Mapping the substrate-binding subsite specificity of a Porphyromonas gingivalis Tpr peptidase.

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion.

Biochemical and electrochemical characterization of biofilms formed on everolimus-eluting coronary stents.

Drug-eluting stents (DES) are mostly used in percutaneous coronary intervention, which is the main treatment for coronary artery occlusion. This procedure aims to restore the natural lumen, while minimizing the risk of restenosis. However, stent insertion increases the risk for infections, due to contamination of the device or insertion hub with normal skin flora. While coronary stent infection is a rare complication, it can be fatal. Currently, there is little information on biofilm formation on everolimus-eluting stents. Although everolimus is not designed as an antimicrobial agent, its antimicrobial activity should be investigated. In this study, biofilm formation on everolimus-eluting and bare metal stents (BMS) is characterized through biochemical and electrochemical methods. DES and BMS are inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, both independently and in co-culture. Biofilms formed on DES were 49.6 %, 12.9 % and 47.5 % higher than on BMS for P. aeruginosa, S. epidermidis and their co-culture, respectively. Further, the charge output for DES was 18.9 % and 59.7 % higher than BMS for P. aeruginosa and its co-culture with S. epidermidis, respectively. This observation is most likely due to higher surface roughness of DES, which favors biofilm formation. This work shows that bioelectrochemical methods can be used for rapid detection of biofilms on drug-eluting and bare metal stents, which may find application in quality assessment of stents and in characterization of stents removed after polymicrobial infections.

Biosynthesis of Gold Nanoparticles by Vascular Cells in vitro.

Biosynthesis of gold nanoparticles (AuNPs) for antimicrobial and chemotherapeutic applications is a well-established process in microbial hosts such as bacterial, fungi, and plants. However, reports on AuNPs biosynthesis in mammalian cells are scarce. In this study, bovine aortic endothelial cells (BAECs) and bovine aortic smooth muscle cells (BASMCs) were examined for their ability to synthesize AuNPs in vitro. Cell culture conditions such as buffer selection, serum concentration, and HAuCl4 concentration were optimized before the biosynthesized AuNPs were characterized through visible spectrometry, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. BAECs and BASMC produced small, spherical AuNPs that are semi-crystalline with a similar diameter (23 ± 2 nm and 23 ± 4 nm). Hydrogen peroxide pretreatment increased AuNPs synthesis, suggesting that antioxidant enzymes may reduce Au3+ ions as seen in microbial cells. However, buthionine sulfoximine inhibition of glutathione synthesis, a key regulator of oxidative stress, failed to affect AuNPs generation. Taken together, these results show that under the right synthesis conditions, non-tumor cell lines can produce detectable concentrations of AuNPs in vitro.

Integrating inflammatory serum biomarkers into a risk calculator for prostate cancer detection.

Improved prostate cancer detection methods would avoid over-diagnosis of clinically indolent disease informing appropriate treatment decisions. The aims of this study were to investigate the role of a panel of Inflammation biomarkers to inform the need for a biopsy to diagnose prostate cancer. Peripheral blood serum obtained from 436 men undergoing transrectal ultrasound guided biopsy were assessed for a panel of 18 inflammatory serum biomarkers in addition to Total and Free Prostate Specific Antigen (PSA). This panel was integrated into a previously developed Irish clinical risk calculator (IPRC) for the detection of prostate cancer and high-grade prostate cancer (Gleason Score ≥ 7). Using logistic regression and multinomial regression methods, two models (Logst-RC and Multi-RC) were developed considering linear and nonlinear effects of the panel in conjunction with clinical and demographic parameters for determination of the two endpoints. Both models significantly improved the predictive ability of the clinical model for detection of prostate cancer (from 0.656 to 0.731 for Logst-RC and 0.713 for Multi-RC) and high-grade prostate cancer (from 0.716 to 0.785 for Logst-RC and 0.767 for Multi-RC) and demonstrated higher clinical net benefit. This improved discriminatory power and clinical utility may allow for individualised risk stratification improving clinical decision making.

Disease-Relevant Single Cell Photonic Signatures Identify S100ß Stem Cells and their Myogenic Progeny in Vascular Lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100ß+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- ß1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100ß+ vSCs and identified the presence of S100ß+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.

Reactive Oxygen Species (ROS), Intimal Thickening, and Subclinical Atherosclerotic Disease.

Arteriosclerosis causes significant morbidity and mortality worldwide. Central to this process is the development of subclinical non-atherosclerotic intimal lesions before the appearance of pathologic intimal thickening and advanced atherosclerotic plaques. Intimal thickening is associated with several risk factors, including oxidative stress due to reactive oxygen species (ROS), inflammatory cytokines and lipid. The main ROS producing systems in-vivo are reduced nicotinamide dinucleotide phosphate (NADPH) oxidase (NOX). ROS effects are context specific. Exogenous ROS induces apoptosis and senescence, whereas intracellular ROS promotes stem cell differentiation, proliferation, and migration. Lineage tracing studies using murine models of subclinical atherosclerosis have revealed the contributory role of medial smooth muscle cells (SMCs), resident vascular stem cells, circulating bone-marrow progenitors and endothelial cells that undergo endothelial-mesenchymal-transition (EndMT). This review will address the putative physiological and patho-physiological roles of ROS in controlling vascular cell fate and ROS contribution to vascular regeneration and disease progression.

A Live Bio-Therapeutic for Mastitis, Containing Lactococcus lactis DPC3147 With Comparable Efficacy to Antibiotic Treatment.

Bovine mastitis is an ongoing significant concern in the dairy and agricultural industry resulting in substantial losses in milk production and revenue. Among the predominant etiological agents of bovine mastitis are Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli. Currently, the treatment of choice for bovine mastitis involves the use of commercial therapeutic antibiotic formulations such as TerrexineTM, containing both kanamycin and cephalexin. Such antibiotics are regularly administered in more than one dose resulting in the withholding of milk for processing for a number of days. Here, we describe the optimization of a formulation of Lactococcus lactis DPC3147, that produces the two-component bacteriocin lacticin 3147, in a liquid paraffin-based emulsion (formulation hereafter designated 'live bio-therapeutic') for the first time and compare it to the commercial antibiotic formulation TerrexineTM, with a view to treating cows with clinical/sub-clinical mastitis. Critically, in a field trial described here, this 'ready-to-use' emulsion containing live L. lactis DPC3147 cells exhibited comparable efficacy to TerrexineTM when used to treat mastitic cows. Furthermore, we found that the L. lactis cells within this novel emulsion-based formulation remained viable for up to 5 weeks, when stored at 4, 22, or 37°C. The relative ease and cost-effective nature of producing this 'live bio-therapeutic' formulation, in addition to its enhanced shelf life compared to previous aqueous-based formulations, indicate that this product could be a viable alternative therapeutic option for bovine mastitis. Moreover, the single-dose administration of this 'live bio-therapeutic' formulation is a further advantage, as it can expedite the return of the milk to the milk pool, in comparison to some commercial antibiotics. Overall, in this field trial, we show that the live bio-therapeutic formulation displayed a 47% cure rate compared to a 50% cure rate for a commercial antibiotic control, with respect to curing cows with clinical/sub-clinical mastitis. The study suggests that a larger field trial to further demonstrate efficacy is warranted.

Microbial bioelectrosynthesis of hydrogen: Current challenges and scale-up.

Sustainable energy supplies are needed to supplement and eventually replace fossil fuels. Molecular hydrogen H2 is a clean burning, high-energy fuel that is also used as reducing gas in industrial processes. H2 is mainly synthesized by steam reforming of natural gas, a non-renewable fuel. There are biosynthetic strategies for H2 production; however, they are associated with poor yield and have high cost. The application of an electrochemical driving force in a microbial electrolysis cell (MEC) improves the yield of biological reactions. The performance of the MEC is influenced by experimental parameters such as the electrode material, reactor design, microbial consortia and the substrate. In this review, factors that affect the performance of MECs are discussed and critically analysed. The potential for scale-up of H2 bioelectrosynthesis is also discussed.

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles.

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16±7nm, and 19±4nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43±19nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.

Fungal biosynthesis of gold nanoparticles: mechanism and scale up.

Gold nanoparticles (AuNPs) are a widespread research tool because of their oxidation resistance, biocompatibility and stability. Chemical methods for AuNP synthesis often produce toxic residues that raise environmental concern. On the other hand, the biological synthesis of AuNPs in viable microorganisms and their cell-free extracts is an environmentally friendly and low-cost process. In general, fungi tolerate higher metal concentrations than bacteria and secrete abundant extracellular redox proteins to reduce soluble metal ions to their insoluble form and eventually to nanocrystals. Fungi harbour untapped biological diversity and may provide novel metal reductases for metal detoxification and bioreduction. A thorough understanding of the biosynthetic mechanism of AuNPs in fungi is needed to reduce the time of biosynthesis and to scale up the AuNP production process. In this review, we describe the known mechanisms for AuNP biosynthesis in viable fungi and fungal protein extracts and discuss the most suitable bioreactors for industrial AuNP biosynthesis.

Effects of atmospheric air plasma treatment of graphite and carbon felt electrodes on the anodic current from Shewanella attached cells.

The attachment of electrochemically active microorganisms (EAM) on an electrode is determined by both the chemistry and topography of the electrode surface. Pre-treatment of the electrode surface by atmospheric air plasma introduces hydrophilic functional groups, thereby increasing cell attachment and electroactivity in short-term experiments. In this study, we use graphite and carbon felt electrodes to grow the model EAM Shewanella loihica PV-4 at oxidative potential (0.2 V vs. Ag/AgCl). Cell attachment and electroactivity are measured through electrodynamic methods. Atmospheric air plasma pre-treatment increases cell attachment and current output at graphite electrodes by 25%, while it improves the electroactivity of the carbon felt electrodes by 450%. Air plasma pre-treatment decreased the coulombic efficiency on both carbon felt and graphite electrodes by 60% and 80%, respectively. Microbially produced flavins adsorb preferentially at the graphite electrode, and air plasma pre-treatment results in lower flavin adsorption at both graphite and carbon felt electrodes. Results show that air plasma pre-treatment is a feasible option to increase current output in bioelectrochemical systems.