Automated Sample Preparation System for Rapid Biological Threat Detection

2005 ◽  
Author(s):  
Jeanne C. Stachowiak ◽  
Erin E. Shugard ◽  
Pamela Caton ◽  
Bruce P. Mosier ◽  
Ron Renzi ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Bacterial Species ◽  
Fused Silica ◽  
Fluorescent Labeling ◽  
Control Flow ◽  
Size Exclusion ◽  
Bacterial Cells ◽  
Fused Silica Capillaries ◽  
Exclusion Chromatography ◽  
Automated Sample Preparation

Rapid, automated sample preparation of bacterial cells and spores is required for threat analysis by remotely deployed chemical and biological warning systems. Sandia is designing, building, and testing an automated front-end sample preparation system based on miniature and microfluidic components, with the goal of concentrating bacterial species collected from the air, harvesting and solubilizing proteins from them, and delivering them to Sandia’s MicroChemLab capillary gel electrophoresis system1,2 for analysis (Fig. 1). Miniature, motorized valves and pumps control flow between system components connected by fused silica capillaries (Fig. 4). Sample processing modules include concentration by dielectrophoresis in an array of insulating posts or by mechanical filtration; heat-activated chemical lysis; mechanical filtration; removal of chemical lysis agents by size exclusion chromatography (SEC); and in-capillary fluorescent labeling.

scholarly journals Antibacterial Peptides Produced by Alcalase from Cowpea Seed Proteins

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 870
Author(s):  
Ali Osman ◽  
Gamal Enan ◽  
Abdul-Raouf Al-Mohammadi ◽  
Seham Abdel-Shafi ◽  
Samar Abdel-Hameid ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Seed Proteins ◽  
Negative Ions ◽  
Size Exclusion ◽  
Ionization Mass ◽  
Bacterial Cells ◽  
Antibacterial Efficiency ◽  
Exclusion Chromatography ◽  
Molecular Masses ◽  
Cowpea Seed

Cowpea seed protein hydrolysates (CPH) were output from cowpea seeds applying alcalase® from Bacillus licheniformis. CPH with an elevated level of hydrolysis was fractionated by size exclusion chromatography (SEC). Both CPH and SEC-portions showed to contain antimicrobial peptides (AMPs) as they inhibited both Gram-positive bacteria, such as Listeria monocytogenes LMG10470 (L. monocytogenes), Listeria innocua. LMG11387 (L. innocua), Staphylococcus aureus ATCC25923 (S.aureus), and Streptococcus pyogenes ATCC19615 (St.pyogenes), and Gram-negative bacteria, such as Klebsiella pnemoniae ATCC43816 (K. pnemoniae), Pseudomonas aeroginosa ATCC26853 (P. aeroginosa), Escherichia coli ATCC25468) (E.coli) and Salmonella typhimurium ATCC14028 (S. typhimurium).The data exhibited that both CPH and size exclusion chromatography-fraction 1 (SEC-F1) showed high antibacterial efficiency versus almost all the assessed bacteria. The MIC of the AMPs within SEC-F1 and CPHs were (25 µg/mL) against P. aeruginosa, E.coli and St. pyogenes. However, higher MICsof approximately 100–150 µg/mL showed for both CPHs and SEC-F1 against both S. aureus and L. innocua; it was 50 µg/mL of CPH against S.aureus. The Electro-spray-ionization-mass-spectrometry (ESI-MS) of fraction (1) revealed 10 dipeptides with a molecular masses arranged from 184 Da to 364 Da and one Penta peptide with a molecular mass of approximately 659 Da inthe case of positive ions. While the negative ions showed 4 dipeptides with the molecular masses that arranged from 330 Da to 373 Da. Transmission electron microscope (TEM) demonstrated that the SEC-F1 induced changes in the bacterial cells affected. Thus, the results suggested that the hydrolysis of cowpea seed proteins by Alcalase is an uncomplicated appliance to intensify its antibacterial efficiency.

scholarly journals Structural Characterization of Human Immunity-Related GTPase Family M (IRGM)

2014 ◽  
Vol 70 (a1) ◽  
pp. C835-C835
Author(s):  
Sarah Sabboobeh ◽  
Bhushan Nagar
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Mechanism Of Action ◽  
Mitochondrial Fission ◽  
Potential Interaction ◽  
Size Exclusion ◽  
Bacterial Cells ◽  
Structure Based Drug Design ◽  
Cellular Autophagy ◽  
Exclusion Chromatography

The Immunity-Related GTPase Family M protein (IRGM) is involved in regulating cellular autophagy. Cellular knockdown of IRGM was shown to allow RNA viruses to hijack the autophagic immune response. Additionally, recent genetic studies have shown that underexpression of IRGM is associated with the incidence of Crohn's disease and infection by Mycobacterium tuberculosis. IRGM is an interferon-induced GTPase with an evolutionary conserved P-loop. It is an effector of the interferon-gamma pathway, but, unlike its protein family members, is not directly activated by the pathway. Its mechanism of action has been proposed to occur by translocation of IRGM to the mitochondria through recognition of cardiolipin, and affecting mitochondrial fission to induce autophagy. This potential interaction with cardiolipin might indicate the presence of a unique GTPase recognition and activation fold within IRGM. Our goal is to determine the X-ray crystal structure of IRGM in an effort to understand its molecular role in normal and diseased states. Additionally, we seek to test its interaction with and mechanism of recognition to mitochondrial cardiolipin as well as other autophagy-inducing binding partners. Currently, we have managed to express human IRGM in bacterial cells and have purified it to homogeneity using affinity and size-exclusion chromatography. These findings will serve to elucidate the mechanism of action of IRGM. Crucially, we hope to gain an understanding of its contributing role to Crohn's disease and tuberculosis infection at the molecular level, potentially paving the way to structure-based drug design and therapeutic opportunities.

scholarly journals DMBT1 inhibition of Pseudomonas aeruginosa twitching motility involves its N-glycosylation and cannot be conferred by the Scavenger Receptor Cysteine-Rich bacteria-binding peptide domain

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jianfang Li ◽  
Stephanie J. Wan ◽  
Matteo M. E. Metruccio ◽  
Sophia Ma ◽  
Kamran Nazmi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Capacity ◽  
Bacterial Species ◽  
Consensus Sequence ◽  
Scavenger Receptor ◽  
Size Exclusion ◽  
Molecular Characteristics ◽  
Twitching Motility ◽  
Binding Peptide ◽  
Exclusion Chromatography

Abstract The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1–220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

scholarly journals Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity

2021 ◽  
Vol 11 ◽  
Author(s):  
Marek Bogacz ◽  
Faiha M. El Abbar ◽  
Claudia A. Cox ◽  
Jiaqi Li ◽  
Jarred S. Fiedler ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Binding Sites ◽  
Rna Binding ◽  
Bacterial Species ◽  
Size Exclusion ◽  
Mrna Targets ◽  
C Terminus ◽  
Exclusion Chromatography ◽  
Mrna Binding ◽  
Rna Binding Sites

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

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