Structural characteristics of Saccharomyces cerevisiae mannoproteins: Impact of their polysaccharide part.

While they have many properties of interest in enology, the structure-function relationships of mannoproteins and the part played by their polysaccharide moiety are not yet well understood. Mannoproteins (MP) extracted with ß-glucanase from a laboratory yeast strain (WT), two of its mutants (Mnn2 with unbranched N-glycosylated chains and Mnn4 without mannosyl-phosphorylation), and an enological strain (Com) were purified and thoroughly characterized. The protein moiety of the four MPs had the same amino acid composition. Glycosyl-linkage and net charge analyses confirmed the expected differences in mutant strain MPs. MP-Com had the highest mannose/glucose ratio followed by MP-WT/MP-Mnn4, and MP-Mnn2 (13.5 > 5.6 ≈ 5.2 > 2.2). The molar mass dependencies of Rg, Rh, and [η], determined through HPSEC-MALLS-QELS-Viscosimetry, revealed specific conformational properties of mannoproteins related to their nature of highly branched copolymers with two branching levels. It also clearly showed structural differences between MP-Com, MP-WT/Mnn4, and MP Mnn2, and differences between two populations within the four mannoproteins.

Validation of Recombinant Chicken Liver Bile Acid Binding Protein as a Tool for Cholic Acid Hosting.

Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.

An immunohistochemical study of lymphatic elements in the human brain.

Almost 150 papers about brain lymphatics have been published in the last 150 years. Recently, the information in these papers has been synthesized into a picture of central nervous system (CNS) "glymphatics," but the fine structure of lymphatic elements in the human brain based on imaging specific markers of lymphatic endothelium has not been described. We used LYVE1 and PDPN antibodies to visualize lymphatic marker-positive cells (LMPCs) in postmortem human brain samples, meninges, cavernous sinus (cavum trigeminale), and cranial nerves and bolstered our findings with a VEGFR3 antibody. LMPCs were present in the perivascular space, the walls of small and large arteries and veins, the media of large vessels along smooth muscle cell membranes, and the vascular adventitia. Lymphatic marker staining was detected in the pia mater, in the arachnoid, in venous sinuses, and among the layers of the dura mater. There were many LMPCs in the perineurium and endoneurium of cranial nerves. Soluble waste may move from the brain parenchyma via perivascular and paravascular routes to the closest subarachnoid space and then travel along the dura mater and/or cranial nerves. Particulate waste products travel along the laminae of the dura mater toward the jugular fossa, lamina cribrosa, and perineurium of the cranial nerves to enter the cervical lymphatics. CD3-positive T cells appear to be in close proximity to LMPCs in perivascular/perineural spaces throughout the brain. Both immunostaining and qPCR confirmed the presence of adhesion molecules in the CNS known to be involved in T cell migration.

Purification of Tannerella forsythia Surface-Layer (S-Layer) Proteins.

The objective of this chapter is to provide a detailed purification protocol for the surface-layer (S-layer) glycoproteins of the periodontal pathogen Tannerella forsythia. The procedure involves detergent based solubilization of the bacterial S-layer followed by cesium chloride gradient centrifugation and gel permeation chromatography. The protocol is suitable for the isolation of S-layer glycoproteins from T. forsythia strains with diverse O-glycan structures, and aid in understanding the biochemical basis and the role of protein O-glycosylation in bacterial pathogenesis.

Slr4, a newly identified S-layer protein from marine Gammaproteobacteria, is a major biofilm matrix component.

S-layers are paracrystalline proteinaceous lattices that surround prokaryotic cells, forming a critical interface between the cells and their extracellular environment. Here, we report the discovery of a novel S-layer protein present in the Gram-negative marine organism, Pseudoalteromonas tunicata D2. An uncharacterized protein (EAR28894) was identified as the most abundant protein in planktonic cultures and biofilms. Bioinformatic methods predicted a beta-helical structure for EAR28894 similar to the Caulobacter S-layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S-layer was found surrounding the outer membrane in wild-type cells and completely removed from cells in an EAR28894 deletion mutant. S-layer material also appeared to be "shed" from wild-type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S-layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio, and found exclusively in marine metagenomes. We propose the name Slr4 for this novel protein family.

Purification and Characterization of a New CRISP-Related Protein from Scapharca broughtonii and Its Immunomodulatory Activity.

More and more attention has been paid to bioactive compounds isolated from marine organisms or microorganisms in recent years. At the present study, a new protein coded as HPCG2, was purified from Scapharca broughtonii by stepwise chromatography methods. The molecular weight of HPCG2 was determined to be 30.71 kDa by MALDI-TOF-MS. The complete amino acid sequence of HPCG2 was obtained by tandem mass spectrometry combined with transcriptome database analysis, and its secondary structure was analyzed using circular dichroism. HPCG2 comprised 251 amino acids and contained 28.4% α-helix, 26% ß-sheet, 18.6% ß-turn, and 29.9% random coil. HPCG2 was predicted to be a cysteine-rich secretory protein-related (CRISP-related) protein by domain prediction. Moreover, HPCG2 was proved to possess the immunomodulatory effect on the murine immune cells. MTT assay showed that HPCG2 promoted the proliferation of splenic lymphocytes and the cytotoxicity of NK cells against YAC-1 cells. Flow cytometry test revealed that HPCG2 enhanced the phagocytic function of macrophages and polarized them into M1 type in RAW264.7 cells. In particular, Western blot analysis indicated that the immunomodulatory mechanism of HPCG2 was associated with the regulation on TLR4/JNK/ERK and STAT3 signaling pathways in RAW 264.7 cells. These results suggested that HPCG2 might be developed as a potential immunomodulatory agent or new functional product from marine organisms.

Mannoproteins from inactivated whole cells of baker's and brewer's yeasts as functional food ingredients: Isolation and optimization.

Because of the structural multiplicity of yeast mannoproteins, they have shown a great interest as food ingredients for a wide range of applications. The yields and the structural properties of mannoproteins varied depending on the isolation methods and their sources (baker's and brewer's Saccharomyces cerevisiae yeasts). Noncovalently bound mannoproteins (6.5 kDa) with a mannan/protein ratio of 0.63 and 2.78 were recovered upon the heat treatment of brewer's and baker's yeasts, respectively, whereas sodium dodecyl sulfate treatment led mainly to the release of nonglycosylated proteins. The highest yield of mannoproteins was achieved upon the enzymatic isolation with Zymolyase® from Arthrobacter luteus. The recovered covalently bound mannoproteins were characterized by a higher mannan/protein ratio (13.1 to 42.7) and a wider molecular weight distribution (5 to 10 kDa; 10 to 100 kDa; 100 to 400 kDa). Predictive models were developed to understand and modulate the effects of isolation parameters on yield, the mannoproteins content, and the mannan/protein ratio. The enzyme concentration was the most significant parameter affecting the yield, whereas the reaction time was the most significant parameter affecting mannan/protein ratio. Comparison of predicted and experimental values validated the established predicted models for the isolation of well-defined mannoproteins from yeast for targeted food applications. PRACTICAL APPLICATION: The increasing demand for clean label health-promoting foods has fueled the development of highly functional ingredients that offer both techno-functionalities and health-promoting properties. This study reveals the efficiency of whole inactivated yeasts as sources of mannoproteins. Given the dependence of the techno-functional and health-promoting properties of mannoproteins on their molecular properties, the investigation of the effects of the yeast sources and the type of isolation methods on the structural properties of mannoproteins would allow the modulation of their properties. Furthermore, the developed predictive models for the enzymatic process are expected to enhance the isolation efficiency of mannoproteins with well-defined structures.

Electrochemical Biosensors Based on S-Layer Proteins.

Designing and development of electrochemical biosensors enable molecule sensing and quantification of biochemical compositions with multitudinous benefits such as monitoring, detection, and feedback for medical and biotechnological applications. Integrating bioinspired materials and electrochemical techniques promote specific, rapid, sensitive, and inexpensive biosensing platforms for (e.g., point-of-care testing). The selection of biomaterials to decorate a biosensor surface is a critical issue as it strongly affects selectivity and sensitivity. In this context, smart biomaterials with the intrinsic self-assemble capability like bacterial surface (S-) layer proteins are of paramount importance. Indeed, by forming a crystalline two-dimensional protein lattice on many sensors surfaces and interfaces, the S-layer lattice constitutes an immobilization matrix for small biomolecules and lipid membranes and a patterning structure with unsurpassed spatial distribution for sensing elements and bioreceptors. This review aims to highlight on exploiting S-layer proteins in biosensor technology for various applications ranging from detection of metal ions over small organic compounds to cells. Furthermore, enzymes immobilized on the S-layer proteins allow specific detection of several vital biomolecules. The special features of the S-layer protein lattice as part of the sensor architecture enhances surface functionalization and thus may feature an innovative class of electrochemical biosensors.

PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Mass production of a S-layer protein of Bacillus thuringiensis and its toxicity to the cattle tick Rhipicephalus microplus.

The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.

ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research.

BACKGROUND: NTRK1, NTRK2 and NTRK3 fusions are present in a plethora of malignancies across different histologies. These fusions represent the most frequent mechanism of oncogenic activation of these receptor tyrosine kinases, and biomarkers for the use of TRK small molecule inhibitors. Given the varying frequency of NTRK1/2/3 fusions, crucial to the administration of NTRK inhibitors is the development of optimal approaches for the detection of human cancers harbouring activating NTRK1/2/3 fusion genes. MATERIALS AND METHODS: Experts from several Institutions were recruited by the European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) to review the available methods for the detection of NTRK gene fusions, their potential applications, and strategies for the implementation of a rational approach for the detection of NTRK1/2/3 fusion genes in human malignancies. A consensus on the most reasonable strategy to adopt when screening for NTRK fusions in oncologic patients was sought, and further reviewed and approved by the ESMO TR and PM WG and the ESMO leadership. RESULTS: The main techniques employed for NTRK fusion gene detection include immunohistochemistry, fluorescence in situ hybridization (FISH), RT-PCR, and both RNA-based and DNA-based next generation sequencing (NGS). Each technique has advantages and limitations, and the choice of assays for screening and final diagnosis should also take into account the resources and clinical context. CONCLUSION: In tumours where NTRK fusions are highly recurrent, FISH, RT-PCR or RNA-based sequencing panels can be used as confirmatory techniques, whereas in the scenario of testing an unselected population where NTRK1/2/3 fusions are uncommon, either front-line sequencing (preferentially RNA-sequencing) or screening by immunohistochemistry followed by sequencing of positive cases should be pursued.

Exploring lectin-like activity of the S-layer protein of Lactobacillus acidophilus ATCC 4356.

The surface layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling, proteinaceous subunits non-covalently bound to the outmost bacterial cell wall envelope and is involved in the adherence of bacteria to host cells. We have previously described that the S-layer protein of L. acidophilus possesses anti-viral and anti-bacterial properties. In this work, we extracted and purified S-layer proteins from L. acidophilus ATCC 4356 cells to study their interaction with cell wall components from prokaryotic (i.e., peptidoglycan and lipoteichoic acids) and eukaryotic origin (i.e., mucin and chitin), as well as with viruses, bacteria, yeast, and blood cells. Using chimeric S-layer fused to green fluorescent protein (GFP) from different parts of the protein, we analyzed their binding capacity. Our results show that the C-terminal part of the S-layer protein presents lectin-like activity, interacting with different glycoepitopes. We further demonstrate that lipoteichoic acid (LTA) serves as an anchor for the S-layer protein. Finally, a structure for the C-terminal part of S-layer and possible binding sites were predicted by a homology-based model.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

One-step production of bioactive human lipopolysaccharide binding protein from LPS-eliminated E. coli.

Human lipopolysaccharide (LPS) binding protein (LBP) is a ∼60 kDa glycosylated protein that mediates potent innate immune against invading Gram-negative bacteria by recognition of LPS in their outer membranes. To date, there is no method for efficient production of bioactive LPS-free LBP at sufficient amounts through prokaryotic expression system. Here we present a simple approach for rapid preparation of human LBP from a LPS-eliminated E. coli strain named ClearColi BL21 (DE)3. Combined with the usage of an ultra-high-affinity CL7/Im7 purification system, we achieved one-step purification of recombinant human LBP with over 90% purity at a yield of ∼4 mg/L when using LB culture medium. The produced LBP retains full LPS binding activity which was validated by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Collectively, we develop a valid method that can be applied to cost-effectively produce and purify LPS-free proteins.

Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency.

5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10-11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.

Investigation onto the correlation between systemic antibodies to surface glycoproteins of infectious laryngotracheitis virus (ILTV) and protective immunity.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes upper respiratory tract disease in chickens and significant losses to the poultry industry worldwide. Both antibody and cell-mediated responses are generated against ILTV infection; however, the correlation of humoral immune response with protection against ILTV infection is debatable. To examine if whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, four ILTV glycoproteins (gD, gE, gG and gJ) were expressed as recombinant proteins and used in conjunction with commercially available recombinant gC and gI in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-ILTV vaccination sera found that gE was the most antigenic glycoprotein and that gC ODs were strongly correlated with those of gI and gJ, while ODs to gG had a relatively poor correlation with those of other glycoproteins. Moderate to poor correlations were found between microscopic tracheal lesion scores and ODs to individual glycoproteins. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation with protective immunity as measured by the severity of clinical signs, gross lesions, and tracheal viral load. Results from this study demonstrated that systemic antibody titers to individual ILTV glycoproteins C, D, E, G, I and J had a relatively poor correlation to protective immunity.

Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2.

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1-3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

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

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

Development of a qPCR for the detection of infectious laryngotracheitis virus (ILTV) based on the gE gene.

1. Infectious laryngotracheitis is a respiratory disease that affects the poultry industry worldwide. It is common in flocks with high-bird density, causing major economic losses. 2. In this study, a SYBR® FAST polymerase chain reaction (PCR) double-strand DNA intercalating agent assay was performed for the detection of infectious laryngotracheitis virus (ILTV) in clinical samples in comparison with a conventional nested-PCR, both based on the glycoprotein E encoding gene. This assay amplified 56 bp and was capable of detecting 19 to 1 copies of virus. 3. In total, 164 clinical samples were obtained from birds with respiratory problems from the period of 2009-2016. In the nested-PCR, there were 45.12% positive samples and 54.88% negative samples, while in the real-time PCR (qPCR), there were 81.1% positive samples and 18.9% negative samples. 4. In conclusion, qPCR from the DNA double-strand intercalating agent SYBR® GREEN FAST was useful for the diagnosis of ILTV because it detected samples that were negative in nested-PCR. This assay has advantages, such as a shortened processing-time, and no need for post-amplification processing (electrophoresis) with additional reagents, such as MgCl2 and agarose. Hence, qPCR proved to be useful, rapid and low cost for use with clinical samples.

The inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant, carbohydrate chains, and proteinaceous components are all responsible for the cross-reactivity of trypanosome variant surface glycoproteins.

Salivarian trypanosomes evade the host immune system by continually swapping their protective variant surface glycoprotein (VSG) coat. Given that VSGs from various trypanosome stocks exhibited cross-reactivity (Camargo et al., Vet. Parasitol. 207, 17-33, 2015), we analyzed here which components are the antigenic determinants for this cross-reaction. Soluble forms of VSGs were purified from four Venezuelan animal trypanosome isolates: TeAp-N/D1, TeAp-ElFrio01, TeAp-Mantecal01, and TeGu-Terecay323. By using the VSG soluble form from TeAp-N/D1, we found that neither the inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant nor the carbohydrate chains were exclusively responsible for its cross-reactivity. Then, all four purified glycoproteins were digested with papain and the resulting peptides were separated by high-performance liquid chromatography. Dot blot evaluation of the fractions using sera from trypanosome-infected animals yielded peptides that possessed cross-reaction activity, demonstrating for the first time that proteinaceous epitopes are also responsible for the cross-reactivity of trypanosome VSGs.