Thermophiles as Potential Source of Novel Endotoxin Antagonists: the Full Structure and Bioactivity of theLipo‐oligosaccharide from Thermomonas hydrothermalis

Thermomonas hydrothermalis is a Gram‐negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell‐wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram‐negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure‐dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system—an antagonistic effect, as shown in studies of LPS structure–activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo‐oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.     

Different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4–20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion‐type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non‐cavitated enamel caries‐like lesions. The optimal period for inducing caries‐like lesions was 8 days. Microsc. Res. Tech. 78:444–451, 2015. © 2015 Wiley Periodicals, Inc.     

The effect of radiopacifiers agents on pH,calcium release,radiopacity, and antimicrobial properties of different calcium hydroxide dressings

The aim of this study was to evaluate the antimicrobial activity, pH level, calcium ion release, and radiopacity of calcium hydroxide pastes associated with three radiopacifying agents (iodoform, zinc oxide, and barium sulfate). For the pH and calcium release tests, 45 acrylic teeth were utilized and immersed in ultrapure water. After 24 h, 72 h, and 7 days the solution was analyzed by using a pH meter and an atomic absorption spectrophotometer. Polyethylene tubes filled with the pastes were used to perform the radiopacity test. For the antimicrobial test, 25 dentin specimens were infected intraorally in order to induce the biofilm colonization and treated with the pastes for 7 days. The Live/Dead technique and a confocal microscope were used to obtain the ratio of live cells. Parametric and nonparametric statistical tests were performed to show differences among the groups (P < 0.05). The pH analysis at 7 days showed significant differences (P < 0.05) among the groups. No differences among the pastes were found in the calcium release test on the 7th day (P > 0.05). The calcium hydroxide/iodoform samples had the highest radiopacity and antimicrobial activity against the biofilm‐infected dentin in comparison to the other pastes (P < 0.05). Calcium hydroxide mixed with 17% iodoform and 35% propylene glycol into a paste had the highest pH, calcium ion release, radiopacity, and the greatest antimicrobial action versus similar samples mixed with BaSO4 or ZnO. Microsc. Res. Tech. 78:620–625, 2015. © 2015 Wiley Periodicals, Inc.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Ion temperature fluctuation measurements using a retarding field analyzer

The retarding field analyzer (RFA) is a widely used diagnostic tool for the ion temperature measurement in the scrape-off-layer (SOL) of the thermonuclear plasma devices. However, the temporal resolution in the standard RFA application is restricted to the ms timescale. In this paper, a dc operation of the RFA is considered, which allows for the measurement of the plasma ion temperature fluctuations. The method is based on the relation for the RFA current-voltage (I-V) characteristic resulted from a common RFA model of shifted Maxwellian distribution of the analyzed ions, and the measurements of two points on the exponentially decaying region of the I-V characteristic with two differently dc biased RFA electrodes. The method has been tested and compared with conventional RFA measurements of the ion temperature in the tokamak ISTTOK SOL plasma. An ion temperature of T(i) = 17 eV is obtained near the limiter position. The agreement between the results of the two methods is within ～25%. The amplitude of the ion temperature fluctuations is found to be around 5 eV at this location. The method has been validated by taking into account the effect of fluctuations in the plasma potential and the noise contamination, proving the reliability of the results obtained. Finally, constrains to the method application are discussed that include a negligible electron emission from the RFA grids and the restriction to operate in the exponentially decaying region of the I-V characteristic.     

Hematopoietic progenitor constituents and adherent cell progenitor morphology isolated from black-rumped agouti (Dasyprocta prymnolopha, Wagler 1831) bone marrow

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black‐rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10⁷ cells (ACP). Thirteen 25‐cm² culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10⁶ cells per milliliter, and placed in liquid nitrogen at ?196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Influence of the starting condition on the kinetics of sensitization and loss of toughness in an AISI 304 steel

This work describes the investigation of the embrittlement of AISI 304 steel sensitized at 650°C by Charpy impact test, comparing two starting conditions: (1) mill annealed and machined (MA-M); and (2) solution treated at 1050°C by 1 h followed by oil quenching (ST). The degree of sensitization for both samples was assessed by Optical Microscopy (OM), Scanning Electron Microscopy (SEM) and by Double Loop Electrochemical Potentiodynamic Reactivation test. The results showed that MA-M samples undergo more severe and rapid embrittlement than ST ones and a higher kinetics of sensitization due to small strains concentrated in grain boundaries and martensite phase produced during the machining operations. The martensite phase is found to be quite stable at the sensitization treatment at 650°C. The increase of microvoids nucleation at the grain boundaries seems to be the mechanism of embrittlement in the sensitized 304 steel.