Dosimetric impact of statistical uncertainty on Monte Carlo dose calculation algorithm in volumetric modulated arc therapy using Monaco TPS for three different clinical cases

AimTo study the dosimetric impact of statistical uncertainty (SU) per plan on Monte Carlo (MC) calculation in Monaco? treatment planning system (TPS) during volumetric modulated arc therapy (VMAT) for three different clinical cases.BackgroundDuring MC calculation SU is an important factor to decide dose calculation accuracy and calculation time. It is necessary to evaluate optimal acceptance of SU for quality plan with reduced calculation time.Materials and methodsThree different clinical cases as the lung, larynx, and prostate treated using VMAT technique were chosen. Plans were generated with Monaco? V5.11 TPS with 2% statistical uncertainty. By keeping all other parameters constant, plans were recalculated by varying SU, 0.5%, 1%, 2%, 3%, 4%, and 5%. For plan evaluation, conformity index (CI), homogeneity index (HI), dose coverage to PTV, organ at risk (OAR) dose, normal tissue receiving dose ≥5 Gy and ≥10 Gy, integral dose (NTID), calculation time, gamma pass rate, calculation reproducibility and energy dependency were analyzed.ResultsCI and HI improve as SU increases from 0.5% to 5%. No significant dose difference was observed in dose coverage to PTV, OAR doses, normal tissue receiving dose ≥5 Gy and ≥10 Gy and NTID. Increase of SU showed decrease in calculation time, gamma pass rate and increase in PTV max dose. No dose difference was seen in calculation reproducibility and dependent on energy.ConclusionFor VMAT plans, SU can be accepted from 1% to 3% per plan with reduced calculation time without compromising plan quality and deliverability by accepting variations in point dose within the target.     

Fas-negative osteosarcoma tumor cells are selected during metastasis to the lungs: the role of the Fas pathway in the metastatic process of osteosarcoma

Low expression of Fas by different tumors including osteosarcoma, correlates with poor prognosis. We found that osteosarcoma lung metastases from patients expressed negligible amounts of Fas, but primary tumors often expressed high Fas levels. The reason for this discrepancy is unknown. We hypothesized that because FasL is constitutively expressed in the lungs, Fas-positive (Fas(+)) tumor cells entering the lungs would bind with FasL and die from Fas-induced apoptosis, resulting in the "selection" of Fas-negative (Fas(-)) cells, which would eventually form metastases. To test this hypothesis, we injected K7 osteosarcoma cells, which express functional Fas in vitro, into mice and confirmed that its bone tumors were Fas(+), but lung metastases were Fas(-). Next, to inhibit Fas signaling without affecting Fas expression, we transfected these cells with a FADD-dominant negative (FDN) plasmid and developed K7/FDN cells. Metastases formed by K7/FDN cells contained Fas(+) tumor cells. Moreover, K7/FDN cells were retained in the lungs longer and formed more lung metastases than K7 cells. In addition, the incidence of lung metastases in FasL-deficient mice injected with K7 cells was higher than that in wild-type mice. Metastases from FasL-deficient mice but not from wild-type mice contained Fas(+) tumor cells. Based on that, we conclude that Fas(-) osteosarcoma cells are selected during lung metastases formation and that inhibition of Fas signaling in tumors or lack of FasL in the host environment allows the proliferation of Fas(+) osteosarcoma cells in the lungs and promotes metastases growth. Therefore, Fas may be considered as a new therapeutic target for osteosarcoma treatment.     

Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.     

Comparative pulmonary toxicity of cadmium and nickel: histopathological and bronchoalveolar lavage analysis

Pulmonary toxicity of cadmium and nickel was evaluated in rat lungs following intratracheal instillation of their chlorides. Concentration of both the metals varied from 0.2-5 mM. Both the metals increased total number of cells, number of polymorphonuclear neutrophils, total protein, sialic acid and the activity of lactate dehydrogenase and beta-glucuronidase in bronchoalveolar lavage 3 days after exposure. Increase in the levels of the selected parameters was more following Cd exposure than in Ni exposed rats. Histologically there was an inflammatory response and interstitial fibroblastic proliferation in the lungs of Cd exposed animals. These changes were mild in Ni-exposed animals and higher concentrations of Ni were needed to produce changes similar to those produced by smaller concentrations of Cd.     

EBV structural antigens, gp350 and gp85, as targets for ex vivo virus-specific CTL during acute infectious mononucleosis: potential use of gp350/gp85 CTL epitopes for vaccine design

For many years, EBV vaccine development efforts have concentrated on the use of structural Ag, gp350, and have been directed toward Ab-mediated blocking virus attachment to the target cell. There is increasing evidence to suggest that the development of neutralizing Abs in vaccinated animals does not always correlate with protection; nevertheless, it has been postulated that gp350-specific T cell-mediated immune responses may have an effector role in protection. This hypothesis has largely remained untested. In the present study, we demonstrate that CTL from acute infectious mononucleosis patients display strong ex vivo reactivity against the EBV structural Ags, gp85 and gp350. Moreover, long-term follow up studies on infectious mononucleosis-recovered individuals showed that these individuals maintain gp350- and gp85-specific memory CTL, albeit at low levels, in the peripheral blood. These results strongly suggest that CTL specific for EBV structural proteins may play an important role in the control of EBV infection during acute infection. More importantly, we also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results have important implications for future EBV vaccine design and provides evidence, for the first time, that CTL epitopes from EBV structural proteins may be used for establishing strong antiviral immunity against EBV infection.     

Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.