A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a β-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the α-branch of mycolic acids, followed by the joining of these two branches.

Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca2+ in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as an antifungal.

Computer simulation for tube-making by the cold roll-forming process

The conventional design of forming rolls depends heavily on the individual skill of roll designers which is based on intuition and knowledge gained from previous work. Roll design is normally a trial an error procedure, however with the progress of computer technology, CAD/CAM systems for the cold roll-forming industry have been developed. Generally, however, these CAD systems can only provide a flower pattern based on the knowledge obtained from previously successful flower patterns. In the production of ERW (Electric Resistance Welded) tube and pipe, the need for a theoretical simulation of the roll-forming process, which can not only predict the occurrence of the edge buckling but also obtain the optimum forming condition, has been recognised. A new simulation system named "CADFORM" has been devised that can carry out the consistent forming simulation for this tube-making process. The CADFORM system applied an elastic-plastic stress-strain analysis and evaluate edge buckling by using a simplified model of the forming process. The results can also be visualised graphically. The calculated longitudinal strain is obtained by considering the deformation of lateral elements and takes into account the reduction in strains due to the fin-pass roll. These calculated strains correspond quite well with the experimental results. Using the calculated strains, the stresses in the strip can be estimated. The addition of the fin-pass roll reduction significantly reduces the longitudinal compressive stress and therefore effectively suppresses edge buckling. If the calculated longitudinal stress is controlled, by altering the forming flower pattern so it does not exceed the buckling stress within the material, then the occurrence of edge buckling can be avoided. CADFORM predicts the occurrence of edge buckling of the strip in tube-making and uses this information to suggest an appropriate flower pattern and forming conditions which will suppress the occurrence of the edge buckling.

Strain Gauges's Analysis On Implant-retained Prosthesis' Cast Accuracy.

A proper cast is essential for a successful rehabilitation with implant prostheses, in order to produce better structures and induce less strain on the implants. The aim of this study was to evaluate the precision of four different mold filling techniques and verify an accurate methodology to evaluate these techniques. A total of 40 casts were obtained from a metallic matrix simulating three unit implant-retained prostheses. The molds were filled using four different techniques in four groups (n = 10): Group 1 - Single-portion filling technique; Group 2 - Two-step filling technique; Group 3 - Latex cylinder technique; Group 4 - Joining the implant analogs previously to the mold filling. A titanium framework was obtained and used as a reference to evaluate the marginal misfit and tension forces in each cast. Vertical misfit was measured with an optical microscope with an increase of 120 times following the single-screw test protocol. Strain was quantified using strain gauges. Data were analyzed using one-way ANOVA (Tukey's test) (α =0.05). The correlation between strain and vertical misfit was evaluated by Pearson test. The misfit values did not present statistical difference (P = 0.979), while the strain results showed statistical difference between Groups 3 and 4 (P = 0.027). The splinting technique was considered to be as efficient as the conventional technique. The strain gauge methodology was accurate for strain measurements and cast distortion evaluation. There was no correlation between strain and marginal misfit.

IGFR-I expression and structural analysis of the hard palatine mucosa in an ethanol-drinking rat strain (UChA and UChB)

The study analyzed the effects of chronic alcohol ingestion on the ultrastructure of the lining epithelium of the hard palatine mucosa of rats UChA and UChB (lines with voluntary alcohol consumption) in order to contribute to the understanding of the consequences of alcohol abuse for the morphology of the digestive system. Thirty female adult animals aged 120 days were divided into three experimental groups. (1) Ten UChA rats (genetically low ethanol consumer) with voluntary intake of 10% v/v (5.45 g/kg/day) ethanol solution and water. (2) Ten UChB (genetically high ethanol consumer) rats with voluntary intake of 10% v/v (7.16 g/kg/day) ethanol solution and water. (3) Ten Wistar rats with voluntary ad libitum water intake (control group). Both groups received Nuvital pellets ad libitum. The IGFR-I expression was intense in both experimental groups. The epithelial cells of the alcoholic rats UChA and UChB showed many alterations such as the presence of lipid droplets, altered nuclei, nuclei in corneum layer and disrupted mitochondria. It was concluded that ethanol intake induces ultrastructural lesions in the hard palatine mucosa. (C) 2011 Elsevier Ltd. All rights reserved.

In-plane displacement and strain image analysis

Measurements in civil engineering load tests usually require considerable time and complex procedures. Therefore, measurements are usually constrained by the number of sensors resulting in a restricted monitored area. Image processing analysis is an alternative way that enables the measurement of the complete area of interest with a simple and effective setup. In this article photo sequences taken during load displacement tests were captured by a digital camera and processed with image correlation algorithms. Three different image processing algorithms were used with real images taken from tests using specimens of PVC and Plexiglas. The data obtained from the image processing algorithms were also compared with the data from physical sensors. A complete displacement and strain map were obtained. Results show that the accuracy of the measurements obtained by photogrammetry is equivalent to that from the physical sensors but with much less equipment and fewer setup requirements. © 2015Computer-Aided Civil and Infrastructure Engineering.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated clones

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

Genome-scale metabolic network reconstruction of Polaromonas sp. strain JS666: analysis of cDCE degradation rates and design of experiments for bioremediation improvement

Release of chloroethene compounds into the environment often results in groundwater contamination, which puts people at risk of exposure by drinking contaminated water. cDCE (cis-1,2-dichloroethene) accumulation on subsurface environments is a common environmental problem due to stagnation and partial degradation of other precursor chloroethene species. Polaromonas sp. strain JS666 apparently requires no exotic growth factors to be used as a bioaugmentation agent for aerobic cDCE degradation. Although being the only suitable microorganism found capable of such, further studies are needed for improving the intrinsic bioremediation rates and fully comprehend the metabolic processes involved. In order to do so, a metabolic model, iJS666, was reconstructed from genome annotation and available bibliographic data. FVA (Flux Variability Analysis) and FBA (Flux Balance Analysis) techniques were used to satisfactory validate the predictive capabilities of the iJS666 model. The iJS666 model was able to predict biomass growth for different previously tested conditions, allowed to design key experiments which should be done for further model improvement and, also, produced viable predictions for the use of biostimulant metabolites in the cDCE biodegradation.

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a -hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to -hydroxybutyrate that may be converted to the energy and carbon storage compound, poly--hydroxybutyrate. Accordingly, we confirmed the formation of poly-?-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor.

Molecular analysis of yellow fever virus 17DD vaccine strain

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared world wide. As part of a broader approach to determine the genetic variability in YF l7D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purifed directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF l7D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot nybridization of virion RNAs of purified 17DD with two other strains of YF virus only fenome-sized molecules for all three viruses. These observations suggest that vaccine phenotype is primarily associated with the accumulation of mutations.

Comparative Analysis by Polymerase Chain Reaction Amplified Minicircles of Kinetoplast DNA of a Stable Strain of Trypanosoma cruzi from São Felipe, Bahia, its Clones and Subclones: Possibility of Predominance of a Principal Clone in this Area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%.This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14.

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Thermomechanical analysis of strain localization in a ductile detachment zone

At the latitude of the Thor-Odin dome (British Columbia) the Columbia River Detachment defines the eastern margin of the Shuswap metamorphic core complex and localizes in a 1 km thick muscovite-bearing quartzite mylonite. We present a combined Ar-40/Ar-39, (micro) structural, and oxygen isotope study of the deformation history in the detachment and evaluate the spatial and temporal relationships between microstructure formation and localization of strain. High-precision Ar-40/Ar-39 geochronology from different levels in the mylonite delineates a pattern of increasingly younger (49.0 to 47.9 Ma) deformation ages in deeper levels of the mylonitic footwall. The correlation of Ar-40/Ar-39 ages with decreasing deformation temperatures (similar to 550 degrees-400 degrees C) in the top 200 m of the mylonite indicates that deformation migrated downward from the contact with the hanging wall. Strain localization was diachronous in progressively deeper levels of the footwall and was likely controlled by fluid-assisted strain hardening due to advective heat removal and contemporaneous reaction weakening due to dissolution-reprecipitation of white mica. The observed constant high-stress microstructures across the entire detachment indicate that flow stress was buffered by the interplay of strain rate and temperature, where high strain rates at elevated temperature produced the same microstructure as lower strain rates under decreasing temperature conditions. The combined data suggest that the complex interplay among temporally nonuniform rates of footwall exhumation, heat advection, and embrittlement by meteoric fluids strongly determines the thermomechanical behavior of extensional detachments.