Monte Carlo dose verification for intensity-modulated arc therapy

Intensity-modulated arc therapy (IMAT), a technique which combines beam rotation and dynamic multileaf collimation, has been implemented in our clinic. Dosimetric errors can be created by the inability of the planning system to accurately account for the effects of tissue inhomogeneities and physical characteristics of the multileaf collimator (MLC). The objective of this study is to explore the use of Monte Carlo (MC) simulation for IMAT dose verification. The BEAM/DOSXYZ Monte Carlo system was implemented to perform dose verification for the IMAT treatment. The implementation includes the simulation of the linac head/MLC (Elekta SL20), the conversion of patient CT images and beam arrangement for 3D dose calculation, the calculation of gantry rotation and leaf motion by a series of static beams and the development of software to automate the entire MC process. The MC calculations were verified by measurements for conventional beam settings. The agreement was within 2%. The IMAT dose distributions generated by a commercial forward planning system (RenderPlan. Elekta) were compared with those calculated by the MC package. For the cases studied, discrepancies of over 10% were found between the MC and the RenderPlan dose calculations. These discrepancies were due in part to the inaccurate dose calculation of the RenderPlan system. The computation time for the IMAT MC calculation was in the range of 20-80 min on 15 Pentium-Ill computers. The MC method was also useful in verifying the beam apertures used in the IMAT treatments.     

Preparation of graded porous titanium coatings on titanium implant materials by plasma spraying

Graded porous titanium coatings have been deposited on titanium substrates for dental implants by plasma spraying in an argon atmosphere. X-ray diffraction (XRD), scanning electron microscopy (SEM), surface roughness measurement, and tensile strength tests were performed on graded porous coatings. The results showed that Ti(3)O(5) was formed in the outermost surface of the porous coatings due to oxidation. The graded porous coatings consisted of three layers. The outer layer was full of macropores with a surface roughness of approximately 100 microm. The diameter of many macropores reached and even surpassed 150 microm, which could be beneficial for tissue to grow into the coating. The middle layer consisted of a mixture of micropores and macropores. The inner layer was a very dense and tight interface layer that included mechanical, physical, and metallurgical bonding. In tensile strength tests, testing bars peeled off the coatings, because the adhesive agent fractured, but the coatings remained intact.     

Effect of Group A Streptococcal Cysteine Protease on Invasion of Epithelial Cells

Cysteine protease of group A streptococci (GAS) is considered an important virulence factor. However, its role in invasiveness of GAS has not been investigated. We demonstrated in this study that two strains of protease-producing GAS had the ability to invade A-549 human respiratory epithelial cells. Isogenic protease mutants were constructed by using integrational plasmids to disrupt the speB gene and confirmed by Southern hybridization and Western immunoblot analyses. No extracellular protease activity was produced by the mutants. The mutants had growth rates similar to those of the wild-type strains and produced normal levels of other extracellular proteins. When invading A-549 cells, the mutants had a two- to threefold decrease in activity compared to that of the wild-type strains. The invasion activity increased when the A-549 cells were incubated with purified cysteine protease and the mutant. However, blockage of the cysteine protease with a specific cysteine protease inhibitor, E-64, decreased the invasion activity of GAS. Intracellular growth of GAS was not found in A-549 cells. The presence or absence of protease activity did not affect the adhesive ability of GAS. These results suggested that streptococcal cysteine protease can enhance the invasion ability of GAS in human respiratory epithelial cells.     

Rapid, accurate genotyping of the common -alpha(4.2) thalassaemia deletion based on the use of denaturing HPLC

AIMS: To develop an alternative assay for specific genotyping of the -alpha(4.2) thalassaemia deletion based on the DNA sequence features surrounding the breakpoint. METHODS: The 5' and 3' ends of the breakpoint regions of the -alpha(4.2) allele and the normal homologous segments were sequenced in Chinese individuals. A sequence haplotype composed of four single nucleotide variations within the X2/X1 box of the -alpha(4.2) breakpoint region was found in all of the 10 Chinese -alpha(4.2) thalassaemia alleles studied. Based on these findings, a novel polymerase chain reaction (PCR)/denaturing high performance liquid chromatography (DHPLC) assay was developed for rapid genotyping of the -alpha(4.2) allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the alpha globin target sequence encompassing these four polymorphic sites, followed by a partially denaturing HPLC analysis using the transgenomic WAVE DNA fragment analysis system. RESULTS: The three major genotypes (-alpha4.2/alphaalpha, -alpha(4.2)/--SEA, and alphaalpha/alphaalpha) could be distinguished through the characteristic chromatograms generated by the WAVE system. The accuracy of this technique was evaluated blindly, and the results were 100% (40 of 40) concordant with the genotypes previously characterised by Southern blotting or Gap-PCR. CONCLUSIONS: This study validates the PCR/DHPLC approach as a simple, rapid, highly accurate, and cost effective method, potentially adaptable for use in epidemiological surveys, genetic screening, and diagnosis of silent alpha+ thalassaemia and Hb H disease.     

Transforming growth factor-beta3-loaded microtextured membranes for skin regeneration in dermal wounds

Adverse effects of wound healing, such as excessive scar tissue formation, wound contraction, or nonhealing wounds represent a major clinical issue in today's healthcare. Transforming growth factor (TGF)-beta3 has specifically been implicated in wound healing. Our hypothesis was that local administration of TGF-beta3 to excisional dermal wounds would diminish wound contraction and scar formation. Microtextured wound covers, containing different concentrations of TGF-beta3, were placed onto full-thickness excisional skin wounds in guinea pigs. Tattooed reference marks were used to quantify wound contraction. Sixty-four male guinea pigs in four study groups (5 ng TGF-beta3, 50 ng TGF-beta3, no growth factor, sham wound) were followed for up to 6 weeks. We analyzed 19 different parameters of wound healing. Results showed that, in some instances, the 50-ng TGF-beta3 group gave less contraction, whereas the 5-ng TGF-beta3 group gave more contraction. These differences confirm that TGF-beta3 has an optimum working concentration, and suggest this concentration to be closer to 50 ng than to 5 ng TGF-beta3. However, only very few significant differences occurred, and thus we conclude that the clinical relevance of our findings is negligible. Earlier studies, reporting clinically improved wound healing by TGF-beta3, could therefore not be confirmed by this study.     

Genetic diversity of the outer surface protein C gene of southern Borrelia isolates and its possible epidemiological,clinical, and pathogenetic implications

The ospC genes of 20 southern Borrelia strains were sequenced. The strains consisted of B. burgdorferi sensu stricto, B. andersonii, B. bissettii, one undescribed genospecies, MI-8, and one probably new Borrelia species, TXW-1. A high degree of similarity exists between B. burgdorferi sensu stricto and B. bissettii and between B. bissettii and B. andersonii. Lateral transfers of the ospC gene probably occurred between B. burgdorferi sensu stricto and B. bissettii and between B. bissettii and B. andersonii. Internal gene recombination appears to occur among them. The highest degree of genetic diversity among them was observed in the two variable domains (V1 and V2), semivariable domain (SV), and the species-specific epitopes (between amino acids 28 and 31). Differences in ospC sequences among southern strains reflect diversity at the strain and genospecies levels. MI-8, which was recognized as an undescribed genospecies in our previous reports, remains distinguishable in our current analysis of ospC genes and is distinct from B. burgdorferi sensu stricto. Interestingly, another undescribed southern isolate, TXW-1, was not amplified under various PCR conditions. Compared to European B. burgdorferi sensu stricto strains, American B. burgdorferi sensu stricto strains show greater genetic heterogeneity. Southern B. burgdorferi sensu stricto, B. andersonii, and B. bissettii isolates were intermixed with each other in the phylogenetic trees. In the derived trees in our work, at least one southeastern strain of B. burgdorferi, MI-2, most closely aligns with a so-called invasive cluster that possesses many proven human-invasive strains. Transmission experiments show that MI-2 and the strains in this group of southern spirochetes are able to infect mice and hamsters and that the typical vector of Lyme disease, Ixodes scapularis, can acquire the spirochetes from infected mammals. Currently, strain MI-2 appears to be the only southern isolate among the 20 we analyzed that clusters with an OspC invasive group and thus might be invasive for humans.     

Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties

Alginate gels have been used in both drug delivery and cell encapsulation applications in the bead form usually produced by dripping alginate solution into a CaCl2 bath. The major disadvantages to these systems are that the gelation rate is hard to control; the resulting structure is not uniform; and mechanically strong and complex-shaped 3-D structures are difficult to achieve. In this work controlled gelation rate was achieved with CaCO3-GDL and CaSO4-CaCO3-GDL systems, and homogeneous alginate gels were formulated as scaffolds with defined dimensions for tissue engineering applications. Gelation rate increased with increasing total calcium content, increasing proportion of CaSO4, increasing temperature and decreasing alginate concentration. Mechanical properties of the alginate gels were controlled by the compositional variables. Slower gelation systems generate more uniform and mechanically stronger gels than faster gelation systems. The compressive modulus and strength increased with alginate concentration, total calcium content, molecular weight and guluronic acid (G) content of the alginate. MC3T3-E1 osteoblastic cells were uniformly incorporated in the alginate gels and cultured in vitro. These results demonstrated how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications.     

Molecular analysis in Japanese patients with Charcot-Marie-Tooth disease: DGGE analysis for PMP22, MPZ,and Cx32/GJB1 mutations

Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder and is traditionally classified into two major types, CMT type 1 (CMT1) and CMT type 2 (CMT2). Most CMT1 patients are associated with the duplication of 17p11.2-p12 (CMT1A duplication) and small numbers of patients have mutations of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), connexin 32 (Cx32/GJB1), and early growth response 2 (EGR2) genes. Some mutations of MPZ and Cx32 were also associated with the clinical CMT2 phenotype. We constructed denaturing gradient gel electrophoresis (DGGE) analysis as a screening method for PMP22, MPZ, and Cx32 mutations and studied 161 CMT patients without CMT1A duplication. We detected 27 mutations of three genes including 15 novel mutations; six of PMP22, three of MPZ, and six of Cx32. We finally identified 21 causative mutations in 22 unrelated patients and five polymorphic mutations. Eighteen of 22 patients carrying PMP22, MPZ, or Cx32 mutations presented with CMT1 and four of them with MPZ or Cx32 mutations presented with the CMT2 phenotype. DGGE analysis was sensitive for screening for those gene mutations, but causative gene mutation was not identified in many of the Japanese patients with CMT, especially with CMT1. Other candidate genes should be studied to elucidate the genetic basis of Japanese CMT patients.     

Reduced p21(WAF1/CIP1) expression is an early event in gallbladder carcinogenesis and is of prognostic significance for patients with carcinomas of the gallbladder

The molecular basis underlying the development and progression of gallbladder carcinoma (GBC) remains poorly understood. To evaluate the roles of p21(WAF1/CIP1) and p53 in gallbladder carcinogenesis and to assess their prognostic significance for patients with GBC, we used immunohistochemistry to examine the expression of p21(WAF1/CIP1) and p53 protein in a series of surgically resected specimens, including normal epithelia, precancerous lesions adenoma, and dysplasia, and carcinomas of the gallbladder. Reduced p21(WAF1/CIP1) expression was frequently observed in carcinomas (18 of 37 lesions; 49%), and even in precancerous lesions adenomas (3 of 7; 43%) and dysplasias (5 of 5; 100%). p53 overexpression was detected in 43% of the adenomas, 60% of the dysplasias and 57% of the carcinomas. There was an inverse relationship between p21(WAF1/CIP1) and p53 expression in GBCs (P =.01). Survival analysis indicated that reduced p21(WAF1/CIP1) expression was significantly associated with shortened disease-free and overall survival (P =.04 and.03, respectively) for patients with stages II to IV GBCs. These observations suggest that reduced p21(WAF1/CIP1) expression and p53 overexpression contribute to GBC from an early stage and that determination of p21(WAF1/CIP1) expression in surgically resected specimens would add prognostic information to conventional pathologic examinations for patients with advanced-stage GBC.