Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases.

Serine hydroxymethyltransferase (SHMT) provides activated one-carbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N(5),N(10)-methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N(5)-CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.     

Presence of beta human papillomaviruses in nonmelanoma skin cancer from organ transplant recipients and immunocompetent patients in the West of Scotland

Background  Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).
Objectives  We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.
Methods  One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.
Results  In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples ( P  =   0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) ( P  <   0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.
Conclusions  A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis.     

Treatment planning for a synchrotron-based radiotherapy modality

Contrast enhanced radiotherapy (CERT) achieves biologically localised dose enhancement through the preferential uptake of high-Z media by the tumour cells. A treatment planning software originally developed for boron neutron capture therapy (BNCT) has been developed to evaluate treatment plans for CERT. A realistic test case of a brain tumour based on actual CT scans was used to calculate dose distributions with and without the presence of an iodinated contrast medium. An enhancement of dose was observed at all depths with the introduction of iodine and the corresponding dose enhancement factors were calculated for various concentrations.     

Irradiation and dosimetry of Nitinol stent for renal artery brachytherapy

This study was conducted to assess the suitability of ⁴⁸V radioactive stent for use in renal artery brachytherapy. A nickel–titanium alloy Nitinol stent was irradiated over the proton energy range of up to 8.5 MeV, to obtain ⁴⁸V. The depth dose distribution analysis of the activated stent was done with TLD-700GR in a Perspex phantom. We investigated a unique mixed gamma/beta brachytherapy source of ⁴⁸V. For a 10 mm outer-diameter ⁴⁸V stent, the average measured dose rate to vessel was 37 mGy/h. The dosimetry results of the ⁴⁸V stent suggest that the stent is suitable for use in renal artery brachytherapy.     

Characteristics of proton beam scanning dependent on Li target thickness from the viewpoint of heat removal and material strength for accelerator-based BNCT

This study demonstrates the characterization of proton spot scanning on a Li target assembly for accelerator-based BNCT from the viewpoint of heat removal and material strength. These characteristics are investigated as to their dependence on the Li target thickness, considering that the Cu backing plate has more suitable heat removal properties than Li. Two situations are considered in this paper, i.e. the cyclic operation of the spot scanning, and a stalled spot scanning cycle where the proton beam stays focused on a single position on the Li target.It was found that the maximum of the Li temperature and the strain of the Cu backing increase as the cycle period increases. A cycle period less than 120 ms (over 8.3 Hz of frequency) enables the Li temperature to be kept below 150 °C and a cycle of less than 115 ms (8.7 Hz) keeps the Cu strain below the critical value for a 230 μm thick Li target, though the values are evaluated conservatively. Against expectation, the Li temperature and Cu strain are larger for a 100 μm thick target than for a 230 μm target. The required cycle period in this case is 23 ms (43 Hz) for maintaining a reasonable Li temperature and 9 ms (110 Hz) to prevent Cu fatigue fracture.For a stall in the spot scanning cycle, the Cu temperature increases as the beam shutdown time increases. The time for Cu to reach its melting point is estimated to be 4.2 ms at the surface, 20 ms at 1 mm depth, for both of 100 and 230 μm thick targets. At least 34 ms is estimated to be enough to make a hole on Cu backing plate. A beam shutdown mechanism with a response time of about 20 ms is therefore required.