Optimizing electrochemically active microorganisms as a key player in the bioelectrochemical system: Identification methods and pathways to large-scale implementation.

The rapid global economic growth driven by industrialization and population expansion has resulted in significant issues, including reliance on fossil fuels, energy scarcity, water crises, and environmental emissions. To address these issues, bioelectrochemical systems (BES) have emerged as a dual-purpose solution, harnessing electrochemical processes and the capabilities of electrochemically active microorganisms (EAM) to simultaneously recover energy and treat wastewater. This review examines critical performance factors in BES, including inoculum selection, pretreatment methods, electrodes, and operational conditions. Further, authors explore innovative approaches to suppress methanogens and simultaneously enhance the EAM in mixed cultures. Additionally, advanced techniques for detecting EAM are discussed. The rapid detection of EAM facilitates the selection of suitable inoculum sources and optimization of enrichment strategies in BESs. This optimization is essential for facilitating the successful scaling up of BES applications, contributing substantially to the realization of clean energy and sustainable wastewater treatment. This analysis introduces a novel viewpoint by amalgamating contemporary research on the selective enrichment of EAM in mixed cultures. It encompasses identification and detection techniques, along with methodologies tailored for the selective enrichment of EAM, geared explicitly toward upscaling applications in BES.

Pretreatments of lignocellulosic and algal biomasses for sustainable biohydrogen production: Recent progress, carbon neutrality, and circular economy.

Lignocellulosic and algal biomasses are known to be vital feedstocks to establish a green hydrogen supply chain toward achieving a carbon-neutral society. However, one of the most pressing issues to be addressed is the low digestibility of these biomasses in biorefinery processes, such as dark fermentation, to produce green hydrogen. To date, various pretreatment approaches, such as physical, chemical, and biological methods, have been examined to enhance feedstock digestibility. However, neither systematic reviews of pretreatment to promote biohydrogen production in dark fermentation nor economic feasibility analyses have been conducted. Thus, this study offers a comprehensive review of current biomass pretreatment methods to promote biohydrogen production in dark fermentation. In addition, this review has provided comparative analyses of the technological and economic feasibility of existing pretreatment techniques and discussed the prospects of the pretreatments from the standpoint of carbon neutrality and circular economy.

Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.

Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with ß-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

ß-glucan Exposure on the Fungal Cell Wall Tightly Correlates with Competitive Fitness of Candida Species in the Mouse Gastrointestinal Tract.

Candida albicans is responsible for ~400,000 systemic fungal infections annually, with an associated mortality rate of 46-75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of Candida species and is a major source of systemic fungal infections. However, the factors that control GI colonization by Candida species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of Candida species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic C. albicans cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly-related Candida species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of Candida strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI-mimicking and stressful environments or with in vitro and in vivo virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed ß-glucan on the surface of fungal cells, we found this parameter, unlike total ß-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed ß-glucan by Dectin-1 receptor appears to severely limit Candida GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis.

Novel agonists and antagonists for human protease activated receptor 2.

Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH(2), SLIGRL-NH(2)), uncertain target selectivity, and poor bioavailability, limiting their usefulness for specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2 agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC(50) 0.28 µM) selectively induced PAR2-, but not PAR1-, mediated intracellular Ca(2+) release in HT29 human colorectal carcinoma cells. Antagonist GB83 (36, IC(50) 2 µM) is the first compound at micromolar concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g., trypsin, 2f-furoyl-LIGRLO-NH(2), 19). The new compounds are selective for PAR2 over PAR1, serum stable, and suitable for modulating PAR2 in disease models.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Noncovalent tripeptidyl benzyl- and cyclohexyl-amine inhibitors of the cysteine protease caspase-1.

Potent and noncovalent inhibitors of caspase-1 were produced by incorporating a secondary amine (reduced amide) isostere in place of the conventional electrophile (e.g., aldehyde) that normally confers high potency to cysteine protease inhibitors. Benzyl- or cyclohexylamines produced potent, reversible, and competitive inhibitors that were selective for caspase-1 (e.g., K(i) = 47 nM) over caspases 3 and 8 with minimal cytotoxicity. Unlike most cysteine protease inhibitors, these compounds do not react covalently and indiscriminately with thiols.

Antifibrotic activity of an inhibitor of histone deacetylases in DOCA-salt hypertensive rats.

BACKGROUND AND PURPOSE: Histone deacetylases (HDACs) silence genes by deacetylating lysine residues in histones and other proteins. HDAC inhibitors represent a new class of compounds with anti-inflammatory activity. This study investigated whether treatment with a broad spectrum HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), would prevent cardiac fibrosis, part of the cardiovascular remodelling in deoxycorticosterone acetate (DOCA)-salt rats. EXPERIMENTAL APPROACH: Control and DOCA-salt rats were treated with SAHA (25 mg x kg(-1) x day(-1) s.c.) for 32 days. Changes in cardiovascular structure and function were assessed by blood pressure in vivo and in Langendorff perfused hearts, ventricular papillary muscle and in aortic rings in vitro. Left ventricular collagen deposition was assessed by histology. KEY RESULTS: Administration of SAHA to DOCA-salt rats attenuated the following parameters: the increased concentration of over 20 pro-inflammatory cytokines in plasma, increased inflammatory cell infiltration and interstitial collagen deposition, increased passive diastolic stiffness in perfused hearts, prolongation of action potential duration at 20% and 90% of repolarization in papillary muscle, development of left ventricular hypertrophy, systolic hypertension and changes in vascular dysfunction. CONCLUSIONS AND IMPLICATIONS: The HDAC inhibitor, SAHA, attenuated the cardiovascular remodelling associated with DOCA-salt hypertensive rats and improved cardiovascular structure and function, especially fibrosis, in the heart and blood vessels, possibly by suppressing inflammation. Control of cardiac histone or non-histone protein acetylation is a potential therapeutic approach to preventing cardiac remodelling, especially cardiac fibrosis.

Structure-activity relationships for substrate-based inhibitors of human complement factor B.

Human complement is a cascading network of plasma proteins important in immune defense, cooperatively effecting recognition, opsonization, destruction, and removal of pathogens and infected/damaged cells. Overstimulated or unregulated complement activation can result in immunoinflammatory diseases. Key serine proteases in this cascade are difficult to study due to their multiprotein composition, short lifetimes, formation on membranes, or serum circulation as inactive zymogens. Factor B is inactive at pH 7, but a catalytically active serine protease under alkaline conditions, enabling structure-activity relationship studies for 63 substrate-based peptide inhibitors with 4-7 residues and a C-terminal aldehyde. A potent factor B inhibitor was hexpeptide Ac-RLTbaLAR-H (IC(50) 250 nM, pH 9.5), which at pH 7 also blocked formation of membrane attack complex via the "alternative pathway" of complement activation and inhibited human complement mediated lysis of rabbit erythrocytes. Inhibitors of factor B may be valuable probes and drug leads for complement mediated immunity and disease.

Complement component C2, inhibiting a latent serine protease in the classical pathway of complement activation.

The innate immune response to infection or injury involves an antigen-antibody triggered classical pathway (CP) of complement activation, in which soluble and cell surface plasma proteins cooperatively effect elimination of foreign organisms and damaged host cells. However, protracted or dysfunctional complement activation can lead to inflammatory diseases. Complement component 2 bound to C4b is cleaved by classical (C1s) or lectin (MASP2) proteases to produce C4bC2a, a very short-lived C3 convertase (t(1/2) 2 min) that in turn cleaves C3 to C3a and C3b, leading ultimately to formation of Membrane Attack Complex (MAC) and lysis of bacteria and damaged cells. C2 has the same serine protease domain as C4bC2a but in an inactive zymogen-like conformation, requiring cofactor-induced conformational change for activity. Here, we show that C2 has catalytic protease activity in its own right above pH 7, in the absence of cofactor, processing C3 and C3-derived chromogenic peptide fragments. In contrast to the instability of C3 convertase (t(1/2) 2 min, pH 7), the C2 enzyme is indefinitely stable under alkaline conditions, facilitating studies of its catalytic properties and development of small molecule inhibitors. We characterize the catalytic activity of C2 against C3 and short paranitroanilide peptide substrates, and identify potent small molecule inhibitors of C2 that also inhibit classical pathway C3 convertase, MAC formation, and hemolysis of sensitized sheep erythrocytes. These results provide a new avenue and valuable new insights to inhibiting CP complement activation relevant to inflammatory diseases.

Unexpected photolytic decomposition of alkyl azides under mild conditions.

Alkyl azides have been found to undergo an unexpectedly facile photodecomposition under mild conditions (laboratory light) and the reaction gives rise to small amounts of aldehydes and monoacyl aminal rearrangement products that can dramatically impact on the uses of azides in chemistry, biology and medicine.

A refined agonist pharmacophore for protease activated receptor 2.

Protease activated receptor 2 (PAR(2)) is a G protein-coupled receptor implicated in inflammation and cancer. Only a few peptide agonists are known with greater potency than the native agonist SLIGRL-NH(2). Here we report 52 peptide agonists of PAR(2), 26 with activity at sub-micromolar concentrations, and one being iodinated for radioligand experiments. Potency was highest when the N- or C-termini of SLIGRL-NH(2) were modified, pointing to a new ligand pharmacophore model that may aid development of drug-like PAR(2) modulators.

Agonists and antagonists of protease activated receptors (PARs).

Protease activated receptors (PARs) are a category of G-protein coupled receptors (GPCRs) implicated in the progression of a wide range of diseases, including thrombosis, inflammatory disorders, and proliferative diseases. Signal transduction via PARs proceeds via an unusual activation mechanism. Instead of being activated through direct interaction with an extracellular signal like most GPCRs, they are self-activated following cleavage of their extracellular N-terminus by serine proteases to generate a new receptor N-terminus that acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. Short synthetic peptides corresponding to this newly exposed N-terminal tethered ligand can activate three of the four known PARs in the absence of proteases, and such PAR activating peptides (PAR-APs) have served as templates for agonist/antagonist development. In fact much of the evidence for involvement of PARs in diseases has relied upon use of PAR-APs, often of low potency and uncertain selectivity. This review summarizes current structures of PAR agonists and antagonists, the need for more selective and more potent PAR ligands that activate or antagonize this intriguing class of receptors, and outlines the background relevant to PAR activation, assay methods, and physiological properties anticipated for PAR ligands.

Inhibitors of TACE and Caspase-1 as anti-inflammatory drugs.

TNF-alpha neutralising agents such as Infliximab (Remicade), Etanercept (Enbrel) and the IL-1 receptor antagonist Anakinra (Kineret), are currently used clinically for the treatment of many inflammatory diseases such as Crohn's disease, rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis and psoriasis. These protein preparations are expensive to manufacture and administer, need to be injected and can cause allergic reactions. An alternative approach to lowering the levels of TNF-alpha and IL-1beta in inflammatory disease, is to inhibit the enzymes that generate these cytokines using cheaper small molecules. This paper is a broad overview of the progress that has been achieved so far, with respect to small molecule inhibitor design and pharmacological studies (in animals and humans), for the metalloprotease Tumour Necrosis Factor-alpha Converting Enzyme (TACE) and the cysteine protease Caspase-1 (Interleukin-1beta Converting Enzyme, ICE). Inhibitors of these two enzymes are currently considered to be good therapeutic targets that have the potential to provide relatively inexpensive and orally bioavailable anti-inflammatory agents in the future.