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Researchers agree that the ideal scaffold for tissue engineering should possess a 3D and highly porous structure, biocompatibility to encourage cell/tissue growth, suitable surface chemistry for cell attachment and differentiation, and mechanical properties that match those of the surrounding tissues. However, there is no consensus on the optimal pore distribution. In this study, we investigated the effect of pore distribution on corrosion resistance and performance of human mesenchymal stem cells (hMSC) using titanium scaffolds fabricated by laser beam powder bed fusion (PBF-LB). We designed two scaffold architectures with the same porosities (i.e., 75 %) but different distribution of pores of three sizes (200, 500, and 700 µm). The pores were either grouped in three zones (graded, GRAD) or distributed randomly (random, RAND). Microfocus X-ray computed tomography revealed that the chemically polished scaffolds had the porosity of 69 ± 4 % (GRAD) and 71 ± 4 % (RAND), and that the GRAD architecture had the higher surface area (1580 ± 101 vs 991 ± 62 mm2) and the thinner struts (221 ± 37 vs 286 ± 14 µm). The electrochemical measurements demonstrated that the apparent corrosion rate of chemically polished GRAD scaffold decreased with the immersion time extension, while that for polished RAND was increased. The RAND architecture outperformed the GRAD one with respect to hMSC proliferation (over two times higher although the GRAD scaffolds had 85 % higher initial cell retention) and migration from a monolayer. Our findings demonstrate that the pore distribution affects the biological properties of the titanium scaffolds for bone tissue engineering.

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Thiamine deficiency (TD) results in lactate acidosis, which is associated with neurodegeneration. The aim of this study was to investigate this alteration in primary rat brain endothelia. Spectrophotometric analysis of culture media revealed that only a higher concentration of pyrithiamine, which accelerates the intracellular blocking of thiamine, significantly elevated the lactate level and lactate dehydrogenase activity within 7 days. The medium without pyrithiamine and with a thiamine concentration comparable to pathophysiological plasma levels mildly reduced only the activity of transketolase. This suggests that significant metabolic changes may not occur at the early phase of TD in cerebral capillary cells, while anaerobic glycolysis in capillaries may be mediated during late stage/chronic TD.

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Psoralens react photochemically with DNA to form interstrand crosslinks as well as two types of monoadduct (furan-side and pyrone-side adducts). To investigate the relative roles of these adducts in toxicity, we have studied the interaction of 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) with bacteriophage T7. These two derivatives differ in the fraction of pyrone-side monoadducts formed, TMP producing very small amounts of this type of adduct. The results show similar phage survival for the two psoralen analogs at equivalent numbers of crosslinks per DNA molecule. However, the survival fraction of treated phage is significantly lower than the fraction of noncrosslinked DNA molecules. Phage survival decreases after secondary irradiation which is used to transform monoadducts into crosslinks, but this decrease is not due solely to crosslinks; at doses beyond that required to transform all crosslinkable monoadducts into crosslinks, phage survival continues to decrease, pointing to the production of other genotoxic lesions during secondary irradiation. These results indicate that, although crosslinks can kill phage T7, as shown by the secondary irradiation results, they are not sufficient in number to explain the psoralen toxicity after primary irradiation. Therefore monoadducts, both furan-side and pyrone-side types, must in large part be responsible for phage inactivation.

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Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species beta 4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

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Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species b4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

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We have studied the toxic effects of alkylating agents with a well characterized model: phage T7. Treatment of bacteriophage T7 with methyl methanesulfonate led to perturbation of phage-specific protein synthesis. Synthesis of class I and II proteins was prolonged, while production of class II and III proteins was delayed. This delay increased for proteins coded by genes located further to the right on the T7 genetic map. In extracts prepared from cells infected by alkylated phage, the specific activity of T7 RNA polymerase was decreased. These results suggest that the toxic action of methyl methanesulfonate is directed towards viral transcription.

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Analysis of thin sections of Escherichia coli B cells infected by normal (nonalkylated) or alkylated bacteriophage T7 showed that alkylation altered phage morphogenesis. To understand these morphogenetic alterations, we have isolated phage-related particles from infected-cell lysates by differential and sucrose gradient centrifugation. Cells infected by normal and by alkylated phage produced mature phage particles, empty heads, and proheads; however, production of proheads and mature phage particles was less in the case of alkylated phage. These lysates also contained sedimentable material which migrated more slowly than empty heads on sucrose gradients. In the case of alkylated phage, this peak contained radioactive material in amounts nearly equal to that in either proheads or empty heads; for normal phage, this peak represented a smaller fraction of the total radioactivity. Examination of the gradient fractions by electron microscopy revealed appreciable quantities of phage tails and tail-related particles. The same gradient fractions contained phage tail proteins: gene products (gps) 11, 12, and 17 as well as smaller amounts of gp 8, the head-tail connector. In addition, these fractions contained two other proteins which we believe to be of bacterial origin. These proteins may be related to tail formation or function as part of the phage receptor. On the basis of our data, we propose an alternative morphogenetic pathway for T7 tail formation, a pathway which would involve formation of a complex of tail proteins prior to association with the phage head.

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Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.

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Experiments were conducted to determine whether phage T7 treated with methyl methanesulfonate used multiplicity reactivation to repair alkylation lesions. This type of repair was found to be operative at high multiplicities in actively growing wild-type Escherichia coli B cells.

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After exposure to a glow discharge, Ultra-Clear ultracentrifuge tubes become wettable and hence suitable for use with conventional gradient formers. Tubes treated by this method can thus replace the formerly widely used cellulose nitrate tubes which are no longer available.

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Alkylation of T7 bacteriophage by methyl methane sulfonate blocked superinfection exclusion. This blockage could be correlated with a delay in the synthesis of phage-specific proteins. Therefore we conclude that protein synthesis directed by the primary infecting phage is required for efficient exclusion of superinfecting phage particles.

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Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204-213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5-10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.

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Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage. Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 degrees C; alkylated phage was then incubated 24 h at 30 degrees C to induce depurination. These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid. After ejection, the phage preparations were analyzed by electron microscopy. DNA lengths in capsid-DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined. To establish the direction of DNA ejection, E. coli RNA polymerase was bound to capsid-DNA complexes. The results showed that DNA was partially ejected from both alkylated and depurinated phages. In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end. We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end. For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.

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Alkylation of T7 bacteriophage considerably delayed phage development and reduced the phage's killing action on host cells. Only a small fraction of infected cells produced phage. For these phages, the latent period was markedly prolonged but the burst was equivalent to or only slightly lower than that of untreated phage. In the progeny of alkylated phage, there was an increase in the fraction of defective particles as well as a change in their morphology. These data show that infection with alkylated T7 bacteriophage is to a large degree abortive; hence, biological consequences of this infection are very different from those characteristic of a normal virus infection.

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Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.

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Marker rescue experiments with alkylated T7 bacteriophage carried out in the presence and in the absence of nalidixic acid suggest that the gradient in rescue is due to two alkylation-induced causes: a DNA injection defect and an interference with DNA synthesis.

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The D-A-P was administered to 60 male sex offenders from a correctional institution. The drawings of the inmates, taken as a whole, seemed to be indicative of narcissism, assertion, sex disturbance, aggression, and anxiety. Significant differences in the drawings of rapists, child molesters, and incest offenders were found. The following D-A-P aspects appeared to be prominent in the drawings of the Ss, in descending order: reinforcement, long nose, large head, legs wide apart, too much hair, waistline emphasis, slash mouth, long feet, pointy fingers, distorted heads, and opposite sex drawn first.

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After treatment with methyl or ethyl methane sulfonate, T7 amber mutants display a reduced capacity for recombination. Moreover, alkylation reduces recombination frequency involving markers on the right-hand side of the genetic map more than it reduces recombination frequency involving markers on the left-hand side. We interpret this to mean that alkylation can stop DNA injection at any point along the DNA molecule, and that T7 phage injects its DNA in a unique fashion starting from the end carrying the genes for early proteins.

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Alkylation by ethyl or methyl methanesulfonate to an extent that inactivates more than 99.5% of T7 coliphages has no effect on phage adsorption on Escherichia coli B cells, but decreases the amount of phage DNA injected into the host cells. Depurination interferes with the injection of the phage DNA. Failure to inject the whole phage genome thus appears to be a cause of the immediate as well as of the delayed inactivation of the T7 coliphage treated by monofunctional alkylating agents; the hypothesis that it is the only cause of inactivation, although not very likely, cannot be excluded at the present time.

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