Slicing Algorithm and Partition Scanning Strategy for 3D Printing Based on GPU Parallel Computing.

Aiming at the problems of over stacking, warping deformation and rapid adjustment of layer thickness in electron beam additive manufacturing, the 3D printing slicing algorithm and partition scanning strategy for numerical control systems are studied. The GPU (graphics processing unit) is used to slice the 3D model, and the STL (stereolithography) file is calculated in parallel according to the normal vector and the vertex coordinates. The voxel information of the specified layer is dynamically obtained by adjusting the projection matrix to the slice height. The MS (marching squares) algorithm is used to extract the coordinate sequence of the binary image, and the ordered contour coordinates are output. In order to avoid shaking of the electron gun when the numerical control system is forming the microsegment straight line, and reduce metal overcrowding in the continuous curve C0, the NURBS (non-uniform rational b-splines) basis function is used to perform curve interpolation on the contour data. Aiming at the deformation problem of large block components in the forming process, a hexagonal partition and parallel line variable angle scanning technology is adopted, and an effective temperature and deformation control strategy is formed according to the European-distance planning scan order of each partition. The results show that the NURBS segmentation fits closer to the original polysurface cut line, and the error is reduced by 34.2% compared with the STL file slice data. As the number of triangular patches increases, the algorithm exhibits higher efficiency, STL files with 1,483,132 facets can be cut into 4488 layers in 89 s. The slicing algorithm involved in this research can be used as a general data processing algorithm for additive manufacturing technology to reduce the waiting time of the contour extraction process. Combined with the partition strategy, it can provide new ideas for the dynamic adjustment of layer thickness and deformation control in the forming process of large parts.

Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII.

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ßßα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase.

Tuberculosis, which is caused by Mycobacterium tuberculosis, remains to be a global health problem. The thick and complex cell envelope has been implicated in many aspects of the pathogenicity of M. tuberculosis. M. tuberculosis UDP-glucose pyrophosphorylase (UGP, coded by galU, Rv0993) is involved in cell envelope precursor synthesis. UGP catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose 1-phosphate (Glc-l-P). Bacterial UGPs are completely unrelated to their eukaryotic counterparts. This enzyme is recognized as a virulence factor in several bacterial species and is conserved among mycobacterial species, which makes it a good target for mycobacterial pathogenicity research. The recombinant M. tuberculosis UGP (rMtUGP) was purified in Escherichia coli and found to be stable and catalytically active. The effects of pH, temperature and Mg2+ on enzyme activity were characterized. In addition, subcellular localization studies revealed that most of M. tuberculosis UGP protein was located in the cell wall. The purification and characterization of M. tuberculosis UGP may help to decipher the pathogenicity of M. tuberculosis.

Biosynthesis of isoprenoids: characterization of a functionally active recombinant 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) from Mycobacterium tuberculosis H37Rv.

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the leading infectious diseases to humans. It is urgent to discover novel drug targets for the development of antitubercular agents. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for isoprenoid biosynthesis has been considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammals. MEP cytidyltransferase (IspD), the third-step enzyme of the pathway, catalyzes MEP and CTP to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and PPi. In the work, ispD gene from M. tuberculosis H37Rv (MtIspD) was cloned and expressed. With N-terminal fusion of a histidine-tagged sequence, MtIspD could be purified to homogeneity by one-step nickel affinity chromatography. MtIspD exists as a homodimer with an apparent molecular mass of 52 kDa. Enzyme property analysis revealed that MtIspD has high specificity for pyrimidine bases and narrow divalent cation requirements, with maximal activity found in the presence of CTP and Mg(2+). The turnover number of MtIspD is 3.4 s(-1). The Km for MEP and CTP are 43 and 92 muM, respectively. Furthermore, MtIspD shows thermal instable above 50 degrees C. Circular dichroism spectra revealed that the alteration of tertiary conformation is closely related with sharp loss of enzyme activity at higher temperature. This study is expected to help better understand the features of IspD and provide useful information for the development of novel antibiotics to treat M. tuberculosis.

MSTF: a domain involved in bacterial metallopeptidases and surface proteins, mycobacteriophage tape-measure proteins and fungal proteins.

Here we report a novel domain, MSTF (domain involved in bacterial metallopeptidases, surface proteins and other proteins, also present in mycobacteriophage tape-measure proteins and fungal proteins), which is present in bacteria, phages and fungi. MSTF is about 67-94 amino acids in length with one HxDHxH motif and some highly conserved residues including His, Gly, Ala and Asp. Secondary structure prediction indicated that this domain contains two alpha-helices and one beta-sheet. Identification of MSTF will provide an opportunity to develop new strategies to combat pathogenic microorganisms, especially Mycobacterium tuberculosis.

Expression, purification and properties of shikimate dehydrogenase from Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for alpha-helix, beta-sheet, beta-turn, and random coil were 29.2 %, 9.3 %, 32.7 %, and 28.8 %, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature 63(o)C. The apparent Michaelis constant for shikimic acid and NADP(+) were calculated to be about 29.5 microM and 63 microM. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of NADP(+) with an enzyme turnover number of 399 s(-1). Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.

Identification of a novel domain--DIM, which defines a new family composed mainly of bacterial membrane proteins.

We report here the identification of a novel domain - DIM (N-terminal domain in bacterial membrane proteins and other proteins) present exclusively in bacterial species including mycobacteria, revealed by PSI-BLAST iterative searches. DIM comprises about 53 amino acids in length with conserved Leu, Ile and Gly residues. Secondary structure prediction indicated that this domain contains two alpha-helices. DIM occurs at the N-terminus of proteins, and was found particularly but not exclusively in proteins with a transmembrane domain, and also in proteins with a FHA domain or RPT repeats. DIM-containing proteins have been reported to be involved in pathogenicity, signal transduction or small solute transport.

[Molecular cloning, expression, purification and properties of isocitrate lyase gene from Mycobacterium tuberculosis H37Rv].

OBJECTIVE: To obtain recombinant protein with enzymatic activities of isocitrate lyase (ICL). METHODS: The icl gene was amplified by polymerase chain reaction (PCR) from Mycobacterium tuberculosis H(37)Rv strain genomic DNA and cloned into pET28-a(+) vector. The recombinant protein was expressed in E.coli BL21 (DE3). Enzyme activity of the protein was assayed after purifying with Ni-NTA resin. RESULTS: The recombinant ICL was purified in a highly active state with a specific activity of about 7.657 x 10(2) micromol x mg(-1) x min(-1). The pH curve indicated that recombinant ICL activity was optimal at pH 7.4. The LC/MS spectrometry showed a 50 603.347 molecular mass of recombinant ICL. The CD spectrum showed that the percentages for alpha- helix, beta- sheet, beta- turn, and random coil were 43.8%, 31.9%, 3.4%, and 20.9%, respectively. CONCLUSIONS: The icl gene of Mycobacterium tuberculosis H(37)Rv was successfully cloned and expressed. The enzymatic properties demonstrated the purified recombinant protein had activities of ICL. This work can facilitate immunologic research and the discovery of novel antimicrobial agents against Mycobacterium tuberculosis.