invasion with Meloidogyne arenaria on the biomass (leaves, stems, roots and total biomass) of Tiny Tim plants is expressed in a significant increasing, mainly due to the development of the parasites. After treatment with different concentrations of NH4VO3 the decreasing in the biomass of leaves, stems and roots is observed which reflects on the total biomass of plants. The concentration of NH4VO3 eliminates the unfavourable changes not only in the chemical content of plants but also in their biomass. It could be taken into consideration as an alternative method used instead of treatment with nematocides.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

Waterbugs (Heteroptera: Nepomorpha and Gerromorpha) as sources of essential n‐3 polyunsaturated fatty acids in Central Siberian ecoregions

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Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis

UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA), the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB) irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations.     

Strengthening Community-Based Vital Events Reporting for Real-Time Monitoring of Under-Five Mortality: Lessons Learned from the Balaka and Salima Districts in Malawi

Background

Malawi ratified a compulsory birth and death registration system in 2009. Until it captures complete coverage of vital events, Malawi relies on other data sources to calculate mortality estimates. We tested a community-based method to estimate annual under-five mortality rates (U5MR) through the Real-Time Monitoring of Under-Five Mortality (RMM) project in Malawi. We implemented RMM in two phases, and conducted an independent evaluation of phase one after 21 months of implementation. We present results of the phase two validation that covers the full project time span, and compare the results to those of the phase one validation.

Methods and Findings

We assessed the completeness of the counts of births and deaths and the accuracy of disaggregated U5MR from the community-based method against a retrospective full pregnancy history for rolling twelve-month periods after the independent evaluation. We used full pregnancy histories collected through household interviews carried out between November 2013 and January 2014 as the validation data source. Health Surveillance Agents (HSAs) across the 160 catchment areas submitted routine reports on pregnancies, births, and deaths consistently. However, for the 15-month implementation period post-evaluation, average completeness of birth event reporting was 76%, whereas average completeness of death event reporting was 67% relative to that expected from a comparable pregnancy history. HSAs underestimated the U5MR by an average of 21% relative to that estimated from a comparable pregnancy history.

Conclusions

On a medium scale, the community-based RMM method in Malawi produced substantial underestimates of annualized U5MR relative to those obtained from a full pregnancy history, despite the additional incentives and quality-control activities. We were not able to achieve an optimum level of incentive and support to make the system work while ensuring sustainability. Lessons learned from the implementation of RMM can inform programs supporting community-based interventions through HSAs in Malawi.