Enhancing polypropylene bioconversion and lipogenesis by Yarrowia lipolytica using a chemical/biological hybrid process.

Plastic waste can serve as a feedstock for microbial bioconversion using a chemical/biological hybrid strategy. We developed a polypropylene (PP) upcycling process that coupled pyrolysis with bioconversion by the oleaginous yeast Yarrowia lipolytica. Using virgin PP, we optimized pH, inoculum density, C/N ratio, and osmolarity and increased the fatty acid titer nearly four-fold to 1.9 g L-1, with 41 percent cellular fatty acid content, the highest content reported to date for plastic-to-lipid microbial bioconversion. The highest fatty acid titer was achieved with an inoculum density of 3 (OD 600 nm), pH = 6.0 and C/N ratio of 80:1. Increasing the medium osmolarity by adding sodium chloride adversely affected cell growth and did not improve the fatty acid titer. The maximum fatty acid titer occurred under conditions that balanced cell growth versus lipogenesis. Using postconsumer PP, the fatty acid titer was significantly lower (0.13 g L-1). Overall, the work demonstrates the potential and the challenges associated with microbial bioconversion of plastics.

4-Ethoxybenzoic acid inhibits Staphylococcus aureus biofilm formation and potentiates biofilm sensitivity to vancomycin.

The adverse health effects of Staphylococcus aureus biofilm infections coupled with an increased global prevalence of antibiotic resistance highlight the need for novel anti-pathogenic, anti-biofilm compounds. The authors recently determined that ethyl-4-ethoxybenzoic acid (EEB) had anti-pathogenic, anti-biofilm activity. Based on this finding, a structure-activity analysis was undertaken to identify more effective compounds. Microtitre crystal violet assays followed by plate counts were conducted to measure the dose-dependent anti-biofilm and antimicrobial activities of 13 phenolic compounds related to EEB. By displaying these characteristics on a two-component plot, 4-ethoxybenzoic acid (4EB) and methyl gallate were identified as two anti-pathogenic, anti-biofilm compounds of interest. To characterize their mechanisms of activity, their effects on cell hydrophobicity, hemolysis activity, membrane integrity, extracellular polymeric substance production and vancomycin sensitivity were examined. Both 4EB and methyl gallate inhibited up to 87% of biofilm formation with minimal impact on the viability of stationary-phase cells or bacterial growth. Combination treatments of 4EB and vancomycin decreased the viability of biofilm-dwelling cells by up to 85% compared with vancomycin alone, indicating a synergistic effect. Methyl gallate did not potentiate vancomycin. 4EB decreased the percentage of hydrophobic cells in culture from 78% to 49%, indicating that 4EB may prevent biofilm formation by altering cell membrane hydrophobicity. These findings suggest that 4EB has potential as an anti-pathogenic, anti-biofilm agent for the prevention of S. aureus biofilms, or as a treatment for established biofilms when combined with antibiotics.

Microbial bioconversion of thermally depolymerized polypropylene by Yarrowia lipolytica for fatty acid production.

Plastic production and waste generation will continue to rise as nations worldwide grow economically. In this work, we detail a pyrolysis-based bioconversion process for polypropylene (PP) to produce value-added fatty acids (FAs). PP pellets were depolymerized by pyrolysis, generating oil that consisted of mainly branched chain fatty alcohols and alkenes. The oil was mixed with biodegradable surfactants and trace nutrients and mechanically homogenized. The resulting medium, OP4, was used for fermentation by Yarrowia lipolytica strain 78-003. Y. lipolytica assimilated > 80% of the substrate over 312 h, including 86% of the fatty alcohols. Y. lipolytica produced up to 492 mg L-1 lipids, compared with 216 mg L-1 during growth in surfactant-based control medium. C 18 compounds, including oleic acid, linoleic acid, and stearic acid, were the predominant products, followed by C 16 compounds palmitic and palmitoleic acids. Two percent of the products was C 20 compounds. The majority of the products were unsaturated FAs. Growth on hydrophobic substrates (OP4 medium, hexadecane) was compared with growth on hydrophilic substrates (glucose, starch). The resulting FA profiles revealed an absence of short-chain fatty acids during growth on hydrophobic media, findings consistent with ex novo FA biosynthesis. Overall, FA profiles by Y. lipolytica during growth in OP4 medium were similar to FA profiles while growing on natural substrates. The process described here offers an alternative approach to managing postconsumer plastic waste.

Rhamnus prinoides (gesho): A source of diverse anti-biofilm activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhamnus prinoides (gesho) is an evergreen shrub from East Africa traditionally used for the treatment of illnesses including atopic dermatitis, ear, nose and throat infections, pneumonia, arthritis, brucellosis, flu, indigestion and fatigue. AIM OF THE STUDY: Several of the conditions for which gesho is traditionally used are associated with communities of surface-attached microorganisms, or biofilms. We hypothesized that gesho has anti-biofilm activity. The principal aim of this study was to evaluate gesho-associated anti-biofilm activity and identify active compounds. MATERIALS AND METHODS: Lyophilized ethanol and aqueous extracts were prepared from dried Rhamnus prinoides stems and leaves. Biofilm inhibition was measured by crystal violet staining and subsequent viability assays were conducted on growth agar. Chemical fractionation, chemical testing, Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) were used to isolate and identify active compounds. RESULTS: Leaf and stem ethanol extracts significantly inhibited Staphylococcus aureus, Bacillus subtilis and Streptococcus mutans biofilm formation up to 99.9% and reduced planktonic cell growth up to 10 log units relative to untreated controls. The anti-biofilm activity of the ethanol stem extracts was due to a biocidal or bacteriostatic mechanism while bacteriostatic or anti-pathogenic mechanisms were attributed to the leaf ethanol extract. Gesho extracts showed activity against all three species tested but the treatment efficacy and mechanism were species dependent. Chemical fractionation and activity screens of the leaf ethanol extract identified ethyl 4-ethoxybenzoate and 4-hydroxy 4-methyl pentanone to be compounds with anti-biofilm activity. Ethyl 4-ethoxybenzoate activity was potentiated by DMSO. Notably, concentrations of both compounds were identified where biofilm formation was prevented without inhibition of cell growth; i.e. anti-pathogenic characteristics were evident. CONCLUSION: Gesho leaf ethanol extract contains chemicals with anti-biofilm and bactericidal activities. This work lends support to the traditional use of gesho for treating topical infections and warrants further investigation into Rhamnus prinoides as a source of antibacterial and anti-biofilm agents.

Molecules and Mechanisms Underlying the Antimicrobial Activity of Escapin, an l-Amino Acid Oxidase from the Ink of Sea Hares.

Many marine animals use chemicals to defend themselves and their eggs from predators. Beyond their ecologically relevant functions, these chemicals may also have properties that make them beneficial for humans, including biomedical and industrial applications. For example, some chemical defenses are also powerful antimicrobial or antitumor agents with relevance to human health and disease. One such chemical defense, escapin, an l-amino acid oxidase in the defensive ink of the sea hare Aplysia californica, and related proteins have been investigated for their biomedical properties. This review details our current understanding of escapin's antimicrobial activity, including the array of molecules generated by escapin's oxidation of its major substrates, l-lysine and l-arginine, and mechanisms underlying these molecules' bactericidal and bacteriostatic effects on planktonic cells and the prevention of formation and removal of bacterial biofilms. Models of escapin's effects are presented, and future directions are proposed.

Discovery of Chromane Propionic Acid Analogues as Selective Agonists of GPR120 with in Vivo Activity in Rodents.

GPR120 (FFAR4) is a fatty acid sensing G protein coupled receptor (GPCR) that has been identified as a target for possible treatment of type 2 diabetes. A selective activator of GPR120 containing a chromane scaffold has been designed, synthesized, and evaluated in vivo. Results of these efforts suggest that chromane propionic acid 18 is a suitable tool molecule for further animal studies. Compound 18 is selective over the closely related target GPR40 (FFAR1), has a clean off-target profile, demonstrates suitable pharmacokinetic properties, and has been evaluated in wild-type/knockout GPR120 mouse oGTT studies.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

Inhibition and Dispersal of Pseudomonas aeruginosa Biofilms by Combination Treatment with Escapin Intermediate Products and Hydrogen Peroxide.

Escapin is an l-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. We initiated the present study to investigate how the combination of EIP and H2O2 affected bacterial biofilms, using Pseudomonas aeruginosa as a model. Specifically, we examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to biofilm formation in untreated controls or with EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to that of the controls. Area layer analysis of biofilms posttreatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, and these concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to the effect of either EIP or H2O2 alone. Collectively, our results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.

Comparison of limited-contact dynamic compression plate and locking compression plate constructs for proximal interphalangeal joint arthrodesis in the horse.

This study compared in vitro monotonic and cyclic mechanical properties of equine proximal interphalangeal joint arthrodeses stabilized using an open or closed technique combined with axial 4.5 mm narrow limited-contact dynamic compression plate (LC-DCP) or 4.5 mm narrow locking compression plate (LCP). Ten forelimb pairs were randomly assigned to LCP or LC-DCP groups. One limb in each pair was assigned to either open or closed technique. Limbs were tested for cyclic fatigue at 20 000 cycles and then single-cycle to failure under 3-point dorsopalmar bending. There was no significant difference in stiffness of constructs during cyclic fatigue testing or on force or stiffness at failure in single cycle to failure testing between open and closed techniques or between plate types. Both implants, surgical technique, or combinations thereof are suitable for clinical use. More work is necessary to define the interaction between implant type and surgical technique.

Comparaison des constructions de plaques de compression dynamique à contact limité et de plaques de compression à verrouillage pour l'arthrodèse de l'articulation interphalangienne proximale chez les chevaux. Cette étude a comparé les propriétés mécaniques monotoniques et cycliques in vitro des arthrodèses interphalangiennes proximales équines stabilisées à l'aide d'une technique ouverte ou fermée combinée à une plaque de compression dynamique axiale étroite à contact limité de 4,5 mm (LC-DCP) ou à une plaque de compression étroite à verrouillage de 4,5 mm (LCP). Dix paires de pattes avant ont été assignées au hasard à des groupes LCP ou LC-DCP. Une patte de chaque paire était assignée soit à la technique ouverte ou fermée. Les pattes ont été évaluées pour la fatigue cyclique à 20 000 cycles, puis à un cycle unique jusqu'à l'échec à l'aide d'une flexion dorsopalmaire à 3 points. Il n'y avait pas de différence significative dans la rigidité des constructions durant l'évaluation de fatigue cyclique ou sur la force ou la rigidité à l'échec durant l'évaluation dans un cycle unique jusqu'à l'échec entre les techniques ouvertes et fermées ou entre les types de plaques. Les deux implants, techniques chirurgicales ou une combinaison des deux sont appropriés à l'usage clinique. Il est nécessaire d'effectuer des recherches additionnelles pour définir l'interaction entre le type d'implant et la technique chirurgicale.(Traduit par Isabelle Vallières).

Red, purple and pink: the colors of diffusion on pinterest.

Many lab studies have shown that colors can evoke powerful emotions and impact human behavior. Might these phenomena drive how we act online? A key research challenge for image-sharing communities is uncovering the mechanisms by which content spreads through the community. In this paper, we investigate whether there is link between color and diffusion. Drawing on a corpus of one million images crawled from Pinterest, we find that color significantly impacts the diffusion of images and adoption of content on image sharing communities such as Pinterest, even after partially controlling for network structure and activity. Specifically, Red, Purple and pink seem to promote diffusion, while Green, Blue, Black and Yellow suppress it. To our knowledge, our study is the first to investigate how colors relate to online user behavior. In addition to contributing to the research conversation surrounding diffusion, these findings suggest future work using sophisticated computer vision techniques. We conclude with a discussion on the theoretical, practical and design implications suggested by this work-e.g. design of engaging image filters.

1,3-Oxazines as BACE1 and/or BACE2 inhibitors: a patent evaluation (WO2012156284).

This patent review covers the contents of Hoffman-La Roche and Siena Biotech's patent application WO2012156284 titled '1,3-Oxazines as BACE1 and/or BACE2 Inhibitors.' Beta-site amyloid precursor protein-converting enzyme (BACE1) and BACE2 activities are reported to support the claimed compounds' use as therapeutics for Alzheimer's disease and type II diabetes, respectively. A common core motif of the claimed compounds is the six-membered 1,3-oxazine system. To gain access to the S3 and S3 subpocket of the BACE1 active site, various linkers are described including nitrogen- and oxygen-based, aryl, and amide-based linkers. Of the 65 compounds claimed, 6 had IC50s less than 100 nM in the BACE1 cell assay. Cellular BACE2 inhibition data are reported for 20 compounds with 2 under 100 nM.

LuxS influences Escherichia coli biofilm formation through autoinducer-2-dependent and autoinducer-2-independent modalities.

Escherichia coli produces biofilms in response to the small molecule autoinducer-2 (AI-2), a product of the LuxS enzyme. LuxS is part of the activated methyl cycle and could also affect biofilm development by AI-2-independent effects on metabolism. A luxS deletion mutant of E. coli W3110 and an inducible plasmid-luxS-complemented strain were used to identify AI-2-independent phenotypes. Differential interference contrast microscopy revealed distinct surface colonization patterns. Confocal microscopy followed by quantitative image analysis determined differences in biofilm topography correlating with luxS expression; deletion mutant biofilms had a 'spreading' phenotype, whereas the complement had a 'climbing' phenotype. Addition of exogenous 4,5-dihydroxy-2,3-pentanedione (DPD), an AI-2 precursor, to the deletion mutant increased biofilm height and biomass, whereas addition of the methyl donor S-adenosyl methionine or aspartate prevented the luxS-complemented strain from producing a thick biofilm. The luxS-complemented strain autoaggregated, indicating that fimbriae production was inhibited, which was confirmed by transmission electron microscopy. DPD could not induce autoaggregation in the deletion mutant, demonstrating that fimbriation was an AI-2-independent phenotype. Carbon utilization was affected by LuxS, potentially contributing to the observed phenotypic differences. Overall, the work demonstrated that LuxS affected E. coli biofilm formation independently of AI-2 and could assist in adapting to diverse conditions.

Discovery of an Orally Available, Brain Penetrant BACE1 Inhibitor that Affords Robust CNS Aß Reduction.

Inhibition of BACE1 to prevent brain Aß peptide formation is a potential disease-modifying approach to the treatment of Alzheimer's disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aß levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aß40 levels when administered orally to rats.

Recurrent Aspergillus contamination in a biomedical research facility: a case study.

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.

Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity.

The diagnosis of bacterial infections remains a major challenge in medicine. Although numerous contrast agents have been developed to image bacteria, their clinical impact has been minimal because they are unable to detect small numbers of bacteria in vivo, and cannot distinguish infections from other pathologies such as cancer and inflammation. Here, we present a family of contrast agents, termed maltodextrin-based imaging probes (MDPs), which can detect bacteria in vivo with a sensitivity two orders of magnitude higher than previously reported, and can detect bacteria using a bacteria-specific mechanism that is independent of host response and secondary pathologies. MDPs are composed of a fluorescent dye conjugated to maltohexaose, and are rapidly internalized through the bacteria-specific maltodextrin transport pathway, endowing the MDPs with a unique combination of high sensitivity and specificity for bacteria. Here, we show that MDPs selectively accumulate within bacteria at millimolar concentrations, and are a thousand-fold more specific for bacteria than mammalian cells. Furthermore, we demonstrate that MDPs can image as few as 10(5) colony-forming units in vivo and can discriminate between active bacteria and inflammation induced by either lipopolysaccharides or metabolically inactive bacteria.

Expansion of SAR studies on triaryl bis sulfone cannabinoid CB2 receptor ligands.

We report further expansion of the structure activity relationship (SAR) on the triaryl bis sulfone class of compounds (I), which are potent CB(2) receptor ligands with excellent selectivity over the CB(1) receptor. This study was extended to B ring changes, followed by simultaneous optimization of the A-, B-, and C-rings. Compound 42 has excellent CB(2) potency, selectivity and rat exposure.

Patterns of antimicrobial therapy in severe nosocomial infections: empiric choices, proportion of appropriate therapy, and adaptation rates--a multicentre, observational survey in critically ill patients.

This prospective, observational multicentre (n=24) study investigated relationships between antimicrobial choices and rates of empiric appropriate or adequate therapy, and subsequent adaptation of therapy in 171 ICU patients with severe nosocomial infections. Appropriate antibiotic therapy was defined as in vitro susceptibility of the causative pathogen and clinical response to the agent administered. In non-microbiologically documented infections, therapy was considered adequate in the case of favourable clinical response <5 days. Patients had pneumonia (n=127; 66 ventilator-associated), intra-abdominal infection (n=23), and bloodstream infection (n=21). Predominant pathogens were Pseudomonas aeruginosa (n=29) Escherichia coli (n=26), Staphylococcus aureus (n=22), and Enterobacter aerogenes (n=21). In 49.6% of infections multidrug-resistant (MDR) bacteria were involved, mostly extended-spectrum beta-lactamase (EBSL)-producing Enterobacteriaceae and MDR non-fermenting Gram-negative bacteria. Prior antibiotic exposure and hospitalisation in a general ward prior to ICU admission were risk factors for MDR. Empiric therapy was appropriate/adequate in 63.7% of cases. Empiric schemes were classified according to coverage of (i) ESBL-producing Enterobacteriaceae and non-fermenting Gram-negative bacteria ("meropenem-based"), (ii) non-fermenting Gram-negative bacteria (schemes with an antipseudomonal agent), and (iii) first-line agents not covering ESBL-Enterobacteriaceae nor non-fermenting Gram-negative bacteria. Meropenem-based schemes allowed for significantly higher rates of appropriate/adequate therapy (p<0.001). This benefit remained when only patients without risk factors for MDR were considered (p=0.021). In 106 patients (61%) empiric therapy was modified: in 60 cases following initial inappropriate/inadequate therapy, in 46 patients in order to refine empiric therapy. In this study reflecting real-life practice, first-line use of meropenem provided significantly higher rates of the appropriate/adequate therapy, irrespective of presence of risk factors for MDR.

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria.

Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.

Agarose stabilization of fragile biofilms for quantitative structure analysis.

Agarose was used to stabilize fragile biofilms cultivated in parallel plate flow cells prior to imaging by confocal laser scanning microscopy. An essential element to the success of the procedure was the application of a ceramic heat pad to the flow cell to maintain agarose fluidity until the biofilm was enveloped. Quantitative digital image analysis demonstrated the effectiveness of this technique for generating reproducible measurements of a three-dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging.

Non-aromatic A-ring replacement in the triaryl bis-sulfone CB2 receptor inhibitors.

The triaryl bis-sulfone 1 was modified by converting the aryl A-ring to a piperidine ring. The piperidine ring was further elaborated to a spirocyclopropyl piperidine moiety. The effect on CB2 binding potency, rat calcium channel affinity, and CYP 2C9 inhibition is described.