Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell ...wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.
Background & Aims: Toll-like receptors (TLRs) represent a class of transmembrane pattern recognition receptors essential for microbial recognition and control of innate immune responses. Commensal ...bacteria play an important role in maintaining tolerance and active stability of the intestinal epithelial barrier by suppressing intestinal inflammation, yet the mechanisms of action are unknown. The aim of this study was to determine the functional relevance of TLR2 to control tight junction (TJ)-associated intestinal epithelial barrier integrity to balance mucosal homeostasis against inflammatory stress-induced damage. Methods: TLR2 ligand (synthetic Pam3 Cys-SK4 PCSK)-induced activation of signaling cascades and TJ-associated distribution was assessed by using Western blotting and confocal microscopy combined with functional transfection and inhibitor studies in model intestinal epithelial cell (IEC) lines (IEC-6, Caco-2) or primary IEC cultured short-term ex vivo. DSS colitis was induced by standard protocol in wild-type, TLR2−/−, and MyD88−/− mice. Spontaneous apoptosis was assessed by terminal deoxinucleotidyl-transferase-mediated dUTP-biotin nick end-labeling. Results: Data from in vitro and ex vivo models of intestinal epithelial cells revealed that TLR2 stimulation effectively preserves TJ-associated barrier assembly against stress-induced damage through promotion of PI3K/Akt-mediated cell survival via MyD88. Furthermore, in vivo studies underscored that TLR2-mediated TJ regulation critically determines susceptibility to intestinal injury and inflammation. Inflammatory stress in mice deficient of TLR2 or MyD88 induced early TJ-associated disruption interrelated with anti-apoptotic failure of the intestinal epithelial barrier. Oral treatment of colitis with the TLR2 ligand PCSK significantly suppressed mucosal inflammation and apoptosis by efficiently restoring TJ-associated integrity of the intestinal epithelium in vivo. Conclusion: TLR2 may provide a target to pharmacologically modulate mucosal injury and intestinal inflammation.
Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, ...deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (~75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.
Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this ...study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona. Three strains, from the genera
, and
, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m
/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m
/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.
Summary
It has been recently shown that Toll‐like receptor (TLR) signalling in murine nonparenchymal liver cells (NPCs) is suppressed in the presence of Hepatitis B virus surface antigen (HBsAg). It ...is not clear, however, whether this is also relevant for the adaptive immune responses and how this effect is mediated. Peripheral blood mononuclear cells (PBMCs) from Hepatitis B virus (HBV) patients and controls were stimulated by TLR ligands in the absence or presence of autologous serum. Interestingly, TLR‐mediated cytokine expression (Interleukin‐6 and ‐10) as well as TLR3‐induced interferon (IFN) expression in PBMCs of HBV patients was significantly higher than in the healthy volunteers, showing a negative correlation with the levels of HBsAg. In addition, TLR3‐mediated IFN‐γ production was inhibited in the presence of HBV‐containing serum. To mechanistically analyse this observation, murine Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs) were stimulated with TLR3 ligands in the presence or absence of HBsAg. Mixed lymphocyte reactions were performed to study T‐cell activation induced by TLR‐stimulated NPCs. Gene expression of cytokines and TLR3 was analysed by quantitative rt‐PCR, and activation of transcription factors was assessed by Western blot or reporter gene assays. TLR‐induced expression of interferon γ, interferon sensitive genes and proinflammatory cytokines in murine KCs and LSECs was efficiently suppressed in the presence of HBsAg, whereas the expression of anti‐inflammatory cytokines was enhanced. Activation of NFκB, IRF‐3 and MAPKs in these liver cells was potently suppressed by HBsAg. T‐cell activation mediated through TLR3‐stimulated KCs or LSECs was suppressed by HBsAg which could be reverted by anti‐IL‐10 antibodies. These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to maintain a persistent infection.
Non-response to combination therapy by patients with hepatitis C virus (HCV) has previously been associated with a strong hepatic upregulation of interferon stimulated genes (ISGs) including ISG15. ...Therefore, the aim of this study was to further elucidate the functional role of this molecule.
ISG15 expression was suppressed by siRNAs or enhanced by over-expression in genomic and subgenomic human or murine HCV replicon systems. In addition, ISG15 expression was analysed in liver samples of patients with HCV prior to antiviral therapy and correlated with clinical and virological parameters.
Short- or long-term knockdown of ISG15 expression suppressed HCV replication comparable to IFNs without evidence for the induction of resistant mutations. Triple therapy consisting of ISG15 knockdown, interferon alpha (IFNalpha) and ribavirin led to complete suppression of the HCV NS5A protein, corresponding to 99% suppression of HCV-RNA compared to 75% suppression by IFNalpha and ribavirin only. Combination treatment of ISG15 knockdown and IFN was associated with enhanced and prolonged expression of selected ISGs. Consistent with these in vitro data, high hepatic ISG15 levels correlated with the unfavourable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN during the initial phase of treatment.
ISG15 plays an important role in the HCV replication cycle. Therefore, therapies based on the suppression of ISG15 may provide a promising strategy to overcome non-response to standard combination treatment in the future. Furthermore, analysis of ISG15 prior to therapy may be useful to predict short-term and long-term outcome and thus tailor antiviral therapy with pegIFN and ribavirin.
Regular consumption of fast-food (FF) as a form of typical Western style diet is associated with obesity and the metabolic syndrome, including its hepatic manifestation nonalcoholic fatty liver ...disease. Currently, it remains unclear how intermittent excess FF consumption may influence liver metabolism. The study aimed to characterize the effects of a single FF binge on hepatic steatosis, inflammation, bile acid (BA), glucose and lipid metabolism.
Twenty-five healthy individuals received a FF meal and were asked to continue eating either for a two-hour period or until fully saturated. Serum levels of transaminases, fasting BA, lipid profile, glucose and cytokine levels as well as transient elastography and controlled attenuation parameter (CAP; to assess hepatic steatosis) were analyzed before (day 0) and the day after FF binge (day 1). Feces was collected prior and after the FF challenge for microbiota analysis.
The FF meal induced a modest increase in CAP, which was accompanied by a robust increase of fasting serum BA levels. Surprisingly, levels of cholesterol and bilirubin were significantly lower after the FF meal. Differentiating individuals with a relevant delta BA (>1 μmol/l) increase vs. individuals without (delta BA ≤1 μmol/l), identified several gut microbiota, as well as gender to be associated with the BA increase and the observed alterations in liver function, metabolism and inflammation.
A single binge FF meal leads to a robust increase in serum BA levels and alterations in parameters of liver injury and metabolism, indicating a novel metabolic aspect of the gut–liver axis.
Summary
The hepatitis C virus (HCV) establishes persistent infections despite strong activation of the innate immune system through TLR3 and other sensors. Therefore, we analysed regulatory ...mechanisms of TLR3‐induced immune responses in nonparenchymal liver cells (NPCs). Effects of Interleukin‐10 (IL‐10), transforming growth factor beta (TGF‐β) and immunoregulatory miR‐155 on poly I:C‐activated murine (C57BL/6) Kupffer cells (KC) and sinusoidal endothelial cells (LSEC) were assessed in vitro. NPCs were assayed for inflammatory and antiviral cytokines and T‐cell (Balb/c)‐activating factors. Gene expression of miR‐155, IL‐10, TGF‐β and interferon sensitive genes (ISGs) in biopsies of patients with HCV was determined by qrt‐PCR. TLR3‐induced antiviral activity in murine NPCs was potently suppressed by IL‐10 and TGF‐β which correlated with decreased TLR3 expression and inhibition of NF‐κB and IRF‐3 activation. T‐cell activation, induced by TLR3‐activated NPCs, was also suppressed by IL‐10 and TGF‐β, which was associated with a down‐regulation of CD80 and CD86. Pretreatment with IL‐10 or TGF‐β suppressed TLR3‐induced miR‐155 expression, which itself positively regulated poly I:C‐mediated immune responses, thus counteracting IL‐10 or TGF‐β‐induced immunosuppression. In addition, hepatic expression of miR‐155 was elevated in chronically infected patients with HCV, was associated with an IL‐28B SNP (rs12979860) and was inversely correlated with HCV serum load and ISG expression levels. As miR‐155 is a key regulator of anti‐inflammatory mechanisms that control innate and adaptive hepatic immune responses during HCV infection, miR‐155 based therapies may represent a novel mechanism to control HCV in the future.
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