Metabolomic Analysis of Cold Acclimation of Arctic Mesorhizobium sp. Strain N33

Arctic Mesorhizobium sp. N₃₃ isolated from nodules of Oxytropis arctobia in Canada’s eastern Arctic has a growth temperature range from 0°C to 30°C and is a well-known cold-adapted rhizobia. The key molecular mechanisms underlying cold adaptation in Arctic rhizobia remains totally unknown. Since the concentration and contents of metabolites are closely related to stress adaptation, we applied GC-MS and NMR to identify and quantify fatty acids and water soluble compounds possibly related to low temperature acclimation in strain N₃₃. Bacterial cells were grown at three different growing temperatures (4°C, 10°C and 21°C). Cells from 21°C were also cold-exposed to 4°C for different times (2, 4, 8, 60 and 240 minutes). We identified that poly-unsaturated linoleic acids 18∶2 (9, 12) & 18∶2 (6, 9) were more abundant in cells growing at 4 or 10°C, than in cells cultivated at 21°C. The mono-unsaturated phospho/neutral fatty acids myristoleic acid 14∶1(11) were the most significantly overexpressed (45-fold) after 1hour of exposure to 4°C. As reported in the literature, these fatty acids play important roles in cold adaptability by supplying cell membrane fluidity, and by providing energy to cells. Analysis of water-soluble compounds revealed that isobutyrate, sarcosine, threonine and valine were more accumulated during exposure to 4°C. These metabolites might play a role in conferring cold acclimation to strain N₃₃ at 4°C, probably by acting as cryoprotectants. Isobutyrate was highly upregulated (19.4-fold) during growth at 4°C, thus suggesting that this compound is a precursor for the cold-regulated fatty acids modification to low temperature adaptation.     

Glycerol Monolaurate and Dodecylglycerol Effects on Staphylococcus aureus and Toxic Shock Syndrome Toxin-1 In Vitro and In Vivo

Background

Glycerol monolaurate (GML), a 12 carbon fatty acid monoester, inhibits Staphylococcus aureus growth and exotoxin production, but is degraded by S. aureus lipase. Therefore, dodecylglycerol (DDG), a 12 carbon fatty acid monoether, was compared in vitro and in vivo to GML for its effects on S. aureus growth, exotoxin production, and stability.

Methodology/Principal Findings

Antimicrobial effects of GML and DDG (0 to 500 µg/ml) on 54 clinical isolates of S. aureus, including pulsed-field gel electrophoresis (PFGE) types USA200, USA300, and USA400, were determined in vitro. A rabbit Wiffle ball infection model assessed GML and DDG (1 mg/ml instilled into the Wiffle ball every other day) effects on S. aureus (MN8) growth (inoculum 3×10⁸ CFU/ml), toxic shock syndrome toxin-1 (TSST-1) production, tumor necrosis factor-α (TNF-α) concentrations and mortality over 7 days. DDG (50 and 100 µg/ml) inhibited S. aureus growth in vitro more effectively than GML (p<0.01) and was stable to lipase degradation. Unlike GML, DDG inhibition of TSST-1 was dependent on S. aureus growth. GML-treated (4 of 5; 80%) and DDG-treated rabbits (2 of 5; 40%) survived after 7 days. Control rabbits (5 of 5; 100%) succumbed by day 4. GML suppressed TNF-α at the infection site on day 7; however, DDG did not (<10 ng/ml versus 80 ng/ml, respectively).

Conclusions/Significance

These data suggest that DDG was stable to S. aureus lipase and inhibited S. aureus growth at lower concentrations than GML in vitro. However, in vivo GML was more effective than DDG by reducing mortality, and suppressing TNF-α, S. aureus growth and exotoxin production, which may reduce toxic shock syndrome. GML is proposed as a more effective anti-staphylococcal topical anti-infective candidate than DDG, despite its potential degradation by S. aureus lipase.     

Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection

Cytosolic fungal fatty acid synthase is composed of two subunits α and β, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14∶0), palmitic acid (16∶0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target.     

Capsaicin protects mice from community-associated methicillin-resistant Staphylococcus aureus pneumonia

Background

α-toxin is one of the major virulence factors secreted by most Staphylococcus aureus strains, which played a central role in the pathogenesis of S. aureus pneumonia. The aim of this study was to investigate the impact of capsaicin on the production of α-toxin by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA 300 and to further assess its performance in the treatment of CA-MRSA pneumonia in a mouse model.

Methodology/Principal Findings

The in vitro effects of capsaicin on α-toxin production by S. aureus USA 300 were determined using hemolysis, western blot, and real-time RT-PCR assays. The influence of capsaicin on the α-toxin-mediated injury of human alveolar epithelial cells was determined using viability and cytotoxicity assays. Mice were infected intranasally with S. aureus USA300; the in vivo protective effects of capsaicin against S. aureus pneumonia were assessed by monitoring the mortality, histopathological changes and cytokine levels. Low concentrations of capsaicin substantially decreased the production of α-toxin by S. aureus USA 300 without affecting the bacterial viability. The addition of capsaicin prevented α-toxin-mediated human alveolar cell (A549) injury in co-culture with S. aureus. Furthermore, the in vivo experiments indicated that capsaicin protected mice from CA-MRSA pneumonia caused by strain USA 300.

Conclusions/Significance

Capsaicin inhibits the production of α-toxin by CA-MRSA strain USA 300 in vitro and protects mice from CA-MRSA pneumonia in vivo. However, the results need further confirmation with other CA-MRSA lineages. This study supports the views of anti-virulence as a new antibacterial approach for chemotherapy.     

Biosynthesis and Characterization of Polyhydroxyalkanoates Copolymers Produced by Pseudomonas putida Bet001 Isolated from Palm Oil Mill Effluent

The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C_8∶0) to oleic acid (C_18∶1) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the ¹H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T _d) of 264.6 to 318.8 (±0.2) ^oC, melting temperature (T _m) of 43. (±0.2) ^oC, glass transition temperature (T _g) of −1.0 (±0.2) ^oC and apparent melting enthalpy of fusion (ΔH _f) of 100.9 (±0.1) J g⁻¹.     

Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community‐acquired methicillin‐resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG‐DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short‐chain fatty acids including acetic, butyric and propionic acids with anti‐USA300 activities are produced by PEG‐DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis‐laden PEG‐DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG‐DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.     

Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of Veillonella parvula and Megasphaera elsdenii Phospholipids

Veillonella parvula ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In Megasphaera elsdenii lipids, which are similar in composition to the lipids of V. parvula, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens. M. elsdenii contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.     

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.     

Growth of Candida ingens on Supernatant from Anaerobically Fermented Pig Waste: Effects of Temperature and pH

Candida ingens, a pellicle-forming yeast utilizing volatile fatty acids, grew over a pH range of 4.1 to 6.0 on nonsterile supernatants from anaerobically fermented pig wastes; growth was inconsistent between pH 4.1 and 4.6. When ambient temperature above the pellicle was 21°C and the temperature of the medium was 29 to 32°C, a pH range of 4.8 to 5.0 gave yields of 1.90 to 3.31 g of dry matter per liter, and 0.059 to 0.065 mol of volatile fatty acids was utilized per liter. There was no advantage in utilization of volatile fatty acids and yield of dry matter in keeping the pH constant during a 24-h growth period. C. ingens grew at pH 4.8 and 5.0 when both ambient and medium temperatures were 30°C. When ambient temperature was 10°C, maximum yield and utilization of volatile fatty acids occurred at a medium temperature of 28 to 30°C.     

Importance of Fatty Acid Compositions in Patients with Peripheral Arterial Disease

Objective

Importance of fatty acid components and imbalances has emerged in coronary heart disease. In this study, we analyzed fatty acids and ankle-brachial index (ABI) in a Japanese cohort.

Methods

Peripheral arterial disease (PAD) was diagnosed in 101 patients by ABI ≤0.90 and/or by angiography. Traditional cardiovascular risk factors and components of serum fatty acids were examined in all patients (mean age 73.2±0.9 years; 81 males), and compared with those in 373 age- and sex-matched control subjects with no evidence of PAD.

Results

The presence of PAD (mean ABI: 0.71±0.02) was independently associated with low levels of gamma-linolenic acid (GLA) (OR: 0.90; 95% CI: 0.85–0.96; P = 0.002), eicosapentaenoic acid∶arachidonic acid (EPA∶AA) ratio (OR: 0.38; 95% CI: 0.17–0.86; P = 0.021), and estimated glomerular filtration rate (OR: 0.97; 95% CI: 0.96–0.98; P<0.0001), and with a high hemoglobin A1c level (OR: 1.34; 95% CI: 1.06–1.69; P = 0.013). Individuals with lower levels of GLA (≤7.95 µg/mL) and a lower EPA∶AA ratio (≤0.55) had the lowest ABI (0.96±0.02, N = 90), while the highest ABI (1.12±0.01, N = 78) was observed in individuals with higher values of both GLA and EPA∶AA ratio (P<0.0001).

Conclusion

A low level of GLA and a low EPA∶AA ratio are independently associated with the presence of PAD. Specific fatty acid abnormalities and imbalances could lead to new strategies for risk stratification and prevention in PAD patients.     

Potential Use of Avocado Oil on Structured Lipids MLM-Type Production Catalysed by Commercial Immobilised Lipases

Structured Lipids are generally constituents of functional foods. Growing demands for SL are based on a fuller understanding of nutritional requirements, lipid metabolism, and improved methods to produce them. Specifically, this work was aimed to add value to avocado oil by producing dietary triacylglycerols (TAG) containing medium-chain fatty acids (M) at positions sn-1,3 and long-chain fatty acids (L) at position sn-2. These MLM-type structured lipids (SL) were produced by interesterification of caprylic acid (CA) (C8:0) and avocado oil (content of C18:1). The regiospecific sn-1,3 commercial lipases Lipozyme RM IM and TL IM were used as biocatalysts to probe the potential of avocado oil to produce SL. Reactions were performed at 30–50°C for 24 h in solvent-free media with a substrate molar ratio of 1∶2 (TAG:CA) and 4–10% w/w enzyme content. The lowest incorporation of CA (1.1% mol) resulted from Lipozyme RM IM that was incubated at 50°C. The maximum incorporation of CA into sn-1,3 positions of TAG was 29.2% mol. This result was obtained at 30°C with 10% w/w Lipozyme TL IM, which is the highest values obtained in solvent-free medium until now for structured lipids of low-calories. This strategy opens a new market to added value products based on avocado oil.     

Complete Genome Sequence of a Pantón-Valentine Leukocidin-Negative Community-Associated Methicillin-Resistant Staphylococcus aureus Strain of Sequence type 72 from Korea

In the past decade, community-associated (CA-) infections with methicillin-resistant Staphylococcus aureus (MRSA) have emerged throughout the world. Different CA-MRSA strains dominate in different geographical locations. Many CA-MRSA lineages contain genes coding for the Pantón-Valentine leukocidin. However, the role of this leukotoxin in CA-MRSA pathogenesis is still controversial. The genome sequences of two key PVL-positive CA-MRSA strains (USA300, USA400) have been reported, but we lack information on the more recently found PVL-negative CA-MRSA strains. One such strain is the PVL-negative ST72, the main cause of CA-MRSA infections in Korea. Here, we report the entire genome sequence of CA-MRSA ST72 and analyze its gene content with a focus on virulence factors. Our results show that this strain does not have considerable differences in virulence factor content compared to other CA-MRSA strains (USA300, USA400), indicating that other toxins do not substitute for the lack of PVL in ST72. This finding is in accordance with the notion that differential expression of widespread virulence determinants, rather than the acquisition of additional virulence factors on mobile genetic elements, such as PVL, is responsible for the increased virulence of CA- compared to hospital-associated MRSA.     

Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.     

Clinical and Epidemiological Factors Associated with Methicillin Resistance in Community-Onset Invasive Staphylococcus aureus Infections: Prospective Multicenter Cross-Sectional Study in Korea

Successful empirical therapy of Staphylococcus aureus infections requires the ability to predict methicillin resistance. Our aim was to identify predictors of methicillin resistance in community-onset (CO) invasive S. aureus infections. Sixteen hospitals across Korea participated in this study from May to December 2012. We prospectively included cases of S. aureus infection in which S. aureus was isolated from sterile clinical specimens ≤72 hours after hospitalization. Clinical and epidemiological data were gathered and compared in methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) cases. Community-associated (CA) infections were defined as in previous studies. In total, there were 786 cases of community-onset S. aureus infection, 102 (13.0%) of which were CA-MRSA. In addition to known risk factors, exposure to 3rd generation cephalosporins in the past 6 months [odds ratio (OR), 1.922; 95% confidence interval (CI), 1.176–3.142] and close contact with chronically ill patients in the past month (OR, 2.647; 95% CI, 1.189–5.891) were independent risk factors for MRSA infection. However, no clinical predictors of CA-MRSA were identified. Methicillin resistance, CO infection, and appropriateness of empirical antibiotics were not significantly related to 30-day mortality. MRSA infection should be suspected in patients recently exposed to 3rd generation cephalosporins or chronically-ill patients. There were no reliable predictors of CA-MRSA infection, and mortality was not affected by methicillin resistance.     

Genome-Wide Association Analyses for Fatty Acid Composition in Porcine Muscle and Abdominal Fat Tissues

Fatty acid composition is an important phenotypic trait in pigs as it affects nutritional, technical and sensory quality of pork. Here, we reported a genome-wide association study (GWAS) for fatty acid composition in the longissimus muscle and abdominal fat tissues of 591 White Duroc×Erhualian F₂ animals and in muscle samples of 282 Chinese Sutai pigs. A total of 46 loci surpassing the suggestive significance level were identified on 15 pig chromosomes (SSC) for 12 fatty acids, revealing the complex genetic architecture of fatty acid composition in pigs. Of the 46 loci, 15 on SSC5, 7, 14 and 16 reached the genome-wide significance level. The two most significant SNPs were ss131535508 (P = 2.48×10⁻²⁵) at 41.39 Mb on SSC16 for C20∶0 in abdominal fat and ss478935891 (P = 3.29×10⁻¹³) at 121.31 Mb on SSC14 for muscle C18∶0. A meta-analysis of GWAS identified 4 novel loci and enhanced the association strength at 6 loci compared to those evidenced in a single population, suggesting the presence of common underlying variants. The longissimus muscle and abdominal fat showed consistent association profiles at most of the identified loci and distinct association signals at several loci. All loci have specific effects on fatty acid composition, except for two loci on SSC4 and SSC7 affecting multiple fatness traits. Several promising candidate genes were found in the neighboring regions of the lead SNPs at the genome-wide significant loci, such as SCD for C18∶0 and C16∶1 on SSC14 and ELOVL7 for C20∶0 on SSC16. The findings provide insights into the molecular basis of fatty acid composition in pigs, and would benefit the final identification of the underlying mutations.     

The Natural Product Magnolol as a Lead Structure for the Development of Potent Cannabinoid Receptor Agonists

Magnolol (4-allyl-2-(5-allyl-2-hydroxyphenyl)phenol), the main bioactive constituent of the medicinal plant Magnolia officinalis, and its main metabolite tetrahydromagnolol were recently found to activate cannabinoid (CB) receptors. We now investigated the structure-activity relationships of (tetrahydro)magnolol analogs with variations of the alkyl chains and the phenolic groups and could considerably improve potency. Among the most potent compounds were the dual CB₁/CB₂ full agonist 2-(2-methoxy-5-propyl-phenyl)-4-hexylphenol (61a, K _i CB₁∶0.00957 µM; K _i CB₂∶0.0238 µM), and the CB₂-selective partial agonist 2-(2-hydroxy-5-propylphenyl)-4-pentylphenol (60, K _i CB₁∶0.362 µM; K _i CB₂∶0.0371 µM), which showed high selectivity versus GPR18 and GPR55. Compound 61b, an isomer of 61a, was the most potent GPR55 antagonist with an IC₅₀ value of 3.25 µM but was non-selective. The relatively simple structures, which possess no stereocenters, are easily accessible in a four- to five-step synthetic procedure from common starting materials. The central reaction step is the well-elaborated Suzuki-Miyaura cross-coupling reaction, which is suitable for a combinatorial chemistry approach. The scaffold is versatile and may be fine-tuned to obtain a broad range of receptor affinities, selectivities and efficacies.     

Competition among Species of Stored-Product Psocids (Psocoptera) in Stored Grain

We evaluated the competition among stored-product psocid species by conducting two series of laboratory experiments. In the first series, three species of Liposcelididae were used: Liposcelis bostrychophila, Liposcelis decolor, and Liposcelis paeta. Five adult females of these species were placed in vials containing wheat, either alone or in all possible combinations of two species. The number of adults in the vials was counted after 35, 70, 105, 140, and 175 days. These tests were performed at 25 and 30°C. At 25°C, there were no differences in numbers of L. bostrychophila when this species was reared either alone or with each of the other two species. At 30°C, L. bostrychophila was the dominant species. The presence of L. bostrychophila had a negative effect on the growth of populations of L. decolor and L. paeta. The presence of L. paeta did not affect growth of populations of L. decolor, although the presence of L. decolor occasionally reduced growth of populations of L. paeta. In the second series of tests, L. bostrychophila adult females were placed in vials of wheat either alone or with adult females of Lepinotus reticulatus, at the ratios of (L. bostrychophila: L. reticulatus) 10∶0, 9∶1, 7∶3, 5∶5, 3∶7, 1∶9, and 0∶10. These tests were carried out only at 30°C, and the observation periods were the same as for the first series of tests. Liposcelis bostrychophila was the dominant species in this case as well, regardless of the ratio of the parental females. At the end of the experimental period, L. reticulatus was present only in vials that contained this species alone. Our results showed that L. bostrychophila outcompetes the other stored-product psocid species tested.     

Mechanisms of lipid peroxide formation in animal tissues

1. Homogenates of rat liver, spleen, heart and kidney form lipid peroxides when incubated in vitro and actively catalyse peroxide formation in emulsions of linoleic acid or linolenic acid. 2. In liver, catalytic activity is distributed throughout the nuclear, mitochondrial and microsomal fractions and is present in the 100000g supernatant. Activity is weak in the nuclear fraction. 3. Dilute (0·5%, w/v) homogenates catalyse peroxidation over the range pH5·0–8·0 but concentrated (5%, w/v) homogenates inhibit peroxidation and destroy peroxide if the solution is more alkaline than pH7·0. 4. Ascorbic acid increases the rate of peroxidation of unsaturated fatty acids catalysed by whole homogenates of liver, heart, kidney and spleen at pH6·0 but not at pH7·4. 5. Catalysis of peroxidation of unsaturated fatty acids by the mitochondrial and microsomal fractions of liver is inhibited by ascorbic acid at pH7·4 but the activity of the supernatant fraction is enhanced. 6. Inorganic iron or ferritin are active catalysts in the presence of ascorbic acid. 7. Lipid peroxide formation in linoleic acid or linolenic acid emulsions catalysed by tissue homogenates is partially inhibited by EDTA but stimulated by o-phenanthroline. 8. Cysteine or glutathione (1mm) inhibits peroxide formation catalysed by whole homogenates, mitochondria or haemoprotein. Inhibition increases with increase of pH.     

The effect of low temperatures on fatty acid biosynthesis in plants

1. Of three systems, bulb tissue, plant leaf tissue and intact green algal (Chlorella vulgaris) cells, only the former shows an increase in rate of formation of unsaturated fatty acids with decrease in temperature. 2. In bulb tissue the oxygen concentration is rate-limiting for synthesis of unsaturated fatty acids at temperatures down to 10°. 3. At elevated oxygen concentrations the formation of unsaturated fatty acids in bulb tissue increases with temperature. 4. The failure of photosynthetic tissues to respond to either lower temperatures or increased oxygen concentrations in the presence of light is attributed to photosynthetic production of excess of oxygen. This is supported by the fact that in the dark a potentiating oxygen effect on the formation of unsaturated fatty acids can be demonstrated. 5. The HCO₃⁻ ion concentration has a small effect on the formation of unsaturated fatty acids. 6. Elevated content of unsaturated acids at lower temperatures in plants is attributed to increases in oxygen concentration in solution.     

Potential of the Virion-Associated Peptidoglycan Hydrolase HydH5 and Its Derivative Fusion Proteins in Milk Biopreservation

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 10⁴ CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents for controlling undesirable bacteria in dairy products.