Leishmaniamamazonensis DNA in wild females of Lutzomyia cruzi (Diptera: Psychodidae) in the state of Mato Grosso do Sul, Brazil

Studies on natural infection by Leishmania spp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmania in wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect Leishmania DNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi ) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruzi were dissected and also submitted to molecular analysis. The infection rate based on the presence of Leishmania DNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensis was identified in Lu. cruzi by the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensis DNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.

The Herpesvirus Nuclear Egress Complex Component, UL31, is Recruited to Sites of DNA Damage Through Poly(ADP-RIBOSE) Binding

Thesis (Master, Biomedical & Molecular Sciences) -- Queen's University, 2016-08-23 15:03:30.807

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Ceramic membranes for separation of proteins and DNA by in situ growth of alumina nanofibres with a nanomesh of metal oxide nanofibres

Ceramic membranes were fabricated by in situ synthesis of alumina nanofibres in the pores of an alumina support as a separation layer, and exhibited a high permeation selectivity for bovine serum albumin relative to bovine hemoglobin (over 60 times) and can effectively retain DNA molecules at high fluxes.

Synchronous fluorescence and UV–vis spectroscopic studies of interactions between the tetracycline antibiotic, aluminium ions and DNA with the aid of the Methylene Blue dye probe

Synchronous fluorescence spectroscopy (SFS) was applied for the investigation of interactions of the antibiotic, tetracycline (TC), with DNA in the presence of aluminium ions (Al3+). The study was facilitated by the use of the Methylene Blue (MB) dye probe, and the interpretation of the spectral data with the aid of the chemometrics method, parallel factor analysis (PARAFAC). Three-way synchronous fluorescence analysis extracted the important optimum constant wavelength differences, Δλ, and showed that for the TC–Al3+–DNA, TC–Al3+ and MB dye systems, the associated Δλ values were different (Δλ = 80, 75 and 30 nm, respectively). Subsequent PARAFAC analysis demonstrated the extraction of the equilibrium concentration profiles for the TC–Al3+, TC–Al3+–DNA and MB probe systems. This information is unobtainable by conventional means of data interpretation. The results indicated that the MB dye interacted with the TC–Al3+–DNA surface complex, presumably via a reaction intermediate, TC–Al3+–DNA–MB, leading to the displacement of the TC–Al3+ by the incoming MB dye probe.

Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4R24C/R24C/Tyr-NrasQ61K mice following neonatal UVR

To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4R24C/R24C/NrasQ61K mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.

Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research

Cell proliferation is a critical and frequently studied feature of molecular biology in cancer research. Therefore, various assays are available using different strategies to measure cell proliferation. Metabolic assays such as AlamarBlue, WST-1, and MTT, which were originally developed to determine cell toxicity, are being used to assess cell numbers. Additionally, proliferative activity can be determined by quantification of DNA content using fluorophores, such as CyQuant and PicoGreen. Referring to data published in high ranking cancer journals, 945 publications applied these assays over the past 14 years to examine the proliferative behaviour of diverse cell types. Within this study, mainly metabolic assays were used to quantify changes in cell growth yet these assays may not accurately reflect cellular proliferation rates due to a miscorrelation of metabolic activity and cell number. Testing this hypothesis, we compared metabolic activity of different cell types, human cancer cells and primary cells, over a time period of 4 days using AlamarBlue and fluorometric assays CyQuant and PicoGreen to determine their DNA content. Our results show certain discrepancies in terms of over-estimation of cell proliferation with respect to the metabolic assay in comparison to DNA binding fluorophores.