Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

<p>Abstract</p> <p>Background</p> <p>Previous reports of site-directed deletion analysis on gamma (γ)-phage lysin protein (PlyG) have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199) abrogates its binding activity specific to the cell wall of <it>Bacillus anthracis</it>. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for <it>B. anthracis</it>. Furthermore, by using <it>B. anthracis </it>(Sterne, 34F2), an animal vaccine and <it>B. cereus</it>-<it>4342</it>, a γ-phage susceptible rare strain as surrogates of <it>B. anthracis</it>, development of proof-of-concepts for <it>B. anthracis </it>are feasible.</p> <p>Results</p> <p>Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6) for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both <it>B. anthracis </it>(Sterne, 34F2) and <it>B. cereus-</it>4342. The peptides however did not bind to <it>B. cereus</it>-11778, <it>B. thuringiensis</it>, and <it>B. cereus</it>-10876 suggesting their specificity for <it>B. anthracis</it>-Sterne and <it>B. cereus</it>-<it>4342</it>. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria.</p> <p>Conclusion</p> <p>Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for <it>B. anthracis</it>.</p

Identification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture

INTRODUCTION: XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture. METHODS: Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray. RESULTS: MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types. DISCUSSION: The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types

New proof-of-concept in viral inactivation: virucidal efficacy of 405 nm light against feline calicivirus as a model for norovirus decontamination

The requirement for novel decontamination technologies for use in hospitals is ever present. One such system uses 405 nm visible light to inactivate microorganisms via ROS-generated oxidative damage. Although effective for bacterial and fungal inactivation, little is known about the virucidal effects of 405 nm light. Norovirus (NoV) gastroenteritis outbreaks often occur in the clinical setting, and this study was designed to investigate potential inactivation effects of 405 nm light on the NoV surrogate, feline calicivirus (FCV). FCV was exposed to 405 nm light whilst suspended in minimal and organically-rich media to establish the virucidal efficacy and the effect biologically-relevant material may play in viral susceptibility. Antiviral activity was successfully demonstrated with a 4 Log10 (99.99%) reduction in infectivity when suspended in minimal media evident after a dose of 2.8 kJ cm−2. FCV exposed in artificial faeces, artificial saliva, blood plasma and other organically rich media exhibited an equivalent level of inactivation using between 50–85% less dose of the light, indicating enhanced inactivation when the virus is present in organically-rich biologically-relevant media. Further research in this area could aid in the development of 405 nm light technology for effective NoV decontamination within the hospital environment

A new proof-of-concept in bacterial reduction : antimicrobial action of violet-blue light (405 nm) in ex vivo stored plasma

Bacterial contamination of injectable stored biological fluids such as blood plasma and platelet concentrates preserved in plasma at room temperature is a major health-risk. Current pathogen-reduction technologies (PRT) rely on the use of chemicals and/or ultraviolet-light, which affects product quality and can be associated with adverse events in recipients. 405nm violet-blue light is antibacterial without the use of photosensitizers, and can be applied at levels safe for human exposure, making it of potential interest for decontamination of biological fluids such as plasma. As a pilot study to test whether 405nm light is capable of inactivating bacteria in biological fluids, rabbit and human plasma were seeded with bacteria and treated with a 405nm light emitting diode (LED) exposure system (patent pending). Inactivation was achieved in all tested samples, ranging from low volumes to pre-bagged plasma. 99.9% reduction of low density bacterial populations (≤103 CFUml-1), selected to represent typical ‘natural’ contamination levels, were achieved using doses of 144 Jcm-2. The penetrability of 405nm light, permitting decontamination of pre-bagged plasma, and the non-requirement for photosensitizing agents, provides a new proof-of-concept in bacterial reduction in biological fluids, especially injectable fluids relevant to transfusion medicine

405-nm light for bacterial reduction in blood plasma : preliminary investigations into antimicrobial efficacy and plasma protein integrity

Background: Pathogen reduction technologies (PRT) for blood products can reduce the incidence of transfusion-transmitted infection and associated wastage of blood products. Visible 405nm-light has been shown to inactivate bacteria in situ in bagged blood plasma without the addition of photo-sensitive chemicals. However, threshold levels for plasma protein compatibility and optimal bactericidal activity are currently unknown. This study investigates different treatment conditions and their suitability for safely inactivating bacteria in blood plasma. Method: Plasma seeded with Staphylococcus aureus (102–105CFU/ml) was exposed to 405nm-light at low and high irradiances (10, 100mW/cm2) with treatment times ranging between 0.2–7-hr (≤252 Jcm-2). SDS-PAGE was then used to assess the light effect in terms of antimicrobial treatment levels on plasma protein integrity. Results: High and low intensity treatment regimens achieved significant bacterial inactivation (P=144 Jcm-2. Conclusion: The results of this study have highlighted the safety potential of 405nm-light treatment on blood plasma. Further research is required to determine the upper and lower threshold treatment levels and functionality of plasma proteins post-exposure for further development of this technology as a PRT tool for application in transfusion medicine

Non-ionizing 405nm light as a potential bactericidal technology for platelet safety : evaluation of in vitro bacterial inactivation and in vivo platelet recovery in severe combined immunodeficient mice

Bacterial contamination of ex vivo stored platelets is a cause of transfusion-transmitted infection. Violet-blue 405 nm light has recently demonstrated efficacy in reducing the bacterial burden in blood plasma, and its operational benefits such as non-ionizing nature, penetrability, and non-requirement for photosensitizing agents, provide a unique opportunity to develop this treatment for in situ treatment of ex vivo stored platelets as a tool for bacterial reduction. Sealed bags of platelet concentrates, seeded with low-level Staphylococcus aureus contamination, were 405 nm light-treated (3-10 mWcm-2) up to 8 hr. Antimicrobial efficacy and dose efficiency was evaluated by quantification of the post-treatment surviving bacterial contamination levels. Platelets treated with 10 mWcm-2 for 8 hr were further evaluated for survival and recovery in severe combined immunodeficient (SCID) mice. Significant inactivation of bacteria in platelet concentrates was achieved using all irradiance levels, with 99.6-100% inactivation achieved by 8 hr (P<0.05). Analysis of applied dose demonstrated that lower irradiance levels generally resulted in significant decontamination at lower doses: 180 Jcm-2/10 mWcm-2 (P=0.008) compared to 43.2 Jcm-2/3 mWcm-2 (P=0.002). Additionally, the recovery of light-treated platelets, compared to non-treated platelets, in the murine model showed no significant differences (P ≥ 0.05). This report paves the way for further comprehensive studies to test 405 nm light treatment as a bactericidal technology for stored platelet

Antibacterial action of visible 405-nm light for bacterial reduction in blood plasma

The introduction of risk prevention measures, such as blood screening and donor deferrals have dramatically reduced the incidence of transfusion-transmitted viral infections. Nevertheless, bacterial contamination of blood transfusion products remains a concern to patient health, and a range of pathogen reduction technologies have been developed to reduce this risk. Visible violet-blue light, in the region of 405-nm, has recently demonstrated potential for in situ treatment of ex vivo stored plasma and platelet products, without the need for additional photosensitizers. This study assessed the broad-spectrum efficacy of 405-nm light against a range of bacteria implicated in transfusion-transmitted infections: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniaeandYersinia enterocolitica. Plasma was seeded with clinically-relevant low-level bacterial contamination (102-103CFUmL-1) and exposed to a 405-nm light dose of 360 Jcm-2 (1-hr at 100mWcm-2) using a small-scale exposure system. Broad spectrum antibacterial efficacy was observed, with 99.0 – 100% inactivation achieved for all bacterial species tested. Bacterial inactivation tests were then scaled-up to expose large volumes of prebagged plasma seeded with S. aureusat ~103 CFUmL-1, to 22mWcm-2 405-nm light, under agitation, (≤396 Jcm-2). Successful bacterial inactivation was observed using the large-scale exposure system, with a dose of 238 Jcm-2 (3-hr at ~22mWcm-2) achieving complete (3.5-log10) reductions in prebagged bacterial-seeded plasma (P=0.001). Results from this study support further development of visible 405-nm light technology as a bactericidal tool for application in transfusion medicine. This abstract reflects the views of the author and should not be construed to represent FDA’s views or policies

Human platelet concentrates treated with microbicidal 405 nm light retain hemostasis activity

Chemical and UV light-based pathogen reduction technologies are currently in use for human platelet concentrates (PCs) to enhance safety from transfusion-transmitted infections. Relative to UV light, 405 nm violet-blue light in the visible spectrum is known to be less harmful. Hence, in this report for the first time, we have assessed the global hemostasis activity of PCs stored in plasma and the activities of six plasma coagulation factors (CFs) as a measure of in vitro hemostatic activity following exposure to the microbicidal 405 nm light. Apheresis PC samples collected from each screened human donor (n = 22) were used for testing of PCs and platelet poor plasma (PPP). Both PCs and PPPs were treated for 5 h with 405 nm light to achieve a previously established microbicidal light dose of 270 J/cm2. Activated partial thromboplastin time and prothrombin time-based potency assays using a coagulation analyzer and hemostatic capacity via Thromboelastography were analyzed. Thromboelastography analysis of the light-treated PCs and plasma present in the PCs showed little difference between the treated and untreated samples. Further, plasma present in the PCs during the light treatment demonstrated a better stability in potency assays for several coagulation factors compared to the plasma alone prepared from PCs first and subjected to the light treatment separately. Overall, PCs stored in plasma treated with 405 nm violet-blue light retain activity for hemostasis

The preclinical validation of 405 nm light parasiticidal efficacy on Leishmania donovani in ex vivo platelets in a rag2−/− mouse model

Violet–blue light of 405 nm in the visible spectrum at a dose of 270 J/cm2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani-spiked platelets were evaluated in an immunodeficient Rag2−/− mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2−/− mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets