Cytosine Ratio Research Articles

A multi-objective butterfly optimization algorithm for protein encoding

The integration of multiple genes to maximize protein expression levels represents an important challenge in synthetic biology. This task relies on the definition of multiple protein-coding sequences, which must be as different as possible to avoid information loss. Proteins can be encoded in different ways, using synonymous codons that translate into the same amino acid. Some codons are better suited to the host than others, thus being preferable the use of the most fitting ones. However, adopting only the most highly adapted codons would lead to very similar coding sequences. An additional criterion is given by the fact that the designed sequences must contain a suitable guanine–cytosine (GC) ratio in accordance with the characteristics of the host organism. Therefore, this biological task requires the simultaneous optimization of several, conflicting objectives. This work proposes a novel multi-objective approach for protein encoding, which tackles the problem according to a new formulation based on three objective functions: codon adaptation index, Hamming distance between sequences, and GC content. Our work extends the recent Butterfly Optimization Algorithm to multi-objective contexts, integrating problem-specific operators to boost solution quality by covering the different aspects required for accurate protein encoding. Two key structures, a taboo list and a best solution list, are defined to conduct improved searches attending to the potential improvements that each solution in the population can promote. Experiments conducted on nine real-world proteins reveal the attainment of relevant solutions from different evaluation perspectives, showing significant improvements over other single and multi-objective methods from the literature.

Complete genome sequence of Limosilactobacillus fermentum JNU532 as a probiotic candidate for the functional food and feed supplements

Lactic acid bacteria (LAB) have been reported to possess various beneficial properties and are commonly used as probiotics. LAB play a crucial role in milk fermentation, industrial lactic acid fermentation, and health and medicine. Limosilactobacillus fermentum isolated from fermented dairy and food products is considered as 'Generally Recognized as Safe' by FDA. Limosilactobacillus fermentum plays an important role in modulation of the intestinal microbiota, enhancing the host immune system and improving feed digestibility. We isolated a probiotic candidate that was identified and named Limosilactobacillus fermentum JNU532. In a previous report, cell-free culture of L. fermentum JNU532 exhibited anti-melanogenic and antioxidant activities. In this study, we present the complete genome assembly of the bacterial strain JNU532. The final genome consists of one circular chromosome (2,077,416 base pairs) with a guanine + cytosine (GC) ratio of 51.5%.

Nanopore sequencing of chloroplast genome of Scapania undulata (L.) Dumort., 1835 (Scapaniaceae, Jungermanniales)

We performed long-read sequencing of the Scapania undulata (L.) Dumort. chloroplast genome with Oxford Nanopore Technology using improved flow cells (10.3). S. undulata is a leafy liverwort living on rocks in or near water in mountains. The size of the complete plastome sequence is 117,651 bp long, the large single copy (LSC) region is 80,606 bp long, the small single copy (SSC) region is 19,401 bp long. The nanopore sequencing allowed for the distinction of inverted repeats A and B (IRA, IRB) which were not identical. Guanine cytosine (GC) ratio is 33.7% for the complete chloroplast genome, for LSC and SSC are 31.7% and 30.6%. The phylogeny reconstruction shows that Scapania undulata is the sister to S. ciliata and S. griffithii.

P14.02 Influence of individual CpG methylation status on outcome in adult patients with glioblastoma multiforme receiving alkylating agent treatment

Abstract Background: Methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promotor causes gene silencing and has been associated with a favourable prognosis in patients with glioblastoma multiforme (GBM) receiving alkylating chemotherapy. However, analysis of MGMT promotor methylation is usually reported as a cut-off depending on the results of the correspondent CpG site testing. This approach disregards a possible heterogeneity concerning the methylation status within the individual CpG sites and its possible association with prognosis in GBM patients. The current study aimed at elucidating the association between methylation of CpG sites 74–98 within the MGMT promotor region and outcome in GBM patients receiving alkylating agents. Material and Methods: Individual methylation status of 230 patients with histologically proven GBM following concomitant radio-chemotherapy with TMZ after stereotactic biopsy or open tumor resection (OTR) was assessed by the Sanger sequencing (Sseq) approach. Methylation of CpG sites 74–98 within the MGMT promotor region was defined according to a ratio of cytosine /thymine peak >50%. The total number of methylated CpG sites as well as clinical factors such as age, Karnofsky Performance Score (KPS) and mode of surgical procedure were correlated with outcome using proportional hazards models. In a subset of 34 patients, a correlation between individual CpG methylation and MGMT mRNA expression was performed. Results: Median progression-free (PFS) and overall survival (OS) were 7.8 and 14.6 months, respectively. Alongside younger age, KPS> 80 and OTR, the cumulative total number of methylated loci within the CpG sites 74–98 was strongly associated with both PFS and OS and retained its prognostic influence on outcome in multivariate models (p <0.001). Furthermore, a linear coherence between the total number of methylated CpG sites 74–98 and survival parameters could be observed. Moreover, low number of methylated CpG sites was observed in tumor specimen with a high mRNA expression and vice versa (Spearman correlation coefficient: -0.62). Conclusion: In contrast to the concept of dichotomizing the MGMT promotor status into ‘methylated’ and ‘non-methylated’, our approach shows a clear heterogeneity within the methylation status of the CpG sites 74–98 within the GBM tumor specimens. Our data suggest a strong correlation between outcome and the total number of methylated CpG sites, thus an up-front analysis of the individual GpC site methylation status prior to initiation of alkylating chemotherapy might help to improve treatment response in GBM patients.

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues

Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase (BChE, EC 3.1.1.8), which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-to-handle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene (hBChE) as published. The ratio of guanine and cytosine in four kinds of bases ((G+C) ratio) was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg mL–1 with an activity of 14.7 U mL–1, which was purified 3.2×103-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range (>60% activity at pH 4.0 to 8.0). Moreover, it had a good adaptability to high temperature (>60% activity at both 50 and 60°C up to 60 min) and good stability at 70°C. The enzyme can be activated by Li+, Co+, Zn2+ and ethylene diamine tetraacetic acid (EDTA), but inhibited by Mg2+, Mn2+, Fe2+, Ag+ and Ca2+. Na+ had little effect on its activity. The values of hBChE of the Michaelis constant (Km) and maximum reaction velocity (Vm) were 89.4 mmol L–1 and 1721 mmol min–1 mg–1, respectively. The bimolecular rate constants (Ki) of the hBChE to four pesticides were similar with that of electric eel AChE (EeAChE) and higher than that of horse BChE (HoBChE). All values of the half maximal inhibitory concentration of a substance (IC50) for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.

A validated quantitative liquid chromatography–tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA

5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography-tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20pM, 2nM and 10nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200ng of DNA. Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells.

Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection

Alterations in global DNA methylation are implicated in several pathobiological processes. The tissues stored as paraffin blocks represent an important source of DNA for retrospective genetic and epigenetic analysis on a large scale. Therefore, we developed the first capillary electrophoresis method able to measure global methylation in formalin-fixed, paraffin-embedded (FFPE) DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed. Analytes were baseline-separated within 8 min by using 300 mM tris(hydroxymethyl)aminomethane phosphate pH 3.75 as the running buffer. With use of electrokinetic injection the limit of detection (LOD) in real sample was 0.1 nM, thus improving by about 400-fold the LOD of the previously described methods based on capillary electrophoresis. Sample extraction and purification were optimized so that evaluation of the DNA methylation degree was possible starting from 0.5-1 μg of DNA with intra- and interassay relative standard deviations for the 5-methylcytosine to total cytosine ratio of 2.0 and 3.2%, respectively. Because of its high accuracy and throughput, our method will be useful for large-scale applications to determine the implications of genomic DNA methylation levels in tumorigenesis.

Clozapine and sulpiride but not haloperidol or olanzapine activate brain DNA demethylation

Cortical GABAergic dysfunction, a hallmark of both schizophrenia (SZ) and bipolar (BP) disorder pathophysiologies may relate to the hypermethylation of GABAergic gene promoters (i.e., reelin and GAD67). Benefits elicited by a combination of atypical antipsychotics with valproate (VPA) (a histone deacetylase inhibitor that may also activate brain DNA demethylation) in SZ or BP disorder treatment prompted us to investigate whether the beneficial action of this association depends on induction of a putative DNA demethylase activity. To monitor this activity, we measured the ratio of 5-methyl cytosine to unmethylated cytosine in reelin and GAD67 promoters in the mouse frontal cortex and striatum. We compared normal mice with mice pretreated with l-methionine (5.2 mmol/kg s.c. twice a day for 7 days) to hypermethylate promoters, including reelin and GAD67. Clinically relevant doses of clozapine (CLZ) (3.8 to 15 micromol/kg twice a day s.c. for 3 days) and sulpiride (SULP) (12.5 to 50 micromol/kg twice a day for 3 days) but not clinically relevant doses of haloperidol (HAL) (1.3 to 4 micromol/kg twice a day s.c. for 3 days) or olanzapine (OLZ) (4 to 15 micromol/kg twice a day for 3 days) exhibited dose-related increases in the cortical and striatal demethylation of hypermethylated reelin and GAD67 promoters. These effects of CLZ and SULP were dramatically potentiated by a clinically relevant VPA dose (0.5 mmol/kg twice a day for 3 days). By activating a DNA demethylase, the association of CLZ or SULP with VPA may facilitate a chromatin remodeling that normalizes the GABAergic gene expression down-regulation detected in the telencephalic regions of SZ and BP patients.

The Mildiomycin Biosynthesis: Initial Steps for Sequential Generation of 5‐Hydroxymethylcytidine 5′‐Monophosphate and 5‐Hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.

Rapid quantification of total genomic DNA methylation degree by short-end injection capillary zone electrophoresis

We propose a new capillary zone electrophoresis method applying short-end injection technique for the fast evaluation of methylcystosine/total cytosine ratio after acidic DNA hydrolysis. By short-end injection and by using a 100 mmol/l Tris solution titrated with 1 mol/l phosphoric acid to pH 3.75 as background electrolyte, cytosine and methylcytosine were separated with a good resolution in less than 1.5 min. Stepwise multiple linear regression with DNA methylation degree as the dependent variable and age, cysteine, homocysteine and methionine as independent variables, showed a negative association with age and that total cysteine is the most important determinant of DNA methylation.

Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects

Alterations in global DNA methylation are implicated in various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA. DNA was hydrolyzed using formic acid. Cytosine and 5-methylcytosine were separated by gradient-elution reversed-phase chromatography with a mobile phase containing nonafluoropentanoic acid (NFPA) as ion-pairing reagent and quantified using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers. Linear calibration curves were obtained in the range 0.111-4.422 ng/microL [mean correlation co-efficient 0.9983 (SD=0.0011), n=9] for cytosine and 0.0048-0.1936 ng/microL [mean correlation coefficient 0.9991 (SD=0.0010), n=9] for 5-methylcytosine. The intra- and inter-assay CVs for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n=9) and 3.5% (n=8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n=6) and 6.5% (n=14), respectively for pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The limit of detection (signal-to-noise ratio 3) was 2 pg on-column for cytosine and 5-methylcytosine. In healthy subjects (n=109), the mCyt/tCyt ratio varied from 2.6% to 4.8% (median 4.1%). DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype (p=0.046). No association with B-vitamin status was observed. This LC-ESI-MS/MS method is easy to perform and offers reproducibility, selectivity and sensitivity for studying DNA methylation. The method allows a sample throughput of approximately 200 samples/week. The MTHFR C677T genotype influences age-related changes in DNA methylation.

Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within ±5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h.

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2- 13C]cytosine and [2- 13C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5–6%.

Methylenetetrahydrofolate reductase 677C→T polymorphism affects DNA methylation in response to controlled folate intake in young women

DNA methylation is critical for normal genomic structure and function and is dependent on adequate folate status. A polymorphism (677C→T) in a key folate enzyme, methylenetetrahydrofolate reductase (MTHFR), may impair DNA methylation when folate intake is inadequate and may increase the risk of reproductive abnormalities. The present study was designed to evaluate the effect of the MTHFR 677C→T polymorphism on changes in global DNA methylation in young women consuming a low folate diet followed by repletion with the current Recommended Dietary Allowance (RDA). Women (age 20–30 years) with the TT (variant; n = 19) or CC ( n = 22) genotype for the MTHFR 677C→T polymorphism participated in a folate depletion-repletion study (7 weeks, 115 μg DFE/day; 7 weeks, 400 μg DFE/day). DNA methylation was measured at baseline, week 7, and week 14 using a [ 3H]methyl acceptance assay and a novel liquid chromatography tandem mass spectrometry assay of the DNA bases methylcytosine and cytosine. [ 3H]Methyl group acceptance tended to increase ( P = 0.08) during depletion in all subjects, indicative of a decrease in global DNA methylation. During repletion, the raw change and the percent change in the methylcytosine/total cytosine ratio increased ( P = 0.03 and P = 0.04, respectively) only in the subjects with the TT genotype. Moderate folate depletion in young women may cause a decrease in overall DNA methylation. The response to folate repletion suggests that following folate depletion women with the MTHFR 677 TT genotype have a greater increase in DNA methylation with folate repletion than women with the CC genotype.

A phase I trial of the DNA-hypomethylating agent 5-Aza-2'-Deoxycytidine in combination with carboplatin both given 4 weekly by intravenous injection in patients with advanced solid tumours

2005 Background: There is increasing evidence that DNA hypermethylation of CpG islands at key genes is associated with chemo resistance. For instance, methylation of a CpG-island at the hMLH1 promoter may be a major mechanism for loss of MLH1 expression associated with chemo resistance. Decitabine (5-Aza-2'-deoxycytidine) is a demethylating agent which has been shown in in vivo studies to reactivate MLH1 expression leading to sensitisation of chemo resistant tumours to a range of cytotoxic drugs. This Phase I study investigated the feasibility of delivering a dose of decitabine which would cause demethylation and hence potential sensitisation without causing prohibitive myelosuppression in combination with carboplatin. Methods: Pts with a variety of malignancies were enrolled. The starting dose of decitabine (on Day 1) was 45mg/m2. This was escalated in steps of 45mg/m2. Carboplatin at a fixed dose of AUC5 was given on Day 8. The interval between decitabine and carboplatin was 8 days which was within the optimal time for demethylation from xenograft studies. Treatment was repeated every 4 weeks. Results: 14 heavily-pretreated pts have been enrolled to date (6M/8F). Median age: 55; mean PS (ECOG): 1. Pts have been treated at 3 dose levels of decitabine: 45, 90 and 135mg/m2. Median number of cycles: 2 (range 0.5–6). 13/14 patients were evaluable for toxicity. MTD was identified as decitabine 135mg/m2 with grade 4 febrile neutropenia (1 pt), and grade 4 neutropenia necessitating delay of cycle 2 for >7 days (1 pt). Other possible treatment-related toxicities included: nausea, vomiting, diarrhoea, fatigue, anorexia, anaemia (all ≤grade 2 and easily manageable). PD studies showed reduction in 5-methylcytosine to total cytosine ratios in DNA isolated from peripheral mononuclear cells at all dose levels, and was maximal between Days 8 and 12 following decitabine infusion Conclusion: This study confirms that the combination of decitabine and carboplatin is feasible. Futher patients are being accrued to assess PD effects in tumour as well as peripheral blood and buccal smears. No significant financial relationships to disclose.

A phase I trial of the DNA-hypomethylating agent 5-Aza-2'-Deoxycytidine in combination with carboplatin both given 4 weekly by intravenous injection in patients with advanced solid tumours

2005 Background: There is increasing evidence that DNA hypermethylation of CpG islands at key genes is associated with chemo resistance. For instance, methylation of a CpG-island at the hMLH1 promoter may be a major mechanism for loss of MLH1 expression associated with chemo resistance. Decitabine (5-Aza-2'-deoxycytidine) is a demethylating agent which has been shown in in vivo studies to reactivate MLH1 expression leading to sensitisation of chemo resistant tumours to a range of cytotoxic drugs. This Phase I study investigated the feasibility of delivering a dose of decitabine which would cause demethylation and hence potential sensitisation without causing prohibitive myelosuppression in combination with carboplatin. Methods: Pts with a variety of malignancies were enrolled. The starting dose of decitabine (on Day 1) was 45mg/m2. This was escalated in steps of 45mg/m2. Carboplatin at a fixed dose of AUC5 was given on Day 8. The interval between decitabine and carboplatin was 8 days which was within the optimal time for demethylation from xenograft studies. Treatment was repeated every 4 weeks. Results: 14 heavily-pretreated pts have been enrolled to date (6M/8F). Median age: 55; mean PS (ECOG): 1. Pts have been treated at 3 dose levels of decitabine: 45, 90 and 135mg/m2. Median number of cycles: 2 (range 0.5–6). 13/14 patients were evaluable for toxicity. MTD was identified as decitabine 135mg/m2 with grade 4 febrile neutropenia (1 pt), and grade 4 neutropenia necessitating delay of cycle 2 for >7 days (1 pt). Other possible treatment-related toxicities included: nausea, vomiting, diarrhoea, fatigue, anorexia, anaemia (all ≤grade 2 and easily manageable). PD studies showed reduction in 5-methylcytosine to total cytosine ratios in DNA isolated from peripheral mononuclear cells at all dose levels, and was maximal between Days 8 and 12 following decitabine infusion Conclusion: This study confirms that the combination of decitabine and carboplatin is feasible. Futher patients are being accrued to assess PD effects in tumour as well as peripheral blood and buccal smears. No significant financial relationships to disclose.

Molecular and Phenotypic Characterization of Strains ofPhotobacterium damselaesubsp.piscicidaIsolated from Hybrid Striped Bass Cultured in Louisiana, USA

Photobacteriosis, which is caused by the bacterium Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) was found to be a primary cause of mortality in hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops) cultured in brackish water in southern Louisiana. The disease was diagnosed on 50 occasions from 1990 through 2000 by the Louisiana Aquatic Diagnostic Laboratory, and mortality ranged from 5% to 90% in individual culture units. Strains of the bacterium from the Louisiana coast of the Gulf of Mexico were identified by means of biochemical tests, guanine–cytosine ratio, and Aquarapid Pp kit. Strains were characterized according to their biochemical phenotype, enzyme activities, plasmid profile, genotypic profile as determined by randomly amplified polymorphic DNA (RAPD) analysis, and antimicrobial susceptibility. When compared with representative isolates from the Chesapeake Bay, Greece, Japan, and Israel, Louisiana Gulf Coast isolates of P. damselae subsp. piscicida were found to be almost identical in biochemical phenotype and enzyme activity. The isolates differed in their plasmid profiles and antimicrobic susceptibilities. Louisiana isolates were found to possess a unique plasmid banding profile relative to strains from other geographic locations. The Louisiana isolates typically produced two large plasmid bands greater than 30 kilo–base pairs (kb) and two smaller bands 8.0 kb and 5.0 kb in size. Isolates from Israel and Greece exhibited similar banding patterns but were different from Louisiana and Japanese isolates. Resistance to Romet and Terramycin by some Louisiana strains was the result of acquisition of an R plasmid. When analyzed by RAPD, the Louisiana Gulf Coast strains were found to belong to clonal lineage group 2, which displays a fingerprint similar to that of the Japanese strains.

Caloramator proteoclasticus sp. nov., a new moderately thermophilic anaerobic proteolytic bacterium.

A new moderately thermophilic proteolytic anaerobe, strain UT, was isolated from mesophilic granular methanogenic sludge. The cells were spore-forming, motile rods that were 0.4 micron wide and 2.4 to 4 microns long and stained gram negative. Electron micrographs of thin sections revealed the presence of an atypical gram-positive cell wall. Optimum growth occurred at 55 degrees C and at pH values between 7.0 and 7.5, with a doubling time of 30 min. The DNA base ratio of guanine plus cytosine was 31 mol%. The bacterium fermented proteins mainly to acetate, hydrogen, formate, and branched-chain fatty acids. Several amino acids, including glutamate, aspartate, arginine, histidine, threonine, methionine, and branched-chain amino acids, were also utilized. Glutamate was degraded to acetate, formate, hydrogen, and alanine. In addition, the strain degraded carbohydrates, including glucose, fructose, mannose, cellobiose, and starch, to acetate, ethanol, formate, lactate, and hydrogen. The results of a 16S rRNA sequence analysis phylogenetically placed strain UT in the low-guanine-plus-cytosine-content subgroup of the gram-positive phylum. We propose to classify the described strain in the genus Caloramator as a new species, Caloramator proteoclasticus. The type strain of C. proteoclasticus, strain U, has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 10124.

A preliminary investigation of the order Bangiales (Bangiophycidae, Rhodophyta) based on sequences of nuclear small‐subunit ribosomal RNA genes

SUMMARYWe investigated phylogenetic relationships among red algae of the order Bangiales by analysis of sequences of the nuclear gene encoding cytosolic small‐subunit ribosomal RNA in Bangia atropurpurea (Roth) C. Ag. and eight samples representing seven species of Porphyra. The ssu‐rDNA range from 1818 to 1845 nucleotides in length, with guanosine plus cytosine ratios between 47.0% and 48.6%. A group IC1 intron occurs in the B atropurpurea ssu‐rDNAs at the same position as in P. spiralis var. amplifolia Oliveira Filho et Coll and several other eukaryote ssu‐rDNAs. The nine sequences form a stable monophyletic group upon phylogenetic analysis. The ssu‐rDNA from B. atropurpurea nests stably within the Porphyra group and is closely related to P. amplissima (Kjellm.) Setchell et Hus in Hus, making the genus Porphyra paraphyletic. No correlation is seen between phylogenetic position and number of cell layers in the Porphyra thallus. We discuss possible taxonomic and evolutionary implications of these observations.