Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex.

Heparan sulfate (HS) proteoglycans are extended (-GlcAß1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1-EXT2 (EXT1-2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold ß1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. The two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide defined by its repeating disaccharide backbone structure of [4)-α-D-GalA-(1,2)-α-L-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identified, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown. Here a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to reflect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into a polymeric product. RGGAT1 is a member of a new GT family categorized as GT116, which does not group into existing GT-A clades and is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan. RGGAT1 has a predicted GT-A fold structure but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type. The identification of RGGAT1 and the 8-member Arabidopsis GT116 family provides a new avenue for studying the mechanism of RG-I synthesis and the function of RG-I in plants.

Multiple Arabidopsis galacturonosyltransferases synthesize polymeric homogalacturonan by oligosaccharide acceptor-dependent or de novo synthesis.

Homogalacturonan (HG), the most abundant pectic glycan, functions as a cell wall structural and signaling molecule essential for plant growth, development and response to pathogens. HG exists as a component of pectic homoglycans, heteroglycans and glycoconjugates. HG is synthesized by members of the GALACTURONOSYLTRANSFERASE (GAUT) family. UDP-GalA-dependent homogalacturonan:galacturonosyltransferase (HG:GalAT) activity has previously been demonstrated for GAUTs 1, 4 and 11, as well as the GAUT1:GAUT7 complex. Here, we show that GAUTs 10, 13 and 14 are also HG:GalATs and that GAUTs 1, 10, 11, 13, 14 and 1:7 synthesize polymeric HG in vitro. Comparison of the in vitro HG:GalAT specific activities of the heterologously-expressed proteins demonstrates GAUTs 10 and 11 with the lowest, GAUT1 and GAUT13 with moderate, and GAUT14 and the GAUT1:GAUT7 complex with the highest HG:GalAT activity. GAUT13 and GAUT14 are also shown to de novo synthesize (initiate) HG synthesis in the absence of exogenous HG acceptors, an activity previously demonstrated for GAUT1:GAUT7. The rate of de novo HG synthesis by GAUT13 and GAUT14 is similar to their acceptor dependent HG synthesis, in contrast to GAUT1:GAUT7 for which de novo synthesis occurred at much lower rates than acceptor-dependent synthesis. The results suggest a unique role for de novo HG synthesis by GAUTs 13 and 14. The reducing end of GAUT13-de novo-synthesized HG has covalently attached UDP, indicating that UDP-GalA serves as both a donor and acceptor substrate during de novo HG synthesis. The functional significance of unique GAUT HG:GalAT catalytic properties in the synthesis of different pectin glycan or glycoconjugate structures is discussed.

In Envelope Additive/Subtractive Manufacturing and Thermal Post-Processing of Inconel 718.

This study investigated the application of an in envelope additive/subtractive (LPBF) manufacturing method (Matsuura LUMEX-Avance-25) to fabricate IN718 benchmarking coupons. The coupons were then examined comprehensively for surface finish both with and without high-speed micro-machining. The microstructure of the manufactured IN718 coupons was investigated thoroughly in the as-fabricated condition and following three different standard and one non-standard post-processing heat treatments. As built coupons revealed columnar grain morphology mainly along the <100> direction with a cellular dendritic sub-grain structure and without any strengthening precipitates. Grain size, aspect ratio, and texture were maintained after each of the applied four heat treatments. Only one of the standard heat treatments resulted in the δ phase formation. The other three heat treatments effectively dissolved the Laves phase preventing the δ formation while promoting the formation of γ'/γ″ precipitates. Despite the observed differences in their microstructures, all of the heat treatments resulted in similar yield and ultimate tensile strength values that ranged between 1103−1205 MPa and 1347−1387 MPa, respectively. These values are above the minimum requirements of 1034 MPa and 1241 MPa for the wrought material. The non-standard heat treatment provided the highest elongation of 24.0 ± 0.1% amongst all the heat-treated specimens without a significant loss in strength, while the standard heat treatment for the wrought parts resulted in the lowest elongation of 18.3 ± 0.7% due to the presence of δ phase.

Critical Review of Plant Cell Wall Matrix Polysaccharide Glycosyltransferase Activities Verified by Heterologous Protein Expression.

The life cycle and development of plants requires the biosynthesis, deposition, and degradation of cell wall matrix polysaccharides. The structures of the diverse cell wall matrix polysaccharides influence commercially important properties of plant cells, including growth, biomass recalcitrance, organ abscission, and the shelf life of fruits. This review is a comprehensive summary of the matrix polysaccharide glycosyltransferase (GT) activities that have been verified using in vitro assays following heterologous GT protein expression. Plant cell wall (PCW) biosynthetic GTs are primarily integral transmembrane proteins localized to the endoplasmic reticulum and Golgi of the plant secretory system. The low abundance of these enzymes in plant tissues makes them particularly difficult to purify from native plant membranes in quantities sufficient for enzymatic characterization, which is essential to study the functions of the different GTs. Numerous activities in the synthesis of the major cell wall matrix glycans, including pectins, xylans, xyloglucan, mannans, mixed-linkage glucans (MLGs), and arabinogalactan components of AGP proteoglycans have been mapped to specific genes and multi-gene families. Cell wall GTs include those that synthesize the polymer backbones, those that elongate side branches with extended glycosyl chains, and those that add single monosaccharide linkages onto polysaccharide backbones and/or side branches. Three main strategies have been used to identify genes encoding GTs that synthesize cell wall linkages: analysis of membrane fractions enriched for cell wall biosynthetic activities, mutational genetics approaches investigating cell wall compositional phenotypes, and omics-directed identification of putative GTs from sequenced plant genomes. Here we compare the heterologous expression systems used to produce, purify, and study the enzyme activities of PCW GTs, with an emphasis on the eukaryotic systems Nicotiana benthamiana, Pichia pastoris, and human embryonic kidney (HEK293) cells. We discuss the enzymatic properties of GTs including kinetic rates, the chain lengths of polysaccharide products, acceptor oligosaccharide preferences, elongation mechanisms for the synthesis of long-chain polymers, and the formation of GT complexes. Future directions in the study of matrix polysaccharide biosynthesis are proposed.

A two-phase model for the non-processive biosynthesis of homogalacturonan polysaccharides by the GAUT1:GAUT7 complex.

Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.

Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of GAlactUronosylTransferase12 (GAUT12) causes increased recalcitrance and decreased growth in Populus.

BACKGROUND: The development of fast-growing hardwood trees as a source of lignocellulosic biomass for biofuel and biomaterial production requires a thorough understanding of the plant cell wall structure and function that underlie the inherent recalcitrance properties of woody biomass. Downregulation of GAUT12.1 in Populus deltoides was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand GAUT12.1 function in biomass recalcitrance and plant growth, here we report the effects of P. trichocarpa GAUT12.1 overexpression in P. deltoides. RESULTS: Increasing GAUT12.1 transcript expression by 7-49% in P. deltoides PtGAUT12.1-overexpression (OE) lines resulted in a nearly complete opposite biomass saccharification and plant growth phenotype to that observed previously in PdGAUT12.1-knockdown (KD) lines. This included significantly reduced glucose, xylose, and total sugar release (12-13%), plant height (6-54%), stem diameter (8-40%), and overall total aerial biomass yield (48-61%) in 3-month-old, greenhouse-grown PtGAUT12.1-OE lines compared to controls. Total lignin content was unaffected by the gene overexpression. Importantly, selected PtGAUT12.1-OE lines retained the recalcitrance and growth phenotypes upon growth for 9 months in the greenhouse and 2.8 years in the field. PtGAUT12.1-OE plants had significantly smaller leaves with lower relative water content, and significantly reduced stem wood xylem cell numbers and size. At the cell wall level, xylose and galacturonic acid contents increased markedly in total cell walls as well as in soluble and insoluble cell wall extracts, consistent with increased amounts of xylan and homogalacturonan in the PtGAUT12.1-OE lines. This led to increased cell wall recalcitrance, as manifested by the 9-15% reduced amounts of recovered extractable wall materials and 8-15% greater amounts of final insoluble pellet in the PtGAUT12.1-OE lines compared to controls. CONCLUSIONS: The combined phenotype and chemotype data from P. deltoides PtGAUT12.1-OE and PdGAUT12.1-KD transgenics clearly establish GAUT12.1 as a recalcitrance- and growth-associated gene in poplar. Overall, the data support the hypothesis that GAUT12.1 synthesizes either an HG-containing primer for xylan synthesis or an HG glycan required for proper xylan deposition, anchoring, and/or architecture in the wall, and the possibility of HG and xylan glycans being connected to each other by a base-sensitive covalent linkage.

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform.

The widespread dissemination of CTX-M extended spectrum ß-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16,800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes.

Design and synthesis of a novel series of [1-(4-hydroxy-benzyl)-1H-indol-5-yloxy]-acetic acid compounds as potent, selective, thyroid hormone receptor ß agonists.

The design, synthesis, and structure activity relationships for a novel series of indoles as potent, selective, thyroid hormone receptor ß (TRß) agonists is described. Compounds with >50× binding selectivity for TRß over TRα were generated and evaluation of compound 1c from this series in a model of dyslipidemia demonstrated positive effects on plasma lipid endpoints in vivo.

Focal Ca2+ transient detection in smooth muscle.

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Benzopyrans are selective estrogen receptor beta agonists with novel activity in models of benign prostatic hyperplasia.

Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor beta (ERbeta) in vitro assays. X-ray crystal structures of SERBA-1 in ERalpha and beta help explain observed beta-selectivity of this ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate in CD-1 mice (ERbeta effect), while having no effect on gonadal hormone levels (ERalpha effect) at 10x the efficacious dose, consistent with in vitro properties of this molecule.