Concentrations Of Peptidoglycan Research Articles

The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram- bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Macrolides Decrease the Proinflammatory Activity of Macrolide-Resistant Streptococcus pneumoniae.

Over the past 2 decades, the prevalence of macrolide-resistant Streptococcus pneumoniae (MRSP) has increased considerably, due to widespread macrolide use. Although macrolide usage has been proposed to be associated with treatment failure in patients with pneumococcal diseases, macrolides may be clinically effective for treating these diseases, regardless of the susceptibility of the causative pneumococci to macrolides. As we previously demonstrated that macrolides downregulate the transcription of various genes in MRSP, including the gene encoding the pore-forming toxin pneumolysin, we hypothesized that macrolides affect the proinflammatory activity of MRSP. Using HEK-Blue cell lines, we found that the supernatants from macrolide-treated MRSP cultures induced decreased NF-κB activation in cells expressing Toll-like receptor 2 and nucleotide-binding oligomerization domain 2 compared to the supernatants from untreated MRSP cells, suggesting that macrolides inhibit the release of these ligands from MRSP. Real-time PCR analysis revealed that macrolides significantly downregulated the transcription of various genes encoding peptidoglycan synthesis-, lipoteichoic acid synthesis-, and lipoprotein synthesis-related molecules in MRSP cells. The silkworm larva plasma assay demonstrated that the peptidoglycan concentrations in the supernatants from macrolide-treated MRSP cultures were significantly lower than those from untreated MRSP cultures. Triton X-114 phase separation revealed that lipoprotein expression decreased in macrolide-treated MRSP cells compared to the lipoprotein expression in untreated MRSP cells. Consequently, macrolides may decrease the expression of bacterial ligands of innate immune receptors, resulting in the decreased proinflammatory activity of MRSP. IMPORTANCE To date, the clinical efficacy of macrolides in pneumococcal disease is assumed to be linked to their ability to inhibit the release of pneumolysin. However, our previous study demonstrated that oral administration of macrolides to mice intratracheally infected with macrolide-resistant Streptococcus pneumoniae resulted in decreased levels of pneumolysin and proinflammatory cytokines in bronchoalveolar lavage fluid samples compared to the levels in samples from untreated infected control mice, without affecting the bacterial load in the fluid. This finding suggests that additional mechanisms by which macrolides negatively regulate proinflammatory cytokine production may be involved in their efficacy in vivo. Furthermore, in this study, we demonstrated that macrolides downregulated the transcription of various proinflammatory-component-related genes in S. pneumoniae, which provides an additional explanation for the clinical benefits of macrolides.

Lonicera japonica Thunb. as a promising antibacterial agent for Bacillus cereus ATCC14579 based on network pharmacology, metabolomics, and in vitro experiments.

Lonicera japonica Thunb. has attracted much attention for its treatment of bacterial and viral infectious diseases, while its active ingredients and potential mechanisms of action have not been fully elucidated. Here, we combined metabolomics, and network pharmacology to explore the molecular mechanism of Bacillus cereus ATCC14579 inhibition by Lonicera japonica Thunb. In vitro inhibition experiments showed that the Lonicera japonica Thunb.'s water extracts, ethanolic extract, luteolin, quercetin, and kaempferol strongly inhibited Bacillus cereus ATCC14579. In contrast, chlorogenic acid and macranthoidin B had no inhibitory effect on Bacillus cereus ATCC14579. Meanwhile, the minimum inhibitory concentrations of luteolin, quercetin, and kaempferol against Bacillus cereus ATCC14579 were 15.625 μg mL-1, 31.25 μg mL-1, and 15.625 μg mL-1. Based on the previous experimental basis, the metabolomic analysis showed the presence of 16 active ingredients in Lonicera japonica Thunb.'s water extracts and ethanol extracts, with differences in the luteolin, quercetin, and kaempferol contents between the water extracts and ethanol extracts. Network pharmacology studies indicated that fabZ, tig, glmU, secA, deoD, nagB, pgi, rpmB, recA, and upp were potential key targets. Active ingredients of Lonicera japonica Thunb. may exert their inhibitory effects by inhibiting ribosome assembly, the peptidoglycan biosynthesis process, and the phospholipid biosynthesis process of Bacillus cereus ATCC14579. An alkaline phosphatase activity assay, peptidoglycan concentration assay, and protein concentration assay showed that luteolin, quercetin, and kaempferol disrupted the Bacillus cereus ATCC14579 cell wall and cell membrane integrity. Transmission electron microscopy results showed significant changes in the morphology and ultrastructure of the cell wall and cell membrane of Bacillus cereus ATCC14579, further confirming the disruption of the cell wall and cell membrane integrity of Bacillus cereus ATCC14579 by luteolin, quercetin, and kaempferol. In conclusion, Lonicera japonica Thunb. can be used as a potential antibacterial agent for Bacillus cereus ATCC14579, which may exert its antibacterial activity by destroying the integrity of the cell wall and membrane.

Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.

Electrical monitoring of infection biomarkers in chronic wounds using nanochannels

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients’ life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20–100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids.Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.

Mechanism Investigation of Excess Sludge Disintegration by Stirred Ball Mill—Utilized Transmission Electron Microscope Observation and Peptidoglycan Concentration Determination

Mechanism Investigation of Excess Sludge Disintegration by Stirred Ball Mill—Utilized Transmission Electron Microscope Observation and Peptidoglycan Concentration Determination

The phosphorylation of phosphoglucosamine mutase GlmM by Ser/Thr kinase STK mediates cell wall synthesis and virulence in Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes serious economic losses in the pig industry. Phosphorylation is an important mechanism of protein modification. Recent studies have reported that the serine/threonine kinase (STK) gene contributes to the growth and virulence of SS2. However, the mechanism underlying the regulatory functions of STK in SS2 has not been thoroughly elucidated to date. In this study, phosphoproteomic analysis was performed to determine substrates of the STK protein. Twenty-two proteins with different cell functions were identified as potential substrates of STK. Phosphoglucosamine mutase (GlmM) was selected for further investigation among them. In vitro phosphorylation assay and immunoprecipitation assay indicated that GlmM was phosphorylated by STK at the Ser-101 site and the phosphorylation level of GlmM can be affected. We observed that compared to the wild-type strain ZY05719, the glmM-deficient strain (ΔglmM) and the glmM S101A point mutation strain (CΔglmM S101A) showed aberrant cell morphology and attenuated virulence, including enlarged cell volume, absent capsule, decreased resistance, lower survival caused by unusual peptidoglycan synthesis, and significantly attenuated pathogenicity in a mouse infection model. Additionally, compared to ZY05719 and CΔglmM, GlmM enzyme acivities and peptidoglycan concentrations of the stk-deficient strain (Δstk), CΔglmM S101A decreased significantly. These experiments revealed that STK phosphorylates GlmM at the Ser-101 site to impact GlmM enzyme activity and control cell wall peptidoglycan synthesis to affect SS2 pathogenicity.

Effects of Peptidoglycan, Lipoteichoic Acid and Lipopolysaccharide on Inflammation, Proliferation and Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. LPS can directly inhibit proliferation and milk fat synthesis of bovine mammary epithelial cells (BMECs) while inducing mastitis, but it is unclear whether PGN and LTA also have such effects. Furthermore, since the three PAMPs usually appear simultaneously in the udder of cows with mastitis, their synergistic effects on proliferation and milk fat synthesis of BMECs are worth investigating. The immortalized BMECs (MAC-T cells) were stimulated for 24 h using various concentrations of PGN, LTA and LPS, respectively, to determine the doses that could effectively cause inflammatory responses. Next, the cells were stimulated for 24 h with no endotoxins (CON), PGN, LTA, LPS, PGN + LTA, and PGN + LTA + LPS, respectively, with the predetermined doses to analyze their effects on proliferation and milk fat synthesis of BMECs. PGN, LTA and LPS successfully induced inflammatory responses of BMECs with doses of 30, 30 and 0.1 μg/mL, respectively. Although the proliferation of BMECs was significantly inhibited in the following order: LTA < PGN + LTA < PGN + LTA + LPS, there was no change in cell morphology and cell death. LTA significantly promoted the expression of fatty acid synthesis-related genes but did not change the content of intracellular triglyceride (TG), compared with the CON group. The mRNA expression of fatty acid synthesis-related genes in the LPS group was the lowest among all the groups. Meanwhile, LPS significantly decreased the content of intracellular non-esterified fatty acids (NEFAs) and TG, compared with the CON group. PGN had no effects on milk fat synthesis. Co-stimulation with PGN, LTA and LPS significantly increased the expression of fat acid synthesis-related genes and the intracellular NEFAs, but decreased intracellular TG, compared with sole LPS stimulation. Collectively, PGN, LTA and LPS showed an additive effect on inhibiting proliferation of BMECs. The promoting role of LTA in fatty acid synthesis might offset the negative effects of LPS in this regard, but co-stimulation with PGN, LTA and LPS significantly decreased intracellular TG content.

Concentrations of bacteria and bacterial and fungal spores calculated from chemical tracers associated with size-segregated aerosol in a composting plant

The lack of information on biological risks in workplaces arises from the difficulty to measure bioaerosol. This study aimed to develop and improve the bioaerosol monitoring technique that uses proper biomarkers as a tool. Muramic and dipicolinic acids, and ergosterol were used as tracers for bacteria cells, bacterial spores, and fungal spores, respectively. Furthermore, 12- and 13-methyltetradecanoic acids (iso- and anteiso- C15:0) were used to study the presence of airborne bacteria and 3-hydroxy fatty acids were used to determine the concentration of peptidoglycan. Airborne particulate matter was sampled in a municipal indoor waste composting facility by multistage impactor samplers, during three main stages of composting process. The microorganism content, in airborne particles with aerodynamic diameter minor then 1 μm and between 1 and 10 μm, was determined starting from the aforementioned biomarker concentrations. For iso- and anteiso- C15:0, a conversion factor to transform its concentration into bacterial content was tentatively proposed. The results show that the chemical method covers some gaps in the information about bioaerosol presence in polluted atmospheres. Differences up to two orders of magnitude are observed, by comparing the results obtained by biomarkers and by cultivation-dependent methods. The microbial content, expressed as a percentage by mass on respect the PM, ranged from 4 to 28% with higher percentages during shredding and mixing stages and lower values during biocell opening operations. Bacterial spores, bacterial cells, and fungal spores detected were high in number, compared with the findings in similar studies elsewhere.

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13C and 15N specific labels in d- and l-alanine and l-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l-alanyl carbonyl‑carbon bridge labels to d-[3-13C]alanyl and l-[ε-15N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.

Estradiol alters the immune-responsiveness of cervical epithelial cells stimulated with ligands of Toll-like receptors 2 and 4.

The mucosa of the female reproductive tract plays a pivotal role in host defence. Pregnancy must alter immunological mechanisms at this interface to protect the conceptus. We sought to determine how estradiol (E2) alters the immune-responsiveness of cervical epithelial cells to ligand stimulation of Toll-like receptor (TLR)-2 and -4. Human ectocervical epithelial cells (HECECs) were cultured and co-incubated with two concentrations of E2 and peptidoglycan (PGN) or lipopolysaccharide (LPS) over durations that ranged between 10 minutes and 18 hours. Cytometric Bead Array was performed to quantify eight cytokines in the supernatant fluid. In response to PGN, HECECs co-incubated with E2 released lesser quantities of IL-1ß and IFNγ, higher levels of RANTES, and variable levels of IL-6 and IL-8 than those not exposed to E2. In contrast, HECECs co-incubated with LPS and E2 secreted increased levels of IL-1ß, IL-6, IL-8, and IFNγ at 2 and 18 hours than HECECs not exposed to E2, and reduced levels of RANTES at same study time-points. Estradiol alters the immune-responsiveness of cultured HECECs to TLR2 and TLR4 ligands in a complex fashion that appears to vary with bacterial ligand, TLR subtype, and duration of exposure. Our observations are consistent with the functional complexity that this mucosal interface requires for its immunological roles.

The Effect of &lt;i&gt;Lactobacillus Plantarum &lt;/i&gt;and Bacterial Peptidoglycan on the Growth of Mouse Tumors &lt;i&gt;in vivo&lt;/i&gt; and &lt;i&gt;in vitro&lt;/i&gt;

Some members of the microbiota have been shown to be effective strategies for inhibiting tumor growth through stimulation of host anti-tumor immune responses. Anti-tumor immune effects were observed when Lactobacillus plantarum (Lp), a member of the gut microbiota, was used to treat colorectal cancer in mice. Moreover, constituents of bacteria, including peptidoglycan (PG), have been shown to exhibit tumoricidal effects. The aim of this study was to investigate the anti-tumor effects of Lp on serum levels of angiogenic and immunostimulatory cytokines in melanoma-bearing mice in vivo; as well as the effect of PG on the growth of mouse melanoma and breast cancer cells in vitro. Fifty C57BL/6 female mice were divided into two groups. Prior to tumor implantation, Lp was administered via oral gavage for 2 weeks to the experimental group. After receiving subcutaneous injections of B16F10 melanoma cells, Lp administration was continued once per week for 3 weeks to the experimental group. After the last bacterial administration, serum levels of Vascular Endothelial Growth Factor (VEGF) and Interleukin-12 (IL-12) were determined by ELISA. Additionally, mice from both groups were monitored for survival. Moreover, B16F10 melanoma and EMT6 breast cancer cells were incubated separately with two PG concentrations for 48 h and percent viability was determined. A significant decrease in the serum levels of VEGF and a significant increase in the serum levels of IL-12 were observed in the group treated with Lp Moreover, 20% survival rate was noted in the group treated with Lp in vitro, a marked decrease in the viability of mouse melanoma and breast cancer cells was observed 48 h post-incubation with PG. It appears that Lp possesses anti-tumor activity, by both stimulating the immune system and suppressing angiogenesis. Moreover, Lp appears to have a direct tumoricidal effect through PG.

Increased Endovascular Staphylococcus aureus Inoculum Is the Link Between Elevated Serum Interleukin 10 Concentrations and Mortality in Patients With Bacteremia

Cell wall peptidoglycan stimulates interleukin 10 (IL-10) production in Staphylococcus aureus bacteremia (SaB) animal models, but clinical data are not available. This study evaluates the impact of intravascular bacterial cell numbers (ie, the level of bacteremia), in patients at the time of clinical presentation on IL-10 production and its association with S. aureus bacteremia (SaB) mortality. Blood and isolates were collected in 133 consecutive SaB patients. Serum IL-10 was quantified by an electrochemoluminescence assay. Bacterial inoculum was measured in patient sera with elevated (n = 8) or low (n = 8) IL-10 using a magnetic bacterial capture assay. Staphylococcus aureus from these 2 groups were introduced into whole blood ex vivo to determine IL-10 production with variable inocula. IL-10 serum concentration was higher in SaB patient mortality (n = 27) vs survival (n = 106) (median, 36.0 pg/mL vs 10.4 pg/mL, respectively, P < .001). Patients with elevated IL-10 more often had endovascular SaB sources. The inoculum level of SaB was higher in patients with elevated serum IL-10 vs patients with low IL-10 (35.5 vs 0.5 median CFU/mL; P = .044). Ex vivo studies showed that 108 CFU/mL yielded greater IL-10 than did 103 CFU/mL (4.4 ± 1.8 vs 1.0 ± 0.6 pg/mL; P < .01). Elevated IL-10 serum concentrations at clinical presentation of SaB were highly associated with mortality. High intravascular peptidoglycan concentration, driven by a higher level of bacteremia, is a key mediator of IL-10 anti-inflammatory response that portends poor clinical outcome. Using IL-10 as an initial biomarker, clinicians may consider more aggressive antimicrobials for rapid bacterial load reduction in high-risk SaB patients.

Use of anaerobic hydrolysis pretreatment to enhance ultrasonic disintegration of excess sludge.

To improve the excess sludge disintegration efficiency, reduce the sludge disintegration cost, and increase sludge biodegradability, a combined pretreatment of anaerobic hydrolysis (AH) and ultrasonic treatment (UT) was proposed for excess sludge. Results showed that AH had an advantage in dissolving flocs, modifying sludge characteristics, and reducing the difficulty of sludge disintegration, whereas UT was advantageous in damaging cell walls, releasing intracellular substances, and decomposing macromolecular material. The combined AH-UT process was an efficient method for excess sludge pretreatment. The optimized solution involved AH for 3 days, followed by UT for 10 min. After treatment, chemical oxygen demand, protein, and peptidoglycan concentrations reached 3,949.5 mg O2/L, 752.5 mg/L and 619.1 mg/L, respectively. This work has great significance for further engineering applications, namely, reducing energy consumption, increasing the sludge disintegration rate, and improving the biochemical properties of sludge.

Regulation of Toll-like receptor expression in human conjunctival epithelial cells.

Previous studies showed marked decrease of multiple Toll-like receptor (TLR) expression in corneal and conjunctival epithelial cells upon culture in vitro. The aim of this study was to identify factor(s) which regulate TLR expression. Primary human conjunctival epithelial cells and immortal conjunctival (IOBA-NHC) and corneal epithelial cell lines (HCET) were used. The effect of various cytokines, hypoxia, mechanical wounding, and airlifting culture on TLR expression was examined by quantitative PCR and western blot analysis. Ligand stimulated TLR activation was analyzed. TLR mRNA expression increased modestly when cultured monolayered cells were stimulated by TNF-α, IL-1α, IL-1β, IL-6, IL-8, IFN-γ (about 2-fold), hypoxia (2.1- to 4.8-fold selectively), and wounding (3.1- to 9.3-fold). In airlifted multilayered cells, TLR expression increased 7.8- to 25.9-fold compared to monolayered cells. Airlifted cells showed increased response to low concentrations of lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation. NFκB inhibition prevented the formation of cell sheets and led to the collapse of already-formed multilayered structure and the simultaneous reduction of TLR mRNA level. In conclusion, our study showed that the conjunctival epithelial cell expressed TLR was sensitive to various stimulants, and a multilayered epithelium-like structure was needed to maintain TLR expression.

Can primary production contribute non-labile organic matter in the sea: Amino acid enantiomers along the coast south of the Changjiang Estuary in May

Amino acid enantiomers (AAEs) in suspended particulate organic matter (OM) were measured along the coast south of the Changjiang Estuary in May to determine the origin and nature of estuarine particulate OM. The degradation index (DI) for amino acid yields revealed that fresh OM corresponded to depletion of dissolved inorganic nitrogen, whereas degraded OM corresponded to excess nutrients. This indicates coupling between OM degradation and nutrient regeneration in surface waters. Two clearly different trends were found for the amino acid content of total particulate nitrogen. Offshore samples were characterized by amino acid enrichment (91%), whereas inshore samples were depleted in amino acids (14%), probably because of terrestrial impacts during the previous winter. Compared to samples previously taken from the lower Changjiang reaches (Shao et al., 2011), samples of surface coastal waters can be clearly identified by the D/L ratio of selected AAE, indicating the potential of AAEs for identifying OM sources. Peptidoglycan estimated on the basis of D-AAEs, ranged from 3 to 275nM. For fresh particulate OM (DI>0.5), the peptidoglycan concentration was positively correlated with OM freshness and particulate-nitrogen-normalized peptidoglycan was comparable to or even higher than that in terrestrial OM. This suggests that estuarine and coastal zones make a significant contribution to non-labile OM production. Further analysis suggests that heterotrophic bacteria and Synechococcus are notable contributors.

The effect of peptidoglycan enriched diets on antimicrobial peptide gene expression in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to investigate the effect of feeding rainbow trout (Oncorhynchus mykiss) peptidoglycan (PG) enriched diets on antimicrobial peptide (AMP) gene expression. Fish were divided into 5 groups and fed diets containing 0, 5, 10, 50 and 100 mg PG/Kg, and sampled 1, 7 and 14 days later. The expression of eight AMP genes (four defensins, two cathelicidins and two liver expressed AMPs) was determined in skin, gill, gut and liver, tissues important for first lines of defence or production of acute phase proteins. Up-regulation of many AMPs was found after feeding the PG enriched diets, with sequential expression seen over the time course studied, where defensins were typically expressed early and cathelicidins and LEAPs later on. A number of clear differences in AMP responsiveness between the tissues examined were also apparent. Of the four PG concentrations used, 5 mg PG/Kg did not always elicit AMP gene induction or to the same degree as seen with the other diets. The three higher dose groups generally showed similar trends although differences in fold change were more pronounced in the 50 and 100 mg PG/Kg groups. Curiously several AMPs were down-regulated after 14 days of feeding in gills, gut and liver. Nevertheless, overall the PG enriched diets had a positive effect on AMP expression. Further investigations now need to be undertaken to confirm whether this higher AMP gene expression correlates with protection against common bacterial diseases and if PG enriched diets have value as a means to temporarily boost the piscine immune system.

Comparison of nuclear magnetic resonance methods for the analysis of organic matter composition from soil density and particle fractions

Environmental contextThe association of specific organic matter (OM) compounds with clay mineral surfaces is believed to protect these compounds from degradation and thus result in long-term protection in soil. The molecular-level composition of soil OM associated with soil fractions was measured and compared using solid-state 13C nuclear magnetic resonance (NMR) and solution-state 1H NMR methods. Combining these methods allowed more detailed characterisation of OM associated with different soil fractions and will improve the understanding of OM dynamics in soil. AbstractOrganic matter (OM) associated with fine soil fractions is hypothesised to be protected from complete biodegradation by soil microbes. It is therefore important to understand the structure and stage of decomposition of OM associated with various soil fractions. Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy has been used extensively to investigate the OM composition of soils and soil fractions. Solution-state 1H NMR spectroscopy has not been used as much but is an emerging tool for analysing soil OM because 1H NMR spectra are often better resolved and provide information that complements the structural information obtained from solid-state 13C NMR experiments. This study compares one-dimensional solution-state 1H NMR and solid-state 13C NMR methods for assessing the degradation and composition of OM in three different soils, and their light and clay-size fractions. The alkyl/O-alkyl degradation parameter was consistent across all NMR methods and showed that OM associated with clay-size fractions were at more advanced stages of degradation as compared to that in light density soil fractions. Solution-state 1H and diffusion edited (DE) 1H NMR results showed the presence of high concentrations of microbial-derived peptidoglycan and peptide side-chains in clay-sized fractions. Lignin was also identified in clay-sized fractions using solid-state 13C and solution-state 1H NMR techniques. The combination of solid-state 13C and solution-state 1H NMR methods provides a more detailed analysis of OM composition and thereby facilitates a better understanding of the fate and preservation of OM in soil.

Pathogen associated molecular pattern-induced TNF, IL-1β, IL-6 and CXCL-8 production from feline whole blood culture

Whole blood culture ( C wb) is a method to evaluate leukocyte response to stimuli. We used C wb to evaluate the inflammatory response to pathogen associated molecular patterns (PAMPs) in cats. Blood was collected from diluted with RPMI and stimulated with various concentrations of lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PG) or control (PBS). Multiple concentrations of LPS, LTA and PG significantly stimulated tumor necrosis factor (TNF), interleukin (IL)-1β and CXC chemokine ligand (CXCL)-8 in feline C wb. All PAMPs failed to stimulate IL-6 production and PG failed to stimulate CXCL-8 production. Lipopolysaccharide was a more potent inducer of IL-1β and CXCL-8 than LTA or PG and LTA is a more potent inducer of CXCL-8 than PG. Based on these data, PAMPs from gram positive and negative bacteria induce TNF, IL-1β and CXCL-8 production in feline whole blood. Cats appear to be relatively more sensitive to gram negative compared to gram positive bacteria.

NOD2 Negatively Regulates Organic Dust-Induced Inflammation in Monocytes/Macrophages

RATIONALE: Chronic organic dust exposure in the agricultural industry results in significant airway disease. Since we have recently demonstrated high concentration of peptidoglycan in large animal farming environments, we examined in vitro, the role of the intracellular peptidoglycan breakdown product sensor, nucleotide oligomerization domain (NOD)2, in regulating organic dust-induced inflammation.METHODS: Organic dust extract (ODE) from swine operations was utilized. Adherent THP-1 monocytes were transfected with NOD2 or non-targeting siRNA by HiPerfect system. Transfected cells were subsequently stimulated with and without 1% ODE and as a control, the NOD2 agonist, muramyl dipetide (MDP), for 24 hours. Transfection efficiency was evaluated by real-time RT-PCR. Next, lung macrophages isolated from wild type and NOD2 knockout mice were ex vivo stimulated with 1% ODE for 24 hours. Cell-free supernatants for mediators were evaluated by ELISA.RESULTS: Relative mRNA expression of NOD2 in MDP- and ODE-stimulated NOD2 knockdown cells was significantly reduced to 32.8% ± 9.6% and 34.85% ± 8.6%, respectively. As anticipated, TNFα and CXCL8 secretion was diminished in NOD2 knockdown cells stimulated with MDP (p < 0.05; N = 3). However, stimulation of NOD2 knockdown cells with ODE resulted in significantly enhanced CXCL8 and IL-6 secretion as compared to control, but no change in TNFα (N = 3). Confirming these findings, murine NOD2-/- lung macrophages demonstrated significantly enhanced KC and MIP-2 (human CXCL8 homologs), IL-6, but not TNFα secretion, with ODE stimulation as compared to wild type lung macrophages (p < 0.05; N = 4 mice/group).CONCLUSIONS: The peptidoglycan breakdown product intracellular sensor, NOD2, appears to negatively regulate organic dust-induced macrophage inflammation in a selective manner. RATIONALE: Chronic organic dust exposure in the agricultural industry results in significant airway disease. Since we have recently demonstrated high concentration of peptidoglycan in large animal farming environments, we examined in vitro, the role of the intracellular peptidoglycan breakdown product sensor, nucleotide oligomerization domain (NOD)2, in regulating organic dust-induced inflammation. METHODS: Organic dust extract (ODE) from swine operations was utilized. Adherent THP-1 monocytes were transfected with NOD2 or non-targeting siRNA by HiPerfect system. Transfected cells were subsequently stimulated with and without 1% ODE and as a control, the NOD2 agonist, muramyl dipetide (MDP), for 24 hours. Transfection efficiency was evaluated by real-time RT-PCR. Next, lung macrophages isolated from wild type and NOD2 knockout mice were ex vivo stimulated with 1% ODE for 24 hours. Cell-free supernatants for mediators were evaluated by ELISA. RESULTS: Relative mRNA expression of NOD2 in MDP- and ODE-stimulated NOD2 knockdown cells was significantly reduced to 32.8% ± 9.6% and 34.85% ± 8.6%, respectively. As anticipated, TNFα and CXCL8 secretion was diminished in NOD2 knockdown cells stimulated with MDP (p < 0.05; N = 3). However, stimulation of NOD2 knockdown cells with ODE resulted in significantly enhanced CXCL8 and IL-6 secretion as compared to control, but no change in TNFα (N = 3). Confirming these findings, murine NOD2-/- lung macrophages demonstrated significantly enhanced KC and MIP-2 (human CXCL8 homologs), IL-6, but not TNFα secretion, with ODE stimulation as compared to wild type lung macrophages (p < 0.05; N = 4 mice/group). CONCLUSIONS: The peptidoglycan breakdown product intracellular sensor, NOD2, appears to negatively regulate organic dust-induced macrophage inflammation in a selective manner.