Optimalni uvjeti za proizvodnju biomase i rekombinantne glicerol kinaze s pomoću kvasca Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT1) was cloned into the expression vector pPICZα A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (φ(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of φ(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R²=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4·10^–5 % biotin, 1 % methanol and 1 % glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.Gen za ekstracelularnu glicerol kinazu iz Saccharomyces cerevisiae (GUT1) kloniran je u ekspresijski vektor pPICZα A i integriran u genom metilotrofnog kvasca Pichia pastoris X-33. Prisutnost GUT1 potvrđena je PCR analizom. Izdvojena su četiri klona, u kojima je ispitana funkcionalnost rekombinantnog enzima. Jedan je od ispitanih klonova imao aktivnost glicerol kinaze od 0,32 U/mL i specifičnu aktivnost proteina od 0,0025 U/mg. Podloga za maksimalnu proizvodnju biomase na tresilici s pomoću rekombinantnog kvasca Pichia pastoris optimirana je uporabom glicerola, volumnog udjela od 2,31 %, kao izvora ugljika. Za optimiranje je upotrijebljena metoda odzivnih površina. U preliminarnim su ispitivanjima, primjenom Plackett-Burmanovog dizajna, određeni najvažniji čimbenici što utječu na proizvodnju biomase, a to su: volumni udio glicerola (φ(Gly)) i vrijeme uzgoja (t). Daljnji su eksperimenti provedeni radi optimiranja proizvodnje biomase, a pratili su centralno složeni dizajn kao funkciju volumnog udjela glicerola i vremena. Volumni je udio glicerola imao pozitivni linearni utjecaj na proizvodnju biomase. Vrijeme uzgoja je također bitno utjecalo (na razini linearno pozitivnih i kvadratnih zavisnosti) na proizvodnju biomase. Eksperimentalni su se podaci dobro uklapali u konveksnu funkciju koja opisuje polinom drugoga reda, u kojem je biomasa funkcija obaju faktora (R²=0,946). Prinos i specifična aktivnost glicerol kinaze ponajprije su ovisili o dodatku glicerola i metanola podlozi. Sastav optimirane podloge za proizvodnju enzima bio je: 1 % kvaščeva ekstrakta, 1 % peptona, 100 mM fosfatnog pufera (pH=6,0), 1,34 % podloge s kvascem i dušikom, 4·10^-5 % biotina, 1 % metanola i 1 % glicerola, pomoću kojih je dobivena aktivnost glicerol kinaze od 0,89 U/mL i koncentracija ukupnih proteina od 14,55 g/L u podlozi nakon 48 sati uzgoja

Assessment of historical fecal contamination in Curitiba, Brazil, in the last\ud 400 years using fecal sterols

A 400-year sedimentary record of the Barigui River was investigated using fecal biomarkers and nutrient distribution. The temporal variability in cholesterol, cholestanol, coprostanol, epicoprostanol, stigmastanol, stigmasterol, stigmastenol, sitosterol, and campesterol between 1600 and 2011 was assessed. Anthropogenic influences, such as deforestation and fecal contamination from humans and livestock, were observed from\ud 1840. The sterol ratios exhibit evidence of hens, horses, cows, and an unknown herbivore, which may be a capybara (Hydrochoerus hydrochaeris), from1820 and has been observed moremarkedly from1970 onward. Human fecal contamination was detected from 1840 and was observed more markedly from 1930 due to population growth. Thus, the sanitation conditions and demographic growth of Curitiba seemed to be the main factors of human sewage pollution, as the coprostanol concentration over timewas strongly correlatedwith the population growth (r= 0.71, p b 0.001) although diagenetic processes have also been observed

A mathematical analysis of the evolution of perturbations in a modified Chaplygin gas model

One approach in modern cosmology consists in supposing that dark matter and dark energy are different manifestations of a single `quartessential' fluid. Following such idea, this work presents a study of the evolution of perturbations of density in a flat cosmological model with a modified Chaplygin gas acting as a single component. Our goal is to obtain properties of the model which can be used to distinguish it from another cosmological models which have the same solutions for the general evolution of the scale factor of the universe, without the construction of the power spectrum. Our analytical results, which alone can be used to uniquely characterize the specific model studied in our work, show that the evolution of the density contrast can be seen, at least in one particular case, as composed by a spheroidal wave function. We also present a numerical analysis which clearly indicates as one interesting feature of the model the appearence of peaks in the evolution of the density constrast.Comment: 21 pages, accepted for publication in General Relativity and Gravitatio

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

<p>Abstract</p> <p>Background</p> <p>Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by <it>ERBB2</it> (<it>HER-2/neu</it>) oncogene expression.</p> <p>Results</p> <p>The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of <it>ERBB2</it>-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of <it>ERBB2</it>. The relative expression balance between AS variants from 3 genes was differentially modulated by <it>ERBB2</it> in this model system.</p> <p>Conclusions</p> <p>In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by <it>ERBB2</it>-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.</p

Association between atrial fibrillation and <i>Helicobacter pylori</i>

The connection between atrial fibrillation (AF) and H. pylori (HP) infection is still matter of debate. We performed a systematic review and metanalysis of studies reporting the association between AF and HF. A systematic review of all available reports in literature of the incidence of HP infection in AF and comparing this incidence with subjects without AF were analysed. Risk ratio and 95% confidence interval (CI) and risk difference with standard error (SE) were the main statistics indexes. Six retrospective studies including a total of 2921 were included at the end of the selection process. Nine hundred-fifty-six patients (32.7%) were in AF, whereas 1965 (67.3%) were in normal sinus rhythm (NSR). Overall, 335 of 956 patients with AF were HP positive (35%), whereas 621 were HP negative (65%). In addition, 643 of 1965 NSR patients (32.7%) were HP positive while 1,322 were negative (67.3%; Chi-square 2.15, p = 0.21). The Cumulative Risk Ratio for AF patients for developing an HP infection was 1.19 (95% CI 1.08–1.41). In addition, a small difference risk towards AF was found (0.11 [SE = 0.04]). Moreover, neither RR nor risk difference were influenced by the geographic area at meta-regression analysis. Finally, there was a weak correlation between AF and HP (coefficient = 0.04 [95% CI −0.01–0.08]). We failed to find any significant correlation between H. pylori infection and AF and, based on our data, it seems unlikely than HP can be considered a risk factor for AF. Further larger research is warranted

Supplement: "Localization and broadband follow-up of the gravitational-wave transient GW150914" (2016, ApJL, 826, L13)

This Supplement provides supporting material for Abbott et al. (2016a). We briefly summarize past electromagnetic (EM) follow-up efforts as well as the organization and policy of the current EM follow-up program. We compare the four probability sky maps produced for the gravitational-wave transient GW150914, and provide additional details of the EM follow-up observations that were performed in the different bands

Measurement of the CP-violating phase ϕs and the Bs0 meson decay width difference with Bs0 → J/ψϕ decays in ATLAS

A measurement of the Bs0 decay parameters in the Bs0 → J/ψϕ channel using an integrated luminosity of 14.3 fb−1 collected by the ATLAS detector from 8 TeV pp collisions at the LHC is presented. The measured parameters include the CP -violating phase ϕs, the decay width Γs and the width difference between the mass eigenstates ΔΓs. The values measured for the physical parameters are statistically combined with those from 4.9 fb−1 of 7 TeV data, leading to the following: ϕ s =−0.090±0.078(stat.)±0.041(syst.)rad ΔΓ s =0.085±0.011(stat.)±0.007(syst.)ps −1 Γ s =0.675±0.003(stat.)±0.003(syst.)ps −1 In the analysis the parameter ΔΓs is constrained to be positive. Results for ϕs and ΔΓs are also presented as 68% and 95% likelihood contours in the ϕs-ΔΓs plane. Also measured in this decay channel are the transversity amplitudes and corresponding strong phases. All measurements are in agreement with the Standard Model predictions