Glycan Sequence‐Dependent Nod2 Activation Investigated by Using a Chemically Synthesized Bacterial Peptidoglycan Fragment Library

Nucleotide oligomerization domain‐containing protein 2 (Nod2), an innate immune receptor, recognizes bacterial cell‐wall peptidoglycan (PGN), the minimum ligand of which is muramyl dipeptide (MDP). Enzymatic digestion of PGN appears to be important for Nod2 recognition. PGN is degraded by muramidase or glucosamidase through a process that produces two types of glycan sequence; glycans containing GlcNAcβ(1→4)MurNAc or MurNAcβ(1→4)GlcNAc. In this report, a range of disaccharide or tetrasaccharide fragments of each sequence were chemically synthesized, and their activities in stimulating human Nod2 (hNod2) were investigated. The results reveal that hNod2 recognitions is dependent on the glycan sequence, as demonstrated by comparing the activities of glycans with the same peptide moieties. (MurNAcβ(1→4)GlcNAc)₂‐containing structures exhibited stronger activity than those containing (GlcNAcβ(1→4)MurNAc)₂. The results suggest that differences in the enzymatic degradation process affect the host's immunomodulation process.     

Structural Insights into Enzymatic Degradation of Oxidized Polyvinyl Alcohol

The ever‐increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel α/β‐hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA‐degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid‐like β‐ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the C?C bond of β‐diketone, although it has a catalytic triad similar to that of most α/β‐hydrolases. Analysis of the crystal structures suggests a double‐oxyanion‐hole mechanism, previously only found in thiolase cleaving β‐ketoacyl‐CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications.     

LPEATs Tailor Plant Phospholipid Composition through Adjusting Substrate Preferences to Temperature

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms.     

Highly efficient selective oxidation of sulfides to sulfoxides by montmorillonite-immobilized metalloporphyrins in the presence of molecular oxygen

Highly efficient and selective oxidation of sulfides to sulfoxides with dioxygen catalyzed by cationic meso-tetrakis (1-methyl-4-pyridyl) (TM4PyP) metalloporphyrins immobilized into montmorillonite (MT) interlayer was achieved. Manganese (II) porphyrin (MnTM4PyP-MT) presented excellent activity for the oxidation of sulfides under ambient conditions. In the model oxidation, thioanisole was converted completely and the selectivity towards sulfoxide was up to 95%. This catalyst also showed high activity and selectivity for the most sulfides. The catalyst could be reused consecutively five times without significant loss of activity.     

Catalytic resolution of dl-tryptophan amides using the resting cells of Flavobacterium aquatile ZJB-09211 in a two-phase system

The catalytic activity of Flavobacterium aquatile ZJB-09211 towards the kinetic resolution of DL-tryptophan amides was significantly enhanced by ethyl acetate. A maximum enzyme activity of 5118.62 U/g was obtained under the optimized conditions consisting of a mixture of ethyl acetate and Tris–HCl buffer (30:70). In a scale-up reaction, the tryptophan amide concentration was improved to 200 mM, with 49.85% (e.e. > 99.95%) of the substrate being converted to l-tryptophan. The addition of an organic solvent to the process therefore provided an effective approach for improving the activity of the amidase that could be applied to other amidase-catalyzed bioprocesses.     

Synthesis and photophysical properties of covalently linked boron dipyrromethene dyads

Two covalently linked boron dipyrromethene (BODIPY) dyads containing meso-phenyl BODIPY and meso-furyl BODIPY units connected via meso-meso and meso-α positions were synthesized by a Pd(0) coupling reaction. The dyads are freely soluble in common organic solvents and their structures were confirmed by HR-MS, 1D and 2D NMR techniques. Absorption studies indicate that the meso-aryl BODIPY and the meso-furyl BODIPY absorb in two different regions and that the meso-furyl BODIPY absorbs at lower energy compared to the meso-aryl BODIPY. The steady state fluorescence studies carried out by exciting the meso-aryl BODIPY unit clearly indicated an efficient singlet-singlet energy transfer from the meso-aryl BODIPY unit to meso-furyl BODIPY unit in both dyads. Furthermore the meso-α linked BODIPY dyad (?_f = 0.41) is more fluorescent than its corresponding BODIPY monomers whereas the meso-meso linked BODIPY dyad (?_f = 0.017) is weakly fluorescent. This unexpected observation was tentatively attributed to the restricted rotation of the BODIPY units in the meso-α linked dyad resulting in enhancement of radiative transitions. The time-resolved fluorescence study also indicated that meso-α linked BODIPY dyad is more fluorescent with singlet state lifetime of 3.7 ns. The DFT studies carried out on dyads are in agreement with the experimental observations.     

Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)–Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-β-xylanases that hydrolyze within the xylan structure, and the β-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.     

Comparison between unsupported mesoporous Co3O4 and supported Co3O4 on mesoporous silica as catalysts for N2O decomposition

Mesoporous Co₃O₄ (meso-Co₃O₄) and Co₃O₄ nanoparticles supported on mesoporous silica SBA-15 (Co/SBA-15) were prepared by hydrothermal synthesis and an impregnation method, respectively. Although the as-prepared meso-Co₃O₄ had mesopores and a higher surface area comparable to that of Co/SBA-15, its catalytic activity for N₂O decomposition was much lower than that of Co₃O₄/SBA-15. The low catalytic activity of meso-Co₃O₄ mainly stems from the drastic decrease of the meso-Co₃O₄ surface area under the reaction condition used. On the other hand, Co/SBA-15 maintained its high surface area and mesopores with the aid of a robust silica support. This finding indicates that Co₃O₄ supported by a support is much more stable and efficient than meso-Co₃O₄ under N₂O decomposition reaction conditions.     

Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

A meso‐diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D ‐amino acids other than its native substrate. Site‐directed mutagenesis similar to that carried out on the residues mutated by Vedha‐Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D ‐amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso‐diaminopimelate (meso‐DAP), D ‐leucine (D ‐leu), and 4‐methyl‐2‐oxopentanoic acid (MOPA) were solved. meso‐DAP was found in an area outside the catalytic cavity; this suggested a possible two‐step substrate‐binding mechanism for meso‐DAP. D ‐leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso‐diaminopimelate dehydrogenases.     

Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm,Eisenia andrei

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.     

A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB

The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d -Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d -Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB.     

Aluminated hierarchical silicalite-2 particles: Catalyst with remarkably increased lifetime for methanol to hydrocarbons

Aluminated hierarchical silicalite-2 with both large particle size and high external surface area was prepared by aluminating silicalite-2 under the protection of TBA⁺ cations in a mild alkaline solution. The textural and acidic properties of thus formed material (Z11-meso) were characterized by SEM, TEM, XRD, N₂ adsorption, FTIR, NH₃-TPD, and ²⁷Al NMR spectroscopy. The resulting hierarchical Z11-meso contained mainly tetrahedral coordinated aluminum species with low Brønsted/Lewis ratio (B/L) while preserved the zeolitic structure. Owning to the high external surface area (S_ext), hierarchical porous structures and low B/L ratio, Z11-meso outperformed the microporous ZSM-11 counterparts in methanol to hydrocarbons in terms of both activity and stability.     

Activation of the glmS Ribozyme Confers Bacterial Growth Inhibition

The ever‐growing number of pathogenic bacteria resistant to treatment with antibiotics call for the development of novel compounds with as‐yet unexplored modes of action. Here, we demonstrate the in vivo antibacterial activity of carba‐α‐d ‐glucosamine (CGlcN). In this mode of action study, we provide evidence that CGlcN‐mediated growth inhibition is due to glmS ribozyme activation, and we demonstrate that CGlcN hijacks an endogenous activation pathway, hence utilizing a prodrug mechanism. This is the first report describing antibacterial activity mediated by activating the self‐cleaving properties of a ribozyme. Our results open the path towards a compound class with an entirely novel and distinct molecular mechanism.     

LytR-CpsA-Psr Glycopolymer Transferases: Essential Bricks in Gram-Positive Bacterial Cell Wall Assembly

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes—the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N-acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N-acetylglucosamine residues of PGN—in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris. Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria.     

Stereospecific preparation of polyacrylamide with low polydispersity by ATRP in the presence of Lewis acid

The stereospecific atom transfer radical polymerizations of acrylamide were achieved in the presence of the Lewis acid Y(OTf)₃ or AlCl₃ as stereospecific catalyst using chloroacetic acid/CuCl/N,N,N′,N′-tetramethyl-ethylenediamine (TMEDA) as initiating system. The addition of Lewis acid Y(OTf)₃ in the ATRP of acrylamide led to an increased polymerization rate and an improved tacticity of polyacrylamide (m ∼ 71%) at the expense of controllability of the molecular weight distribution. In the case of AlCl₃, the polymerizations were committed to afford the resultant polyacrylamide with lower polydispersity index ranging from 1.03 to 1.42 and well-controlled tacticity with meso content ranging from 57 to 76% depending on the different reaction conditions used. Lower temperature or higher concentration of the feeding Lewis acid helped to obtain the polyacrylamide with increased tacticity which revealed a decreased glass transition temperature (T_g).     

miR-29a Participated in Nacre Formation and Immune Response by Targeting Y2R in Pinctada martensii

miR-29a is a conserved miRNA that participates in bone formation and immune response in vertebrates. miR-29a of Pinctada martensii (Pm-miR-29a) was identified in the previous research though deep sequencing. In this report, the precise sequence of mature Pm-miR-29a was validated using miRNA rapid amplification of cDNA ends (miR-RACE) technology. The precursor sequence of Pm-miR-29a was predicted to have 87 bp. Stem loop qRT-PCR analysis showed that Pm-miR-29a was easily detected in all the tissues, although expressions in the mantle and gill were low. The microstructure showed the disrupted growth of the nacre after Pm-miR-29a over-expression, which was induced by mimic injection into P. martensii. Results of the target analysis indicated that neuropeptide Y receptor type 2 (Y2R) was the potential target of Pm-miR-29a. Meanwhile, Pm-miR-29a mimics could obviously inhibit the relative luciferase activity of the reporter containing 3′ UTR (Untranslated Regions) of the Y2R gene. Furthermore, the expression of Y2R was downregulated whereas expressions of interleukin 17 (IL-17) and nuclear factor κB (NF-κB) were upregulated after Pm-miR-29a over-expression in the mantle and gill, thereby suggesting that Pm-miR-29a could activate the immune response of the pearl oyster. Results showed that Pm-miR-29a was involved in nacre formation and immune response by regulating Y2R in pearl oyster P. martensii.