Gel-based Techniques Research Articles

Cargo quantification of functionalized DNA origami for therapeutic application.

In recent years, notable advances in nanotechnology-based drug delivery have emerged. A particularly promising platform in this field is DNA origami-based nanoparticles, which offer highly programmable surfaces, providing precise control over the nanoscale spacing and stoichiometry of various cargo. These versatile particles are finding diverse applications ranging from basic molecular biology to diagnostics and therapeutics. This growing interest creates the need for effective methods to quantify cargo on DNA origami nanoparticles. Our study consolidates several previously validated methods focusing on gel-based and fluorescence-based techniques, including multiplexed quantification of protein, peptide, and nucleic acid cargo on these nanoparticles. This work may serve as a valuable resource for groups researchers keen on utilizing DNA origami-based nanoparticles in therapeutic applications.

Detecting antimony(III) on-site using novel gel-based techniques: Colorimetric diffusive equilibrium in thin films for two-dimensional imaging and surface-enhanced Raman scattering for sensitive quantification

Antimony (Sb) pollution has raised increasing public concerns and its rapid on-site screening is central for the risk assessment. Herein, we proposed two gel-based methods based on colorimetric diffusive equilibrium in thin films (DET) and surface-enhanced Raman scattering (SERS), for two-dimensional imaging and sensitive detection of Sb(III) by revisiting the phenylfluorone (PhF) complexation reaction. PhF was well dispersed in the polyvinyl alcohol (PVA) hydrogel and reacted with Sb(III) in the DET gel to form a strong PhF-Sb(III) complex. The distribution of Sb(III) was easily visualized at a submillimeter resolution using computer imaging densitometry, with a detection limit (LOD) of ∼100 nmol L−1. Field application in the Sb mine area reveals limited dissolved Sb(III) penetrating the redox barrier below the sediment-water interface by 20 mm in rivers and tailing pond sediments. To improve the detection sensitivity and apply the principle to trace Sb quantification, a SERS platform was established by anchoring PhF on the hydrogel-stabilized Ag nanoparticles via C–O–Ag bonding to specifically detect Raman-inactive Sb(III). Benefiting from the high SERS activity of PhF and enrichment ability of hydrogel, Sb(III) was quantified with a LOD of 1.2–10.7 nmol L−1 depending on the sample volume. The coexisting ions at a 100-fold higher concentration than Sb(III) resulted in only 3.3–10.4 % variation in SERS intensity, indicating a negligible interference on the SERS platform. The platform exhibited a RSD of 6.6–13.1 % and acceptable recoveries for various environmental matrices, highlighting its promise in on-site application.

S-Locus Genotyping in Japanese Plum by High Throughput Sequencing Using a Synthetic S-Loci Reference Sequence.

Self-incompatibility in Prunus species is governed by a single locus consisting of two highly multi-allelic and tightly linked genes, one coding for an F-box protein-i.e., SFB in Prunus- controlling the pollen specificity and one coding for an S-RNase gene controlling the pistil specificity. Genotyping the allelic combination in a fruit tree species is an essential procedure both for cross-based breeding and for establishing pollination requirements. Gel-based PCR techniques using primer pairs designed from conserved regions and spanning polymorphic intronic regions are traditionally used for this task. However, with the great advance of massive sequencing techniques and the lowering of sequencing costs, new genotyping-by-sequencing procedures are emerging. The alignment of resequenced individuals to reference genomes, commonly used for polymorphism detection, yields little or no coverage in the S-locus region due to high polymorphism between different alleles within the same species, and cannot be used for this purpose. Using the available sequences of Japanese plum S-loci concatenated in a rosary-like structure as synthetic reference sequence, we describe a procedure to accurately genotype resequenced individuals that allowed the analysis of the S-genotype in 88 Japanese plum cultivars, 74 of them are reported for the first time. In addition to unraveling two new S-alleles from published reference genomes, we identified at least two S-alleles in 74 cultivars. According to their S-allele composition, they were assigned to 22 incompatibility groups, including nine new incompatibility groups reported here for the first time (XXVII-XXXV).

Proteome response of winter-hardy wheat to cold acclimation

The proteome analysis of winter crops during cold acclimation and overwintering can provide important information for designing breeding processes. The current experiment was carried out to investigate the proteome changes in frost-tolerant winter wheat (cv. Norstar) during different cold acclimation (CA) periods under field conditions in a cold and high-altitude region by two-dimensional gel-based proteomic techniques. The results showed that frost tolerance significantly increased by CA and the lethal freezing temperatures (LT50) 10, 14, and 18 weeks after seed sowing were -28?C, -22?C, and -10?C, respectively. By the beginning of the reproductive stage (double ridge stage), the LT50 values had decreased significantly. Around 1000 protein spots were distinguished by Coomassie staining on the gels. The changes in the proteins during the CA often occurred in those with a functional role in photosynthesis, energy production (glycolysis), transcription, chaperone-like activities, membrane and cytoskeleton reorganisation, transport, redox adjustments, and signalling. The results revealed that changes in chloroplast proteins, certain transcription factors such as MADS-box transcription factor 26, and antioxidant enzymes (ascorbate peroxidase) show a similar trend to freezing tolerance, and their expression decreases with the onset of reproductive growth and the loss of freezing tolerance. During the acclimation period, most of the changes were focused on defence systems and cytoskeleton rearrangement, while, photosynthesis, and energy production became the main priority at the beginning of reproductive growth.

Subcellular Fractionation for DIGE-Based Proteomics.

Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation, and this has been successfully achieved using gel-based techniques, particularly that of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often underrepresented but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from the skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative 2D-DIGE analysis.

Tracking Daratumumab Clearance Using Mass Spectrometric Approaches: Implications on M Protein Monitoring and Reusing Daratumumab

Background:MASS-FIX is a screening method for serum and urine monoclonal proteins in multiple myeloma and related plasma cell disorders, which uses immunoglobulin enrichment coupled with matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF). In addition to superior sensitivity over conventional gel-based techniques, MASS-FIX can distinguish therapeutic monoclonal antibodies (MoAb) from patient's M protein. As the utilization of therapeutic MoAbs increases, it is essential to understand the persistence pattern of these therapeutic antibodies in the serum. We designed this study to evaluate the duration of daratumumab detection by MASS-FIX in the serum of treated patients.Methods:We used a prospectively maintained database at Mayo clinic to identify patients with multiple myeloma and related plasma cell disorders who were treated with a daratumumab-containing regimen anytime during their disease course and had serial MASS-FIX data available after discontinuation of daratumumab. A univariate analysis was performed to assess for factors that may impact the clearance of daratumumab.Results:We included 125 patients with plasma cell disorders who received daratumumab as first or subsequent line of treatment between March 15 th, 2016, and March 4 th, 2020. The median age was 60.2 years and 57% were male. The most common diagnoses were multiple myeloma (70%) and light chain amyloidosis (18%). Daratumumab-based treatments were initiated after a median of 28.8 (IQR: 6.4-76.3) months from initial diagnosis. The most common regimen used was daratumumab, bortezomib and dexamethasone (23%); 26% underwent transplant after daratumumab-based induction. The median duration of treatment with a daratumumab-based regimen was 208 (IQR: 99-479) days. The median follow-up from the time of daratumumab discontinuation was 457 (95% CI: 346-NR) days. By last follow up, daratumumab was not detected by MASS-FIX in 93 (74%) patients but remained detectable in 32 (26%) patients. The median time from daratumumab discontinuation to disappearance of daratumumab by MASS-FIX was 160 (IQR: 107-233) days. On univariate analysis, the presence of ≥0.5 grams of urine protein was associated with earlier disappearance of daratumumab on MASS-FIX [risk ratio (RR): 2.0, P=0.02). The median time from daratumumab discontinuation to disappearance of daratumumab on MASS-FIX was 116 (95%CI: 76-160) days in patients with urine protein ≥0.5 grams and 203 (95%CI: 162-216) days in patients with urine protein <0.5 grams (P=0.02). There was no association between the time to disappearance of daratumumab by MASS-FIX and old age ≥70 (RR: 0.9, P=0.81], male gender (RR: 0.9, P=0.60), eGFR <60 (RR: 1.0, P=0.98), daratumumab schedule (every 1/2 weeks vs >2weeks) (RR: 1.0, P=0.97), treatment duration (<200 days vs ≥200 days) ( RR: 1.0, P=0.95), or transplantation status (RR: 1.0, P=0.98).Conclusion:The therapeutic monoclonal antibody daratumumab remains detectable in the serum of treated patients by MASS-FIX for several months after discontinuation and the duration varies between individual patients. This data has implications for diagnostic and monitoring testing and may provide guidance for reuse of daratumumab in clinical trials and practice. Proteinuria is associated with earlier disappearance of daratumumab by MASS-FIX and may have implications in patients with amyloidosis and monoclonal immunoglobulin deposition disease (MIDD). Further studies are needed to identify additional factors associated with the timing of disappearance. DisclosuresMurray: Mayo Clinic: Other: Has received patents for the Mass-Fix technology which has been licensed to the Binding Site with potential royalties.. Dispenzieri: Takeda: Research Funding; Alnylam: Research Funding; Pfizer: Research Funding; Oncopeptides: Consultancy; Sorrento Therapeutics: Consultancy; Janssen: Consultancy, Research Funding. Kapoor: Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; AbbVie: Research Funding. Gertz: Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; Ionis Pharmaceuticals: Other: Advisory Board; Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy; Aurora Biopharma: Other: Stock option; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring. Dingli: Alexion: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; GSK: Consultancy. Kumar: Antengene: Consultancy, Honoraria; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Carsgen: Research Funding; Tenebio: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Recent progress in cytometric technologies and their applications in ecotoxicology and environmental risk assessment.

Environmental toxicology focuses on identifying and predicting impact of potentially toxic anthropogenic chemicals on biosphere at various levels of biological organization. Presently there is a significant drive to gain deeper understanding of cellular and sub-cellular mechanisms of ecotoxicity. Most notable is increased focus on elucidation of cellular-response networks, interactomes, and greater implementation of cell-based biotests using high-throughput procedures, while at the same time decreasing the reliance on standard animal models used in ecotoxicity testing. This is aimed at discovery and interpretation of molecular pathways of ecotoxicity at large scale. In this regard, the applications of cytometry are perhaps one of the most fundamental prospective analytical tools for the next generation and high-throughput ecotoxicology research. The diversity of this modern technology spans flow, laser-scanning, imaging, and more recently, Raman as well as mass cytometry. The cornerstone advantages of cytometry include the possibility of multi-parameter measurements, gating and rapid analysis. Cytometry overcomes, thus, limitations of traditional bulk techniques such as spectrophotometry or gel-based techniques that average the results from pooled cell populations or small model organisms. Novel technologies such as cell imaging in flow, laser scanning cytometry, as well as mass cytometry provide innovative and tremendously powerful capabilities to analyze cells, tissues as well as to perform in situ analysis of small model organisms. In this review, we outline cytometry as a tremendously diverse field that is still vastly underutilized and often largely unknown in environmental sciences. The main motivation of this work is to highlight the potential and wide-reaching applications of cytometry in ecotoxicology, guide environmental scientists in the technological aspects as well as popularize its broader adoption in environmental risk assessment.

Can Serum Nitrosoproteome Predict Longevity of Aged Women?

Aging is characterized by increase in reactive oxygen (ROS) and nitrogen (RNS) species, key factors of cardiac failure and disuse-induced muscle atrophy. This study focused on serum nitroproteome as a trait of longevity by adopting two complementary gel-based techniques: two-dimensional differential in gel electrophoresis (2-D DIGE) and Nitro-DIGE coupled with mass spectrometry of albumin-depleted serum of aged (A, n = 15) and centenarian (C, n = 15) versus young females (Y, n = 15). Results indicate spots differently expressed in A and C compared to Y and spots changed in A vs. C. Nitro-DIGE revealed nitrosated protein spots in A and C compared to Y and spots changed in A vs. C only (p-value < 0.01). Nitro-proteoforms of alpha-1-antitripsin (SERPINA1), alpha-1-antichimotripsin (SERPINA3), ceruloplasmin (CP), 13 proteoforms of haptoglobin (HP), and inactive glycosyltransferase 25 family member 3 (CERCAM) increased in A vs. Y and C. Conversely, nitrosation levels decreased in C vs. Y and A, for immunoglobulin light chain 1 (IGLC1), serotransferrin (TF), transthyretin (TTR), and vitamin D-binding protein (VDBP). Immunoblottings of alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR) and thioredoxin reductase 1 (TRXR1) indicated lower levels of ADH5 in A vs. Y and C, whereas TRXR1 decreased in A and C in comparison to Y. In conclusion, the study identified putative markers in C of healthy aging and high levels of ADH5/GSNOR that can sustain the denitrosylase activity, promoting longevity.

Characterization of variations within the rumen metaproteome of Holstein dairy cattle relative to morning feed offering

Few studies have utilized proteomic techniques to progress our knowledge of protein-mediated pathways within the rumen microbial community, and no previous research has used these techniques to investigate the patterns or variations of these proteins within this community. It was hypothesized that there would be fluctuations of rumen microbial protein abundances due to feed intake-mediated nutrient availability and that these could be identified using non gel-based proteomic techniques. This study investigated the fluctuations of bovine rumen metaproteome utilizing three mid to late-lactation Holsteins. Rumen fluid was collected at three timepoints on three days relative to their first morning feed offering (0 h, 4 h, and 6 h). Samples were pooled within timepoint within cow across day, analyzed using LC-MS/MS techniques, and analyzed for variations across hour of sampling using PROC MIXED of SAS with orthogonal contrasts to determine linear and quadratic effects. A total of 658 proteins were characterized across 19 microbial species, with 68 proteins identified from a variety of 15 species affected by time of collection. Translation-related proteins such as 50S and 30S ribosomal protein subunit variants and elongation factors were positively correlated with hour of sampling. Results suggest that as nutrients become more readily available, microbes shift from conversion-focused biosynthetic routes to more encompassing DNA-driven pathways.

The Queen Conch (Lobatus gigas) Proteome: A Valuable Tool for Biological Studies in Marine Gastropods

Queen conch (Lobatus gigas) is a marine gastropod endemic to the Caribbean. This species is a cultural symbol, being a significant local food source and the second largest commercial fishery in the region. However, over-exploitation and natural habitat degradation have exerted high survival pressure on this species. This work aims to provide novel proteomic data to highlight the metabolism of the species and to provide an important tool for the understanding of queen conch biology and physiology. Herein, we profiled the whole proteome from 3 organs (gills, digestive gland and muscle) of L. gigas combining gel-free and gel-based techniques. Overall 420 clusters of proteins were identified corresponding to the minimum identification requirement of protein sequence redundancy. Gene ontology and KEGG analysis highlighted 59 metabolic pathways between identified proteins. The most relevant routes according to the number of sequences found per pathway were purine and thiamine metabolism, closely related to nucleotide and carbohydrate metabolism. We also emphasize the high number of proteins associated to the biosynthesis of antibiotics (93 proteins and a total of 28 enzymes), which were among the top-twenty pathways identified by KEGG analysis. The proteomics approach allowed the identification and description of putative markers of oxidative stress, xenobiotic metabolism, heat shock response and respiratory chain for the first time in the species, which could be extremely useful in future investigations for diagnosing and monitoring L. gigas population health.

HRM Efficiency and Limitations for High-Throughput SSR Genotyping: A Case Study Using Grapevine Flavor-Linked Markers

Genetic diversity studies based on variation of simple sequence repeats (SSRs), have been greatly relied on easy to use gel-based techniques which, however, can be both time-consuming as well as inefficient for accurate genotyping [1].

Approaches to the discovery of non-invasive urinary biomarkers of prostate cancer.

Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. Prostate specific antigen (PSA) measured in blood has been used for decades as an aid for physicians to detect the presence of prostate cancer. However, the PSA test has limited sensitivity and specificity, leading to unnecessary biopsies, overdiagnosis and overtreatment of patients. For these reasons, there is an urgent need for more accurate PCa biomarkers that can detect PCa with high sensitivity and specificity. Urine is a unique source of potential protein biomarkers that can be measured in a non-invasive way. This review comprehensively summarizes state of the art approaches used in the discovery and validation of urinary biomarkers for PCa. Numerous strategies are currently being used in the discovery of urinary biomarkers for prostate cancer including gel-based separation techniques, mass spectrometry, activity-based proteomic assays and software approaches. Antibody-based approaches remain preferred method for validation of candidate biomarkers with rapidly advancing multiplex immunoassays and MS-based targeted approaches. In the last decade, there has been a dramatic acceleration in the development of new techniques and approaches in the discovery of protein biomarkers for prostate cancer including computational, statistical and data mining methods. Many urinary-based protein biomarkers have been identified and have shown significant promise in initial studies. Examples of these potential biomarkers and the methods utilized in their discovery are also discussed in this review.

Subcellular Fractionation for DIGE-Based Proteomics.

Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation and this has been successfully achieved using gel-based techniques, particularly that of two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often under-represented, but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative DIGE analysis.

Identification of novel biomarkers for treatment monitoring in canine leishmaniosis by high-resolution quantitative proteomic analysis.

The objective of this study was to use the Tandem Mass Tag (TMT) isobaric label-based proteomic approach, in order to identify new potential biomarkers for the treatment monitoring of canine leishmaniosis that could not be identified by the use of gel-based techniques. For this purpose serum samples were obtained from 5 clinically diseased dogs before and one month after the treatment of canine leishmaniosis. The non-depleted serum samples were subjected to reduction, alkylation and trypsin digestion, and the resulting peptides were labeled using 6-plex TMT reagents. To obtain information about protein identities and relative quantification, liquid chromatography-MS analysis of multiplexed TMT-labeled peptides was employed. This gel-free, label-based quantitative proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p<0.05) in expression (two downregulated and 21 upregulated ranging from 1.25 to 2.5 fold change). Comparison of gel-free TMT-based quantification and a gel-based approach previously applied to the same samples resulted in the identification of some common markers (Apo-A1, vitamin D binding protein and RBP4). However, 20 additional differentially represented proteins were highlighted by the gel-free approach, 13 of which have not been previously reported in canine leishmaniosis. In conclusion, the TMT-based proteomic approach allowed identification of new serum proteins that significantly change in concentration after canine leishmaniosis treatment. These proteins are involved in various physiopathological processes such as inflammatory, coagulation or defense mechanisms, and could potentially be suitable biomarkers for treatment monitoring of this parasitic disease.

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from genome sequences, though there are over lapped proteins. Based on the demonstrated application of data stored in the database for functional analyses, it is suggested that these data will be useful for analyses of biological mechanisms in soybean. Furthermore, coupled with recent advances in information and communication technology, the usefulness of this database would increase in the analyses of biological mechanisms.

A comparison of tissue preparation methods for protein extraction of cocoa (Theobroma cacao L.) pod

Cocoa, Theobroma cacao L. is one of the main tropical industrial crops. Cocoa has a very high level of interfering substances, such as polysaccharides and phenolic compounds that could prevent the isolation of suitable protein. Efficient methods of protein extraction are a priority to successfully apply proteomic analyses. We compared and evaluated two methods (A and B) of tissue preparation for total protein extract by phenol/SDS extraction protocol. The difference in the application of the two methods was that extensively washed dry powder of pod tissue were made in Method A, whereas that crude extract were prepared Method B. Extracted proteins were examined using one-dimensional electrophoresis (1-D). Results show that each extraction method isolated a unique subset of cocoa pod proteome. Principal component analysis showed little variation in the data obtained using Method A, while that in Method s B showed no low reproducibility, thus demonstrating that Method A is a reliable for preparing cocoa pod proteins. The protocol is expected to be applicable to other recalcitrant plant tissues and to be of interest to laboratories involved in plant proteomics analyses. A combination of extraction approaches is recommended for increasing proteome coverage when using gel-based isolation techniques.

Proteomics and SSH Analyses of ALA-Promoted Fruit Coloration and Evidence for the Involvement of a MADS-Box Gene, MdMADS1.

Skin color is a key quality attribute of fruits and how to improve fruit coloration has long been a major concern. 5-Aminolevulinic acid (ALA), a natural plant growth regulator, can significantly increase anthocyanin accumulation in fruit skin and therefore effectively improve coloration of many fruits, including apple. However, the molecular mechanism how ALA stimulates anthocyanin accumulation in fruit skin remains unknown. Here, we investigated the impact of ALA on apple skin at the protein and mRNA levels. A total of 85 differentially expressed proteins in apple skins between ALA and water treatment (control) were identified by complementary gel-based and gel-free separation techniques. Most of these differentially expressed proteins were up-regulated by ALA. Function analysis suggested that 87.06% of the ALA-responsive proteins were associated with fruit ripening. To further screen ALA-responsive regulators, we constructed a subtracted cDNA library (tester: ALA treatment; driver: control) and obtained 104 differentially expressed unigenes, of which 38 unigenes were indicators for the fruit ripening-related genes. The differentially changed proteins and transcripts did not correspond well at an individual level, but showed similar regulated direction in function at the pathway level. Among the identified fruit ripening-related genes, the expression of MdMADS1, a developmental transcription regulator of fruit ripening, was positively correlated with expression of anthocyanin biosynthetic genes (MdCHS, MdDFR, MdLDOX, and MdUFGT) in apple skin under ALA treatment. Moreover, overexpression of MdMADS1 enhanced anthocyanin content in transformed apple calli, which was further enhanced by ALA. The anthocyanin content in MdMADS1-silenced calli was less than that in the control with ALA treatment, but higher than that without ALA treatment. These results indicated that MdMADS1 is involved in ALA-induced anthocyanin accumulation. In addition, anthocyanin-related verification in apple calli suggested that the regulation of MdMADS1 on anthocyanin biosynthesis was partially independent of fruit ripening process. Taken together, our findings provide insight into the mechanism how ALA regulates anthocyanin accumulation and add new information on transcriptase regulators of fruit coloration.

Proteomic comparison of near-isogenic barley (Hordeum vulgare L.) germplasm differing in the allelic state of a major senescence QTL identifies numerous proteins involved in plant pathogen defense

Senescence is the last developmental phase of plant tissues, organs and, in the case of monocarpic senescence, entire plants. In monocarpic crops such as barley, it leads to massive remobilization of nitrogen and other nutrients to developing seeds. To further investigate this process, a proteomic comparison of flag leaves of near-isogenic late- and early-senescing barley germplasm was performed. Protein samples at 14 and 21 days past anthesis were analyzed using both two-dimensional gel-based and label-free quantitative mass spectrometry-based (‘shotgun’) proteomic techniques. This approach identified >9000 barley proteins, and one-third of them were quantified. Analysis focused on proteins that were significantly (p < 0.05; difference ≥1.5-fold) upregulated in early-senescing line ‘10_11’ as compared to late-senescing variety ‘Karl’, as these may be functionally important for senescence. Proteins in this group included family 1 pathogenesis-related proteins, intracellular and membrane receptors or co-receptors (NBS-LRRs, LRR-RLKs), enzymes involved in attacking pathogen cell walls (glucanases), enzymes with possible roles in cuticle modification, and enzymes involved in DNA repair. Additionally, proteases and elements of the ubiquitin-proteasome system were upregulated in line ‘10_11’, suggesting involvement of nitrogen remobilization and regulatory processes. Overall, the proteomic data highlight a correlation between early senescence and upregulated defense functions. This correlation emerges more clearly from the current proteomic data than from a previously performed transcriptomic comparison of ‘Karl’ and ‘10_11’. Our findings stress the value of studying biological systems at both the transcript and protein levels, and point to the importance of pathogen defense functions during developmental leaf senescence.

Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.

Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.

Fecal Carriage of Staphylococcus aureus in the Hospital and Community Setting: A Systematic Review.

Background and rationale: Staphylococcus aureus fecal carriage has been identified as a potential source for nosocomial transmission and a risk factor for disease development. This systematic review determined the overall S. aureus [including methicillin susceptible and resistant S. aureus (MSSA and MRSA)] fecal carriage rates within the community and healthcare settings.Methodology: Peer-reviewed articles indexed in Medline, Scopus, Academic Search Premier, Africa-Wide Information, CINAHL, and Web of Science were identified using applicable and controlled vocabulary through to 11 November 2015. Eligible studies were ascertained by three independent reviewers. Random-effects meta-analyses of proportions were performed to determine S. aureus, MSSA and MRSA fecal carriage rates reported by eligible studies.Results: Twenty six studies were included in this review. The pooled estimates for S. aureus, MSSA and MRSA fecal carriage were 26% (95% confidence interval (CI): 16.8–36.3%), 86% (95% confidence interval (CI): 65.9–97.9%) and 10% (95% CI: 0.7–27.0%), respectively. Fecal S. aureus carriage rates increased on average from 10 to 65% during the first 8 weeks of life, followed by an average carriage rate of 64% at 6 months and 46% at 1 year of life. Genotyping techniques were employed mainly in studies conducted in developed countries and comprised largely of gel-based techniques. Six studies reported on the role of S. aureus fecal strains in diarrhea (n = 2) and the risk for acquiring infections (n = 4). Eight of the 26 studies included in this review performed antibiotic susceptibility testing of S. aureus fecal isolates.Conclusion: This study provides evidence that screening for S. aureus fecal carriage, at least in populations at high risk, could be an effective measure for the prevention of S. aureus transmission and infection in the healthcare and community setting. More well-structured studies need to be conducted and sequence-based genotyping techniques should be employed for the comparison of isolates on a global scale in both developing and developed countries.