Pilot scale production of a phospholipid-enriched dairy ingredient by means of an optimised integrated process employing enzymatic hydrolysis, ultrafiltration and super-critical fluid extraction

Pilot scale production of a dairy ingredient enriched in phospholipids (PLs) was generated from a buttermilk powder (BMP) substrate utilising a combined process of targeted enzymatic hydrolysis of the innate milk proteins followed by ultrafiltration with a 50 kDa membrane. An 8.5 fold increase in PL was achieved in the 50 kDa retentate (50 R) compared to the BMP, 11.05 ± 0.02% and 1.30 ± 0.00% total PL, respectively. Simultaneously, total lipid content in the retentate increased 8.7 fold with reference to the BMP, 60.07 ± 0.54% and 6.84 ± 0.17% total lipid respectively. Protein reduced to 10.58 ± 0.09% (50 R) from 31.40 ± 0.57% in BMP. Supercritical CO2 fluid extraction (SFE) was employed to generate a purified lipid fraction. SFE with ethanol as a co-solvent yielded a purified lipid extract with enriched PLs level of 56.24 ± 0.07% on a dry matter basis. Industrial relevance: PLs have many associated health and nutritional benefits including those related to cognitive development and repair. Generation of an ingredient enriched in dairy PLs would be advantageous from an industrial view to allow fortification of nutritionals, both infant and geriatric, in promoting brain health. The present work demonstrates a novel pilot scale process for the generation of a PL enriched ingredient from a BMP substrate. Utilising a combined process of targeted protein hydrolysis followed by selective removal by ultrafiltration of the smaller molecular weight peptide material, an ingredient with 8.5 fold increase in PL material was achieved. SFE technology was utilised to generate a purified lipid extract with greater PL levels for future applications in biological assays to determine these pathways. The need for investigate and further develop the knowledge relating to the modes of action of these bioactive compounds would be beneficial from a nutritional perspective

Pilot scale production of a phospholipid-enriched dairy ingredient by means of an optimised integrated process employing enzymatic hydrolysis, ultrafiltration and super-critical fluid extraction

Pilot scale production of a dairy ingredient enriched in phospholipids (PLs) was generated from a buttermilk powder (BMP) substrate utilising a combined process of targeted enzymatic hydrolysis of the innate milk proteins followed by ultrafiltration with a 50 kDa membrane. An 8.5 fold increase in PL was achieved in the 50 kDa retentate (50 R) compared to the BMP, 11.05 ± 0.02% and 1.30 ± 0.00% total PL, respectively. Simultaneously, total lipid content in the retentate increased 8.7 fold with reference to the BMP, 60.07 ± 0.54% and 6.84 ± 0.17% total lipid respectively. Protein reduced to 10.58 ± 0.09% (50 R) from 31.40 ± 0.57% in BMP. Supercritical CO2 fluid extraction (SFE) was employed to generate a purified lipid fraction. SFE with ethanol as a co-solvent yielded a purified lipid extract with enriched PLs level of 56.24 ± 0.07% on a dry matter basis. Industrial relevance: PLs have many associated health and nutritional benefits including those related to cognitive development and repair. Generation of an ingredient enriched in dairy PLs would be advantageous from an industrial view to allow fortification of nutritionals, both infant and geriatric, in promoting brain health. The present work demonstrates a novel pilot scale process for the generation of a PL enriched ingredient from a BMP substrate. Utilising a combined process of targeted protein hydrolysis followed by selective removal by ultrafiltration of the smaller molecular weight peptide material, an ingredient with 8.5 fold increase in PL material was achieved. SFE technology was utilised to generate a purified lipid extract with greater PL levels for future applications in biological assays to determine these pathways. The need for investigate and further develop the knowledge relating to the modes of action of these bioactive compounds would be beneficial from a nutritional perspective

Unlikely Alliances and Their Implications for Resource Management in the American West

Collaborative, or participatory governance is an increasingly common means of addressing natural resource issues, especially in the American West where patchworks of public, private, and tribal interests characterize the region’s resources. In this context, unlikely alliances, or partnerships among diverse actors who have historically been at odds, have a growing potential to shape social and ecological outcomes, for better or worse. While these unlikely alliances have received greater attention in recent years, relatively little research has worked to synthesize the concept across diverse contexts and disciplines. Based on a review of the literature on unlikely alliances in natural resource governance, we develop a framework that synthesizes the individual motivations and contextual factors that influence their formation, as well as the social and ecological outcomes that they create. We use this framework to analyze six illustrative cases of unlikely alliances. Our analysis of these cases suggests that unlikely alliances in the American West are likely to arise in the presence of a crisis, when appropriate leadership is present, when some of the actors have interacted effectively in the past, and when actors need to pool resources. The cases also illustrate some common outcomes, including environmental improvement, transformation of social networks, policy change, and shifts in power relationships. We discuss the implications of unlikely alliances for the social-ecological future of the American West. Our paper highlights the role of unlikely alliances in shaping patterns of natural resource governance, and provides a focus for further research in this realm

Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers

<p>Abstract</p> <p>Background</p> <p><it>Haemophilus ducreyi</it>, the causative agent of the sexually transmitted disease chancroid, contains a <it>flp </it>(fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. <it>H. ducreyi </it>mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A <it>tadA </it>mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a <it>flp1flp2 </it>mutant is virulent in rabbits. Whether a <it>flp </it>deletion mutant would cause disease in humans is not clear.</p> <p>Results</p> <p>We constructed 35000HPΔ<it>flp1-3</it>, a deletion mutant that lacks expression of all three Flp proteins but has an intact <it>tad </it>secretion system. 35000HPΔ<it>flp1-3 </it>was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with <it>flp1-3 </it>in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔ<it>flp1-3 </it>(ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (<it>P </it>= 0.52). Mutant papules were significantly smaller (mean, 11.2 mm²) than were parent papules (21.8 mm²) 24 h after inoculation (<it>P </it>= 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (<it>P </it>= 0.001).</p> <p>Conclusion</p> <p>These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of <it>flp1-3 </it>is also necessary for <it>H. ducreyi </it>to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo.</p

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis

Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system

CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems

The Human Skin Microbiome Associates with the Outcome of and Is Influenced by Bacterial Infection

The influence of the skin microbiota on host susceptibility to infectious agents is largely unexplored. The skin harbors diverse bacterial species that may promote or antagonize the growth of an invading pathogen. We developed a human infection model for Haemophilus ducreyi in which human volunteers are inoculated on the upper arm. After inoculation, papules form and either spontaneously resolve or progress to pustules. To examine the role of the skin microbiota in the outcome of H. ducreyi infection, we analyzed the microbiomes of four dose-matched pairs of “resolvers” and “pustule formers” whose inoculation sites were swabbed at multiple time points. Bacteria present on the skin were identified by amplification and pyrosequencing of 16S rRNA genes. Nonmetric multidimensional scaling (NMDS) using Bray-Curtis dissimilarity between the preinfection microbiomes of infected sites showed that sites from the same volunteer clustered together and that pustule formers segregated from resolvers (P = 0.001, permutational multivariate analysis of variance [PERMANOVA]), suggesting that the preinfection microbiomes were associated with outcome. NMDS using Bray-Curtis dissimilarity of the endpoint samples showed that the pustule sites clustered together and were significantly different than the resolved sites (P = 0.001, PERMANOVA), suggesting that the microbiomes at the endpoint differed between the two groups. In addition to H. ducreyi, pustule-forming sites had a greater abundance of Proteobacteria, Bacteroidetes, Micrococcus, Corynebacterium, Paracoccus, and Staphylococcus species, whereas resolved sites had higher levels of Actinobacteria and Propionibacterium species. These results suggest that at baseline, resolvers and pustule formers have distinct skin bacterial communities which change in response to infection and the resultant immune response