Biochemical and Domain Analyses of FSUAxe6B,a Modular Acetyl Xylan Esterase,Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser₄₄, His₂₇₃, Glu₁₉₄, and Asp₂₇₀, with both Glu₁₉₄ and Asp₂₇₀ functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.The development of strategies for biomass conversion to fuels (biofuels) is a subject of keen interest as we search for energy resources alternative to fossil fuels (39). Plant cell matter accounts for 150 to 200 billion tons of biomass on our planet annually (31). It is technically possible, but economically far from realization, to convert plant cell wall to biofuels (41). Thus, currently, plant cell wall utilization as a source of biofuels is mostly at the laboratory scale, although there is a great impetus to move production to the industrial scale.The main components of the plant cell wall are cellulose, hemicellulose, and lignin. These components form complex structures that provide the plant with physical strength (42). Biologically, there are two major steps in the production of alcohols from plant-based feedstock. The first step is an enzymatic hydrolysis of the plant cell wall components to fermentable sugars, and the second step is fermentation of the resultant sugars into alcohols. A major limitation of the process is the lack of highly efficient biocatalysts required for the first step. However, it is known that microbes, either as individuals or consortia, that harbor genes encoding enzymes that hydrolyze plant cell wall polysaccharides abound in nature. Research efforts directed at deepening knowledge of how multiple enzymes participate synergistically to degrade the plant cell wall will accelerate the capacity to achieve the goal of converting biomass to biofuels on a large scale (12, 27). However, improvement of “enzyme cocktails” developed for depolymerization of lignocellulosic biomass will be dependent on a better understanding of the structure/function of individual enzymes that together constitute the arsenal of enzymes (hydrolyzome) used by naturally occurring organisms known to be highly efficient in plant cell wall degradation.Ruminant animals harbor a variety of plant cell wall-degrading bacteria in their first stomach or rumen (26). These animals digest forages with the aid of a microbial consortium that is able to metabolize plant cell wall polysaccharides to short-chain fatty acids, the main energy source for the ruminant host. Fibrobacter succinogenes is a ubiquitous rumen bacterium and has been estimated in previous reports to occupy 0.1% to 1.0% of the microbial population in the cattle rumen, based on the quantification of 16S rRNA genes as a marker (25, 43). F. succinogenes is a significant cellulolytic rumen bacterium, and it has the ability to grow on crystalline cellulose as a sole source of carbon and energy (17). Additionally, it has been demonstrated that this bacterium can solubilize hemicelluloses, although it only partially utilized the constituent monosaccharides released (34). As further evidence, F. succinogenes failed to grow on xylose (33), a constituent of most hemicelluloses. Since F. succinogenes is a highly versatile microbe capable of degrading both cellulose and hemicellulose, strains of this bacterium are attractive models to study natural strategies for efficient deconstruction of plant cell wall polysaccharides.Through analysis of the genome sequence of F. succinogenes S85, a gene cluster that encodes more than 10 hemicellulose-targeting enzymes was identified. Most of the enzymes in the cluster are modular polypeptides, a common feature in many carbohydrate-active enzymes. Kam and coworkers (23) previously identified two acetyl xylan esterases (Axe6A and Axe6B) in this cluster and predicted that each gene encoded a polypeptide composed of two domains: an esterase catalytic domain and a family 6 carbohydrate-binding module (CBM6). Whereas Axe6A was fairly well characterized, difficulties in expression of recombinant Axe6B restricted its characterization (23). In this report, overproduction of recombinant F. succinogenes S85 Axe6B (FSUAxe6B) is demonstrated, and furthermore, it is shown that rather than having two domains, the polypeptide harbors three domains composed of an esterase, CBM6, and a region of unknown function. Bioinformatics analysis suggested that the unknown domain observed in FSUAxe6B is, so far, distributed only in F. succinogenes S85; thus, it was designated F. succinogenes-specific paralogous module 1 or FPm-1. Twenty-four polypeptides, with the majority containing glycoside hydrolase family motifs and CBMs, were found to harbor this peptide at the extreme C-terminal region. In addition to assigning a carbohydrate binding function to FPm-1, critical residues that confer esterase activity to the N-terminal half of FSUAxe6B were also identified through site-directed mutagenesis.     

Male-Biased Autosomal Effect of 16p13.11 Copy Number Variation in Neurodevelopmental Disorders

Copy number variants (CNVs) at chromosome 16p13.11 have been associated with a range of neurodevelopmental disorders including autism, ADHD, intellectual disability and schizophrenia. Significant sex differences in prevalence, course and severity have been described for a number of these conditions but the biological and environmental factors underlying such sex-specific features remain unclear. We tested the burden and the possible sex-biased effect of CNVs at 16p13.11 in a sample of 10,397 individuals with a range of neurodevelopmental conditions, clinically referred for array comparative genomic hybridisation (aCGH); cases were compared with 11,277 controls. In order to identify candidate phenotype-associated genes, we performed an interval-based analysis and investigated the presence of ohnologs at 16p13.11; finally, we searched the DECIPHER database for previously identified 16p13.11 copy number variants. In the clinical referral series, we identified 46 cases with CNVs of variable size at 16p13.11, including 28 duplications and 18 deletions. Patients were referred for various phenotypes, including developmental delay, autism, speech delay, learning difficulties, behavioural problems, epilepsy, microcephaly and physical dysmorphisms. CNVs at 16p13.11 were also present in 17 controls. Association analysis revealed an excess of CNVs in cases compared with controls (OR = 2.59; p = 0.0005), and a sex-biased effect, with a significant enrichment of CNVs only in the male subgroup of cases (OR = 5.62; p = 0.0002), but not in females (OR = 1.19, p = 0.673). The same pattern of results was also observed in the DECIPHER sample. Interval-based analysis showed a significant enrichment of case CNVs containing interval II (OR = 2.59; p = 0.0005), located in the 0.83 Mb genomic region between 15.49–16.32 Mb, and encompassing the four ohnologs NDE1, MYH11, ABCC1 and ABCC6. Our data confirm that duplications and deletions at 16p13.11 represent incompletely penetrant pathogenic mutations that predispose to a range of neurodevelopmental disorders, and suggest a sex-limited effect on the penetrance of the pathological phenotypes at the 16p13.11 locus.     

Preface

The aim of the study was to introduce a convenient method for identification of differences among individual animals in genes supposed to influence meat performance in pigs. The set of seven candidate genes (IGF2, FOS, MC4R, DGAT1, MYF4, MYF, and MC3R) was used. To determine the genotypes, multiplex polymerase chain reaction (PCR) and minisequencing using SNaPshot system (Applied Biosystems; Forster City, CA, USA) were applied. The efficiency of this gene panel for routine testing in pigs was verified in the Black Pied P?e?tice pig breed by the statistical general linear model. The results showed that both the method and the gene panel are convenient for meat quality testing and offer reproducible results.     

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.     

Analysis of the hydrogenotrophic microbiota of wild and captive black howler monkeys (Alouatta pigra) in palenque national park, Mexico

Intestinal methanogenesis is one of the major pathways for consumption of hydrogen produced by bacterial fermentation and is considered to affect the efficiency of host energy harvest; however, little information is available regarding the hydrogenotrophic pathways of nonhuman primates in the wild, in general, and of howler monkeys, in particular. Microbial fermentation of plant structural carbohydrates is an important feature in wild howlers owing to the high fiber and low available energy content of leaves, which make up the primary component of their diet. In contrast, captive howlers may consume greater quantities of fruits and vegetables that are higher in water, lower in fiber, and, along with commercial monkey chow commonly added to captive monkey diets, more readily digestible than the natural diet. In this study, we analyzed the composition of methanogens and sulfate-reducing bacteria (SRB) from fecal samples of black howler monkeys (Alouatta pigra) in the wild and in captivity. The hydrogenotrophic microbiota of three groups of monkeys was evaluated by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, small clone library construction, and quantitative real-time PCR. Abundance of methanogens was lower than SRB in all howler monkey groups studied. DGGE banding patterns were highly similar within each wild and captive group but distinct among groups. Desulfovibrionales-enriched DGGE showed reduced microbial diversity in the captive animals compared with their wild counterparts. Taken together, the data demonstrate that environmental or dietary changes of the host imposed by captivity likely influence the composition of intestinal hydrogenotrophs in black howler monkeys.     

Functional Potential of Soil Microbial Communities in the Maize Rhizosphere

Microbial communities in the rhizosphere make significant contributions to crop health and nutrient cycling. However, their ability to perform important biogeochemical processes remains uncharacterized. Here, we identified important functional genes that characterize the rhizosphere microbial community to understand metabolic capabilities in the maize rhizosphere using the GeoChip-based functional gene array method. Significant differences in functional gene structure were apparent between rhizosphere and bulk soil microbial communities. Approximately half of the detected gene families were significantly (p<0.05) increased in the rhizosphere. Based on the detected gyrB genes, Gammaproteobacteria, Betaproteobacteria, Firmicutes, Bacteroidetes and Cyanobacteria were most enriched in the rhizosphere compared to those in the bulk soil. The rhizosphere niche also supported greater functional diversity in catabolic pathways. The maize rhizosphere had significantly enriched genes involved in carbon fixation and degradation (especially for hemicelluloses, aromatics and lignin), nitrogen fixation, ammonification, denitrification, polyphosphate biosynthesis and degradation, sulfur reduction and oxidation. This research demonstrates that the maize rhizosphere is a hotspot of genes, mostly originating from dominant soil microbial groups such as Proteobacteria, providing functional capacity for the transformation of labile and recalcitrant organic C, N, P and S compounds.     

Osteoblasts: novel roles in orchestration of skeletal architecture

Osteoblasts are located on bone surfaces and are the cells responsible for bone formation through secretion of the organic components of bone matrix. Osteoblasts are derived from mesenchymal osteoprogenitor cells found in bone marrow and periosteum. Following a period of secretory activity, osteoblasts undergo either apoptosis or terminal differentiation to form osteocytes surrounded by bone matrix. Osteoblasts secrete a characteristic mixture of extracellular matrix proteins including type I collagen as the major component as well as proteoglycans, glycoproteins and gamma-carboxylated proteins. Cells of the osteoblast lineage also provide factors essential for differentiation of osteoclasts (bone-resorbing cells). By regulating osteoclast differentiation and activity in response to systemic influences, osteoblasts not only play a central role in regulation of skeletal architecture, but also in calcium homeostasis. Inadequate osteoblastic bone formation in relation to osteoclastic resorption results in osteoporosis, a disease characterised by enhanced skeletal fragility. Cellfacts: Osteoblasts are the cells responsible for bone formation. Osteoblasts indirectly control levels of bone resorption. Osteoblasts play a key role in the pathophysiology of osteoporosis and the resulting fractures, which constitute a major public health burden in developed countries.     

Laserspray Ionization, a New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.Tissue imaging by mass spectrometry (MS) is proving useful in areas such as detecting tumor margins, determining sites of high drug uptake, and mapping signaling molecules in brain tissue (1–8). Imaging using secondary ion mass spectrometry is well established but is only marginally useful with intact molecular mass measurements from biological tissue (9–11). Matrix-assisted laser desorption/ionization (MALDI)-MS operating under vacuum conditions has been used for tissue imaging with success, especially for abundant components such as membrane lipids, drug metabolites, and proteins (12–14). Spatial resolution of ∼20 μm has been achieved (15), and the MALDI-MS method has been applied in an attempt to shed light on Parkinson disease (16, 17), muscular dystrophy (18), obesity, and cancer (12, 19).Unfortunately, there are disadvantages in using vacuum-based MS for tissue imaging in relation to analysis of unadulterated tissue. Also, the mass spectrometers used in these studies frequently have much lower mass resolution and mass accuracy than are available with atmospheric pressure ionization (API)¹ instruments and are not as widely available. Because the vacuum ionization methods produce singly charged ions, mass-selected fragmentation methods provide only limited information, especially for proteins. In addition, no advanced fragmentation such as electron transfer dissociation (ETD) (20–22) is available for confident protein confirmation or identification. Atmospheric pressure (AP) MALDI can be coupled to high performance mass spectrometers but suffers from sensitivity issues for tissue imaging where high spatial resolution is desired (23). AP MALDI also primarily produces singly charged ions (24, 25). Thus, mass and cross-section analysis of intact proteins has yet to be accomplished using AP MALDI because of intrinsic mass range limitations of API instruments, which frequently have a mass-to-charge (m/z) limit of <4000. Thus, new improved methods of mass-specific tissue imaging, especially at AP, are needed.The potential of laserspray ionization (LSI) (Scheme 1) (26–33) for protein tissue analysis is reported here. LSI has advantages relative to other MS-based methods, including speed of analysis, laser ablation of small volumes, more relevant AP conditions, extended mass range and improved fragmentation through multiple charging, and the ability to obtain cross-section data for proteins on appropriate instrumentation. The applicability of LSI for high mass compounds on high performance API mass spectrometers (Orbitrap Exactive and SYNAPT G2) has been demonstrated producing ESI-like multiply protonated ions (26–28). The first experiments showing sequence analysis by ETD using the LSI method were successfully carried out on a Thermo Fisher Scientific (San Jose, CA) LTQ-ETD mass spectrometer (26). Nearly complete sequence coverage was obtained for ubiquitin, an important regulatory protein. Applying ETD fragmentation to LSI-MS analyses potentially provides a new method for studying biological processes, including the mapping of phosphorylation, glycosylation, and ubiquitination sites from intact proteins and directly from tissue.Open in a separate windowScheme 1.Overview of LSI-MS operated in transmission geometry.Furthermore, unlike ESI and related ESI-based methods such as desorption-ESI (34), the LSI method has been shown to allow analysis of lipids in tissue from ablated areas <80 μm (30). In comparison with literature reports for AP MALDI at the same stage of development (35), LSI is more than an order of magnitude more sensitive and is capable of analyzing proteins on high resolution mass spectrometers as was demonstrated by obtaining full-acquisition mass spectra at 100,000 mass resolution (FWHH, m/z 200) after application of only 20 fmol of bovine pancreas insulin in the matrix 2,5-dihydroxyacetophenone (2,5-DHAP) onto a glass microscope slide (33). The analysis speed of LSI was demonstrated by obtaining mass spectra of five samples in 8 s (32). Here, we show the utility of LSI for intact peptide and protein analyses directly from mouse brain tissue. The ability to obtain a protein mass spectrum directly from mouse brain tissue in a single laser shot at 100,000 mass resolution and with ETD fragmentation is demonstrated.     

Automatic detection of the carotid artery boundary on cross-sectional MR image sequences using a circle model guided dynamic programming

Background  

Systematic aerobe training has positive effects on the compliance of dedicated arterial walls. The adaptations of the arterial structure and function are associated with the blood flow-induced changes of the wall shear stress which induced vascular remodelling via nitric oxide delivered from the endothelial cell. In order to assess functional changes of the common carotid artery over time in these processes, a precise measurement technique is necessary. Before this study, a reliable, precise, and quick method to perform this work is not present.     

Discovery and SAR of a novel series of non-MPEP site mGlu5 PAMs based on an aryl glycine sulfonamide scaffold

Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu₅) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site mGlu₅ PAM chemotype.