BIOCHEMICAL DETERMINANTS IN THE CYTOTOXIC ACTION OF 9-BETA- D- ARABINOFURANOSYLADENINE

Studies with 9-(beta)-D-arabinfuranosyladenine (ara-A) have illustrated that there are numerous cell functions which are affected by the nucleoside analog and its metabolites. Although the precise mechanism responsible for the cytotoxicity of this drug is not known, it is presently thought tha

Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues--synthesis and biochemical characterization.

Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.

Synthesis and Biochemical Characterization of Tricyclothymidine Triphosphate (tc-TTP)

Tricyclo-DNA (tc-DNA) is a conformationally restricted oligonucleotide analogue that exhibits promising properties as a robust antisense agent. Here we report on the synthesis and biochemical characterization of tc-TTP, the triphosphate of a tc-DNA nucleoside containing the base thymine. Tc-TTP turned out to be a substrate for the Vent (exo−) DNA polymerase, a polymerase that allows for multiple incorporations of tc-T nucleotides under primer extension reaction conditions. However, the substrate acceptance is rather low, as also observed for other sugar-modified analogues. Tc-TTP and tc-nucleotide-containing templates do not sustain enzymatic polymerization under physiological conditions; this indicates that tc-DNA-based antisense agents will not enter natural metabolic pathways that lead to long-term toxicity.

Tumour genomic and microenvironmental heterogeneity for integrated prediction of 5-year biochemical recurrence of prostate cancer: a retrospective cohort study.

BACKGROUND Clinical prognostic groupings for localised prostate cancers are imprecise, with 30-50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors. METHODS We used DNA-based indices alone or in combination with intra-prostatic hypoxia measurements to develop four prognostic indices in 126 low-risk to intermediate-risk patients (Toronto cohort) who will receive image-guided radiotherapy. We validated these indices in two independent cohorts of 154 (Memorial Sloan Kettering Cancer Center cohort [MSKCC] cohort) and 117 (Cambridge cohort) radical prostatectomy specimens from low-risk to high-risk patients. We applied unsupervised and supervised machine learning techniques to the copy-number profiles of 126 pre-image-guided radiotherapy diagnostic biopsies to develop prognostic signatures. Our primary endpoint was the development of a set of prognostic measures capable of stratifying patients for risk of biochemical relapse 5 years after primary treatment. FINDINGS Biochemical relapse was associated with indices of tumour hypoxia, genomic instability, and genomic subtypes based on multivariate analyses. We identified four genomic subtypes for prostate cancer, which had different 5-year biochemical relapse-free survival. Genomic instability is prognostic for relapse in both image-guided radiotherapy (multivariate analysis hazard ratio [HR] 4·5 [95% CI 2·1-9·8]; p=0·00013; area under the receiver operator curve [AUC] 0·70 [95% CI 0·65-0·76]) and radical prostatectomy (4·0 [1·6-9·7]; p=0·0024; AUC 0·57 [0·52-0·61]) patients with prostate cancer, and its effect is magnified by intratumoral hypoxia (3·8 [1·2-12]; p=0·019; AUC 0·67 [0·61-0·73]). A novel 100-loci DNA signature accurately classified treatment outcome in the MSKCC low-risk to intermediate-risk cohort (multivariate analysis HR 6·1 [95% CI 2·0-19]; p=0·0015; AUC 0·74 [95% CI 0·65-0·83]). In the independent MSKCC and Cambridge cohorts, this signature identified low-risk to high-risk patients who were most likely to fail treatment within 18 months (combined cohorts multivariate analysis HR 2·9 [95% CI 1·4-6·0]; p=0·0039; AUC 0·68 [95% CI 0·63-0·73]), and was better at predicting biochemical relapse than 23 previously published RNA signatures. INTERPRETATION This is the first study of cancer outcome to integrate DNA-based and microenvironment-based failure indices to predict patient outcome. Patients exhibiting these aggressive features after biopsy should be entered into treatment intensification trials. FUNDING Movember Foundation, Prostate Cancer Canada, Ontario Institute for Cancer Research, Canadian Institute for Health Research, NIHR Cambridge Biomedical Research Centre, The University of Cambridge, Cancer Research UK, Cambridge Cancer Charity, Prostate Cancer UK, Hutchison Whampoa Limited, Terry Fox Research Institute, Princess Margaret Cancer Centre Foundation, PMH-Radiation Medicine Program Academic Enrichment Fund, Motorcycle Ride for Dad (Durham), Canadian Cancer Society.

A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis

BACKGROUND A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. RESULTS Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. CONCLUSIONS Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).

Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF-β expression and connective tissue features.

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor β (TGF-β) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-β1 (P=0.009), TGF-β2 (P=0.004) and additional inflammatory markers, and increased TGF-β1 (P=0.0009) and TGF-β2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age- and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-β signaling and offers TGF-β as a marker of FMD.

Methylation of PITX2, HOXD3, RASSF1 and TDRD1 predicts biochemical recurrence in high-risk prostate cancer

PURPOSE To explore differential methylation of HAAO, HOXD3, LGALS3, PITX2, RASSF1 and TDRD1 as a molecular tool to predict biochemical recurrence (BCR) in patients with high-risk prostate cancer (PCa). METHODS A multiplexed nested methylation-specific PCR was applied to quantify promoter methylation of the selected markers in five cell lines, 42 benign prostatic hyperplasia (BPH) and 71 high-risk PCa tumor samples. Uni- and multivariate Cox regression models were used to assess the importance of the methylation level in predicting BCR. RESULTS A PCa-specific methylation marker HAAO in combination with HOXD3 and a hypomethylation marker TDRD1 distinguished PCa samples (>90 % of tumor cells each) from BPH with a sensitivity of 0.99 and a specificity of 0.95. High methylation of PITX2, HOXD3 and RASSF1, as well as low methylation of TDRD1, appeared to be significantly associated with a higher risk for BCR (HR 3.96, 3.44, 2.80 and 2.85, correspondingly) after correcting for established risk factors. When DNA methylation was treated as a continuous variable, a two-gene model PITX2 × 0.020677 + HOXD3 × 0.0043132 proved to be the best predictor of BCR (HR 4.85) compared with the individual markers. This finding was confirmed in an independent set of 52 high-risk PCa tumor samples (HR 11.89). CONCLUSIONS Differential promoter methylation of HOXD3, PITX2, RASSF1 and TDRD1 emerges as an independent predictor of BCR in high-risk PCa patients. A two-gene continuous DNA methylation model "PITX2 × 0.020677 + HOXD3 × 0.0043132" is a better predictor of BCR compared with individual markers.

Möller-Barlow disease in archaeology: Preliminary study of biochemical detection

Human bone is the most direct source for reconstructing health and living conditions of ancient populations. However, many diseases remain undetected in palaeopathology. Möller-Barlow disease (scurvy) is a historically well-documented metabolic disease and must have been common in clinical and sub-clinical severity. Due to long incubation periods and the subtle nature of bone changes osteological evidence is relatively rare (Brickley & Ives 2008). Möller-Barlow disease is caused by deficiency of dietary vitamin C (ascorbic acid) and evokes symptoms like fatigue, haemorrhage, inflammations, delayed wound healing and pain. Vitamin C is a cofactor for the hydroxylation of the amino acids proline and lysine which are essential for the production of intact connective tissue by cross-linking the propeptides in collagen. In a preliminary study we tested the detectability of Möller-Barlow disease by analysis of relative quantitative variability of hydroxylated amino acids in collagen (Pendery & Koon 2013). Samples (N=9) were taken from children with (n=3, cranium, femur, tibia) and without (n=4, cranium, femur, tibia) apparent bone reactions indicative of Möller-Barlow disease, as well as from adults with lethal traumata (n=2; negative controls). The skeletal remains originated from two early medieval cemeteries from Switzerland. Gas chromatographic (GC) analysis revealed minor differences between the samples. So far children with no pathologic alterations had fairly same values as negative controls while children with bone reactions paradoxically exhibited even slightly higher values of hydroxyproline and hydroxylysine. Future research demands for larger sample size and has to discuss sampling strategies. Beside possible misdiagnosis of Möller-Barlow disease it is arguable if only the newly built bone should be analysed even though this could lead to problems related to small sample quantity. It also remains to be seen to which extent varying turnover rates of different skeletal elements, especially in children, must be taken into account.

Biochemical changes in barley plants after excessive supply of copper and manganese

The present study was undertaken to identify changes in some important proteins involved in CO2 fixation (Rubisco, Rubisco activase (RA), Rubisco binding protein (RBP)), NH4+ assimilation (glutamine synthetase (GS) and glutamate synthase (GOGAT)), using immunoblotting, and in the antioxidative defense as a result of Cu or Mn excess in barley leaves (Hordeum vulgare L. cv. Obzor). Activities and isoenzyme patterns of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT), as well as the levels of ascorbate (ASC), non-protein sulfhydryl groups, hydrogen peroxide and oxidative damage to proteins were determined. Data were correlated to the accumulation of Cu or Mn in the leaves after 5 days supply of heavy metal (HM) excess in the nutrient solution. In the highest Cu excess (1500 μM), Rubisco LS and SS were reduced considerably whereas under the highest Mn concentrations (18,300 μM) only minor changes in Rubisco subunits were detected. The RBP was diminished under the highest concentrations of both Cu or Mn. The bands of RA changed differently comparing Cu and Mn toxicity. GS decreased and GOGAT was absent under the highest concentration of Cu. At Mn excess Fd-GOGAT diminished whereas GS was not apparently changed. The development of toxicity symptoms corresponded to an accumulation of Cu or Mn in the leaves and to a gradual increase in protein carbonylation, a lower SOD activity and elevated CAT and GPX activities. APX activity was diminished under Mn toxicity and was not changed under Cu excess. Generally, changes in the isoenzyme profiles were similar under both toxicities. An accumulation of H2O2 was observed only at Mn excess. Contrasting changes in the low-molecular antioxidants were detected when comparing both toxicities. Cu excess affected mainly the non-protein SH groups, while Mn influenced the ASC content. Oxidative stress under Cu or Mn toxicity was most probably the consequence of depletion in low-molecular antioxidants as a result of their involvement in detoxification processes and disbalance in antioxidative enzymes. The link between heavy metal accumulation in leaves, leading to different display of oxidative stress, and changes in individual chloroplast proteins is discussed in the article.

Evaluation of serum biochemical marker concentrations and survival time in dogs with protein-losing enteropathy

OBJECTIVE: To evaluate serum concentrations of biochemical markers and survival time in dogs with protein-losing enteropathy (PLE). DESIGN: Prospective study. ANIMALS: 29 dogs with PLE and 18 dogs with food-responsive diarrhea (FRD). PROCEDURES: Data regarding serum concentrations of various biochemical markers at the initial evaluation were available for 18 of the 29 dogs with PLE and compared with findings for dogs with FRD. Correlations between biochemical marker concentrations and survival time (interval between time of initial evaluation and death or euthanasia) for dogs with PLE were evaluated. RESULTS: Serum C-reactive protein concentration was high in 13 of 18 dogs with PLE and in 2 of 18 dogs with FRD. Serum concentration of canine pancreatic lipase immunoreactivity was high in 3 dogs with PLE but within the reference interval in all dogs with FRD. Serum α1-proteinase inhibitor concentration was less than the lower reference limit in 9 dogs with PLE and 1 dog with FRD. Compared with findings in dogs with FRD, values of those 3 variables in dogs with PLE were significantly different. Serum calprotectin (measured by radioimmunoassay and ELISA) and S100A12 concentrations were high but did not differ significantly between groups. Seventeen of the 29 dogs with PLE were euthanized owing to this disease; median survival time was 67 days (range, 2 to 2,551 days). CONCLUSIONS AND CLINICAL RELEVANCE: Serum C-reactive protein, canine pancreatic lipase immunoreactivity, and α1-proteinase inhibitor concentrations differed significantly between dogs with PLE and FRD. Most initial biomarker concentrations were not predictive of survival time in dogs with PLE.

Biochemical MRI predicts hip osteoarthritis in an experimental ovine femoroacetabular impingement model

BACKGROUND Cam-type femoroacetabular impingement (FAI) resulting from an abnormal nonspherical femoral head shape leads to chondrolabral damage and is considered a cause of early osteoarthritis. A previously developed experimental ovine FAI model induces a cam-type impingement that results in localized chondrolabral damage, replicating the patterns found in the human hip. Biochemical MRI modalities such as T2 and T2* may allow for evaluation of the cartilage biochemistry long before cartilage loss occurs and, for that reason, may be a worthwhile avenue of inquiry. QUESTIONS/PURPOSES We asked: (1) Does the histological grading of degenerated cartilage correlate with T2 or T2* values in this ovine FAI model? (2) How accurately can zones of degenerated cartilage be predicted with T2 or T2* MRI in this model? METHODS A cam-type FAI was induced in eight Swiss alpine sheep by performing a closing wedge intertrochanteric varus osteotomy. After ambulation of 10 to 14 weeks, the sheep were euthanized and a 3-T MRI of the hip was performed. T2 and T2* values were measured at six locations on the acetabulum and compared with the histological damage pattern using the Mankin score. This is an established histological scoring system to quantify cartilage degeneration. Both T2 and T2* values are determined by cartilage water content and its collagen fiber network. Of those, the T2* mapping is a more modern sequence with technical advantages (eg, shorter acquisition time). Correlation of the Mankin score and the T2 and T2* values, respectively, was evaluated using the Spearman's rank correlation coefficient. We used a hierarchical cluster analysis to calculate the positive and negative predictive values of T2 and T2* to predict advanced cartilage degeneration (Mankin ≥ 3). RESULTS We found a negative correlation between the Mankin score and both the T2 (p < 0.001, r = -0.79) and T2* values (p < 0.001, r = -0.90). For the T2 MRI technique, we found a positive predictive value of 100% (95% confidence interval [CI], 79%-100%) and a negative predictive value of 84% (95% CI, 67%-95%). For the T2* technique, we found a positive predictive value of 100% (95% CI, 79%-100%) and a negative predictive value of 94% (95% CI, 79%-99%). CONCLUSIONS T2 and T2* MRI modalities can reliably detect early cartilage degeneration in the experimental ovine FAI model. CLINICAL RELEVANCE T2 and T2* MRI modalities have the potential to allow for monitoring the natural course of osteoarthrosis noninvasively and to evaluate the results of surgical treatments targeted to joint preservation.