Analysis, evaluation, and optimization of kinetic parameters for performance appraisal and design of UASB reactors

Studies have been undertaken to explore the applicability of different kinetic models for the performance appraisal of upflow anaerobic sludge blanket (UASB) reactors treating wastewater in the range of 300-4000 mg COD/l. Three kinetic models namely, Monod, Grau second-order, and Haldane model are considered for the analysis. Both linear and nonlinear regressions have been performed to examine the best-fit among the kinetic models. In this process, five error analysis methods have been used to analyze the data. Apart from optimization of kinetic coefficients with minimization of associated errors, prediction of effluent COD has also been undertaken to verify the applicability of kinetic models. In both the cases, Grau second-order model is found to be the best class of fit for wide range of data sets in UASB reactor.     

Interactions of a designed peptide with lipopolysaccharide: Bound conformation and anti-endotoxic activity

Designed peptides that would selectively interact with lipopolysaccharide (LPS) or endotoxin and fold into specific conformations could serve as important scaffolds toward the development of antisepsis compounds. Here, we describe solution structure of a designed amphipathic peptide, H₂N-YVKLWRMIKFIR-CONH₂ (YW12D) in complex with endotoxin as determined by transferred nuclear Overhauser effect spectroscopy. The conformation of the isolated peptide is highly flexible, but undergoes a dramatic structural stabilization in the presence of LPS. Structure calculations reveal that the peptide presents two amphipathic surfaces in its bound state to LPS whereby each surface is characterized by two positive charges and a number of aromatic and/or aliphatic residues. ITC data suggests that peptide interacts with two molecules of lipid A. In activity assays, YW12D exhibits neutralization of LPS toxicity with very little hemolysis of red blood cells. Structural and functional properties of YW12D would be applicable in designing low molecular weight non-toxic antisepsis molecules.     

Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: A high-resolution NMR study

Recently, γ-secretase modulators (GSM) have been shown to interact directly with the amyloid precursor protein (APP) and simultaneously inhibit the activity of the Presenilin domain of γ-secretase. A clear understanding of the molecular recognition pathways by which GSM can target both γ-secretase and Aβ precursor protein can lead to the development of more effective inhibitors. To examine whether this direct interaction with APP affects the downstream Aβ fibril formation, we chose to investigate three different molecules in this study: Sulindac sulfide, Semagacestat and E2012 from the class of generation I GSMs, γ-secretase inhibitors (GSI), and generation II GSM molecules, respectively. Firstly, through NMR based ligand titration, we identified that Sulindac sulfide and Semagacestat interact strongly with Aβ40 monomers, whereas E2012 does not. Secondly, using saturation transfer difference (STD) NMR experiments, we found that all three molecules bind equally well with Aβ40 fibrils. To determine if these interactions with the monomer/fibril lead to a viable inhibition of the fibrillation process, we designed an NMR based time-dependent assay and accurately distinguished the inhibitors from the non-inhibitors within a short period of 12 h. Based on this pre-seeded fibril assay, we conclude that none of these molecules inhibit the ongoing fibrillation, rather ligands such as Semagacestat and E2012 accelerated the rate of aggregation.     

Anchoring of palladium(II) in chemically modified mesoporous silica: An efficient heterogeneous catalyst for Suzuki cross-coupling reaction

The synthesis and characterization of a highly efficient and reusable catalyst, Pd(II) immobilized in mesoporous silica MCM-41, are described. Pd(II) Schiff-base moiety has been anchored onto mesoporous silica surface via silicon alkoxide chemistry. The catalyst has been characterized by small-angle X-ray diffraction (SAX), FTIR and electronic spectroscopy as well as elemental analysis. The catalyst is used in Suzuki cross-coupling reaction of various aryl halides, including less reactive chlorobenzene, and phenylboronic acid to give biaryls in excellent yields without any additive or ligand. High selectivity for the bi-aryl products containing both electron-donating and electron-withdrawing substituents, mild reaction conditions and possibility of easy recycle makes the catalyst highly desirable to address the industrial needs and environmental concerns.     

Degradation of polypropylene–poly-L-lactide blend by bacteria isolated from compost

Abstract

Polypropylene (PP) degrading bacteria (P1 to P16) were isolated from compost using enrichment technique. Five isolates (P3, P6, P8, P10, and P13) were selected based on their degradation abilities. These isolates were identified as Bacillus spp. through biochemical characteristics and 16S rDNA sequence analysis. The isolates were tested for their ability to degrade blends of PP and poly-L-lactide (PLLA) (PP80 and PP80C6) in minimal media as well as in soil. In minimal media, the growth of bacteria increased with time, showing utilization of blend as carbon source. The protein content was estimated at the end of 15?days and maximum amount was secreted by isolate P8 indicating maximum potential to degrade polymers compared to other isolates. Scanning electron microscopy (SEM) results revealed the formation of biofilm on the polymer surface. Fourier-transform infrared spectroscopy (FTIR) analysis showed the formation of new bond at 2123?cm^?1 and breakage of old C=O ester bond at 1757?cm^?1 in case of polymer PP80C6. Thermogravimetric analysis (TGA) showed decrease in thermal stability of polymers after degradation. The carbon dioxide evolved from sample was measured and biodegradation degree was also calculated. The degree of biodegradation shown by the isolate P8 was 12% and the P6 was 10%. The results demonstrated that Bacillus species isolated from composted samples in this study provided promising evidence for the biodegradation of polypropylene and poly-L-lactide (PP-PLLA) blends in the environment.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Influence of Temperature and Growth Phase on Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117–124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42°C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42°C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25°C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37°C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.     

Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths

Aim: To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. Methods and Results: Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria‐Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37°C but not at 4°C. Transmission electron microscopy, enzyme‐linked immunosorbent assay, and mRNA analysis further supported these observations. Conclusion: Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. Significance and Impact of the study: As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody‐based detection of L. monocytogenes.     

Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides: STRUCTURES AND ACTIVITIES IN LIPOPOLYSACCHARIDE*?

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.LPS² or endotoxin, a major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is critically involved in health and diseases of humans (1, 2). LPS is essential for bacterial survival through establishing an efficient permeability barrier against a variety of antimicrobial compounds including hydrophobic antibiotics, detergents, host-defense proteins, and antimicrobial peptides (3, 4). Several studies have demonstrated that LPS catalyzes folding of outer membrane proteins as a chaperone (5–7).LPS, a potent inducer of innate immune systems, hence called endotoxin, is primarily responsible for lethality in sepsis and septic shock syndromes associated with serious Gram-negative infections (8–10). Circulating LPS in bloodstream is intercepted by the phagocytic cells of the innate immune system. Once induced by LPS, these phagocytes produce proinflammatory cytokines, e.g. tumor necrosis factor-α, interleukin-6, and interleukin-1β, through the activation of a Toll-like pattern recognition receptor (11, 12). The release of cytokines in response to microbial invasion is a natural function of the innate immunity. However, an uncontrolled and overwhelming production of these cytokines may cause “endotoxic shock” or septic shock, typified by endothelial tissue damage, loss of vascular tone, coagulopathy, and multiple organ failure, often resulting in death (9, 10). Sepsis is the major cause of mortality in the intensive care unit, accounting for 200,000 deaths every year in the United States alone (13). It was demonstrated that release of LPS from antibiotic-treated Gram-negative bacteria can indeed enhance sepsis (14). Therefore, an effective antibiotic should not only exert antibacterial activities but also have the ability to sequester LPS and ameliorate its toxicity. Therefore, an amalgamated property of LPS-neutralizing and antimicrobial activity would be highly desirable for antimicrobial agents. Polymyxin B is a prototypical antimicrobial and antiendotoxic antibiotic; however, its neurotoxicity and nephrotoxicity limit its application to topical use (15). The increasing emergence of bacterial strains that are resistant to conventional antibiotics has initiated vital structure/function studies of membrane-perturbing cationic antimicrobial peptides (16–20). More recent studies have been conducted to understand interactions between antimicrobial peptides with LPS to gain insights into the mechanism of outer membrane perturbation, antibacterial activities, and LPS neutralization (21–26). These studies have delineated the role of amino acid sequence properties, LPS-peptide interactions by biophysical methods, and global structural parameters, obtained by CD and FTIR.Designing synthetic peptides and elucidation of three-dimensional structures in complex with LPS would be useful for the purpose of rational development of non-toxic antisepsis and antimicrobial therapeutics. Such studies will also be potentially instructive in establishing rules by which folded structures can be stabilized on the LPS surface. Extensive work in the field of peptide design primarily focuses on mimicking secondary structures and tertiary folds of proteins. Usually, short linear peptides are often structurally flexible; however, the functions of these peptides are highly dependent on their ability to adopt folded structures upon complex formation with their cognate receptors. In this regard, designed peptides that would yield high resolution structures in complex with LPS have not been well pursued. LPS, being a negatively charged amphiphilic molecule, interacts with naturally occurring peptides or protein fragments containing basic/polar and hydrophobic amino acids, although there are considerable variations in lengths, sequences, and amino acid compositions among these peptides (27, 28).Here, we have determined the three-dimensional structures of a series of 12-residue peptides in the context of LPS. To the best of our knowledge, these results show, for the first time, that atomic resolution structures of designed peptides obtained in LPS could be correlated with their antiendotoxic activities. Furthermore, the LPS-induced structures of active, inactive, and short peptide motif, presented here, may provide building blocks for the designing novel proteins for the outer membrane.