Peptidomic Analysis of Human Cell Lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEIC293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the PI position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human β-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. β-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

A New Extraction Approach To Correct The Effect Of Apparent Increase In The Secoiridoid Content After Filtration Of Virgin Olive Oil.

In the current study, a new approach has been developed for correcting the effect that moisture reduction after virgin olive oil (VOO) filtration exerts on the apparent increase of the secoiridoid content by using an internal standard during extraction. Firstly, two main Spanish varieties (Picual and Hojiblanca) were submitted to industrial filtration of VOOs. Afterwards, the moisture content was determined in unfiltered and filtered VOOs, and liquid-liquid extraction of phenolic compounds was performed using different internal standards. The resulting extracts were analyzed by HPLC-ESI-TOF/MS, in order to gain maximum information concerning the phenolic profiles of the samples under study. The reduction effect of filtration on the moisture content, phenolic alcohols, and flavones was confirmed at the industrial scale. Oleuropein was chosen as internal standard and, for the first time, the apparent increase of secoiridoids in filtered VOO was corrected, using a correction coefficient (Cc) calculated from the variation of internal standard area in filtered and unfiltered VOO during extraction. This approach gave the real concentration of secoiridoids in filtered VOO, and clarified the effect of the filtration step on the phenolic fraction. This finding is of great importance for future studies that seek to quantify phenolic compounds in VOOs.

Polyphenol Extraction Optimisation From Ceylon Gooseberry (dovyalis Hebecarpa) Pulp.

Originally from Asia, Dovyalis hebecarpa is a dark purple/red exotic berry now also produced in Brazil. However, no reports were found in the literature about phenolic extraction or characterisation of this berry. In this study we evaluate the extraction optimisation of anthocyanins and total phenolics in D. hebecarpa berries aiming at the development of a simple and mild analytical technique. Multivariate analysis was used to optimise the extraction variables (ethanol:water:acetone solvent proportions, times, and acid concentrations) at different levels. Acetone/water (20/80 v/v) gave the highest anthocyanin extraction yield, but pure water and different proportions of acetone/water or acetone/ethanol/water (with >50% of water) were also effective. Neither acid concentration nor time had a significant effect on extraction efficiency allowing to fix the recommended parameters at the lowest values tested (0.35% formic acid v/v, and 17.6 min). Under optimised conditions, extraction efficiencies were increased by 31.5% and 11% for anthocyanin and total phenolics, respectively as compared to traditional methods that use more solvent and time. Thus, the optimised methodology increased yields being less hazardous and time consuming than traditional methods. Finally, freeze-dried D. hebecarpa showed high content of target phytochemicals (319 mg/100g and 1,421 mg/100g of total anthocyanin and total phenolic content, respectively).

Intensification Of Bioactive Compounds Extraction From Medicinal Plants Using Ultrasonic Irradiation.

Extraction processes are largely used in many chemical, biotechnological and pharmaceutical industries for recovery of bioactive compounds from medicinal plants. To replace the conventional extraction techniques, new techniques as high-pressure extraction processes that use environment friendly solvents have been developed. However, these techniques, sometimes, are associated with low extraction rate. The ultrasound can be effectively used to improve the extraction rate by the increasing the mass transfer and possible rupture of cell wall due the formation of microcavities leading to higher product yields with reduced processing time and solvent consumption. This review presents a brief survey about the mechanism and aspects that affecting the ultrasound assisted extraction focusing on the use of ultrasound irradiation for high-pressure extraction processes intensification.

A Modified Acidic Approach For Dna Extraction From Plant Species Containing High Levels Of Secondary Metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Supercritical Carbon Dioxide Extraction Of Capsaicinoids From Malagueta Pepper (capsicum Frutescens L.) Assisted By Ultrasound.

Extracts from malagueta pepper (Capsicum frutescens L.) were obtained using supercritical fluid extraction (SFE) assisted by ultrasound, with carbon dioxide as solvent at 15MPa and 40°C. The SFE global yield increased up to 77% when ultrasound waves were applied, and the best condition of ultrasound-assisted extraction was ultrasound power of 360W applied during 60min. Four capsaicinoids were identified in the extracts and quantified by high performance liquid chromatography. The use of ultrasonic waves did not influence significantly the capsaicinoid profiles and the phenolic content of the extracts. However, ultrasound has enhanced the SFE rate. A model based on the broken and intact cell concept was adequate to represent the extraction kinetics and estimate the mass transfer coefficients, which were increased with ultrasound. Images obtained by field emission scanning electron microscopy showed that the action of ultrasonic waves did not cause cracks on the cell wall surface. On the other hand, ultrasound promoted disturbances in the vegetable matrix, leading to the release of extractable material on the solid surface. The effects of ultrasound were more significant on SFE from larger solid particles.

Molecularly Imprinted Solid Phase Extraction Of Fluconazole From Pharmaceutical Formulations.

This work encompasses a direct and coherent strategy to synthesise a molecularly imprinted polymer (MIP) capable of extracting fluconazole from its sample. The MIP was successfully prepared from methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile (porogenic solvent) in the presence of fluconazole as the template molecule through a non-covalent approach. The non-imprinted polymer (NIP) was prepared following the same synthetic scheme, but in the absence of the template. The data obtained from scanning electronic microscopy, infrared spectroscopy, thermogravimetric and nitrogen Brunauer-Emmett-Teller plot helped to elucidate the structural as well as the morphological characteristics of the MIP and NIP. The application of MIP as a sorbent was demonstrated by packing it in solid phase extraction cartridges to extract fluconazole from commercial capsule samples through an offline analytical procedure. The quantification of fluconazole was accomplished through UPLC-MS, which resulted in LOD≤1.63×10(-10) mM. Furthermore, a high percentage recovery of 91±10% (n=9) was obtained. The ability of the MIP for selective recognition of fluconazole was evaluated by comparison with the structural analogues, miconazole, tioconazole and secnidazole, resulting in percentage recoveries of 51, 35 and 32%, respectively.

Extraction, purification and biochemical characterization of a peroxidase from Copaifera langsdorffii leaves

The aim of this work is to obtain, purify and characterize biochemically a peroxidase from Copaifera langsdorffii leaves (COP). COP was obtained by acetone precipitation followed by ion-exchange chromatography. Purification yielded 3.5% of peroxidase with the purification factor of 46.86. The COP optimum pH is 6.0 and the temperature is 35 ºC. COP was stable in the pH range of 4.5 to 9.3 and at temperatures below 50.0 ºC. The apparent Michaelis-Menten constants (Km) for guaiacol and H2O2 were 0.04 mM and 0.39 mM respectively. Enzyme turnover was 0.075 s-1 for guaiacol and 0.28 s-1 for hydrogen peroxide. Copaifera langsdorffii leaves showed to be a rich source of active peroxidase (COP) during the whole year. COP could replace HRP, the most used peroxidase, in analytical determinations and treatment of industrial effluents at low cost.

Diagnosis and treatment of extraction sockets in preparation for implant placement: report of three cases

This article addresses diagnostic parameters that should be assessed in the treatment of extraction sockets with dental implant placement by presenting three case reports that emphasize the relevance of the amount of remaining bone walls. Diagnosis was based on the analysis of clinical and radiographic parameters (e.g.: bone defect morphology, remaining bone volume, presence of infections on the receptor site). Case 1 presents a 5-wall defect in the maxillary right central incisor region with severe root resorption, which was treated with immediate implant placement. Cases 2 and 3 present, respectively, two- and three-wall bone defects that did not have indication for immediate implants. These cases were first submitted to a guided bone regeneration (GBR) procedure with bone graft biomaterial and membrane barriers, and the implants were installed in a second surgical procedure. The analysis of the preoperative periodontal condition of the adjacent teeth and bone defect morphology is extremely important because these factors determine the choice between immediate implant or GBR treatment followed by implant installation in a subsequent intervention.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308

The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.

ScriptLattes: an open-source knowledge extraction system from the Lattes platform

The Lattes platform is the major scientific information system maintained by the National Council for Scientific and Technological Development (CNPq). This platform allows to manage the curricular information of researchers and institutions working in Brazil based on the so called Lattes Curriculum. However, the public information is individually available for each researcher, not providing the automatic creation of reports of several scientific productions for research groups. It is thus difficult to extract and to summarize useful knowledge for medium to large size groups of researchers. This paper describes the design, implementation and experiences with scriptLattes: an open-source system to create academic reports of groups based on curricula of the Lattes Database. The scriptLattes system is composed by the following modules: (a) data selection, (b) data preprocessing, (c) redundancy treatment, (d) collaboration graph generation among group members, (e) research map generation based on geographical information, and (f) automatic report creation of bibliographical, technical and artistic production, and academic supervisions. The system has been extensively tested for a large variety of research groups of Brazilian institutions, and the generated reports have shown an alternative to easily extract knowledge from data in the context of Lattes platform. The source code, usage instructions and examples are available at http://scriptlattes.sourceforge.net/.

Vegetable-origin foam employed in dye extraction in tanning and leather processing facilities

This study addressed the use of conventional and vegetable origin polyurethane foams to extract C. I. Acid Orange 61 dye. The quantitative determination of the residual dye was carried out with an UV/Vis absorption spectrophotometer. The extraction of the dye was found to depend on various factors such as pH of the solution, foam cell structure, contact time and dye and foam interactions. After 45 days, better results were obtained for conventional foam when compared to vegetable foam. Despite presenting a lower percentage of extraction, vegetable foam is advantageous as it is considered a polymer with biodegradable characteristics.

Design, construction and evaluation of a simple pressurized solvent extraction system

This work describes the construction and testing of a simple pressurized solvent extraction (PSE) system. A mixture of acetone:water (80:20), 80 ºC and 103.5 bar, was used to extract two herbicides (Diuron and Bromacil) from a sample of polluted soil, followed by identification and quantification by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). The system was also used to extract soybean oil (70 ºC and 69 bar) using pentane. The extracted oil was weighed and characterized through the fatty acid methyl ester analysis (myristic (< 0.3%), palmitic (16.3%), stearic (2.8%), oleic (24.5%), linoleic (46.3%), linolenic (9.6%), araquidic (0.3%), gadoleic (< 0.3%), and behenic (0.3%) acids) using high-resolution gas chromatography with flame ionization detection (HRGC-FID). PSE results were compared with those obtained using classical procedures: Soxhlet extraction for the soybean oil and solid-liquid extraction followed by solid-phase extraction (SLE-SPE) for the herbicides. The results showed: 21.25 ± 0.36% (m/m) of oil in the soybeans using the PSE system and 21.55 ± 0.65% (m/m) using the soxhlet extraction system; extraction efficiency (recovery) of herbicides Diuron and Bromacil of 88.7 ± 4.5% and 106.6 ± 8.1%, respectively, using the PSE system, and 96.8 ± 1.0% and 94.2 ± 3.9%, respectively, with the SLP-SPE system; limit of detection (LOD) and limit of quantification (LOQ) for Diuron of 0.012 mg kg-1 and 0.040 mg kg-1, respectively; LOD and LOQ for Bromacil of 0.025 mg kg-1 and 0.083 mg kg-1, respectively. The linearity used ranged from 0.04 to 1.50 mg L-1 for Diuron and from 0.08 to 1.50 mg L-1 for Bromacil. In conclusion, using the PSE system, due to high pressure and temperature, it is possible to make efficient, fast extractions with reduced solvent consumption in an inert atmosphere, which prevents sample and analyte decomposition.

Extraction and concentration of biogenic calcium oxalate from plant leaves

The objective of this study was to extract and concentrate calcium oxalate (CaOx) crystals from plant leaves that form the above mentioned crystals. The chemical and physical studies of CaOx from plant to be performed depend on an adequate amount of the crystals. The plant used in this study was croton (Codiaeum variegatum). The leaves were ground in a heavy duty blender and sieved through a 0.20 mm sieve. The suspension obtained was suspended in distilled water. The crystals were concentrated at the bottom of a test tube. The supernatant must be washed until it is free of plant pigments and other organic substances. Biogenic CaOx crystals have well-defined and sharp peaks, indicating very high crystallinity. Moreover, the CaOx crystals were not damaged during the extraction procedure, as can be seen on the scanning electron microscope images. The porposed method can be considered efficient to extract and concentrate biogenic calcium oxalate.