Surface-mediated spontaneous emulsification of the acylated peptide, semaglutide.

Acylated peptides composed of glucagon-like peptide-1 receptor agonists modified with a fatty acid side chain are an important class of therapeutics for type 2 diabetes and obesity but are susceptible to an unusual physical instability in the presence of hydrophobic surfaces, i.e., spontaneous emulsification, also known as ouzo formation in practice. In this work, light scattering, small-angle X-ray scattering, and circular dichroism measurements are used to characterize the physical properties of the semaglutide colloidal phase, including size distribution, shape, secondary structure, internal structure, and internal composition, as a function of solution physico-chemical conditions. The existence and size of the colloids formed are successfully predicted by a classical Rayleigh model, which identifies the parameters controlling their size and formation. Colloid formation is found to be catalyzed by hydrophobic surfaces, and formation rates are modeled as an autocatalytic reaction, enabling the formation of a master curve for various surfaces that elucidates the mechanism. Surfaces differ due to differences in surface wettability, which can be correlated with Hansen solubility parameters. This work provides insights into this unusual colloidal phenomenon and guides the peptide synthesis process and drug product formulation in the pharmaceutical industry.

Lentils protein isolate as a fermenting substrate for the production of bioactive peptides by lactic acid bacteria and neglected yeast species.

In the current trend where plant-based foods are preferred over animal-based foods, pulses represent an alternative source of protein but also of bioactive peptides (BPs). We investigated the pattern of protein hydrolysis during fermentation of red lentils protein isolate (RLPI) with various lactic acid bacteria and yeast strains. Hanseniaspora uvarum SY1 and Fructilactobacillus sanfranciscensis E10 were the most proteolytic microorganisms. H. uvarum SY1 led to the highest antiradical, angiotensin-converting enzyme-inhibitory and antifungal activities, as found in low molecular weight water soluble extracts (LMW-WSE). The 2039 peptide sequences identified by LMW-WSE were screened using BIOPEP UWM database, and 36 sequences matched with known BPs. Fermentation of RLPI by lactic acid bacteria and yeasts generated 12 peptides undetected in raw RLPI. Besides, H. uvarum SY1 led to the highest abundance (peak areas) of BPs, in particular with antioxidant and ACE-inhibitory activities. The amino acid sequences LVR and LVL, identified in the fermented RLPI, represent novel findings, as they were detected for the first time in substrates subjected to microbial fermentation. KVI, another BP highly characteristic of RLPI-SY1, was previously observed only in dried bonito. 44 novel potential BPs, worthy of further characterization, were correlated with antifungal activity.

Disposable electrochemical platform based on solid-binding peptides and carbon nanomaterials: an alternative device for leishmaniasis detection.

Neglected tropical diseases are those caused by infectious agents or parasites and are considered endemic in low-income populations. These diseases also have unacceptable indicators and low investment in research, drug production, and control. Tropical diseases such as leishmaniasis are some of the main causes of morbidity and mortality around the globe. Electrochemical immunosensors are promising tools for diagnostics against these diseases. One such benefit is the possibility of assisting diagnosis in isolated regions, where laboratory infrastructure is lacking. In this work, different peptides were investigated to detect antibodies against Leishmania in human and canine serum samples. The peptides evaluated (395-KKG and 395-G) have the same recognition site but differ on their solid-binding domains, which ensure affinity to spontaneously bind to either graphene oxide (GO) or graphene quantum dots (GQD). Cyclic voltammetry and differential pulse voltammetry were employed to investigate the electrochemical behavior of each assembly step and the role of each solid-binding domain coupled to its anchoring material. The graphene affinity peptide (395-G) showed better reproducibility and selectivity when coupled to GQD. Under the optimized set of experimental conditions, negative and positive human serum samples responses were distinguished based on a cut-off value of 82.5% at a 95% confidence level. The immunosensor showed selective behavior to antibodies against Mycobacterium leprae and Mycobacterium tuberculosis, which are similar antibodies and potentially sources of false positive tests. Therefore, the use of the graphene affinity peptide as a recognition site achieved outstanding performance for the detection of Leishmania antibodies.

The role of glucose-dependent insulinotropic polypeptide 3 (G1P-3) and nucleolar phosphoprotein-1 (NPM1) in pathogenesis of psoriasis.

Background Psoriasis is a multifactorial, hyperproliferative, chronic inflammatory skin disease affecting males and females equally. Aims To study the expression of certain non-coding RNAs, Interferon Alpha Inducible Protein 6 (IFI6), previously named Glucose-dependent Insulinotropic Polypeptide 3 (G1P-3), and nucleolar phosphoprotein (in serum and tissue), and to attempt to elucidate their role in the pathogenesis of psoriasis, which in turn might help in treatment. Methods Twenty patients with psoriasis and 20 healthy subjects were included in this study. Serum and skin biopsies were obtained from all participants. Molecular biology techniques were employed to estimate the expression levels of long noncoding G1P-3 and nucleolar phosphoprotein in serum and skin biopsy. Results Psoriasis patients had a mean age of 41.85 ± 12.29. The median serum G1P-3 level of the patients' group (3.330) was significantly higher than that of the control group (1.085) (P ≤ 0.001). Tissue G1P-3 level of the patients' group (6.495) was also significantly higher compared to that of controls (1.040) (P ≤ 0.001). Similarly, for nucleolar phosphoprotein, the median serum level of patients' group (2.030) was significantly higher than that of controls (1.040) (P ≤ 0.001) and median tissue level (5.425) was also significantly higher than that of controls (1.040) (P ≤ 0.001). Limitations In this study, only outpatients were included and follow-up was not well-handled. For future work, follow-up can be considered. Conclusion Long non-coding G1P-3 as well as nucleolar phosphoprotein may be considered as genetic markers for psoriasis susceptibility. In future, these might provide a novel direction for advances in psoriasis treatment.

Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry.

BACKGROUND: Mycobacterium leprae, the causative agent of Hansen's disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells. OBJECTIVE: To design a LAMA2-based peptide that targets PGL-1 from M. leprae. METHODS: We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations. FINDINGS: We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four ß-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1. MAIN CONCLUSIONS: Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Multiplex peptide microarray profiling of antibody reactivity against neglected tropical diseases derived B-cell epitopes for serodiagnosis in Zimbabwe.

INTRODUCTION: Peptides (B-cell epitopes) have broad applications in disease diagnosis and surveillance of pathogen exposure. In this framework, we present a pilot study to design and produce a peptide microarray for the integrated surveillance of neglected tropical diseases. The peptide microarray was evaluated against peptides derived from Ascaris lumbricoides, Necator americanus, Schistosoma haematobium, Schistosoma mansoni, Trichuris trichiura, Bacillus anthracis, Mycobacterium leprae, Wuchereria bancrofti, Rabies lyssavirus, Chlamydia trachomatis and Trypanosoma brucei. METHODS: S. haematobium was diagnosed using the urine filtration technique. S. mansoni, A. lumbricoides, N. americanus and T. trichiura were diagnosed using the Kato Katz and formal ether concentration techniques. Immunogenic peptides were retrieved from the Tackling Infection to Benefit Africa infectious diseases epitope microarray. Further peptides were predicted using ABCpred. IgG and IgM reactivity against the derived peptides were evaluated using peptide microarray multiplex immunoassays. Positive response was defined as fluorescence intensity ≥ 500 fluorescence units. Immunodominant peptides were identified using color-coded heat maps and bar graphs reflecting the obtained fluorescence signal intensities. Receiver Operating Characteristic analysis and Mann-Whitney-U test were performed to determine the diagnostic validity of the peptides. RESULTS: Species-specific responses with at least one peptide derived from each NTD pathogen were observed. The reactive peptides included; for S. haematobium, XP_035588858.1-206-220 and XP_035588858.1-206-220 immunodominant for IgG and IgM respectively, for S. mansoni, P20287.1-58-72 immunodominant for both antibodies and for T. trichiura, CDW52482.1-326-340 immunodominant for IgG and CDW57769.1-2017-2031 and CDW57769.1-1518-1532 immunodominant for IgM. According to ROC analysis most of the peptides selected were inaccurate; with AUC < 0.5. Some peptides had AUC values ranging from 0.5 to 0.5875 for both IgM and IgG suggesting no discrimination. CONCLUSION: Multiplex peptide microarrays are a valuable tool for integrated NTDs surveillance and for screening parasites exposure in endemic areas. Species sero-reactivity observed in the study maybe indicative of exposure to the different NTDs parasites. However, although peptides with the least cross reactivity were selected there is need to validate the sero-reactivity with recombinant antigens and immune-blotting techniques such as western blotting.

TN strain proteome mediated therapeutic target mapping and multi-epitopic peptide-based vaccine development for Mycobacterium leprae.

Leprosy is a significant universal health problem that is remarkably still a concern in developing countries due to infection frequency. New therapeutic molecules and next-generation vaccines are urgently needed to accelerate the leprosy-free world. In this direction, the present study was performed using two routes: proteome-mediated therapeutic target identification and mapping as well as multi-epitopic peptide-based novel vaccine development using state of the art of computational biology for the TN strain of M. leprae. The TN strain was selected from 65 Mycobacterium strains, and TN strain proteome mediated 83 therapeutic protein targets were mapped and characterized according to subcellular localization. Also, drug molecules were mapped with respect to protein targets localization. The Druggability potential of proteins was also evaluated. For multi-epitope peptide-based vaccine development, the four common types of B and T cell epitopes were identified (SLFQSHNRK, VVGIGQHAA, MMHRSPRTR, LGVDQTQPV) and combined with the suitable peptide linker. The vaccine component had an acceptable protective antigenic score (0.9751). The molecular docking of vaccine components with TLR4/MD2 complex exhibited a low ACE value (-244.12) which signifies the proper binding between the two molecules. The estimated free Gibbs binding energy ensured accurate protein-protein interactions (-112.46 kcal/mol). The vaccine was evaluated through adaptive immunity stimulation as well as immune interactions. The molecular dynamic simulation was carried out by using CHARMM topology-based parameters to minimize the docked complex. Subsequently, the Normal Mode Analysis in the internal coordinates showed a low eigen-value (1.3982892e-05), which also signifies the stability of molecular docking. Finally, the vaccine components were adopted for reverse transcription and codon optimization in E. coli strain K12 for the pGEX-4T1 vector, which supports in silico cloning of the vaccine components against the pathogen. The study directs the experimental study for therapeutics molecules discovery and vaccine candidate development with higher reliability.

Antigenic sites in SARS-CoV-2 spike RBD show molecular similarity with pathogenic antigenic determinants and harbors peptides for vaccine development.

The spike protein of coronavirus is key target for drug development and other pharmacological interventions. In current study, we performed an integrative approach to predict antigenic sites in SARS-CoV-2 spike receptor binding domain and found nine potential antigenic sites. The predicted antigenic sites were then assessed for possible molecular similarity with other known antigens in different organisms. Out of nine sites, seven sites showed molecular similarity with 54 antigenic determinants found in twelve pathogenic bacterial species (Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Borrelia burgdorferi, Clostridium perfringens, Clostridium tetani, Helicobacter Pylori, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholera and Yersinia pestis), two malarial parasites (Plasmodium falciparum and Plasmodium knowlesi) and influenza virus A. Most of the bacterial antigens that displayed molecular similarity with antigenic sites in SARS-CoV-2 RBD (receptor binding domain) were toxins and virulent factors. Antigens from Mycobacterium that showed similarity were mainly involved in modulating host cell immune response and ensuring persistence and survival of pathogen in host cells. Presence of a large number of antigenic determinants, similar to those in highly pathogenic microorganisms, not merely accounts for complex etiology of the disease but also provides an explanation for observed pathophysiological complications, such as deregulated immune response, unleashed or dysregulated cytokine secretion (cytokine storm), multiple organ failure etc., that are more evident in aged and immune-compromised patients. Over-representation of antigenic determinants from Plasmodium and Mycobacterium in all antigenic sites suggests that anti-malarial and anti-TB drugs can prove to be clinical beneficial for COVID-19 treatment. Besides this, anti-leprosy, anti-lyme, anti-plague, anti-anthrax drugs/vaccine etc. are also expected to be beneficial in COVID-19 treatment. Moreover, individuals previously immunized/vaccinated or had previous history of malaria, tuberculosis or other disease caused by fifteen microorganisms are expected to display a considerable degree of resistance against SARS-CoV-2 infection. Out of the seven antigenic sites predicted in SARS-CoV-2, a part of two antigenic sites were also predicted as potent T-cell epitopes (KVGGNYNYL444-452 and SVLYNSASF366-374) against MHC class I and three (KRISNCVADYSVLYN356-370, DLCFTNVYADSFVI389-402, and YRVVVLSFELLHA508-520) against MHC class II. All epitopes possessed significantly lower predicted IC50 value which is a prerequisite for a preferred vaccine candidate for COVID-19.

Molecular motifs encoding self-assembly of peptide fibers into molecular gels.

Peptides are a promising class of gelators, due to their structural simplicity, biocompatibility and versatility. Peptides were synthesized based on four amino acids: leucine, phenylalanine, tyrosine and tryptophan. These peptide gelators, with systematic structural variances in side chain structure and chain length, were investigated using Hansen solubility parameters to clarify molecular features that promote gelation in a wide array of solvents. It is of utmost importance to combine both changes to structural motifs and solvent in simultaneous studies to obtain a global perspective of molecular gelation. It was found that cyclization of symmetric dipeptides, into 2,5-diketopiperazines, drastically altered the gelation ability of the dipeptides. C-l-LL and C-l-YY, which are among the smallest peptide LMOGs reported to date, are robust gelators with a large radius of gelation (13.44 MPa1/2 and 13.90 MPa1/2, respectively), and even outperformed l-FF (5.61 MPa1/2). Interestingly, both linear dipeptides (l-FF and l-LL) gelled similar solvents, yet when cyclized only cyclo-dityrosine was a robust gelator, while cyclo-diphenylalanine was not. Changes in the side chains drastically affected the crystal morphology of the resultant gels. Symmetric cyclo dipeptides of leucine and tyrosine were capable of forming extremely high aspect ratio fibers in numerous solvents, which represent new molecular motifs capable of driving self-assembly.

Inhibitory effect of four novel synthetic peptides on food spoilage yeasts.

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 µg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 µg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.

New therapeutic patents used for the treatment of leprosy: a review.

Leprosy is a granulomatous disease, infectious and transmissible, which affects the skin and peripheral nerves, having Mycobacterium leprae as causative agent. The manifestation of this disease causes cutaneous lesions, peripheral neuropathies and, in more extreme cases, may generate deformities and disabilities in affected individuals. Patents were identified using the descriptor 'leprosy' and code A61K of the international patent classification, which indicates only products that meet human needs. The analysis was made using the WIPO, ESPACENET and USPTO databases, until the month of September 2016. Through this review, we found a variety of in vitro, pre-clinical and clinical studies relating to the treatment of leprosy with different types of compounds and forms of administration. New treatment proposals should include pain reduction capabilities, prevention or limitation of the appearance of cutaneous lesions, as well as prevention of the progression of the disease to more severe stages that may lead to loss of function or potentiate the individual's immune response to the M. leprae bacillus in order to prevent bacterial spread. We concluded that any patents developed with natural products were not found in the treatment of leprosy. All the deposited products were synthetic origin, mostly tested in humans and of varied forms of administration.

Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

Autoimmunity to Tropomyosin-Specific Peptides Induced by Mycobacterium leprae in Leprosy Patients: Identification of Mimicking Proteins.

Background: It has been shown earlier that there is a rise in the levels of autoantibodies and T cell response to cytoskeletal proteins in leprosy. Our group recently demonstrated a rise in both T and B cell responses to keratin and myelin basic protein in all types of leprosy patients and their associations in type 1 reaction (T1R) group of leprosy. Objectives: In this study, we investigated the association of levels of autoantibodies and lymphoproliferation against myosin in leprosy patients across the spectrum and tried to find out the mimicking proteins or epitopes between host protein and protein/s of Mycobacterium leprae. Methodology: One hundred and sixty-nine leprosy patients and 55 healthy controls (HC) were enrolled in the present study. Levels of anti-myosin antibodies and T-cell responses against myosin were measured by ELISA and lymphoproliferation assay, respectively. Using 2-D gel electrophoresis, western blot and MALDI-TOF/TOF antibody-reactive spots were identified. Three-dimensional structure of mimicking proteins was modeled by online server. B cell epitopes of the proteins were predicted by BCPREDS server 1.0 followed by identification of mimicking epitopes. Mice of inbred BALB/c strain were hyperimmunized with M. leprae soluble antigen (MLSA) and splenocytes and lymph node cells of these animals were adoptively transferred to naïve mice. Results: Highest level of anti-myosin antibodies was noted in sera of T1R leprosy patients. We observed significantly higher levels of lymphoproliferative response (p < 0.05) with myosin in all types of leprosy patients compared to HC. Further, hyperimmunization of inbred BALB/c strain of female mice and rabbit with MLSA revealed that both hyperimmunized rabbit and mice evoked heightened levels of antibodies against myosin and this autoimmune response could be adoptively transferred from hyperimmunized to naïve mice. Tropomyosin was found to be mimicking with ATP-dependent Clp protease ATP-binding subunit of M. leprae. We found four mimicking epitopes between these sequences. Conclusion: These data suggest that these mimicking proteins tropomyosin and ATP-dependent Clp protease ATP-binding subunit of M. leprae or more precisely mimicking epitopes (four B cell epitopes) might be responsible for extensive tissue damage during type1 reaction in leprosy.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

Topical peptides as cosmeceuticals.

Peptides are known to have diverse biological roles, most prominently as signaling/regulatory molecules in a broad variety of physiological processes including defense, immunity, stress, growth, homeostasis and reproduction. These aspects have been used in the field of dermatology and cosmetology to produce short, stable and synthetic peptides for extracellular matrix synthesis, pigmentation, innate immunity and inflammation. The evolution of peptides over the century, which started with the discovery of penicillin, has now extended to their usage as cosmeceuticals in recent years. Cosmeceutical peptides may act as signal modulators of the extracellular matrix component, as structural peptides, carrier peptides and neurotransmitter function modulators. Transdermal delivery of peptides can be made more effective by penetration enhancers, chemical modification or encapsulation of peptides. The advantages of using peptides as cosmeceuticals include their involvement in many physiological functions of the skin, their selectivity, their lack of immunogenicity and absence of premarket regulatory requirements for their use. However, there are disadvantages: clinical evidence for efficacy is often weak, absorption may be poor due to low lipophilicity, high molecular weight and binding to other ingredients, and prices can be quite high.

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis.

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0(+)CD69(+)CD4(+) memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.

Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae.

In recent years, Bacillus species have received considerable attention for the biological control of many fungal diseases. In this study, Bacillus amyloliquefaciens Q-426 was tested for its potential use against a variety of plant pathogens. Our screen for genes involved in the biosynthesis of antifungal agents revealed that the fen and bmy gene clusters are present in the Q-426 genome. Lipopeptides such as bacillomycin D, fengycin A, and fengycin B were purified from the bacterial culture broth and subsequently identified by ESI-mass spectrometry. The minimal inhibitory concentration of fengycin A against Fusarium oxysporum f. sp. spinaciae W.C. Snyder & H.N. Hansen O-27 was determined to be 31.25 µg ml(-1) . However, exposure of fungal cells to 50 µg ml(-1) of fengycin A did not allow permeation of fluorescein diacetate into the cytoplasm through the cell membrane. Moreover, leakage of intracellular inorganic cations, nucleic acid and protein were also not detected, indicating that the fungal cell membrane is not the primary target of action for fengycin A. Profound morphological changes were observed in the F. oxysporum strain and spore germination was completely inhibited, suggesting that 50 µg ml(-1) of fengycin A acts, at least, as a fungistatic agent.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection.

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.