Penicillium verrucosum in wheat and barley indicates presence of ochratoxin A

AIMS: The aims of this study were to isolate and identify ochratoxin A (OTA) producing fungi in cereals containing OTA and to determine the best selective and indicative medium for recovery of OTA producing fungi. METHODS AND RESULTS: Seventy-six wheat, barley and rye samples from Europe containing OTA and 17 samples without OTA were investigated using three different media, dichloran yeast sucrose agar (DYSG), dichloran rose bengal yeast extract sucrose agar (DRYES) and dichloran 18% glycerol agar (DG18). Hundred kernels were plated on each medium and the kind and number of fungal OTA producers were recorded as percentage of infestation. Penicillium verrucosum was the sole OTA producer found in cereals. The average percentage of infestation of P. verrucosum counts was recorded as 28.3% on DYSG, 10.3% on DRYES and 9.9% on DG18 on the OTA containing samples and 0.8% on DYSG, 0.4% on DRYES and 0.6% on DG18 for the samples without OTA. CONCLUSIONS: Penicillium verrucosum was the sole OTA producer in European cereals. Determination of P. verrucosum infestation and infection was best detected on DYSG after 7 days at 20 degrees C. The percentage of infestation of P. verrucosum found on DYSG and OTA content in cereals were correlated. More than 7% infestation of P. verrucosum indicated OTA contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method could be used as a cereal quality control.     

Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum

Our results demonstrate high concentrations of the UV absorbing mycotoxin citrinin in the outer layer of spores from three citrinin-producing strains of Penicillium verrucosum, which is released in an aqueous environment. An important function of the toxin could be to act as a sun protectant in order to create favorable conditions during the initial germination process. When spores from these strains of P. verrucosum were examined by confocal microscopy, a clearly visible fluorescent layer associated with the cell wall was observed. The strains were grown on agar plates, and the mycelial mat was washed with saline. This suspension contained at least 95% of the spores and particulate material, which was removed by filtration after counting the conidia. An aliquot of this filtrate was extracted and citrinin was purified by high pressure liquid chromatography. The absorbance at 319 nm was used to calculate the amount of UV absorbing material released from the spores. Based on the spore numbers in the suspension of the saline extract, we estimated that this material released was 1.4–4.1 pg per spore or 8–24% of the spore weight. Citrinin (and minor amounts of ochratoxin A and some other unidentificable fluorescent compounds) were observed in the filtrate when subjected to thin layer chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

Predicting noncompliant levels of ochratoxin A in cereal grain from Penicillium verrucosum counts

AIMS: To model the probability of exceeding the European legislative limit of 5 microg ochratoxin A (OTA) per kilogram grain in relation to Penicillium verrucosum levels and storage conditions, and to evaluate the possibilities of using P. verrucosum colony counts for predicting noncompliant OTA levels. METHODS AND RESULTS: Cereal samples were inoculated with P. verrucosum spores and stored for up to 9 months at temperatures and water activities ranging from 10-25 degrees C and aw 0.77-0.95. A logistic regression analysis showed that the probability of exceeding 5 microg OTA kg(-1) grain was related to colony counts of P. verrucosum and water activity. The sensitivity and specificity of various P. verrucosum count thresholds for predicting noncompliant OTA levels were estimated, using data from the storage trial and natural cereal samples. CONCLUSION: The risk of exceeding 5 microg OTA kg(-1) grain increased with increasing levels of P. verrucosum, and with increasing water activities. A threshold of 1000 CFU P. verrucosum per gram grain is suggested to predict whether or not the legislative limit is exceeded. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided a tool to evaluate the levels of P. verrucosum in grain in relation to OTA levels. Hence, mycological analyses can be used to identify cereal samples with high risk of containing OTA levels above the legislative limit.     

Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice

To survey protein expression patterns in the reduced culm number (RCN) rice, a comparative shotgun proteomic analysis was conducted. For large-scale protein identification, multidimensional protein identification technology (MudPIT) coupled with pre-fractionation of plant shoot proteins led to the identification of 3004 non-redundant rice proteins. By statistically comparing relative amounts of 1353 reproducibly identified proteins between the RCN rice and the wild-type rice, 44 differentially expressed proteins were detected, where 42 proteins were increased and 2 proteins were decreased in the RCN rice. These proteins appear to have roles in glycolysis, trichloroacetic acid cycle, secondary metabolism, nutrient recycling, and nucleotide metabolism and repair. Consequently, we hypothesized that the RCN rice might fail to maintain sugar nutrient homeostasis. This was confirmed with the observation that the sucrose concentration was increased significantly in the RCN rice compared with the wild-type rice. Also, the RCN rice showed a hypersensitive response to exogenous sucrose treatment.     

Comparative proteomic analysis of the insulin‐induced L6 myotube secretome

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti‐inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC‐ESI‐MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.     

Comprehensive proteomic analysis of human bile

Bile serves diverse functions from metabolism to transport. In addition to acids and salts, bile is composed of proteins secreted or shed by the hepatobiliary system. Although there have been previous efforts to catalog biliary proteins, an in-depth analysis of the bile proteome has not yet been reported. We carried out fractionation of non-cancerous bile samples using a multipronged approach (SDS-PAGE, SCX and OFFGEL) followed by MS analysis on an LTQ-Orbitrap Velos mass spectrometer using high resolution at both MS and MS/MS levels. We identified 2552 proteins - the largest number of proteins reported in human bile till date. To our knowledge, there are no previous studies employing high-resolution MS reporting a more detailed catalog of any body fluid proteome in a single study. We propose that extensive fractionation coupled to high-resolution MS can be used as a standard methodology for in-depth characterization of any body fluid. This catalog should serve as a baseline for the future studies aimed at discovering biomarkers from bile in gallbladder, hepatic, and biliary cancers.     

蛹虫草原基分化的差异蛋白质组学研究

蛹虫草子实体形成及发育的蛋白分子机制尚不清楚,本研究引入SWATH非标记定量蛋白质组学技术,对蛹虫草Cordyceps militaris 905菌株的菌丝体（mycelium,My）、原基（primordium,Po）、生长期子实体（developmental fruiting body,DF）和成熟期子实体（mature fruiting body,MF）进行了比较蛋白质组学分析。经搜库比对,从蛹虫草的My、Po、DF和MF中依次鉴定蛋白1 136个、1 090个、1 018个和997个（global FDR 1%）,经维恩分析后获得C. militaris 905蛹虫草表达蛋白1 578个。在此基础上,SWATH非标记技术定量蛋白1 109个。本研究获得了蛹虫草Po期与My期、DF期与Po期、MF期与DF期的差异表达蛋白,依次为115个、352个和104个,并对菌丝体分化形成原基的差异表达蛋白进行了重点解析。GO注释结果表明,Po期与My期差异表达蛋白以有机含氮类化合物代谢为主,其中AMP（活性成分虫草素合成的中间产物）从头生物合成途径富集最为显著。约1/5的差异表达蛋白参与氧化还原反应,还原酶活性的蛋白在原基中几乎都上调表达,而氧化功能的蛋白受到抑制,表明蛹虫草原基分化可能受到氧化应激的诱导。蛋白互作网络分析结果进一步表明,氧化还原反应与核苷类物质代谢相关联,可能通过影响AMP从头生物合成途径来调控虫草素的生物合成。对蛹虫草子实体系统的蛋白质组学研究和解析有利于揭示子实体形成的蛋白分子机制,为蛹虫草的基础和栽培研究提供了理论支撑。     

Label‐free quantitative proteomic analysis of tolerance to drought in Pisum sativum

Abiotic stresses caused by adverse environmental conditions are responsible for heavy economic losses on pea crop, being drought one of the most important abiotic constraints. Development of pea cultivars well adapted to dry conditions has been one of the major tasks in breeding programs. The increasing food requirements drive the necessity to broaden the molecular basis of tolerance to drought to develop pea cultivars well adapted to dry conditions. We have used a shotgun proteomic approach (nLC‐MSMS) to study the tolerance to drought in three pea genotypes that were selected based on differences in the level of water deficit tolerance. Multivariate statistical analysis of data unraveled 367 significant differences of 700 identified when genotypes and/or treatment were compared. More than half of the significantly changed proteins belong to primary metabolism and protein regulation categories. We propose different mechanisms to cope drought in the genotypes studied. Maintenance of the primary metabolism and protein protection seems a strategy for drought tolerance. On the other hand susceptibility might be related to maintenance of the homeostatic equilibrium, a very energy consuming process. Data are available via ProteomeXchange with identifier PXD004587.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.     

Increasing information from shotgun proteomic data by accounting for misassigned precursor ion masses

Although mass spectrometers are capable of providing high mass accuracy data, assignment of true monoisotopic precursor ion mass is complicated during data-dependent ion selection for LC-MS/MS analysis of complex mixtures. The complication arises when chromatographic peak widths for a given analyte exceed the time required to acquire a precursor ion mass spectrum. The result is that many measured monoisotopic masses are misassigned due to calculation from a single mass spectrum with poor ion statistics based on only a fraction of the total available ions for a given analyte. Such data in turn produces errors in automated database searches, where precursor m/z value is one search parameter. We propose here a postacquisition approach to correct misassigned monoisotopic m/z values that involves peak detection over the entire elution profile and correction of the precursor ion monoisotopic mass. As a result of using this approach to reprocess shotgun proteomic data we increased peptide sequence assignments by 10% while reducing the estimated false positive ratio from 1 to 0.2%. We also show that 4% of the salvaged identifications may be accounted for by correction of mixed tandem mass spectra resulting from fragmentation of multiple peptides simultaneously, a situation which we refer to as accidental CID.     

Shotgun proteomic analysis of Chlamydia trachomatis

Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328 chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67 proteins previously annotated as hypothetical or conserved hypothetical. Taken together, >/=35% of the predicted proteome for C. trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date.     

Changes in nucleotide pools during conidia formation of Penicillium chrysogenum in solid culture

Abstract Using Cellophane-sheet techniques, changes in nucleotide pool size during conidia formation of Penicillium chrysogenum on agar plates were studied. On conidia-forming medium (nutritionally poor medium), the pool size of nucleoside triphosphates, especially GTP, decreased dramatically at mid-growth phase, concomitantly with the exhaustion of extracellular phosphate. Onset of conidia formation was observed just after the GTP pool decrease.     

Shotgun proteomic analysis of the Bombyx mori anterior silk gland: An insight into the biosynthetic fiber spinning process

The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid–crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle‐specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090.     

Comparison between procedures using SDS for shotgun proteomic analyses of complex samples

Filter-aided sample preparation (FASP) and a new sample preparation method using a modified commercial SDS removal spin column are quantitatively compared in terms of their performance for shotgun proteomic experiments in three complex proteomic samples: a Saccharomyces cerevisiae lysate (insoluble fraction), a Caenorhabditis elegans lysate (soluble fraction), and a human embryonic kidney cell line (HEK293T). The characteristics and total number of peptides and proteins identified are compared between the two procedures. The SDS spin column procedure affords a conservative fourfold improvement in throughput, is more reproducible, less expensive (i.e. requires less materials), and identifies between 30 and 107% more peptides at q≤0.01, than the FASP procedure. The peptides identified by SDS spin column are more hydrophobic than species identified by the FASP procedure as indicated by the distribution of GRAVY scores. Ultimately, these improvements correlate to as great as a 50% increase in protein identifications with two or more peptides.     

Field ecology of the ochratoxin A-producing Penicillium verrucosum: Survival and resource colonisation in soil

A field experiment was conducted to elucidate the survival of P. verrucosum in infested bulk soil(T1) and infested soil with waste grain (T2). The infested soil and reference soil (T3) was filled into steel cylinders, which were buried and sampled 13 times during a period from October 1994 to March 1996.The abundance of P. verrucosum and indigenous soil fungi were assessed by dilution plating on a selective and diagnostic medium (DYSG). Kernel infection was examined in T2. According to our results, P. verrucosum seems well adapted to survival in arable soil and little affected by indigenous fungi. During the first autumn and winter the grain caused a proliferation of P. verrucosum while its abundance in bulk soil was more constant except for a decrease in February 95, which is ascribed to frost/thaw alternations. In T2, P. verrucosum initially infected more than 50% of the kernels but during the first few months it was ousted by other fungi. A hypothesis regarding waste grain as the natural niche for the fungus in the field was therefore partly rejected. A gradual decrease in the abundance of P. verrucosum in soil during spring, a die-off in the dry summer and aproliferation during the second winter were found in both T1 and T2. Our results cannot provide the reason for the increase during the second winter. On an overall basis, however, they show that P. verrucosum can survive in the field, proliferate on soil organic matter and probably become an integral part of the soil ecosystem. This may constitute a risk of grain contamination when given appropriate environmental conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses.