Mycobacterial Antigen Driven Activation of CD14++CD16? Monocytes Is a Predictor of Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14⁺⁺CD16⁻ monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment.     

Extreme thermophilic ethanol production from rapeseed straw: Using the newly isolated Thermoanaerobacter pentosaceus and combining it with Saccharomyces cerevisiae in a two‐step process

The newly isolated extreme thermophile Thermoanaerobacter pentosaceus was used for ethanol production from alkaline‐peroxide pretreated rapeseed straw (PRS). Both the liquid and solid fractions of PRS were used. T. pentosaceus was able to metabolize the typical process inhibitors present in lignocellulosic hydrolysate, namely 5‐hydroxymethyl furfural (HMF) and furfural, up to concentrations of 1 and 0.5 g L^?1, respectively. Above these levels, xylose consumption was inhibited up to 70% (at 3.4 g‐furfural L^?1) and 75% (at 3.4 g‐HMF L^?1). T. pentosaceus was able to grow and produce ethanol directly from the liquid fraction of PRS, without any dilution or need for additives. However, when the hydrolysate was used undiluted the ethanol yield was only 37% compared to yield of the control, in which pure sugars in synthetic medium were used. The decrease of ethanol yield was attributed to the high amounts of salts resulting from the alkaline‐peroxide pretreatment. Finally, a two‐stage ethanol production process from PRS using Saccharomyces cerevisiae (utilization of hexoses in the first step) and T. pentosaceus (utilization of pentoses in the second step) was developed. Results showed that the two strains together could achieve up to 85% of the theoretical ethanol yield based on the sugar composition of the rapeseed straw, which was 14% and 50% higher compared to the yield with the yeast or the bacteria alone, respectively. Biotechnol. Bioeng. 2013; 110: 1574–1582. © 2012 Wiley Periodicals, Inc.     

Tumour necrosis factor-alpha (TNFalpha) and interleukin-10 are crucial mediators in post-operative systemic inflammatory response and determine the occurrence of complications after major abdominal surgery

BACKGROUND: The course of serum cytokine levels in patients with postoperative systemic inflammatory response syndrome (SIRS) after major abdominal surgery remains currently unclear. METHODS: Blood was sampled pre- and post-operatively and on days 1 and 2 in 40 patients undergoing major abdominal surgery. Concentrations of tumour necrosis factor-alpha (TNFalpha), interleukin (IL) -6, IL-8, and IL-10 were measured by the LINCOplex assay; those of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) by an enzyme immunoassay. RESULTS: Compared to their pre-operative values, sTREM-1 was elevated on day 2; TNFalpha on day 1; IL-6 and IL-10 post-operatively and on days 1 and 2; and IL-8 post-operatively and on day 1. The duration of operation correlated with TNFalpha and IL-10 at all sampling times, and with IL-6 post-operatively. There were no differences in cytokine concentrations between patients who exhibited post-operative complications and those who did not. IL-10/TNFalpha below 30 was found in all patients with complications (100%) and in 20 patients without complications (64.5%, p: 0.043). CONCLUSIONS: SIRS following major surgery is characterised by complex alterations in cytokine concentrations. The balance between TNFalpha and IL-10 seems to determine the occurrence of post-operative complications.     

Rhizosphere Microbial Community Composition Affects Cadmium and Zinc Uptake by the Metal-Hyperaccumulating Plant Arabidopsis halleri

The remediation of metal-contaminated soils by phytoextraction depends on plant growth and plant metal accessibility. Soil microorganisms can affect the accumulation of metals by plants either by directly or indirectly stimulating plant growth and activity or by (im)mobilizing and/or complexing metals. Understanding the intricate interplay of metal-accumulating plants with their rhizosphere microbiome is an important step toward the application and optimization of phytoremediation. We compared the effects of a “native” and a strongly disturbed (gamma-irradiated) soil microbial communities on cadmium and zinc accumulation by the plant Arabidopsis halleri in soil microcosm experiments. A. halleri accumulated 100% more cadmium and 15% more zinc when grown on the untreated than on the gamma-irradiated soil. Gamma irradiation affected neither plant growth nor the 1 M HCl-extractable metal content of the soil. However, it strongly altered the soil microbial community composition and overall cell numbers. Pyrosequencing of 16S rRNA gene amplicons of DNA extracted from rhizosphere samples of A. halleri identified microbial taxa (Lysobacter, Streptomyces, Agromyces, Nitrospira, “Candidatus Chloracidobacterium”) of higher relative sequence abundance in the rhizospheres of A. halleri plants grown on untreated than on gamma-irradiated soil, leading to hypotheses on their potential effect on plant metal uptake. However, further experimental evidence is required, and wherefore we discuss different mechanisms of interaction of A. halleri with its rhizosphere microbiome that might have directly or indirectly affected plant metal accumulation. Deciphering the complex interactions between A. halleri and individual microbial taxa will help to further develop soil metal phytoextraction as an efficient and sustainable remediation strategy.     

Cationic amphipathic peptide analogs of cathelicidin LL‐37 as a probe in the development of antimicrobial/anticancer agents

Cathelicidin LL‐37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL‐37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13‐amino acid cathelicidin 17‐29 amide segment F¹⁷KRIV²¹QR²³IK²⁵DF²⁷LR‐NH₂ were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R²³ and K²⁵ in the hydrophilic side, V²¹and F²⁷ in the hydrophobic side of the interphase, and F¹⁷ that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17‐29 fragment to investigate how they affect activity. Substitution of the amino‐terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac‐FKRIVQRIL²⁵DFLR‐NH₂ resulted in significant cytotoxicity against A549 cancer cells with an IC₅₀ value 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac‐FKRIVQI²³IKK²⁶FLR‐NH₂, which incorporates the IIKK motif and the peptides FKRIVQL²³L²⁴KK²⁶L²⁷LR‐NH₂ and Ac‐FKRIVQL²³L²⁴KK²⁶L²⁷LR‐NH₂, which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram‐negative bacteria (MIC 3–7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC₅₀ 12.9–9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17‐29 segment, may lead to the preparation of effective biological compounds.     

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.     

Electricity generation and microbial community response to substrate changes in microbial fuel cell

The effect of substrate changes on the performance and microbial community of two-chamber microbial fuel cells (MFCs) was investigated in this study. The MFCs enriched with a single substrate (e.g., acetate, glucose, or butyrate) had different acclimatization capability to substrate changes. The MFC enriched with glucose showed rapid and higher power generation, when glucose was switched with acetate or butyrate. However, the MFC enriched with acetate needed a longer adaptation time for utilizing glucose. Microbial community was also changed when the substrate was changed. Clostridium and Bacilli of phylum Firmicutes were detected in acetate-enriched MFCs after switching to glucose. By contrast, Firmicutes completely disappeared and Geobacter-like species were specifically enriched in glucose-enriched MFCs after feeding acetate to the reactor. This study further suggests that the type of substrate fed to MFC is a very important parameter for reactor performance and microbial community, and significantly affects power generation in MFCs.     

Microwave pretreatment of rape straw for bioethanol production: focus on energy efficiency

The energy efficiency of microwave-assisted dilute sulfuric acid pretreatment of rape straw for the production of ethanol was investigated. Different microwave energy inputs and solid loadings were tested to find economic pretreatment conditions. The lowest energy consumption was observed when solid loading and energy input were fixed at 50% (w/w) and 54 kJ (900 W for 1 min), respectively, and amounted to 5.5 and 10.9 kJ to produce 1 g of glucose after enzymatic hydrolysis and 1 g ethanol after fermentation, respectively. In general, 1 g ethanol can produce about 30 kJ of energy, and therefore, the energy input for the pretreatment was only 35% of the energy output. The approach developed in this study resulted in 92.9% higher energy savings for producing 1 g ethanol when compared with the results of microwave pretreatments previously reported.     

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.