Sodium Dodecyl Sulfate Extraction Research Articles

LAMA5-inspired adhesive dodecapeptide facilitates efficient dentine regeneration: An in vitro and in vivo study.

The primary goal of this study was to investigate the potential effects of A5G81 in inducing reparative dentine (RD) formation both in vitro and in vivo. Cell adhesion was observed by crystal violet staining and quantified by Sodium Dodecyl Sulphate (SDS) extraction. Cell proliferation was investigated using Cell Counting Kit-8 (CCK-8) assay. Spreading of cytoskeleton was visualized using immunofluorescence staining. Protein expression level of Akt signalling pathway was compared in a human Akt pathway phosphorylation array. Genes that were up or downregulated by A5G81 were identified by RNA sequencing. The mRNA expression of odontoblastic markers was detected by quantitative real-time polymerase chain reaction (qPCR). Moreover, mineralization of human dental pulp cells (hDPCs) was visualized by alizarin red staining and quantified using cetylpyridinium chloride (CPC). A direct pulp-capping model was established in SD rats and the RD formation at 2 weeks after operation was observed using HE staining. A5G81 (optimal coating concentration: 0.5 mg/mL) promoted hDPCs adhesion and proliferation to a level that was similar to Type I collagen (COL-1). Meanwhile, A5G81 activated Akt signalling pathway, albeit to a lesser extent than COL-1. An inhibition test indicated that A5G81 induced hDPCs adhesion by activating PI3K pathway. A5G81 induced the expression of ECM remodelling genes and odontoblastic genes, which were demonstrated by RNA-seq and qPCR, respectively. In addition, A5G81 efficiently accelerated the mineralization of hDPCs in both immobilized and soluble forms, a property that makes it more applicable in dental clinic. Finally, the pulp-capping study in rats suggested that use of A5G81 could successfully induce the formation of RD within 2 weeks. Coating of A5G81 to non-tissue culture-treated polystyrene facilitates spreading, proliferation and differentiation of hDPCs, resulting in rapid RD formation in artificially exposed pulp.

Optimised protocols for RNA extraction from a broad taxonomic range of algae

AbstractDespite advancements in RNA extraction methods, RNA extraction from sources rich in polyphenols and polysaccharides such as algae and seagrasses remains a challenge. Here we present a RNA extraction strategy using a hexadecyltrimethylammonium bromide (CTAB) extraction buffer and demonstrate its effectiveness on a broad range of red, green, and brown algae, as well as on the cyanobacterium Arthrospira platensis and the seagrass Zostera marina. For the vast majority of tested samples we achieved high yields of RNA comparable to those obtained from higher plants by commercially available kits (ranging from 3.9 to 125.9 µg RNA g−1 fresh weight). Analysis by UV/Vis spectrometry and capillary electrophoresis revealed high purity and integrity of obtained RNA extracts. For highly challenging species of brown algae like Fucus vesiculosus, Fucus serratus and Dictyosiphon foeniculaceus, we established an alternative procedure using a sodium dodecyl sulfate (SDS) extraction buffer in combination with a commercial kit. With this protocol, even higher RNA yields up to 317.0 µg g−1 fresh weight were extracted from polysaccharide-rich brown algae tissues. This study can serve as a guideline and starting point for the development of RNA extraction protocols for so far unstudied algal species from very diverse taxa.

Molecular characterization of the phytopathogen C. theae (Petch) – Causative of birds eye spot infection in Camellia sinensis host through DNA polymorphism and proteome analysis

The pathogenic fungus Cercospora infecting plants causes high degree symptomatic disease in several important crops including tea (Camellia sinensis). Genetic diversity studies on plant pathogens are essential to tackle plant diseases, hence in the proposed study, Cercospora theae isolated from different regions was characterized using the molecular tool like RFLP of the rDNA regions amplified using ITS primers. Protein profiling for all the isolates was also done by optimizing the protein extraction using sodium dodecyl sulphate (SDS), urea and Trichloroacetic acid (TCA) protocols. Genetic relatedness with the identical size of molecular weight among the three C. theae isolates of different regions was studied from the 5.8S rDNA-ITS amplified regions. Digestion of the amplified regions with seven specific endonucleases exposed the same banding pattern among the isolates. The sequence data from the three isolates showed maximum homology with the Genbank sequence of Cercospora spp. (HQ450006, JQ754039, JQ753964, JN942274, JN942272, FJ460222 and EU581822). Further, an optimized protocol for protein profiling of C. theae was determined to extract the majority of the proteins. The results also revealed that the most suitable protocol for studying the proteomics of C. theae was the SDS extraction method.

Enzyme-Linked Immunosorbent Assay for Pork Determination in Raw and Heated Meats: Combination of Monoclonal Antibodies to Denatured Porcine Myoglobin and Sodium Dodecyl Sulfate Extraction

Several immunoassays for monitoring pork contamination have been developed, but they have limitations in accurate detection because they have been affected by protein denaturation and loss of extraction efficiency due to cooking. In this study, a sandwich enzyme-linked immunosorbent assay (s-ELISA) combined with an extraction method using sodium dodecyl sulfate (SDS) was developed for pork determination in raw and heated meats. To establish monoclonal antibodies (mAbs), SDS-denatured porcine myoglobin (Mb) and synthetic peptides with amino acid sequences of porcine Mb were used for immunization. The s-ELISA quantitatively detected the porcine Mb without cross-reaction to beef and chicken Mbs and lamb and goat meats. The 50% maximal effective concentration of porcine Mb for s-ELISA was 90 ng/mL, and the recoveries of porcine Mb spiked into raw and heated beef and chicken were 94–158%. This s-ELISA detected 1% (w/w) pork mixed with raw and heated beef. For the determination of Mbs in various parts of raw pork, including fatty bellies, the results correlated well with those obtained by high-performance liquid chromatography. Our s-ELISA could be available for authentication of meat products and prevention of mislabeling in pork products.

Oral immunization with soybean storage protein containing amyloid β4‐10 prevents spatial learning decline

AbstractBackgroundAmyloid‐β protein (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD). Because AD pathologies begin two decades before the onset of dementia, prevention of Aβ amyloidosis has been proposed as a mean to block the pathological cascade. We generated a transgenic plant‐based vaccine, a soybean storage protein containing Aβ4‐10, named Aβ+, for oral Aβ immunization.MethodOne mg of Aβ+ or control protein (Aβ‐) with 10 mg cholera toxin subunit B was orally administered via a catheter to TgCRND8 mice once a week from 9 weeks. Extended Morris water maze tests for 10 consecutive days were examined every 4 weeks. Mice were analyzed at 23 weeks (Aβ+ n = 9, Aβ– n = 11), 43 weeks (Aβ+ n = 7, Aβ– n = 6), and 59 weeks (Aβ+ n = 6, Aβ– n = 7).ResultAβ+ immunization raised both anti‐Aβ antibodies and cellular immune responses. Plasma from Aβ+ treated mice showed staining of Aβ oligomers. Spatial learning decline was prevented in the Aβ+ immunized group from 21 weeks. In Tris‐buffered saline (TBS), sodium dodecyl sulfate (SDS), and formic acid (FA) serial extractions, all sets of Aβ species from Aβ monomer, low to high molecular weight Aβ oligomers, and Aβ smears had different solubility in TgCRND8 brains. Aβ oligomers decreased in TBS fractions, corresponding to an increase in high molecular weight Aβ oligomers in SDS extracts and Aβ smears in FA fraction of the Aβ+ treated group. There was significant inhibition of histological Aβ burden, especially in diffuse plaques, and suppression of microglial inflammation. Processing of amyloid‐β protein precursor was not different between Aβ+ and Aβ‐ groups. No evidence of amyloid‐related inflammatory angiopathy was observed.ConclusionAβ+ oral immunization could be a promising, cheap, and long‐term safe disease‐modifying therapy to prevent the pathological process in AD.

Dysregulation of H11 Kinase in the Failing Human Left Ventricular Myocardium

Mitochondrial dysfunction and ongoing cardiomyocyte loss from apoptosis contribute to the progressive LV dysfunction characteristic of the heart failure (HF) state. H11 kinase (H11K) is a 22-kD serine/threonine kinase protein abundantly expressed in the heart. Dysregulation of H11K occurs in HF but its potential contribution to the progression of HF is not fully understood. Cardiac deletion of H11K in mice with LV pressure overload has been shown to result in LV dilation, reduced hypertrophy, impaired contractile function, increased interstitial fibrosis and faster transition to HF. In this study, we examined the expression of H11K in LV myocardium and separately in cytosolic and mitochondrial fractions of explanted failing human hearts. Sodium-dodecyl sulfate (SDS) extracts were prepared from LV tissue of 7 explanted failed human hearts due to idiopathic dilated cardiomyopathy (IDC), 7 due to ischemic cardiomyopathy (ICM) and of 7 non-failing human donor hearts (DNR). Western blotting and specific antibodies were used to assess protein levels of H11K normalized to the internal control GAPDH in homogenate and cytosolic fractions and protein levels of H11K normalized to the internal control porin in mitochondrial fractions. There were no differences in GAPDH and porin among the 3 groups. H11K protein levels were significantly increased in LV homogenate and cytosolic fractions but significantly decreased in mitochondrial fractions of both IDC and ICM failed human hearts compared to DNR hearts (Table). The failing human LV manifests increased H11K protein levels independent of HF etiology. The reduced H11K protein level in mitochondria and its increase in the cytosolic compartment of failing cardiomyocytes represents an adverse maladaptation capable of reducing ATP synthesis by mitochondria and activation of cardiomyocyte pro-apoptotic cell death pathways in the cytosol respectively thus likely contributing to the progression of HF.

A Simple Method for the Design and Development of Flavivirus NS1 Recombinant Proteins Using an In Silico Approach.

Even in countries that are currently not facing a flavivirus epidemic, the spread of mosquito-borne flaviviruses presents an increasing public threat, owing to climate change, international travel, and other factors. Many of these countries lack the resources (viral strains, clinical specimens, etc.) needed for the research that could help cope with the threat imposed by flaviviruses, and therefore, an alternative approach is needed. Using an in silico approach to global databases, we aimed to design and develop flavivirus NS1 recombinant proteins with due consideration towards antigenic variation. NS1 genes analyzed in this study included a total of 6,823 sequences, from Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), and Yellow fever virus (YKV). We extracted and analyzed 316 DENV NS1 sequence types (STs), 59 JEV STs, 75 WNV STs, 30 YFV STs, and 43 ZIKV STs using a simple algorithm based on phylogenetic analysis. STs were reclassified according to the variation of the major epitope by MHC II binding. 78 DENV epitope type (EpT), 29 JEV EpTs, 29 WNV EpTs, 12 YFV EpTs, and 5 ZIKV EpTs were extracted according to their major epitopes. Also, frequency results showed that there were dominant EpTs in all flavivirus. Fifteen STs were selected and purified for the expression of recombinant antigen in Escherichia coli by sodium dodecyl sulfate extraction. Our study details a novel in silico approach for the development of flavivirus diagnostics, including a simple way to screen the important peptide regions.

Evaluation of DNA Extraction Methods for Detection of <i>Leishmania</i> by Polymerase Chain Reaction

There are several methods used to obtain DNA from cells; however, the quantity, integrity, and purity of DNA vary among the methods, which may interfere with the polymerase chain reaction (PCR) results. The objective was to determine the most efficient and cost-effective method that provides the best DNA yield and PCR results. Three methods of DNA isolation were compared: 20% sodium dodecyl sulfate (SDS), guanidine isothiocyanate-phenol-chloroform (GTPC), and DNA extraction using a commercial kit (GE Healthcare GenomicPrep Blood DNA Isolation KitTM). Human peripheral blood samples were inoculated with 104 promastigotes of Leishmania infantum. DNA was quantified and PCR was performed with 13A/13B primers. The results showed that a higher DNA yield was obtained using the GTPC technique (214.51 ng/μL), followed by SDS (26.16 ng/μL) and the commercial kit (10.99 ng/μL). We concluded that while all of the techniques were effective for obtaining DNA, the GTPC method provided the best yield and the brightest bands.

Dysregulation of H11 Kinase in the Failing Left Ventricular Myocardium

Background: Increased expression of H11 kinase (H11K) has been observed in Left Ventricular (LV) myocardium of failed human hearts. Its potential role as a contributor to the progression of Heart Failure (HF) remains uncertain. In the present study we examined the expression of H11K in cytosolic and mitochondrial fractions of failing human and dog LV myocardium and assessed its interaction with Akt (cell-survival enzyme) and p38MAPK (programmed cell death enzyme). Methods: Total RNA and Sodium-Dodecyl Sulfate (SDS) extracts were prepared from homogenate of LV specimens of 6 dogs with intracoronary microembolization-induced HF, 6 normal (NL) dogs, 7 explanted failed human hearts due to Idiopathic Dilated Cardiomyopathy (IDC), 7 failed human hearts due to Ischemic Cardiomyopathy (ICM) and 7 non-failing human donor hearts (DNR). SDS extracts were also prepared from cytosolic and mitochondrial fractions isolated from LV specimens. Results: H11K mRNA and protein levels normalized to GAPDH increased significantly in LV tissue from ICM and IDC hearts compared to DNR hearts and in HF dogs compared with NL dogs. H11K protein levels increased in cytosolic fractions but decreased in mitochondrial fractions of both failed human and dog hearts compared to DNR hearts and NL dog hearts. Immunoprecipitation studies in specimens from HF dogs showed that H11K cytosolic fractions interacted predominantly with p38MAPK and least with Akt when compared with NL dogs. Conclusions: Enhanced interaction of H11K with p38MAPK in HF can promote cell death thus contributing to progressive LV dysfunction. Therapeutic modalities that restore interaction of H11K with Akt and augment H11K translocation to mitochondria can potentially and partially reverse the progression of LV dysfunction by promoting cell survival.

Oral Immunization with Soybean Storage Protein Containing Amyloid-β 4-10 Prevents Spatial Learning Decline.

Amyloid-β (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD). Because AD pathologies begin two decades before the onset of dementia, prevention of Aβ amyloidosis has been proposed as a mean to block the pathological cascade. Here, we generate a transgenic plant-based vaccine, a soybean storage protein containing Aβ4–10, named Aβ+, for oral Aβ immunization. One mg of Aβ+ or control protein (Aβ–) was administered to TgCRND8 mice once a week from 9 weeks up to 58 weeks. Aβ+ immunization raised both anti-Aβ antibodies and cellular immune responses. Spatial learning decline was prevented in the Aβ+ immunized group in an extended reference memory version of Morris water maze test from 21 to 57 weeks. In Tris-buffered saline (TBS), sodium dodecyl sulfate (SDS), and formic acid (FA) serial extractions, all sets of Aβ species from Aβ monomer, low to high molecular weight Aβ oligomers, and Aβ smears had different solubility in TgCRND8 brains. Aβ oligomers decreased in TBS fractions, corresponding to an increase in high molecular weight Aβ oligomers in SDS extracts and Aβ smears in FA fraction of the Aβ+ treated group. There was significant inhibition of histological Aβ burden, especially in diffuse plaques, and suppression of microglial inflammation. Processing of amyloid-β protein precursor was not different between Aβ+ and Aβ– groups. No evidence of amyloid-related inflammatory angiopathy was observed. Thus, Aβ+ oral immunization could be a promising, cheap, and long-term safe disease-modifying therapy to prevent the pathological process in AD.

A comparative study for the isolation and characterization of mannoproteins from Saccharomyces cerevisiae yeast cell wall

Cell wall yeast, recovered upon the production of yeast extract, was investigated as an abundant source of mannoproteins. Selected isolation methods were evaluated for the recovery of mannoproteins, in terms of yield, mannan and protein recovery yield, mannoproteins content and mannan to protein ratio in extracted mannoproteins. The results showed that heat treatment and sodium dodecyl sulfate (SDS) extraction led to a lower yield compared to the enzymatic treatment; and no glycosylated proteins could be obtained upon the SDS extraction. As compared to other methods, the enzymatic approach, based on the use of Zymolyase®, exhibiting a high β-1,3-glucanase activity, resulted in the highest yield, mannoproteins content and mannan to protein ratio. A 5-level, 2-variable central composite rotatable design contributed to the better understanding of the effects of independent variables, reaction time and enzyme units, on the efficiency of the enzymatic treatment and to the better modulation of their actions. The effects of enzyme units and reaction time on the mannoproteins content and the mannan to protein ratio exhibited the same patterns. Comparison of predicted and experimental values validated the established predicted models, which can be used to identify the conditions for the isolation of mannoproteins with well-defined molecular properties.

Efficacy of sodium dodecyl sulphate and natural extracts against E. coli biofilm

The aim of this study was to determine and compare the efficacy of a standard cleaning agent, sodium dodecyl sulphate, and natural extracts from pomegranate peel grape skin and bay laurel leaf against E. coli biofilm. The biofilm was exposed for 10 minutes to three different concentrations of each tested compound. The results show that bay laurel leaf extract is the most efficient with 43% biofilm biomass reduction, followed by pomegranate peel extract (35%); sodium dodecyl sulphate and grape skin extract each have 30% efficacy. Our study demonstrated that natural extracts from selected plants have the same or even better efficacy against E. coli biofilm removal from surfaces than the tested classical cleaning agent do. All this indicates that natural plant extracts, which are acceptable from the health and environment points of view, can be potential substitutes for classical cleaning agents.

In vitro digestion method for estimation of copper bioaccessibility in Açaí berry.

Copper is an essential trace element for humans and its deficiency can lead to numerous diseases. A lot of mineral supplements are available to increase intake of copper. Unfortunately, only a part of the total concentration of elements is available for human body. Thus, the aim of the study was to determine bioaccessibility of copper in Açai berry, known as a “superfood” because of its antioxidant qualities. An analytical methodology was based on size exclusion chromatography (SEC) coupled to a mass spectrometer with inductively coupled plasma (ICP MS) and on capillary liquid chromatography coupled to tandem mass spectrometer with electrospray ionization (µ-HPLC-ESI MS/MS). To extract various copper compounds, berries were treated with the following buffers: ammonium acetate, Tris–HCl, and sodium dodecyl sulfate (SDS). The best extraction efficiency of copper was obtained for SDS extract (88 %), while results obtained for Tris–HCl and ammonium acetate were very similar (47 and 48 %, respectively). After SEC–ICP–MS analysis, main signal was obtained for all extracts in the region of molecular mass about 17 kDa. A two-step model simulated gastric (pepsin) and gastrointestinal (pancreatin) digestion was used to obtain the knowledge about copper bioaccessibility. Copper compounds present in Açai berry were found to be highly bioaccessible. The structures of five copper complexes with amino acids such as aspartic acid, tyrosine, phenylalanine, were proposed after µ-HPLC-ESI MS/MS analysis. Obtained results show that copper in enzymatic extracts is bound by amino acids and peptides what leads to better bioavailability of copper for human body.Graphical abstract

Optimization and comparison of bottom-up proteomic sample preparation for early-stage Xenopus laevis embryos

Xenopus laevis is an important model organism in developmental biology. While there is a large literature on changes in the organism's transcriptome during development, the study of its proteome is at an embryonic state. Several papers have been published recently that characterize the proteome of X. laevis eggs and early-stage embryos; however, proteomic sample preparation optimizations have not been reported. Sample preparation is challenging because a large fraction (~90% by weight) of the egg or early-stage embryo is yolk. We compared three common protein extraction buffer systems, mammalian Cell-PE LB(TM) lysing buffer (NP40), sodium dodecyl sulfate (SDS), and 8M urea, in terms of protein extraction efficiency and protein identifications. SDS extracts contained the highest concentration of proteins, but this extract was dominated by a high concentration of yolk proteins. In contrast, NP40 extracts contained ~30% of the protein concentration as SDS extracts, but excelled in discriminating against yolk proteins, which resulted in more protein and peptide identifications. We then compared digestion methods using both SDS and NP40 extraction methods with one-dimensional reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS). NP40 coupled to a filter-aided sample preparation (FASP) procedure produced nearly twice the number of protein and peptide identifications compared to alternatives. When NP40-FASP samples were subjected to two-dimensional RPLC-ESI-MS/MS, a total of 5171 proteins and 38,885 peptides were identified from a single stage of embryos (stage 2), increasing the number of protein identifications by 23% in comparison to other traditional protein extraction methods.

Rapid identification of Bacillus anthracis by cell wall and capsule components direct fluorescent antibody assay

During the outbreak of anthrax, early diagnosis is critical for effective treatment. Numerous attempts have been made to design antigen based detection tests and to rapidly identify truly anthrax specific antigens for B. anthracis . In Indonesia, standard identification of B. anthracis relies on a combination of time consuming steps including bacterial culture and Ascoli precipitin test, which can take several days to provide a diagnosis. In this study, two component (cell wall and capsule) direct fluorescent antibody assay (DFA) were developed to rapidly identify and to directly detect capsulated B. anthracis . The component used in cell wall DFA (CW-DFA) assay is polysaccharide-peptidoglycan complex, which was prepared from B. anthracis culture by cell lysis, guanidine and sodium dodecyl sulphate (SDS) extraction. The component used in capsule DFA (CAP-DFA) is poly-D-glutamic acid (PGA) which were prepared by extraction of B. anthracis capsule. Component of polysaccharide-peptidoglycan complex and PGA conjugated with hemocyanin were then used as immunogen for immunizing rabbits using Freund’s complete/incomplete adjuvant. The hyperimmune sera were then collected, purified and conjugated to Fluorecent Iso Thiocyanate (FITC). B. anthracis isolates and non B. anthracis isolates were tested by the CW-DFA and CAP-DFA Assays. B. cereus, B. subtilis , other Bacillus sp. and other Gram positive rod bacteria were negative, while capsulated B anthracis gave positive results. The two component (CW DFA and CAP-DFA) assay are specific rapid confirmatory test for capsulated B. anthracis . Key Words : Bacillus anthracis , Cell Wall and Capsule Direct Fluorescent Antibody Assay

Quantitative analysis of genomic element interactions by molecular colony technique

Distant genomic elements were found to interact within the folded eukaryotic genome. However, the used experimental approach (chromosome conformation capture, 3C) enables neither determination of the percentage of cells in which the interactions occur nor demonstration of simultaneous interaction of >2 genomic elements. Each of the above can be done using in-gel replication of interacting DNA segments, the technique reported here. Chromatin fragments released from formaldehyde–cross-linked cells by sodium dodecyl sulfate extraction and sonication are distributed in a polyacrylamide gel layer followed by amplification of selected test regions directly in the gel by multiplex polymerase chain reaction. The fragments that have been cross-linked and separate fragments give rise to multi- and monocomponent molecular colonies, respectively, which can be distinguished and counted. Using in-gel replication of interacting DNA segments, we demonstrate that in the material from mouse erythroid cells, the majority of fragments containing the promoters of active β-globin genes and their remote enhancers do not form complexes stable enough to survive sodium dodecyl sulfate extraction and sonication. This indicates that either these elements do not interact directly in the majority of cells at a given time moment, or the formed DNA–protein complex cannot be stabilized by formaldehyde cross-linking.

Cloning and expression of a surface immunogenic protein in Streptococcus dysgalactiae isolated from fish and its application in enzyme‐linked immunosorbent assays to diagnose S. dysgalactiae infections in fish

Lancefield group C Streptococcus dysgalactiae (GCSD) causes severe necrotic lesions in the caudal peduncle in the genus Seriola farmed in Japan. To develop a sero-diagnostic method for GCSD infection in farmed fish, we attempted to identify a surface immunogenic protein that induces an antibody after infection with GCSD by immunoblot analysis using sera collected from infected fish. A protein obtained from sodium dodecyl sulfate (SDS) extracts of GCSD was identified as S.dysgalactiae surface immunogenic protein (Sd-Sip). Sd-Sip exhibited more than 94% homology with a surface antigen or a hypothetical protein from S.dysgalactiae mammalian isolates at the nucleotide sequence level. Expression of the recombinant Sd-Sip (rSd-Sip) was confirmed by immunoblot analysis, that is, its reactivity to GCSD-infected sera. Antibody detection ELISA using rSd-Sip and their usefulness for diagnosis of GCSD infection were examined. GCSD-infected sera collected from farmed amberjack, Seriola dumerili (Risso), showed strong reaction with immobilized rSd-Sip. Meanwhile, sera immunized by other pathogenic bacteria of fish were showed ELISA values similar to those of non-infected sera. These results of this study suggest that the antibody detection ELISA using rSd-Sip is an effective diagnostic method for GCSD infection in fish.

Fruit differential protein patterns in strawberry cultivars susceptible or resistant to grey mould

The total proteins of four strawberry cultivars with differing resistance and/or susceptibility to grey mould were extracted by two different methods, phenol and sodium dodecyl sulphate (SDS). Compared to phenol extraction, SDS extraction produced higher protein yields but poor quality protein bands with a high degree of streaking indicating possible contamination. In contrast, the total proteins obtained by phenol extraction could be used in two-dimensional polyacrylamide gel electrophoresis (2-DE) because of their higher quality, and the protein spots were well separated for all tested genotypes. From all the cultivars studied in this research, we obtained 89 differential (unmatched) protein spots that may be related to fruit disease resistance or susceptibility. Using the SPSS 11.0 software, we obtained a cluster of the four strawberry cultivars. The cultivars “APF029-4” and “SJ8976-1” were in the same subcluster with the highest Pearson correlation coefficient (0.705). Afterwards, they were amalgamated into the second group with “Joliette”, a disease-resistant cultivar, which means that they might have a similar resistance to grey mould.

Determination of Ethopabate Residues in Chicken Muscles, Liver, and Eggs after Aqueous SDS Extraction by Micellar Liquid Chromatography with Fluorescence Detection with Application to Baby Food

A simple and sensitive liquid chromatographic method was optimized and validated for the analysis of ethopabate residues in chicken muscles, liver, and eggs. Aqueous sodium dodecyl sulfate (SDS) solution was used for extraction. Analytical separation was performed in less than 8 min using a C18 column (150 × 4.6 mm internal diameter, 5 μm particle size) with fluorescence detection at 271/364 nm. A micellar solution composed of 0.1 M SDS, 10 % 1-propanol, and 0.3 % triethylamine in 0.02 M phosphoric acid adjusted to pH 4 was used as the mobile phase. The method was fully validated in accordance with International Conference on Harmonization guidelines. The micellar method was successfully applied to quantitatively determine ethopabate residues in spiked chicken muscles, liver, and eggs. It was also extended to the determination of ethopabate residues in chicken-based baby food. The recoveries obtained were in the 93.81–115.67 % range. The limit of detection was found to be 1.6 ng/g. High extraction efficiency for ethopabate was obtained without matrix interference and in the subsequent chromatographic determination. No organic solvent was used during the pretreatment step.

A non-destructive genotyping system from a single seed for marker-assisted selection in watermelon

Genomic tools for watermelon breeding are becoming increasingly available. A high throughput genotyping system would facilitate the use of DNA markers in marker-assisted selection. DNA extraction from leaf material requires prior seed germination and is often time-consuming and cost prohibitive. In an effort to develop a more efficient system, watermelon seeds of several genotypes and various seed sizes were sampled by removing ⅓ or ½ sections from the distal ends for DNA extraction, while germinating the remaining proximal parts of the seed. Removing ⅓ of the seed from the distal end had no effect on seed germination percentage or seedling vigor. Different DNA extraction protocols were tested to identify a method that could yield DNA of sufficient quality for amplification by polymerase chain reaction. A sodium dodecyl sulfate extraction protocol with 1% polyvinylpyrrolidone yielded DNA that could be amplified with microsatellite primers and was free of pericarp contamination. In this study, an efficient, non-destructive genotyping protocol for watermelon seed was developed.