Prior Extraction Research Articles

Screening and determination of sulphonamide residues in bovine milk samples using a flow injection system

A new, simple, rapid and sensitive flow injection spectrophotometric method was developed for the screening and determination of sulphonamides in bovine milk samples. The method is based on the condensation of sulphathiazole, sulphamethazine, and sulphadimethoxine with p-dimethylaminobenzaldehyde (p-DAB) in acid medium, in the presence of sodium dodecyl sulphate (SDS), producing a yellow compound (λmax=465nm). Optimisation of the experimental parameters was performed using a multivariate methodology. The linear range was 90–500μg/L and the limits of detection and quantification were in the ranges 25–29μg/L and 84–88μg/L, respectively. The procedure was applied for the determination of sulphonamide antibiotics in bovine milk samples submitted to a prior extraction procedure based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methodology. Recoveries of 60.5–70.5% were achieved for milk samples spiked with 0.09, 0.1, 0.2, and 0.3μg/g of each sulphonamide.

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture.

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

Direct chemiluminescent enzyme immunoassay for atrial natriuretic peptide in mammalian plasma using a PEGylated antibody

Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized and secreted by cardiac tissues and plays a pivotal role in maintaining cardiovascular homeostasis. Clinically, ANP is used as a marker of cardiovascular diseases, including heart failure. Although multiple ANP assays are currently available, a more sensitive assay is required for the direct measurement of plasma ANP where there is limited plasma availability, especially in mouse experiments. In the current study, we developed a plate-based sandwich chemiluminescent enzyme immunoassay for the measurement of plasma ANP in rats and mice without the need for prior extraction. To minimize nonspecific binding, we performed a single-step PEGylation procedure targeting the immobilized antibody, which markedly improved the assay’s sensitivity and linearity. The linear range was 0.1 to 250pM, and the minimum detection limit was 0.13pM, 5-fold lower than the lowest value of the commercially available kits. ANP was directly measured in plasma samples without detectable cross-reactivity with B- and C-type natriuretic peptides. The accuracy of the assay was confirmed by spike recovery tests and dilution tests and by comparison with a conventional radioimmunoassay. Based on the species cross-reactivity, this assay can be used to measure human ANP.

Image receptive fields for artificial neural networks

This paper describes the structure of the Image Receptive Fields Neural Network (IRF-NN) introduced recently by our team. This structure extends simplified learning introduced by Extreme Learning Machine and Reservoir Computing techniques to the field of images. Neurons are organized in a single hidden layer feedforward network architecture with an original organization of the [email protected]?s input weights. To represent color images efficiently, without prior feature extraction, the weight values linked to a neuron are determined by a 2-D Gaussian function. The activation of a neuron by an image presents the properties of a nonlinear localized receptive field, parameterized with a small number of degrees of freedom. A network composed of a large number of neurons, each associated with a randomly initialized and constant receptive field, induces a remarkable representation of the images. Supervised training determines only the output weights of the network. It is therefore extremely fast, without retropropagation or iterations, adapted to large sets of images. The network is easy to implement, presents excellent generalization performances for classification applications, and allows the detection of unknown inputs. The efficiency of this technique is illustrated with several benchmarks, photo and video datasets.

Junction-aware shape descriptor for 3D articulated models using local shape-radius variation

An articulated model is composed of a set of rigid parts connected by some flexible junctions. The junction, as a critical local feature, provides valuable information for many 3D semantic analysis applications such as feature recognition, semantic segmentation, shape matching, motion tracking and functional prediction. However, efficient description and detection of junctions still remain a research challenge due to high complexity of 3D articulated deformation. This paper presents a new junction-aware shape descriptor for a 3D articulated model defined by a closed mesh surface. The core idea is to exploit the local shape-radius variation for encoding junction information on the shape boundary surface, where the shape-radius at each point on the surface is the radius of corresponding medial balls within the shape. The presented descriptor is typically computed using a center-surround filter operator, which calculates the Gaussian-weighted average of shape-radius in the neighborhood of each point on the surface. Our descriptor is robust to articulation and can reflect the junction feature well without any explicit shape decomposition or prior skeleton extraction procedure. The experimental results and several potential applications are proposed for demonstrating the effectiveness of our method.

1008-LBP: Comparison of Single Nucleotide Polymorphism Genotyping of CYP2C19∗2 and CYP2C19∗3 alleles by Loop-mediated Isothermal Amplification and Real-time PCR Melting Curve Analysis

To validate the effectiveness of CYP2C19 -LAMP assay for the detection of CYP2C19 ∗ 2 and CYPC19 ∗ 3 in comparison with real time PCR melting curve analysis. CYP2C19 -LAMP primers were essentially adapted from Iwasaki M et al. [1] . The LAMP reaction was optimized at a temperature range from 58 °C to 62 °C. DNA were extracted from whole blood samples collected from forty patients suffering from acute coronary syndrome in a single center of Hong Kong. With the addition of SYBR Green, LAMP results were examined visually in ambient light and also under UV illumination at 365 nm. Real-time PCR melting curve analysis was done by LightMix Kit human CYP2C19 ∗ 2 and CYP2C19 ∗ 3 (TIB Molecular Biology). The two variants exhibit Tm 48.1[o]C and 59.2[o]C and were detected by LightCycler 480 II. CYP2C19 -LAMP assay successfully detected CYP2C19 ∗ 2 and CYPC19 ∗ 3 mutants. Our study found that the assay time of CYP2C19 -LAMP assay was only 30 min whereas real time PCR melting curve analysis was 60 min. We observed that the temperature requirement was very stringent and any temperature outside 60 °C might result in non-specific amplifications. In the LAMP assay, non-specific amplifications were observed with an error rate of 5% (8/160) and resulted in 15% (6/40) being mistyped. These non-specific amplifications, however, could be resolved with duplicated runs. A trial of one heat treated heparinized blood sample was found to be sustainable in LAMP reactions without prior DNA extraction. We showed that CYP2C19 -LAMP assay was a simple, rapid and cost- effective method for CYP2C19 genotyping. Owing to the stringent temperature requirement in LAMP assay, the test is recommended to be run in duplicate with better quality control in obtaining a more reliable result by a thermal cycler.

639. miR-Direct: A Sensitive and Accurate Method for Analysis of Circulating MicroRNAs Without Prior RNA Extraction

639. miR-Direct: A Sensitive and Accurate Method for Analysis of Circulating MicroRNAs Without Prior RNA Extraction

Direct-multiplex PCR assay for meat species identification in food products

This is the first time that direct PCR – DNA amplification without prior DNA extraction – was successfully developed and fully validated for rapid and economical simultaneous identification of six commonly consumed meat species. To achieve this, six species-specific primers were selected from previous reports and newly designed from the mitochondrial cytochrome b (cyt b), cytochrome oxidase I (COI), and 12s rRNA gene. The assay generated PCR products of 100, 119, 133, 155, 253, and 311bp for pork, lamb/mutton, chicken, ostrich meat, horsemeat and beef, respectively. Validation showed that the assay is robust, rapid, economical, reproducible, specific, and sensitive down to 12,500 mitochondrial copy (equating to seven fg). It could be used with a variety of raw meats and products, including highly degraded and processed food samples. This proposed method will be greatly beneficial to the consumers, food industry, and law enforcement.

Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to identifying bacterial and yeast strains. The aim of this study was to evaluate the clinical performance of the VITEK(®) MS system in the identification of bacteria and yeast strains routinely isolated from clinical samples. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria and yeasts regardless of phylum or source of isolation. Discordant results were resolved with 16S rDNA or internal transcribed spacer (ITS) gene sequencing. Colonies (a single deposit on a MALDI disposable target without any prior extraction step) were analyzed using the VITEK(®) MS system. Peptide spectra acquired by the system were compared with the VITEK(®) MS IVD database Version 2.0, and the identification scores were recorded. Of the 1,181 isolates (1,061 bacterial isolates and 120 yeast isolates) analyzed, 99.5% were correctly identified by MALDI-TOF mass spectrometry; 95.7% identified to the species level, 3.6% identified to the genus level, and 0.3% identified within a range of species belonging to different genera. Conversely, 0.1% of isolates were misidentified and 0.4% were unidentified, partly because the species were not included in the database. Re-testing using a second deposit provided a successful identification for 0.5% of isolates unidentified with the first deposit. Our results show that the VITEK(®) MS system has exceptional performance in identifying bacteria and yeast by comparing acquired peptide spectra to those contained in its database. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive method for bacterial and yeast identification. Our results demonstrate that the VITEK(®) MS system is a fast and reliable technique, and has the potential to replace conventional phenotypic identification for most bacterial and yeast strains routinely isolated in clinical microbiology laboratories.

Ultrasound-Guided Needle Localization during Open Parotid Sialolithotomy.

Our objective is to describe a novel operative technique for localization of parotid sialolithiasis, demonstrate the feasibility of the technique, and discuss its indications. Prospective study. Tertiary-level academic center. Patients with symptomatic parotid sialolithiasis who had failed prior sialendoscopic extraction underwent ultrasound needle localization and open sialolithotomy. Data were prospectively collected. Independent variables included size of and location of sialoliths. Eleven patients were treated using transcutaneous ultrasound-guided needle placement and injection of methylene blue prior to external sialolithotomy. Follow-up ranged from 6 to 12 months. Ten (91.9%) patients had stones within the proximal one-third of the ductal lumen, and 1 (9.1%) had stones present within both the proximal one-third and middle one-third of the ductal lumen. The average surgical time was 53 ± 10.8 minutes. The average sialolith length was 7.6 ± 2 mm. The average sialolith width was 6 ± 1.9 mm. All 11 (100%) cases were successful for stone retrieval. Ten (91%) patients had complete symptom resolution, and 1 (9.1%) patient had partial resolution of symptoms. No patients had major complications. Three (27.3%) patients had minor complications. After failing a purely endoscopic approach, sialoliths of the parotid gland pose a problem for precise localization and treatment. Ultrasound has been demonstrated to be reliable for identifying sialoliths. We propose a novel technique and assert that ultrasound-guided needle localization is a reliable aid to effective external parotid sialolithotomy, especially for larger stones >4 mm that are not amenable to sialendoscopic retrieval.

Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

Prospects and difficulties in TiO2 nanoparticles analysis in cosmetic and food products using asymmetrical flow field-flow fractionation hyphenated to inductively coupled plasma mass spectrometry

In this work, we proposed an analytical approach based on asymmetrical flow field-flow fractionation combined to an inductively coupled plasma mass spectrometry (AsFlFFF–ICP-MS) for rutile titanium dioxide nanoparticles (TiO2NPs) characterization and quantification in cosmetic and food products. AsFlFFF–ICP-MS separation of TiO2NPs was performed using 0.2% (w/v) SDS, 6% (v/v) methanol at pH 8.7 as the carrier solution. Two problems were addressed during TiO2NPs analysis by AsFlFFF–ICP-MS: size distribution determination and element quantification of the NPs. Two approaches were used for size determination: size calibration using polystyrene latex standards of known sizes and transmission electron microscopy (TEM). A method based on focused sonication for preparing NPs dispersions followed by an on-line external calibration strategy based on AsFlFFF–ICP-MS, using rutile TiO2NPs as standards is presented here for the first time. The developed method suppressed non-specific interactions between NPs and membrane, and overcame possible erroneous results obtained when quantification is performed by using ionic Ti solutions. The applicability of the quantification method was tested on cosmetic products (moisturizing cream). Regarding validation, at the 95% confidence level, no significant differences were detected between titanium concentrations in the moisturizing cream prior sample mineralization (3865±139mg Ti/kg sample), by FIA–ICP-MS analysis prior NPs extraction (3770±24mg Ti/kg sample), and after using the optimized on-line calibration approach (3699±145mg Ti/kg sample). Besides the high Ti content found in the studied food products (sugar glass and coffee cream), TiO2NPs were not detected.

Exploring computer simulation to assess counseling skills amongst pharmacy undergraduates

Abstract: This study illustrated the genetic algorithms (GA) is useful tool in wavelength selection for the simultaneous spectrophotometric determination of paracetamol (PAR) and chlorzoxazone (CZX) in a multicomponent pharmaceutical dosage form by partial least-squares calibration procedure. Artificial, a training set of the mixtures of PAR and CZX corresponding to central composite design was built to be used as calibration set for PLS and GA-PLS. Synthetic mixtures were used to verify the models. Reasonably good recoveries were obtained and it leads to 32% improvement of predictive ability for PAR and 35% for CZX, respectively. A commercial sample containing two drugs was assayed, and there was statistically no significant difference (P < 0.05) between the results with GA-PLS method and those obtain with the official method. The proposed method has been proved to be beneficial for the determination of both drugs in pharmaceutical tablets without prior extraction. Keywords : Genetic algorithms, Partial least-squares, Paracetamol, Chlorzoxazone

Leukotriene B4 levels in human atherosclerotic plaques and abdominal aortic aneurysms.

BackgroundLeukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome.MethodsAtherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls.ResultsLTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017).ConclusionsLTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

Direct Determination of Oleic Acid in Soybean Oil by Capacitively Coupled Contactless Conductivity Detection Capillary Electrophoresis in an Oil-Miscible KOH/1-Propanol/Methanol Medium

The aim of this work was to develop a quick direct analytical technique for the determination of oleic acid content in soybean oil by non-aqueous capillary electrophoresis with capacitively coupled contactless conductivity detection. The oil-miscible background electrolyte was a mixture of methanol/1-propanol (1:6 v/v) containing 4 × 10-2 mol L-1 KOH and 10% (v/v) ethylene glycol. Samples of 50 g L-1 soybean oil were prepared directly in the background electrolyte added with 1.33 × 10-3 g L-1 of salicylic acid as internal standard. Quantitative tests were performed by adding to the samples pure oleic acid in the range from 0.53 to 2.13 × 10-3 mol L-1. Under negative polarity anionic solutes moved faster than the electro-osmotic flow so that oleic acid was detected in 16 minutes. The limits of detection and quantification were, respectively, 24 and 81 µmol L-1. Such results demonstrate that, unlike required by other methods, low levels of oleic acid can be quantitatively determined in soybean oil without prior extraction.

The CCP3 Collaboration Project – Main Achievements

Abstract The CO 2 Capture Project (CCP) is a partnership of six energy companies working to advance the technologies that will underpin the deployment of industrial-scale CO 2 capture and storage (CCS). Harnessing the unique expertise of its members, the CCP has worked in collaboration with government bodies and more than 60 academic institutions, industry and leading environmental groups. Initiated in 2000, the overall objective of CCP is to deliver major cost reductions for CO 2 capture and demonstrate that geological storage is efficient, verifiable and secure. Phase 3 of The CO 2 Capture Project (CCP3) has been undertaken during 2009-2014 and with continuing support of R&D work for capture and storage technologies at multiple levels of development, including exploratory research to demonstration. Demonstration of the next generation technologies being developed by CCP is a key aspect of this third phase of the project and several capture and storage technologies have been or will be deployed (or made deployment-ready) within the CCP3 timeframe: • The regenerator of a large pilot Fluid Catalytic Cracking (FCC) unit in a Brazilian refinery owned by Petrobras was retrofit to oxy-firing with CO 2 recycle. A successful demonstration was completed in 2012. • The retrofit to oxy-firing of a commercial Once Through Steam Generator (OTSG) used in oil sands extraction operations in Canada (duty of 50 million BTU/hr) was performed in 2013, following completion of a feasibility study by Praxair. A demonstration run is scheduled in early 2014 and the main results will be included in the conference paper. • Field deployment and performance assessments of new CO 2 storage monitoring techniques at third party pilot / demonstration sites include: a modular borehole monitoring (MBM) assembly at Citronelle Dome (Alabama), Borehole Gravity at Cranfield Field (Mississippi) and InSAR at Decatur (Illinois) and electromagnetics at Aquistore (Saskatchewan). Other R&D projects completed in 2013 or early 2014 with co-funding of the CCP include: • Investigation of novel oxygen carriers (none Nickel-based) for Chemical Looping Combustion. • Pilot testing of commercial burners for refinery crude process heaters in oxy-firing mode. • Prolonged laboratory test of palladium alloy membranes in Water Gas Shift operating conditions and design of a pilot membrane module. • Pilot and bench-scale testing of several novel solvents for post-combustion application, including non- aqueous and enzyme-enhanced solvents. • Geomechanical hysteresis of top seal from prior oil extraction processes and potential impacts on CO2 storage containment • Fundamental studies of subsurface physico-chemical processes to simulation of through seal CO 2 leakage and intervention options. • Design of a bench-field scale “fracture sealing” experiment to test candidate sealant performance Selected scenarios for application of the CCP capture technologies are: • Oil refinery (capture from steam boilers, process heaters, fluid catalytic crackers, hydrogen plants) • Steam production for extraction of heavy oils • Natural Gas Combined Cycle (NGCC) power station These represent major sources of CO 2 emissions for the CCP member companies and some of their customers. Storage venues may range from saline formations to CO 2 EOR and possibly unconventional gas reservoirs. Relevant findings and technologies may be leveraged for oil and gas exploration and production characterization and surveillance. An assessment of the performance and economics of state-of-the-art post combustion, oxy-firing and precombustion for the above applications has been carried out. Foster Wheeler was commissioned to conduct technical evaluation studies, while an in-house economic model was developed, which included capture, transportation, and storage costs. The objective of these studies was to provide material for a CO 2 capture handbook to be used to support CO 2 capture technology selection in the oil and gas industry. The technical/economical evaluation of novel technologies developed in CCP3 was undertaken using the same procedure (third party performance evaluation and in-house economic model). The main results will be included in the conference paper. The CCP3 also maintains Policy and Communications capabilities with on-going stakeholder interactions and continues to make accurate and current technical information publicly available including a number if Technical Factsheets and Annual Reports.

Cholesterol and total lipid content in raw and heat processed commercially produced meat from two farms

The present study was conducted to present information about the cholesterol and total lipid content in fresh and roasted chicken meat with skin (breast and drumstick meat), most commonly consumed in Serbia. In addition, to assess the possible effects of carcass weight and nutrition on total lipid and cholesterol content in examined meat cuts. A total number of 48 samples of breast and drumstick muscle of broilers from two farms (Farm I and II), fed ad libitum by commercial diets for growing broilers, were taken in summer, 2012 and autumn, 2013. Total lipid and cholesterol content were determined in raw and heat-processed breast and drumstick muscle with skin. Total lipid content was determined by extraction of fat by petrol ether (Soxhlet) after acid hydrolysis of samples (SRPS ISO 1443/1992).Cholesterol determination was performed after direct saponification (without prior lipid extraction) by using HPLC/PDA system. Generally, all parameters measured were influenced by interaction of care and management and broiler performance at the 5% level or less. The total lipid content in samples of raw breast muscle of chicken from Farm I were the highest in summer (5.53%), (4.2%, in autumn), compared to samples of chickens from Farm II (3.05% in summer and 2.61% in autumn). The total lipid content in samples of raw drumstick were significantly differ (p < 0.001) between Farm I (9.63%) and Farm II (5.19%), only in summer. Cholesterol content (mg/100 g) in the raw breast muscle from Farm I was 53.9 (autumn) and 62.1 (summer), while in samples from Farm II was 46.97 (autumn) and 49.53 (summer). There was significant difference (p < 0.001) in cholesterol content in raw breast muscle of chickens in summer from two farms. In raw drumstick from Farm I the average cholesterol content (mg/100 g) was 70.24 (autumn) and 83.95 (summer), while in samples of chickens from Farm II was 65.05 (autumn) and 60.92 (summer). These differences were significant for cholesterol levels in drumstic between farms in summer (p < 0.001). In heat-processed meat belonging to chickens from Farm I, breast and drumstick contained higher quantities of total lipids compared to samples from Farm II. These differences was significant (p < 0.01) in drumstick between Farm I and II (autumn) and were 13.37%, and 11.10%, respectively. The average cholesterol content (mg/100 g) in samples of heat-processed meat of chicken from Farm I varied between 70.32 (autumn) and 87.37 (summer) and from 75.23 (autumn) to 78.92 (summer) in drumstick, versus 64.33 (summer) and 66.24 (autumn) in breast muscle and from 81.31(summer) to 91.6 (autumn) in drumstick samples of chickens from Farm II. We conclude that factors, such as feed composition, genotype (breed) and gender, influence the total lipid and cholesterol content in the meat. There were no obviously effects of slaughter traits on cholesterol and total lipid content. The results presented here, also shows that further investigations have to be conducted on greater number of samples.

Determination of Impurities in Bioproduced Succinic Acid

At present, significant research resources are directed towards development of renewable products for replacing petrochemicals such as succinic acid. The critical component of this research is the identification of impurities which have a detrimental impact on further processing of succinic acid. We have adapted derivatization with gas chromatography - mass spectrometry to identify and quantify more than 120 impurities in several succinic acid samples. This study focused on petroleum based succinic acid as well as bio-based samples that use a modified E. coli strain for fermentation. To enable an accurate quantification of both the target product and common impurities, we evaluated the acetonitrile extraction efficiency as an alternative to direct derivatization, and then compared several derivatization agents for trimethylsilylation. A prior acetonitrile extraction was shown to be essential to detect impurities in trace concentrations. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was most efficient for derivatization of saccharides and low molecular weight monocarboxylic acids. However, the presence of pyridine was necessary for derivatization of saccharides and polyalcohols with BSTFA, whereas low molecular weight acids had to be quantified without pyridine. Fourteen representative bioproduced succinic acid samples differing in production stage, and cultivation method were characterized. The screening of initial process (1st stage of synthesis) samples showed monocarboxylic acids as most abundant and suggested occurrence of saccharides. Thus we have developed method allowing for quantification of carboxylic acids and saccharides with limits of detection between 0.02-0.3 ng. In initial process bacterial samples and also petrochemical sample, formic, acetic, lactic, oxalic, benzoic, citric and malic acids as well as glycerol, butanediol, and glucose were found in a range of 0.02-1160 μg/g. In final processed samples, formic and acetic acid, and glucose were found in concentration lower than 0.001% demonstrating effectiveness of process as well as applicability of the method as quality control of the process.

Neoarchean metagabbro and charnockite in the Yinshan block, western North China Craton: Petrogenesis and tectonic implications

Properly calibrating the magmatic record of mantle and crustal origin in Archean granite-greenstone terranes is crucial for understanding the petrogenetic and geodynamic processes that generated early continental landmasses. This geochronological and geochemical study documents a Neoarchean bimodal metaplutonic suite of metagabbro and charnockite from the Yinshan block, North China Craton. The meta-gabbroic rocks show an SiO2 range from 45.9 to 51.7% and high MgO content from 6.7 to 16.4%, with enrichment in Ba and light rare earth elements (LaN/YbN=3.11–5.99) and depletion in high field strength elements. Together with their enriched whole-rock Nd (ɛNd(t)=−0.23 to 1.49) and zircon Hf (ɛHf(t)=0.1–6.9) isotopic signatures relative to the Archean depleted mantle in the craton, these rocks are supposed to originate from the second-stage high-temperature partial melting of refractory depleted mantle that experienced prior basaltic magma extraction and subsequent metasomatism by subduction-related fluids. The associated charnockites range in SiO2 from 61.3 to 69.6% and exhibit a magnesian, calc-alkalic and metaluminous character, with variable Sr/Y and LaN/YbN ratios. These elemental features, plus their evolved isotopic compositions (ɛNd(t)=0.93–1.85, zircon ɛHf(t)=−0.3 to 2.5), are consistent with partial melting of newly underplated mafic lower crustal protolith. In combination with widespread occurrence of metasomatized lithospheric mantle-derived magmas (e.g., high-Mg basalts, sanukitoid suites) and juvenile crust-extracted potassic granites in the NCC during Late Neoarchean time, such a mafic and felsic magma association not only attests to the establishment of a craton-scale subduction-related metasomatized sub-continental lithospheric mantle, but also encapsulates a scenario of coupled lithospheric mantle-crust formation at ∼2.7Ga and juvenile crustal reworking at ∼2.5Ga within a modern-style convergent continental margin possibly featuring episodic slab break-off events.

The detection of different Dirofilaria species using direct PCR technique

A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 μl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.