Assessing the composition of the plasma membrane of Leishmania (Leishmania) infantum and L. (L.) amazonensis using label-free proteomics.

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.

Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples.

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

Casein-Conjugated Gold Nanoparticles for Amperometric Detection of Leishmania infantum.

Sensitive and reliable approaches targeting the detection of Leishmania are critical for effective early diagnosis and treatment of leishmaniasis. In this frame, this paper describes a rapid quantification assay to detect Leishmania parasites based on the combination of the electrocatalytic ability of gold nanoparticles (AuNPs) to act as a catalyst for the hydrogen formation reaction along with the specificity of the interaction between casein and the major surface protease of the Leishmania parasite, GP63. First, pure and casein-modified AuNPs were prepared and characterized by scanning electron microscopy and ultraviolet-visible spectroscopy. Then, casein-conjugated AuNPs were incubated with Leishsmania parasites in solution; the formed complex was collected by centrifugation, treated by acidic solution, and the pelleted AuNPs were placed on screen-printed carbon electrodes (SPCEs) and chronoamperometric measurements were carried out. Our results suggest that it is possible to detect Leishmania parasites, with a limit less than 1 parasite/mL. A linear response over a wide concentration interval, ranging from 2 × 10-2 to 2 × 105 parasites/mL, was achieved. Additionally, a pretreatment of Leishmania parasites with Amphotericin B, diminished their interaction with casein. This findings and methodology are very useful for drug efficacy assessment.

The first direct activity assay for the mitochondrial protease OMA1.

Mitochondria continually undergo fission and fusion which allow mitochondria to rapidly change their shape, size, and function throughout the cell life cycle. OMA1, a zinc metalloproteinase enzyme, is a key regulator of the mitochondrial fusion machinery. The paucity of information regarding OMA1 regulation and function largely stems from the fact that there is no direct method to quantitatively measure its activity. Using a fluorescence-based reporter assay, we developed a sensitive method to measure OMA1 enzymatic activity in whole cell lysates.

Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis.

Common assays for endoprotease activity of meprin α and ß are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and ß suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin ß were Km = 184 ±â€¯32 µM and kcat = 20 ±â€¯4 s-1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin ß activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and ß. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.

Protein Profile Analysis of Two Australian Snake Venoms by One- Dimensional Gel Electrophoresis and MS/MS Experiments.

The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes.

Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

Use of Zymography in Trypanosomiasis Studies.

Zymography assay is a semiquantitative technique, very sensitive, and commonly used to determine metalloproteinase levels in different types of biological samples, including tissues, cells, and extracts of protein. Samples containing metalloproteinases are loaded onto a polyacrylamide gel containing sodium dodecyl sulphate (SDS) and a specific substrate (gelatin, casein, collagen, etc.). Then proteins are allowed to migrate under an electric current and the distance of migration is inversely correlated with the molecular weight. After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity (metalloproteinase activity). In the context of infections caused by trypanosomatids (Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei), the characterization of metalloproteinase by zymography can contribute to the comprehension of the pathogenesis mechanisms and host-parasite interaction.

Molecular Methods for Identification of Clostridium tetani by Targeting Neurotoxin.

Tetanus is a potentially fatal muscle spasm disease. It is an important public health problem, especially in rural/tribal areas of developing countries. Tetanus toxin, a neurotoxin (tetanospasmin ), is the most important virulence factor that plays a key role in the pathogenicity of tetanus . Confirmation of virulence by confirming the production of tetanospasmin by infecting species forms the most important part in the diagnosis of tetanus . Various molecular methods have been devised for confirmation of diagnosis by targeting different genes. The most common molecular methods are tetanospasmin producing (TetX) gene-targeted methods using TetX-specific primers. Here, we describe various molecular methods targeting TetX gene such as polymerase chain reaction, pulsed-field gel electrophoresis, Southern blotting, loop-mediated isothermal amplification assay, etc. to confirm the virulence of Cl. tetani.

Colonization with enterotoxigenic Bacteroides fragilis is associated with early-stage colorectal neoplasia.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) is a toxin-producing bacteria thought to possibly promote colorectal carcinogenesis by modulating the mucosal immune response and inducing epithelial cell changes. Here, we aim to examine the association of colonic mucosal colonization with ETBF and the presence of a range of lesions on the colonic neoplastic spectrum. METHODS: Mucosal tissue from up to four different colonic sites was obtained from a consecutive series of 150 patients referred for colonoscopy. The presence and relative abundance of the B. fragilis toxin gene (bft) in each tissue sample was determined using quantitative PCR, and associations with clinicopathological characteristics were analysed. FINDINGS: We found a high concordance of ETBF between different colonic sites (86%). Univariate analysis showed statistically significant associations between ETBF positivity and the presence of low-grade dysplasia (LGD), tubular adenomas (TA), and serrated polyps (P-values of 0.007, 0.027, and 0.007, respectively). A higher relative abundance of ETBF was significantly associated with LGD and TA (P-values of < 0.0001 and 0.025, respectively). Increased ETBF positivity and abundance was also associated with left-sided biopsies, compared to those from the right side of the colon. CONCLUSION: Our results showing association of ETBF positivity and increased abundance with early-stage carcinogenic lesions underlines its importance in the development of colorectal cancer, and we suggest that detection of ETBF may be a potential marker of early colorectal carcinogenesis.

Screening for enterotoxigenic Bacteroides fragilis in stool samples.

Bacteroides fragilis is a commensal bacterium found in the gut of most humans, however enterotoxigenic B. fragilis strains (ETBF) have been associated with diarrhoea and colorectal cancer (CRC). The purpose of this study was to establish a method of screening for the Bacteroides fragilis toxin (bft) gene in stool samples, as a means of determining if carriage of ETBF is detected more often in CRC patients than in age-matched healthy controls. Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls, were screened by standard and quantitative PCR using primers specific for the detection of the bft gene. Bacterial template DNA from stool samples was prepared by two methods: a sweep, where all colonies growing on Bacteroides Bile Esculin agar following stool culture for 48 h at 37 °C in an anaerobic environment were swept into sterile water and heat treated; and a direct DNA extraction from each stool sample. The bft gene was detected more frequently from DNA isolated from bacterial sweeps than from matched direct DNA extractions. qPCR was found to be more sensitive than standard PCR in detecting bft. The cumulative total of positive qPCR assays from both sample types revealed that 19 of the CRC patients had evidence of the toxin gene in their stool sample (27%), compared to seven of the age-matched controls (10%). This difference was significant (P = 0.016). Overall, ETBF carriage was detected more often in CRC patient stool samples compared to controls, but disparate findings from the different DNA preparations and testing methods suggests that poor sensitivity may limit molecular detection of ETBF in stool samples.

Urinary Peptide Analysis Differentiates Pancreatic Cancer From Chronic Pancreatitis.

OBJECTIVES: Differentiation of pancreatic cancer (PCA) from chronic pancreatitis (CP) is challenging. We searched for peptide markers in urine to develop a diagnostic peptide marker model. METHODS: Capillary electrophoresis-mass spectrometry was used to search for peptides in urine of patients with PCA (n = 39) or CP (n = 41). Statistical different peptides were included in a peptide multimarker model. Peptide markers were sequence identified and validated by immunoassay and immunohistochemistry (IHC). RESULTS: Applied to a validation cohort of 54 patients with PCA and 52 patients with CP, the peptide model correctly classified 47 patients with PCA and 44 patients with CP (area under the curve, 0.93; 87% sensitivity; 85% specificity). All 5 patients with PCA with concomitant CP were classified positive. Urine proteome analysis outperformed carbohydrate antigen 19-9 (area under the curve, 0.84) by a 15% increase in sensitivity at the same specificity. From 99 healthy subjects, only four were misclassified. Fetuin-A was the most prominent peptide marker source for PCA as verified by immunoassay and IHC. In silico protease mapping of the peptide markers' terminal sequences pointed to increased meprin-A activity in PCA, which in IHC was associated with neoangiogenesis. CONCLUSIONS: Urinary proteome analysis differentiates PCA from CP and may serve as PCA screening tool.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2ß1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.

Interrogating the Venom of the Viperid Snake Sistrurus catenatus edwardsii by a Combined Approach of Electrospray and MALDI Mass Spectrometry.

The complete sequence characterization of snake venom proteins by mass spectrometry is rather challenging due to the presence of multiple isoforms from different protein families. In the present study, we investigated the tryptic digest of the venom of the viperid snake Sistrurus catenatus edwardsii by a combined approach of liquid chromatography coupled to either electrospray (online) or MALDI (offline) mass spectrometry. These different ionization techniques proved to be complementary allowing the identification a great variety of isoforms of diverse snake venom protein families, as evidenced by the detection of the corresponding unique peptides. For example, ten out of eleven predicted isoforms of serine proteinases of the venom of S. c. edwardsii were distinguished using this approach. Moreover, snake venom protein families not encountered in a previous transcriptome study of the venom gland of this snake were identified. In essence, our results support the notion that complementary ionization techniques of mass spectrometry allow for the detection of even subtle sequence differences of snake venom proteins, which is fundamental for future structure-function relationship and possible drug design studies.

"BINACLE" assay for in vitro detection of active tetanus neurotoxin in toxoids.

Tetanus neurotoxin (TeNT) consists of two protein chains connected by a disulfide linkage: The heavy chain mediates the toxin binding and uptake by neurons, whereas the light chain cleaves synaptobrevin and thus blocks neurotransmitter release.Chemically inactivated TeNT (tetanus toxoid) is utilized for the production of tetanus vaccines. For safety reasons, each toxoid bulk has to be tested for the "absence of toxin and irreversibility of toxoid". To date, these mandatory tests are performed as toxicity tests in guinea pigs. A replacement by an animal-free method for the detection of TeNT would be desirable. The BINACLE (BINding And CLEavage) assay takes into account the receptor-binding as well as the proteolytic characteristics of TeNT: The toxin is bound to immobilized receptor molecules, the light chains are then released by reduction and transferred to a microplate containing synaptobrevin, and the fragment resulting from TeNT-induced cleavage is finally detected. This assay offers a higher specificity for discriminating between toxic TeNT and inactivated toxoid molecules than other published assays. Validation studies have shown that the BINACLE assay allows the sensitive and robust detection of TeNT in toxoids, and thus may indeed represent a suitable alternative to the prescribed animal safety tests for toxoids from several European vaccine manufacturers. Product-specific validations (and possibly adaptations) of the assay protocol will be required. A European collaborative study is currently being initiated to further examine the applicability of the method for toxoid testing. The final aim is the inclusion of the method into the European Pharmacopoeia.

endoProteoFASP: a novel FASP approach to profile salivary peptidome and disclose salivary proteases.

The salivary peptidome, which can represent up to 20% of total secreted proteins in human saliva, is highly influenced by proteolytic events. However, the development of strategies to understand the dynamics underlying the generation of salivary peptides has been a challenging task. In order to disclose in more detail the proteolytic events taking place in saliva, we aimed to characterize salivary peptidome and predict salivary proteases by applying, for the first time, a filter-aided sample preparation (FASP) approach to saliva. Thus, as a proof-of-concept of this application, harvested saliva samples from healthy individuals were incubated in 30 kDa cut-off spin filters for 18 or 115 h, at 37 °C, to promote saliva autolysis and the attained peptidome was characterized and compared with the naturally occurring one. In ex vivo conditions, proline-rich proteins, P-B peptide, histatin 1 and statherin were found to be the most susceptible salivary proteins to proteolysis. Peptide fragments were mainly attributed to the activity of cathepsin L1 and K at 18 h, whereas at 115 h, the attained peptide fragments were attributed to the activity of cathepsins K and L1, and MEP1A. Overall, the described endoProteoFASP approach makes the most of saliva׳s own protease pool and avoids the use of synthetic peptides and exogenous proteases to understand the proteolytic events occurring in the oral fluid. Hence, it could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from different pathophysiological conditions.

Generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells.

No methods for isolating induced alveolar epithelial progenitor cells (AEPCs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs), we identified carboxypeptidase M (CPM) as a surface marker of NKX2-1(+) "ventralized" anterior foregut endoderm cells (VAFECs) in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM(+) cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM(+) cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

Regulation of endothelin-converting enzyme-1 (ECE-1) by the calcimimetic R-568.

Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and Western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP.

[A KEL*02mod allele responsible for an apparent maternity exclusion]. / Un allèle KEL*02mod responsable d'une exclusion apparente de maternité

The patient's rare KEL:1,-2 phenotype was highlighted in course of a routine preoperative erythrocyte typing. Unexpectedly, her two daughters presented a KEL:-1,2 phenotype what appeared first as an apparent maternity exclusion. Flow cytometry, genotyping and adsorption-elution analyses were then performed for those three patients. KEL genotyping showed that the patient's genotype was KEL*01/KEL*02 whereas that of her daughters was KEL*02/KEL*02. By using polyclonal anti-KEL2 reagent, weak amount of KEL2 was identified on the patient's erythrocytes, a result which was confirmed by both flow cytometry and adsorption-elution assays, suggesting that patient's phenotype was in fact KEL:1,2w. These results are in favour of a weak expressed KEL*02 allele (KEL*2mod) transmission coding for a KEL2 antigen detected in some technical conditions only. Those results allowed to explain the apparent maternity exclusion based on initial KEL phenotype. This study also seems to confirm the presence of a compensatory mechanism of the KELmod allele deficient expression in heterozygote patients. A KEL phenotype retrospective study of 80,000 subjects showed a local KEL:1,-2 frequency four times lower than that described in literature. Moreover, a significant number of those individuals would in reality be KEL:1,2w, what still would decrease the real frequency of the KEL:1,2 subjects.