NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis

MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis

Cold Exposure Drives Weight Gain and Adiposity following Chronic Suppression of Brown Adipose Tissue.

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (~20 °C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold exposure (20 °C) and β3-adrenoreceptor agonism (YM-178) under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28 °C to 20 °C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration, thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in the subcutaneous inguinal white adipose tissue (IWAT) in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e., G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e., eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality

Strength of Silica Fume Incorporated Mortar Specimens Exposed to High Temperature

ABSTRACT: The effect of high temperature on the strength of mortar specimens having W/B ratios of 0.4, 0.5 and 0.7 was investigated. Silica fume (SF) was replaced with portland cement at 0, 5 and 10% by weight. After 7, 28 and 90-day standard moist curing, followed by a 7-day drying period in the laboratory, the specimens were exposed to 300 and 600 o C for 24 hours. Regardless of the duration of moist curing, SF addition resulted in higher compressive strength of specimens cured and tested at 20 o C. The effect was more pronounced in specimens having higher SF content. Flexural strength upon SF incorporation was decreased at all W/B ratios. Exposure to 300 o C increased the compressive strength of nearly all specimens. However, the effect of it on flexural strength was somewhat contradictory. All control and SF incorporated specimens showed a dramatic decrease in strength upon exposure to 600 o C. However, the residual strengths of SF mortars were greater than control mixtures. Keywords: Residual strength, high temperature, silica fume. ÖZET: Yüksek sıcaklığın S/B oranı 0.4, 0.5 ve 0.7 olan harç numunelerinin dayanıma olan etkisi incelenmiştir. Karışımlara çimento yerine %0, 5 ve 10 oranında silis dumanı (SD) eklenmiştir. 7, 28 ve 90 gün standard bakım sonunda, numuneler 7 gün laboratuvarda kurutulmuş ve daha sonra 24 saat 300 ve 600 o C sıcaklıkta tutulmuştur. Yüksek sıcaklığa maruz kalmayan numunelerde, bakım süresinden bağımsız olarak SD içeren numuneler daha yüksek basınç dayanımı göstermiştir. Bu etki SD içeriğinin artması ile çoğalmıştır. Bu numunelerin eğilme dayanımında SD eklenmesi ile azalma oluşmuştur. 300 o C sıcaklığın etkisi ile tüm numunelerin basınç dayanımları artmıştır. Bu sıcaklık, eğilme dayanımında çelişkili sonuçlara yol açmıştır. 600 o C sıcaklığın etkisi ile tüm numunelerin dayanımında çarpıcı azalmalar meydana gelmiştir. Fakat SD içeren numunelerin kalan dayanımı, kontrol numunelerinden daha yüksektir

Investigation of high-temperature resistance of natural pozzolan-based geopolymers produced with oven and microwave curing

In this study, high-temperature resistances of oven- and microwave-cured pumice-, perlite-, fly ash- and burnt clay-based geopolymer were investigated and, the results were compared with that of the cement mortars. The effect of high-temperature on the matrix microstructure was investigated by SEM and changes in the crystal structure were monitored by XRD analyses. High-temperature experiments were carried out with 3 h exposure to 450, 600, 750 and 900 °C. The results indicated that both oven- and microwave-cured geopolymer mortars are more resistant to high-temperatures at all temperatures than their portland cement counterpart. However, it was also determined that pumice- and perlite-based mortars could not maintain their volumetric stability at elevated temperatures. © 2022 Elsevier LtdTürkiye Bilimsel ve Teknolojik Araştırma Kurumu, TÜBİTAK: 119M950The authors acknowledge the contribution of Ege University Central Research, Test and Analysis Laboratory for SEM analysis and Izmir Katip Celebi University Central Research Laboratory for XRD analysis.This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) [Grant No 119M950]

Investigation of Flexural Strength and Toughness of Fly Ash Based Geopolymer SIFCON

Geopolymers are environmentally friendly construction materials produced by the use of silica and alumina-rich powder materials with high alkaline solutions. Reinforcing the matrix phase with continuous or discontinuous fibers has been applied for many years to increase the tensile strength and energy absorption capacity of composite materials developed for different applications. SIFCON composites are one of them, and they are produced by placing the fibers in the mold and then filling the mold with very fluid matrix phase. However, there is a need for research on the use of geopolymer matrix in SIFCON production. In this study, fly ash-based geopolymer SIFCON composites containing 5% by volume steel fibers were produced and their flexural strength, compressive strength and toughness properties were investigated on both samples that were not exposed to high temperature and exposed to 900°C. In addition, cement-based conventional SIFCON composites with similar flexural strengths with the geopolymer SIFCON were used for comparison. It was determined that the fly ash-based geopolymer SIFCON had higher flexural strength and toughness values than that of the cement-based SIFCON after exposing to high temperature. In addition, for a given flexural strength, geopolymer SIFCON showed a higher compressive strength than that of conventional SIFCON. © 2022, TUBITAK. All rights reserved

Effect of fly ash addition on the mechanical properties of tile adhesive

WOS: 000230076700009Statistical relationship between various strengths of tile adhesives in which cement or sand was partially replaced with fly ash was studied. A low-lime fly ash was used in five different replacement levels from 5% to 30% by weight of either cement or sand. The tensile adhesion, flexural and compressive strengths of adhesives were determined at 2, 7 and 28 days. In small substitution levels, sand replacement increased the tensile adhesion strength. No strong relationship was found between tensile adhesion strength and flexural or compressive strength of the specimens in which the fly ash was used as sand replacement (r 0.896). Flexural and cornpressive strength values showed quite strong relationship (r > 0.949). This may be due to the fact that both of these strength values were obtained on the same specimens. (c) 2005 Elsevier Ltd. All rights reserved

Mechanical properties of four timber species commonly used in Turkey

The mechanical properties of 4 timber species (poplar, fir, pine and hornbeam) commonly used in Turkey were investigated. The compressive strength, flexural strength and toughness were determined both perpendicular and parallel to the grain. The modulus of elasticity of timber specimens was also determined parallel to the grain for the compressive test and perpendicular to the grain for the flexural test. It was found that loading direction affects all mechanical properties remarkably. Among the timbers tested, maximum and minimum mechanical performances were obtained with the 2 hardwoods, i.e. hornbeam and poplar, respectively. The mechanical performance of the softwoods, i.e. fir and pine, was between that of the 2 hardwoods. Hornbeam showed the minimum anisotropy. Except for hornbeam, the timbers showed very low compressive strength when loaded perpendicular to the grain and very low flexural strength when loaded parallel to the grain. ©TÜ? ITAK