Low Expression Loci and the Use of Pronase in Flow Cytometry Crossmatch.

Pronase treatment of lymphocytes has been used to improve the specificity and sensitivity of flow cytometric crossmatch, especially B-cell flow cytometric crossmatch, due to the presence of Fc receptors on the cell surface. Some limitations have been reported in the literature: false negatives due to the reduction of major histocompatibility complex expression and false-positive T cells in HIV+ patients due to exposure to cryptic epitopes. This study aimed to evaluate the effect of pronase in our assays, using untreated and treated cells with 2.35 U/mL of pronase to improve flow cytometric crossmatch specificity and sensitivity. The study was carried out with donor-specific IgG antibodies (DSAs) to low expression loci (HLA-C, -DQ, or -DP) because, in our laboratory practice, patients with virtual crossmatch (LABScreen single antigen assays) to DSA against antigen HLA-A, B, and DR are excluded from cellular crossmatch. Our results showed that, for T-cell flow cytometry crossmatch (FCXM), a cutoff value of 1171 median fluorescence intensity (MFI), an area under the curve (AUC) of 0.926 (P < .0001), and 0.834 (P < .0001), a sensitivity of 100% and 85.7%, and a specificity of 77.5% and 74.4%, without and with pronase treatment, respectively. For B-cell FCXM without pronase treatment, the best cutoff was 2766 MFI, an AUC of 0.731 (P < .0001), a sensitivity of 69.6%, and a specificity of 66.7%, whereas for B cells treated with pronase, the cutoff value was 4496 MFI, an AUC of 0.852 (P < .0001), a sensitivity of 86.4%, and a specificity of 77.8%. Our analysis of 128 FCXM showed a better performance using the untreated lymphocytes for FCXM with the prerequisite of a higher cutoff value (≈5000 MFI) to reach a better sensitivity and specificity due to the loss of HLA expression.

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

Quantitative selenium speciation in feed by enzymatic probe sonication and ion chromatography-inductively coupled plasma mass spectrometry.

A rapid, sensitive and species preservative analytical method for the simultaneous determination of six selenium (Se) species has been developed. Enzymatic probe sonication (EPS) was investigated as a novel and alternative technology for the extraction of Se species from feed matrices and the results were compared with the conventional hot water extraction, enzymatic hydrolysis and sequential extraction. The critical parameters of EPS such as enzyme types, extraction time, temperature, ultrasonic power and sample/enzyme ratio were varied with control. The Se species were separated and quantitatively determined by ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). Under current optimised conditions, six inorganic and organic Se species were completely separated within 15 min in a single chromatographic run. The spectral interferences from the argon plasma 40Ar2, 40Ar37Cl or 1H79Br were effectively removed by employing the kinetic energy discrimination (KED) mode. Quantitative extraction for total Se (>94.8%) and more than 89.0% for the sum of different Se chemical forms without species transformation were obtained in only 60 s by applying the EPS treatment using aqueous protease XIV. The limits of detection (LODs) and quantification (LOQs) for Se species were in the ranges of 0.21-0.56 µg kg-1 and 0.69-1.87 µg kg-1, respectively. The proposed method was successfully applied to the speciation of Se in several reference materials and feed samples collected from the markets and local farms.

Drug Affinity Responsive Target Stability (DARTS ) Assay to Detect Interaction Between a Purified Protein and a Small Molecule.

Drug affinity responsive target stability (DARTS) assay is used to detect the interaction between a ligand and a protein based on the observation that some ligands can protect the target protein from degradation by proteases when mixed in a solution. To set up the assay, a ligand is first mixed with a purified candidate target protein or a total cell lysate that contains a candidate target protein. Then, different amounts of protease are added to the mixture to allow the enzyme to digest the protein in the mixture. After protease digestion, the candidate target protein is detected by assays such as western blot, silver staining, or Coomassie blue staining. In theory, the candidate protein should be protected by the ligand from protease digestion, which is reflected by higher abundance of the candidate protein in mixtures containing the ligand compared with the control treatment. There are a few significant advantages of DARTS: (a) the ligand does not need to be modified so the native ligand could be used; (b) the candidate target protein could be either purified protein or protein that is present in the total cell lysate; and (c) the assay can be used together with proteomics analysis to identify an unknown target protein. The assay is especially valuable to test the interaction between the ligand and membrane proteins that are often challenging to purify. In this chapter, we use Endosidin2 (ES2) and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1) as an example to show the step-by-step procedure of the DARTS assay.

Development of Small-diameter Polyester Vascular Grafts Coated with Silk Fibroin Sponge.

In recent years, the demand for functional small-diameter (< 6 mm) artificial vascular grafts has greatly increased due to an increase in the number of patients with vascular heart disease. However, currently, there are no available commercial small-diameter grafts. The objective of this research was to develop a porous silk fibroin (SF)-coated poly(ethylene terephthalate) (PET) graft with a diameter < 6 mm. The graft was compared with a gelatin-coated PET graft because the latter PET graft with a diameter ~ 6 mm was widely used as a commercial vascular graft. Initially, porous SF was prepared using Glyc as the porogen [termed SF(Glyc)] and the PET grafts were prepared through the double-Raschel knitting method. Subsequently, the degradation of the SF coating was monitored using protease XIV in vitro and was compared with that observed in gelatin-coated PET grafts. Finally, these grafts were also implanted into rats for an in vivo comparison. In degradation experiments, after 7 days, the SF was clearly digested by protease XIV, but the gelatin on the graft was still remained at the outer surface. In implantation experiments in rats, the SF(Glyc)-coated PET graft was rapidly degraded in vivo and remodeling to self-tissues was promoted compared with the gelatin-coated PET graft. Thrombus formation and intimal hyperplasia were observed in the gelatin-coated PET graft; however, such side reactions were not observed in the SF(Glyc)-coated PET graft. Thus, the porous SF(Glyc)-coated PET graft with a small diameter < 6 mm may be useful as a commercial vascular graft.

Cyclic Peptide-Based Sirtuin Substrates.

In the current study, four side chain-to-side chain cyclic peptides (three 5-mers and one 4-mer) harboring Nε-acetyl-lysine or Nε-myristoyl-lysine were found to be in vitro substrates of the human SIRT1/2/3-catalyzed deacylation with good substrate activities, as judged by the kcat/KM ratios.

Release, Separation, and Recovery of Monomeric Reducing N-Glycans with Pronase E Combined with 9-Chloromethyl Chloroformate and Glycosylasparaginase.

The glycan moiety of glycoproteins plays key roles in various biological processes. However, there are few versatile methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis. In this study, we developed a new method to achieve the release, separation, and recovery of monomeric reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and glycosylasparaginase (GA). Ovalbumin, ribonuclease B, ginkgo, and pine nut glycoproteins were used as materials and sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl, and enzymatically hydrolyzed with GA. The products produced by this method were then detected by electrospray ionization mass spectrometry, high-performance liquid chromatography (HPLC), and online hydrophilic interaction chromatography (HILIC-MS) separation. The results showed that all N-glycans with essentially one amino acid obtained with Pronase E were labeled with Fmoc-Cl and could be efficiently separated and detected via HPLC and HILIC-MS. Finally, the isolated Asn-glycan derivatives were digested with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This method was simple, inexpensive, and broadly applicable and could therefore be quite important for analysis of the structure-function relationships of glycans.

Bacteriocin production by Lactobacillus plantarum ST16Pa in supplemented whey powder formulations.

Whey, the main by-product of the dairy industry, is frequently disposed of in the environment without any treatment due to the high cost of this process. Alternatively, whey can be used as a medium to culture lactic acid bacteria and produce value-added products such as bacteriocins. In this work, we attempted to improve bacteriocin production by Lactobacillus plantarum ST16Pa in a whey powder formulation supplemented with additional sources of carbon, nitrogen, and vitamin B12 at different levels and varying the agitation intensity according to a Plackett-Burman experimental design. Only the addition of tryptone positively influenced the production of this bacteriocin. The results allowed us to identify a supplemented whey formulation, comprising 150 g/L of whey total solids plus 10 g/L of tryptone and soybean extract, whose fermentation by Lb. plantarum ST16Pa in shake flasks under agitation at 150 rpm led to a cell-free supernatant with an antimicrobial activity against Listeria innocua 6a CLIST 2865 (inhibition zone of 13.23 mm) close to that previously obtained in de Man, Rogosa and Sharpe medium by other authors. These results are significant considering that the same strain cultured in cheese whey did not previously display any antimicrobial activity.

Pronase independent flow cytometry crossmatching of rituximab treated patients.

ABO-incompatible (ABOi) kidney transplantation has become an established strategy to increase the number of available living donors. At our center, the conditioning protocol for ABOi patients is based on anti-A/B antibody removal and depletion of B cells with the anti-CD20 mAb rituximab (Mabthera®). It is known that even low amounts of remaining rituximab in serum of patients results in false positive B cell cross match results, masking detection of potentially harmful donor human leukocyte antigen (HLA) specific antibodies. Treatment of donor cells with high concentrations (>1 mg/mL) of pronase is currently standard procedure for elimination of rituximab (RIT) interference. It is, however, troublesome that recent reports indicate that pronase treatment per se can induce incorrect flow cytometry cross match (FCXM) results. The aim of this study was to evaluate an alternative pronase-free FCXM for crossmatching of patients treated with rituximab. FCXM with an anti-RIT monoclonal antibody (mAb) pre-blocking step were evaluated on normal human sera (NHS) and patient sera supplemented with RIT. NHS supplemented with RIT or patient sera, without donor specific antibodies (DSA), resulted in high B cell median channel shift (>200 IgG) above background. This shift was eliminated by a serum pre-blocking step with 2-fold excess of anti-RIT (clone MB2A4). Blocking with anti-RIT did not influence the T cells crossmatch results. We present data supporting proof-of-concept that blocking with anti-RIT antibody prior to XM can enable reliable detection of anti-HLA class I and II donor specific antibodies without use of pronase treated donor cells.

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target.

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.

Application of Electron Paramagnetic Resonance and Solid-state 13C Nuclear Magnetic Resonance of Cross-polarization/Magic Angle Spinning to Study Enzymatic Degradation of Silk Fabrics.

The enzymatic degradation of silk by protease XIV has been investigated by using scanning electron microscopy (SEM), Fourier transfer infrared spectroscopy (FTIR), solid-state 13C nuclear magnetic resonance of cross-polarization/magic angle spinning (13C CP/MAS solid state NMR) and electron paramagnetic resonance (EPR). Micro-morphology of protease XIV aged samples showed that microfilaments were stripped out from the surface of silk fibers. The results of FTIR and 13C CP/MAS solid-state NMR indicated that the enzymatic degradation process could be divided into two stages. The EPR spectra indicated that the enzymatic degradation process was related to the free radical with the g-factor value of 2.0043. We also proposed that at the first degradation stage, the free radicals were apt to lose activities due to the loose structure of the non-crystalline region; at the second degradation stage, the free radicals produced in the crystalline region tended to be stored.

Purification and characterisation of a pronase-inducible lectin isolated from human serum.

A new lectin was purified to electrophoretic homogeneity from pronase treated human serum by a single-step of affinity chromatography on concanavalin A-Sepharose 4B. The isolated lectin agglutinated five types of vertebrate RBC, with highest titer against hen RBC. This activity was independent of divalent cations, insensitive to EDTA and specific to mannosamine, glucosamine as well as galactosamine. Purified lectin gave a single symmetrical peak in its native form with a molecular mass estimate of 6kDa in FPLC analysis and 6.5kDa by MALDI-TOF MS. SDS-PAGE analysis of the lectin revealed that it is a homo-oligomer of a 3kDa subunit protein. Isolated lectin did possess both, hemagglutinating and phenoloxidase activities, but did not exhibit any antibacterial or antifungal activities. In addition, this lectin could oxidize all nine different phenolic substrates tested, with hydroquinone proving to be the best among them. Phenoloxidase inhibitors namely, phenylthiourea and tropolone inhibited this oxidation activity.

Enzyme-assisted extraction of phenolics from winemaking by-products: Antioxidant potential and inhibition of alpha-glucosidase and lipase activities.

Phenolics in food and agricultural processing by-products exist in the soluble and insoluble-bound forms. The ability of selected enzymes in improving the extraction of insoluble-bound phenolics from the starting material (experiment I) or the residues containing insoluble-bound phenolics (experiment II) were evaluated. Pronase and Viscozyme improved the extraction of insoluble-bound phenolics as evaluated by total phenolic content, antioxidant potential as determined by ABTS and DPPH assays, and hydroxyl radical scavenging capacity, reducing power as well as evaluation of inhibition of alpha-glucosidase and lipase activities. Viscozyme released higher amounts of gallic acid, catechin, and prodelphinidin dimer A compared to Pronase treatment. Furthermore, p-coumaric and caffeic acids, as well as procyanidin dimer B, were extracted with Viscozyme but not with Pronase treatment. Solubility plays an important role in the bioavailability of phenolic compounds, hence this study may assist in better exploitation of phenolics from winemaking by-products as functional food ingredients and/or supplements.

Insights into the biological features of the antigenic determinants recognized by four monoclonal antibodies in redia and adult stages of the liver fluke Fasciola hepatica.

Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites, are suitable features that suggest their potential use for developing an epitope-based vaccine for fasciolosis control.

Development of a quantitative fluorescence-based ligand-binding assay.

A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays.

Flow cytometry crossmatch reactivity with pronase-treated T cells induced by non-HLA autoantibodies in human immunodeficiency virus-infected patients.

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However, we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study, 25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1193±631days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells.

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade.

OBJECTIVE: Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion. APPROACH AND RESULTS: A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. CONCLUSIONS: Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis.

The vesicant sulfur mustard (SM) is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons (OPCW). Bioanalytical procedures are mandatory for proving an alleged use and incorporation of SM into the body. We herein present the development and application of a novel optimized procedure suitable for qualitative verification analysis of plasma targeting the SM-adduct of human serum albumin (HSA) alkylated at the cysteine(34) residue. Diluted human plasma is directly mixed with pronase in an ultrafiltration device (10kDa cut-off) for proteolysis (4h, 37°C). Following ultrafiltration the filtrate is diluted and analyzed by microbore liquid chromatography-electrospray ionization high resolution tandem-mass spectrometry (µLC-ESI HR MS/MS) targeting the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP). A hybrid quadrupole time-of-flight mass spectrometer provided high mass spectrometric resolution in the MS/MS mode enabling highest selectivity and sensitivity (lower limit of detection corresponding to 9.8nM SM in plasma). Kinetics of HETE-CP formation from heparin-, citrate-, and EDTA-plasma as well as serum are presented and the influence of different EDTA and pronase concentrations was characterized. The novel procedure was applied to plasma samples provided by the OPCW as well as to patients plasma derived from real cases of SM-poisoning.

Site-Specific Protein N- and O-Glycosylation Analysis by a C18-Porous Graphitized Carbon-Liquid Chromatography-Electrospray Ionization Mass Spectrometry Approach Using Pronase Treated Glycopeptides.

The analysis of N- and O-glycopeptides remains challenging due to the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of the glycoprotein. Trypsin is by far the most commonly used protease in glycoproteomic studies; however, it often results in long peptides that can harbor more than one glycan which may hamper site identification. The use of unspecific proteases such as Pronase can largely overcome this problem by generating glycopeptides with a small peptide portion. While the resulting glycopeptides are very useful for tandem mass spectrometric investigation, the analysis with conventional 1D-LC-ESI-MS/MS approaches can lead to incomplete glycosylation coverage because of the very heterogeneous physicochemical properties of the glycopeptides depending on the peptide sequence as well as the size and charges of the glycan moiety. Here, we describe a universal workflow for site-specific N- and O-glycopeptide analysis of Pronase treated glycoproteins with integrated, sequential C18 reverse phase and porous graphitized carbon-LC-ESI-QTOF-MS/MS employing a combination of lower- and enhanced-energy collision-induced dissociation. The approach was evaluated on glycoprotein standards and also applied to investigate the glycosylation of human IgG3 providing details on the hitherto uncharacterized glycosylation site Asn392 of the CH3 domain. This analytical tool can be applied to a variety of glycoproteins for site-specific N- and O-glycopeptide analysis, resulting in a good glycopeptide coverage within a single sample run and, thus, requiring only small amounts of sample.

Antibacterial potential of a small peptide from Bacillus sp. RPT-0001 and its capping for green synthesis of silver nanoparticles.

Infirmity and death from diseases caused by unsafe food are a continual hazard to communal health safety and socio-economic growth throughout the world. Chemical preservatives are associated with health hazards and toxicity issues. In the study reported here, 200 soil isolates from Western Himalayan region in India were screened for potential antibacterial activity against food-borne pathogens. This study led to the isolation of a bacterial strain belonging to the Genus Bacillus and was designated as RPT-0001. The associated antibacterial activity was sensitive to pronase E treatment. Bioassay-guided fractionation using reverse phase high performance liquid chromatography (RP-HPLC) led to isolation of the antibacterial peptide designated as RPT-0001. The molecular weight of RPT-0001 was determined by electro-spray ionization mass spectroscopy (ESI-MS) as 276.9 Da. RPT-0001 was inhibitory to both Gram-negative and Grampositive food-borne bacteria tested. The characteristics of RPT-0001 do not match with that of any other known antibacterial peptides produced by Bacillus sp. or related genera. Purified RPT-0001 was successfully used in synthesis of silver nanoparticles effective against food-borne pathogenic bacteria. The antibacterial peptide and silver nanoparticles synthesized utilizing it as a capping and reducing agent hold promising potential in food preservation, in packaging material and as a therapeutic agent in the treatment of foodborne infections.