MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

Spatial distribution of arsenic and temporal variation of its concentration in rice

P>In order to gain insights into the transport and distribution of arsenic (As) in intact rice (Oryza sativa) plants and its unloading into the rice grain, we investigated the spatial distribution of As and the temporal variation of As concentration in whole rice plants at different growth stages. To the best of our knowledge, this is the first time that such a study has been performed.

Inductively coupled plasma mass spectroscopy (ICP-MS) and high-performance liquid chromatography (HPLC)-ICP-MS were used to analyze total As concentration and speciation. Moreover, synchrotron-based X-ray fluorescence (SXRF) was used to investigate in situ As distribution in the leaf, internode, node and grain.

Total As concentrations of vegetative tissues increased during the 2 wk after flowering. The concentration of dimethylarsinic acid (DMA) in the caryopsis decreased progressively with its development, whereas inorganic As concentration remained stable. The ratios of As content between neighboring leaves or between neighboring internodes were c. 0.6. SXRF revealed As accumulation in the center of the caryopsis during its early development and then in the ovular vascular trace.

These results indicate that there are different controls on the unloading of inorganic As and DMA; the latter accumulated mainly in the caryopsis before flowering, whereas inorganic As was mainly transported into the caryopsis during grain filling. Moreover, nodes appeared to serve as a check-point in As distribution in rice shoots.

Dietary exposure to aflatoxin and fumonisin among Tanzanian children as determined using biomarkers of exposure

SCOPE: The study aims to evaluate the status of dietary exposure to aflatoxin and fumonisin in young Tanzanian children, using previously validated biomarkers of exposure. METHODS AND RESULTS: A total of 148 children aged 12-22 months, were recruited from three geographically distant villages in Tanzania; Nyabula, Kigwa, and Kikelelwa. Plasma aflatoxin-albumin adducts (AF-alb) and urinary fumonisin B1 (UFB1) were measured by ELISA and LC-MS, respectively. AF-alb was detectable in 84% of children, was highest in fully weaned children (p <0.01) with higher levels being associated with higher maize intake (p <0.05). AF-alb geometric mean (95% CI) was 43.2 (28.7-65.0), 19.9 (13.5-29.2), and 3.6 (2.8-4.7) pg/mg albumin in children from Kigwa, Nyabula, and Kikelelwa, respectively. UFB1 was detectable in 96% of children and the level was highest in children who had been fully weaned (p <0.01). The geometric UFB1 mean (95% CI) was 327.2 (217.1-493.0), 211.7 (161.1-278.1), and 82.8 (58.3-117.7) pg/mL in Kigwa, Nyabula, and Kikelelwa, respectively. About 82% of all the children were exposed to both mycotoxins. CONCLUSION: Young children in Tanzania are chronically exposed to both aflatoxin and fumonisin through contaminated diet, although the level of exposure varies markedly between the three villages studied.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Deoxynivalenol exposure assessment in young children in Tanzania

Scope: This study assessed deoxynivalenol (DON) exposure in children from three geographic locations within Tanzania, over three time points in 1 year, using a urinary biomarker of exposure.

Methods and results: A total of 166 children aged 6-14 months were studied at a maize harvest and followed up twice at 6-month intervals. On two consecutive days, morning urine was collected from each child and urinary DON was measured using an LC-MS method, with and without beta-glucuronidase hydrolysis in order to assess free DON (fDON) and glucuronide DON. Overall, urinary DON increased significantly along with the three visits (geometric mean 1.1, 2.3, and 5.7 ng/mL, at visits 1, 2, and 3, respectively, p <0.01). fDON was 22% of urinary total DON. Urinary DON excretion rate was 74% in village Kikelelwa based on food DON level and food consumption. Assuming 360 mL of urine excreted per day, 10, 19, and 29% of children at visits 1, 2, and 3, respectively, exceeded the provisional maximum tolerable daily intake of 1000 ng/kg b.w./day.

Conclusion: Young children in Tanzania are chronically exposed to DON due to eating contaminated maize, although exposure levels varied markedly by region and season.

A Prospective Study of Growth and Biomarkers of Exposure to Aflatoxin and Fumonisin during Early Childhood in Tanzania

BACKGROUND: Aflatoxin and fumonisin are toxic food contaminants. Knowledge about effects of their exposure and co-exposure on child growth is inadequate.

OBJECTIVE: To investigate the association between child growth and aflatoxin and fumonisin exposure in Tanzania.

METHODS: A total of 166 children were recruited at 6 to 14 months of age and studied at recruitment, and at the sixth and twelfth month following recruitment. Blood and urine samples were collected and analysed for plasma aflatoxin albumin adducts (AF-alb) using ELISA, and urinary fumonisin B1 (UFB1) using LC-MS, respectively. Anthropometric measurements were taken and growth index Z-scores were computed.

RESULTS: AF-alb geometric mean concentrations (95% confidence intervals) were 4.7 (3.9, 5.6), 12.9 (9.9, 16.7) and 23.5 (19.9, 27.7) pg/mg albumin at recruitment, six months, and 12 months from recruitment, respectively. At these respective sampling times, geometric mean UFB1 concentrations (95% CI) were 313.9 (257.4, 382.9), 167.3 (135.4, 206.7) and 569.5 (464.5, 698.2) pg/mL urine, and the prevalence of stunted children were 44%, 55% and 56%, respectively. UFB1 concentrations at recruitment were negatively associated with length for age Z-scores (LAZ) at six months (p = 0.016) and at 12 months from recruitment (p = 0.014). The mean UFB1 of the three sampling times (at recruitment, at six and 12 months from recruitment) in each child was negatively associated with LAZ (p < 0.001) and length velocity (p = 0.004) at 12 months from recruitment. The negative association between AF-alb and child growth did not reach statistical significance.

CONCLUSIONS: Exposure to fumonisin alone, or co-exposure with aflatoxins may contribute to child growth impairment.

Multiple mycotoxin exposure determined by urinary biomarkers in rural subsistence farmers in the former Transkei, South Africa

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean±standard deviation 0.342±0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4±49.4 ng/mg creatinine) after hydrolysis with β-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with β-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529±1.60 ng/mg creatinine), FB1 (96%; 1.52±2.17 ng/mg creatinine), α-zearalenol (92%; 0.614±1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3±27.1 ng/mg creatinine), β-zearalenol (75%; 0.702±2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041±0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

Using SILAC proteomics to investigate the effect of the mycotoxin, alternariol, in the human H295R steroidogenesis model

The mycotoxin alternariol (AOH) is an important contaminant of fruits and cereal products. The current study sought to address the effect of a non-toxic AOH concentration on the proteome of the steroidogenic H295R cell model. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture (SILAC) coupled to 1D-SDS-PAGE-LC-MS/MS was applied to subcellular-enriched protein samples. Gene ontology (GO) and ingenuity pathway analysis (IPA) were further carried out for functional annotation and identification of protein interaction networks. Furthermore, the effect of AOH on apoptosis and cell cycle distribution was also determined by the use of flow cytometry analysis. This work identified 22 proteins that were regulated significantly. The regulated proteins are those involved in early stages of steroid biosynthesis (SOAT1, NPC1, and ACBD5) and C21-steroid hormone metabolism (CYP21A2 and HSD3B1). In addition, several proteins known to play a role in cellular assembly, organization, protein synthesis, and cell cycle were regulated. These findings provide a new framework for studying the mechanisms by which AOH modulates steroidogenesis in H295R cell model. 

Identification and Functional Analysis of a Novel Tryptophyllin Peptide from the Skin of the Red-eye Leaf Frog, Agalychnis callidryas

Amphibian skin has proved repeatedly to be a largely untapped source of bioactive peptides and this is especially true of members of the Phyllomedusinae subfamily of frogs native to South and Central America. Tryptophyllins are a group of peptides mainly found in the skin of members of this genus. In this study, a novel tryptophyllin (TPH) type 3 peptide, named AcT-3, has been isolated and structurally-characterised from the skin secretion and lyophilised skin extract of the red-eye leaf frog, Agalychnis callidryas. The peptide was identified in and purified from the skin secretion by reverse-phase HPLC. MALDI-TOF mass spectrometry and MS/MS fragmentation sequencing established its primary structure as: pGlu-Gly-Lys-Pro-Tyr-Trp-Pro-Pro-Pro-Phe-Leu-Pro-Glu, with a non-protonated molecular mass of 1538.19Da. The mature peptide possessed the canonical N-terminal pGlu residue that arises from post-translational modification of a Gln residue. The deduced open-reading frame consisted of 63 amino acid residues encoding a highly-conserved signal peptide of approximately 22 amino acid residues, an intervening acidic spacer peptide domain, a single AcT-3 encoding domain and a C terminal processing site. A synthetic replicate of AcT-3 was found to antagonise the effect of BK on rat tail artery smooth muscle and to contract the intestinal smooth muscle preparations. It was also found that AcT-3 could dose-dependently inhibit the proliferation of human prostate cancer cell lines after 72h incubation.

Levels of Arsenic and Other Trace Elements in Southern Libyan Agricultural Irrigated Soil and Non-irrigated Soil Projects

The levels of As and various other trace elements found in the irrigated agricultural soil (Tsoil) of southern Libya were compared with non-irrigated soil (Csoil) from the same sampling campaign collected between April and May 2008. The soil samples represented agronomic practice in the southern Libyan regions of Maknwessa (MAK), Aril (ARL) and Taswaa (TAS), and were analyzed by Inductively coupled plasma mass spectrometry (ICP-MS) for Co, Ni, Cu, Se, Mo, Zn, As, Pb, Cd and P. Concentrations of P and As in TAS and MAK were found to be higher in Tsoil compared to Csoil, while the opposite was true for ARL. In general, As concentrations in these areas were 2-3 times lower than the global average. In ARL, the average P concentrations of the Csoil samples were significantly higher than those of Tsoil samples: this site is composed mainly of pasture for animal production, where phosphate fertilizers are used regularly. Distance from the source of irrigation was found to be of an important influence on the heavy metal concentration of the soil, with greater concentrations found closer to the irrigation source. It can be concluded from the results that irrigation water contains elevated levels of As, which finds its way into the soil profile and can lead to accumulation of As in the soil over time.

A new analytical approach for the detection of palm oil in vegetable oil blends

The new Food Information Regulation (1169/2011), dictates that in a refined vegetable oil blend, the type of oil must be clearly identified in the package in contract with current practice where is labelled under the generic and often misleading term “vegetable oil”. With increase consumer awareness in food authenticity, as shown in the recent food scandal with horsemeat in beef products, the identification of the origin of species in food products becomes increasingly relevant. Palm oil is used extensively in food manufacturing and as global demand increases, producing countries suffer from the aftermath of intensive agriculture. Even if only a small portion of global production, sustainable palm oil comes in great demand from consumers and industry. It is therefore of interest to detect the presence of palm oil in food products as consumers have the right to know if it is present in the product or not, mainly from an ethical point of view. Apart from palm oil and its derivatives, rapeseed oil and sunflower oil are also included. With DNA-based methods, the gold standard for the detection of food authenticity and species recognition deemed not suitable in this analytical problem, the focus is inevitably drawn to the chromatographic and spectroscopic methods. Both chromatographic (such as GC-FID and LC-MS) and spectroscopic methods (FT-IR, Raman, NIR) are relevant. Previous attempts have not shown promising results due to oils’ natural variation in composition and complex chemical signals but the suggested two-step analytical procedure is a promising approach with very good initial results.

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5-3' exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R(2) > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC-MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.

Osteogenic cell response to 3-D hydroxyapatite scaffolds developed via replication of natural marine sponges

Bone tissue engineering may provide an alternative to autograft, however scaffold optimisation is required to maximize bone ingrowth. In designing scaffolds, pore architecture is important and there is evidence that cells prefer a degree of non-uniformity. The aim of this study was to compare scaffolds derived from a natural porous marine sponge (Spongia agaricina) with unique architecture to those derived from a synthetic polyurethane foam. Hydroxyapatite scaffolds of 1 cm3 were prepared via ceramic infiltration of a marine sponge and a polyurethane (PU) foam. Human foetal osteoblasts (hFOB) were seeded at 1x105 cells/scaffold for up to 14 days. Cytotoxicity, cell number, morphology and differentiation were investigated. PU-derived scaffolds had 84-91% porosity and 99.99% pore interconnectivity. In comparison marine sponge-derived scaffolds had 56-61% porosity and 99.9% pore interconnectivity. hFOB studies showed that a greater number of cells were found on marine sponge-derived scaffolds at than on the PU scaffold but there was no significant difference in cell differentiation. X-ray diffraction (XRD) and inductively coupled plasma mass spectrometry (ICP-MS) showed that Si ions were released from the marine-derived scaffold. In summary, three dimensional porous constructs have been manufactured that support cell attachment, proliferation and differentiation but significantly more cells were seen on marine-derived scaffolds. This could be due both to the chemistry and pore architecture of the scaffolds with an additional biological stimulus from presence of Si ions. Further in vivo tests in orthotopic models are required but this marine-derived scaffold shows promise for applications in bone tissue engineering.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.