Identification of the sex-determining region in flathead grey mullet (Mugil cephalus).

Elucidation of the sex-determination mechanism in flathead grey mullet (Mugil cephalus) is required to exploit its economic potential by production of genetically determined monosex populations and application of hormonal treatment to parents rather than to the marketed progeny. Our objective was to construct a first-generation linkage map of the M. cephalus in order to identify the sex-determining region and sex-determination system. Deep-sequencing data of a single male was assembled and aligned to the genome of Nile tilapia (Oreochromis niloticus). A total 245 M. cephalus microsatellite markers were designed, spanning the syntenic tilapia genome assembly at intervals of 10 Mb. In the mapping family of full-sib progeny, 156 segregating markers were used to construct a first-generation linkage map of 24 linkage groups (LGs), corresponding to the number of chromosomes. The linkage map spanned approximately 1200 cM with an average inter-marker distance of 10.6 cM. Markers segregating on LG9 in two independent mapping families showed nearly complete concordance with gender (R2  = 0.95). The sex determining locus was fine mapped within an interval of 8.6 cM on LG9. The sex of offspring was determined only by the alleles transmitted from the father, thus indicating an XY sex-determination system.

Biomarker analysis in oesophagogastric cancer: Results from the REAL3 and TransMAGIC trials.

BACKGROUND: REAL3 (Randomised ECF for Advanced or Locally advanced oesophagogastric cancer 3) was a phase II/III trial designed to evaluate the addition of panitumumab (P) to epirubicin, oxaliplatin and capecitabine (EOC) in untreated advanced oesophagogastric adenocarcinoma, or undifferentiated carcinoma. MAGIC (MRC Adjuvant Gastric Infusional Chemotherapy) was a phase III study which demonstrated that peri-operative epirubicin, cisplatin and infused 5-fluorouracil (ECF) improved survival in early oesophagogastric adenocarcinoma. PATIENTS AND METHODS: Analysis of response rate (RR; the primary end-point of phase II) and biomarkers in the first 200 patients randomised to EOC or modified dose (m) EOC+P in REAL3 was pre-planned to determine if molecular selection for the on-going study was indicated. KRAS, BRAF and PIK3CA mutations and PTEN expression were assessed in pre-treatment biopsies and results correlated with response to mEOC+P. Association between these biomarkers and overall survival (OS) was assessed in MAGIC patients to determine any prognostic effect. RESULTS: RR was 52% to mEOC+P, 48% to EOC. Results from 175 assessable biopsies: mutations in KRAS (5.7%), BRAF (0%), PIK3CA (2.5%) and loss of PTEN expression (15.0%). None of the biomarkers evaluated predicted resistance to mEOC+P. In MAGIC, mutations in KRAS, BRAF and PIK3CA and loss of PTEN (phosphatase and tensin homolog) were found in 6.3%, 1.0%, 5.0% and 10.9%, respectively, and were not associated with survival. CONCLUSIONS: The RR of 52% in REAL3 with mEOC+P met pre-defined criteria to continue accrual to phase III. The frequency of the mutations was too low to exclude any prognostic or predictive effect.

The effect of caloric restriction and glycemic load on measures of oxidative stress and antioxidants in humans: results from the CALERIE Trial of Human Caloric Restriction.

Decreasing oxidative stress and increasing antioxidant defense has been hypothesized as one mechanism by which caloric restriction (CR) increases longevity in animals. A total of 46 moderately overweight volunteers (BMI: 25-30 kg/m2), ages 20-42 yr were randomized to either high glycemic (HG) or low glycemic (LG) dietary load CR regimen at either 10% (n=12) or 30% (n=34) of basal caloric intake. All food was provided to participants for 6 mo. Overall, after controlling for CR levels and dietary regimen for 6 mo, plasma glutathione peroxidase activity increased (p=0.04) and plasma protein carbonyl levels decreased (p=0.02) and a non-significant decrease in plasma 8-epi-prostaglandin F2α level was observed (p=0.09). No significant change was observed in other plasma antioxidants such as superoxide dismutase and catalase. These findings indicate that short term CR (10% or 30%) in moderately overweight subjects modulates some but not all measures of antioxidant defense and oxidative stress.

Chimaerism detection in bovine twins, triplets and quadruplets using sex chromosome-linked markers.

The phenomenon of chimaerism occurs in the majority of cattle twin pregnancies. The objectives of this study were to develop a powerful diagnostic test for chimaerism in bovine male and female co-twins using X and Y chromosome-linked markers and to determine the extent of chimaerism in twins, triplets and quadruplets. We developed a multiplex PCR set of three polymorphic markers on chromosome X (DIK2865, DIK2283, AGLA257), where the presence of >1 and >2 alleles per marker is sufficient to prove chimaerism in males and females, respectively. In addition, a specific segment on chromosome Y (BOV97M) is included in the set to indicate chimaerism in females. Visualization of chimaeric alleles was best for DNA extracted from blood, fair for DNA from vaginal smears and failed for DNA extracted from hair. The power of chimaerism identification using this set of markers for DNA extracted from blood was calculated as 99% in males and virtually 100% in females. All females and males in heterosexual twins, triplets and quadruplets displayed evidence of a chimaeric allele in at least one and maximum of three of three X chromosome markers analysed. In addition, all females showed the presence of the BOV97M segment and were validated as chimaeric by the standard clinical diagnosis of impaired vaginal length. Quantitative PCR analysis of BOV97M copies in all twins vs. their sires showed a mean ratio of 45-68% in females and 39-49% in males, indicating a substantial symmetrical exchange of cells among all co-twins. The proposed analysis of X and Y chromosome-linked markers is advantageous to previous methods based on Y chromosome sequences only, because it detects chimaerism in both male and female co-twins.

B-type natriuretic peptide can detect silent myocardial ischaemia in asymptomatic type 2 diabetes.

OBJECTIVE: To find out whether B-type natriuretic peptide (BNP) detects silent myocardial ischaemia in patients with type 2 diabetes, since many of these patients have silent ischaemia leading to unexpected cardiac deaths. DESIGN: Prospective cross-sectional study with consecutive recruitment of patients. SETTING: Outpatient, single centre. PATIENTS: 219 patients with type 2 diabetes. Patients were excluded if they had a history or evidence of cardiac failure. OUTCOME MEASURES: BNP, echocardiography and exercise tolerance test (ETT). BNP was compared with the ETT result in all patients and specifically in those who had no apparent ischaemic heart disease (IHD). RESULTS: 121 patients had no history of IHD or cardiac failure and of these patients 85 had a clearly abnormal or normal ETT result. BNP was higher in patients with an abnormal than with a normal ETT (mean 58.2 (SD 46.3) v 24.4 (SD 15.7) pg/ml, p < 0.001). In univariate analysis BNP was an independent predictor of an abnormal ETT (p < 0.001). In multivariate analysis BNP remained an independent predictor of the ETT result. BNP concentration over 20 pg/ml predicted an abnormal ETT result with a sensitivity of 87% and specificity of 37%, and BNP over 40 pg/ml had a sensitivity of 63% and a specificity of 81%. CONCLUSION: BNP is of value in predicting silent ischaemia on exercise testing in asymptomatic patients with type 2 diabetes.

Evolutionary regulation of the blind subterranean mole rat, Spalax, revealed by genome-wide gene expression.

We applied genome-wide gene expression analysis to the evolutionary processes of adaptive speciation of the Israeli blind subterranean mole rats of the Spalax ehrenbergi superspecies. The four Israeli allospecies climatically and adaptively radiated into the cooler, mesic northern domain (N) and warmer, xeric southern domain (S). The kidney and brain mRNAs of two N and two S animals were examined through cross-species hybridizations with two types of Affymetrix arrays (mouse and rat) and muscle mRNA of six N and six S animals with spotted cDNA mouse arrays. The initial microarray analysis was hypothesis-free, i.e., conducted without reference to the origin of animals. Principal component analysis revealed that 20-30% of the expression signal variability could be explained by the differentiation of N-S species. Similar N-S effects were obtained for all tissues and types of arrays: two Affymetrix microarrays using probe oligomer signals and the spotted array. Likewise, ANOVA and t test statistics demonstrated significant N-S ecogeographic divergence and region-tissue specificity in gene expression. Analysis of differential gene expression between species corroborates previous results deduced by allozymes and DNA molecular polymorphisms. Functional categories show significant N-S ecologic putative adaptive divergent up-regulation of genes highlighting a higher metabolism in N, and potential adaptive brain activity and kidney urine cycle pathways in S. The present results confirm ecologic-genomic separation of blind mole rats into N and S. Gene expression regulation appears to be central to the evolution of blind mole rats.

Frequency and predictors of hypoglycaemia in Type 1 and insulin-treated Type 2 diabetes: a population-based study.

AIMS: To ascertain the frequency and identify predictors of self-reported hypoglycaemia in Type 1 and insulin-treated Type 2 diabetes. METHODS: A random sample of 267 people with insulin-treated diabetes were recruited from a population-based diabetes register in Tayside, Scotland. Each subject prospectively recorded the number of mild and severe hypoglycaemic episodes experienced over a 1-month period. Ordinal logistic regression was performed to identify potential predictors of hypoglycaemia. RESULTS: Five hundred and seventy-two hypoglycaemic events were reported by 155 patients. The participants with Type 1 diabetes had a total of 336 hypoglycaemic events with a rate of 42.89 events per patient per year. Of these, nine were severe hypoglycaemic events, with a rate of 1.15 events per patient per year. Participants with insulin-treated Type 2 diabetes experienced a total of 236 hypoglycaemic events with a rate of 16.37 events per patient per year. Of these, five were severe hypoglycaemic events, which would be equivalent to 0.35 events per patient per year. Predictors of hypoglycaemia in Type 1 diabetes were a history of previous hypoglycaemia (P = 0.006) and co-prescribing of any oral drug (P = 0.048). In patients with insulin-treated Type 2 diabetes, a history of previous hypoglycaemia (P < 0.0001) and duration of insulin treatment (P = 0.014) were significant predictors. CONCLUSION: The incidence of self-reported severe hypoglycaemia in insulin-treated Type 2 diabetes is lower than in Type 1 diabetes but does occur more often than previously reported and with sufficient frequency to cause significant morbidity. Duration of insulin treatment is a key predictor of hypoglycaemia in insulin-treated Type 2 diabetes.

Spironolactone reduces brachial pulse wave velocity and PIIINP levels in hypertensive diabetic patients.

AIMS: To assess whether spironolactone has beneficial effects on blood pressure (BP), N-terminal propeptide of type III procollagen (PIIINP) and pulse wave velocity (PWV) in hypertensive, type II diabetics. METHODS: Ten patients with type II diabetes and hypertension were enrolled in a randomized, double-blind crossover study comparing 4 months' treatment with spironolactone and placebo with a 4-week washout phase. BP, PIIINP and carotid-radial PWV were measured at the end of each treatment phase. RESULTS: Compared with placebo, spironolactone reduced systolic BP by 15.6 +/- 46.1 mmHg (P = 0.005, 95% CI 2.7-28.5 mmHg), PIIINP by 0.6 +/- 0.3 microg l(-1) (P = 0.04, 95% CI 0.02-1.1 microg l(-1)) and PWV by 0.6 +/- 0.2 m s(-1) (P = 0.008, 95% CI 0.18-1.02 m s(-1)). CONCLUSIONS: Spironolactone is effective at reducing systolic BP and brachial artery stiffness as indicated by PWV. It also reduces PIIINP in type II diabetic patients with hypertension.

Spironolactone impairs endothelial function and heart rate variability in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Aldosterone blockade has followed in the footsteps of ACE inhibition in reducing mortality in patients with heart failure. This is associated with its beneficial effects on endothelial function and heart rate variability. Diabetes is another area, where angiotensin II withdrawal has proven to be of particular value. We postulated that aldosterone blockade with spironolactone might also have beneficial effects on the prognostic markers of endothelial function and heart rate variability in diabetic patients. METHODS: We assessed endothelial function by forearm venous occlusion plethysmography in 42 patients with type 2 diabetes mellitus after 1 month of treatment with spironolactone or placebo allocated in a randomised double-blind trial. Of the 42 patients, 20 were on ACE inhibitor therapy. We also assessed heart rate variability, HbA1c and plasma angiotensin II levels at the end of each treatment period. RESULTS: Compared to placebo, spironolactone decreased forearm blood flow response to acetylcholine by 44.56+/-14.56% (p=0.003) in the group as a whole and by 57.61+/-15.56% (p<0.001) in the 20 patients on ACE inhibition. Spironolactone also worsened heart rate variability parameters, with root mean squared standard deviation decreased by 1.99+/-0.93 ms (p=0.03), low-frequency normalised power increased by 2.00+/-0.91 normalised units (nu) (p=0.03), high-frequency normalised power decreased by 1.98+/-0.94 nu (p=0.04) and the low frequency : high frequency ratio increased by 0.40+/-0.19 (p=0.04). HbA1c and angiotensin II increased during treatment with spironolactone by 0.26+/-0.07% (p=0.001) and 8.12+/-1.94 pg/ml (p=0.001) respectively. CONCLUSIONS/INTERPRETATION: Spironolactone worsened endothelial function and heart rate variability in patients with type 2 diabetes. These findings are possibly due to the worsening of glycaemic control and increase in plasma angiotensin II that were seen with spironolactone treatment. Thus the prescription of spironolactone to diabetic patients without heart failure does not seem to be justified.

Cardiovascular autonomic function in patients with hemodynamic instability at induction of capnoperitoneum: a case-control study.

BACKGROUND: This study hypothesized that patients in whom bradycardia and hypotension develop with induction of positive-pressure capnoperitoneum have an underlying autonomic cardiovascular dysfunction. METHODS: A case-control study was conducted to examine the baseline autonomic function of patients in whom bradycardia and hypotension develop with induction of positive-pressure capnoperitoneum. The control group consisted of patients who maintained normal cardiac rhythm and blood pressure during the same procedure. Two groups of tests were performed: bedside stress tests of cardiovascular autonomic function (response graded 1 (normal) to 4 (severely abnormal) and heart rate variability analysis (spectral and time domain components). RESULTS: The study evaluated 6 patients in the bradycardia group and 10 in the control group. The group in whom bradycardia had developed scored significantly worse on the bedside stress tests than the control group (for grades I to IV: chi2 = 6.5, p = 0.022; for trend: chi2 = 5.6, p = 0.018). In contrast, both groups had similar baseline autonomic tone, as measured by heart rate variability. CONCLUSIONS: Patients in whom bradycardia and hypotension develop with induction of positive-pressure capnoperitoneum have cardiovascular autonomic dysfunction, which is identifiable by bedside stress tests of autonomic function.

An ordered comparative map of the cattle and human genomes.

A cattle-human whole-genome comparative map was constructed using parallel radiation hybrid (RH) mapping in conjunction with EST sequencing, database mining for unmapped cattle genes, and a predictive bioinformatics approach (COMPASS) for targeting specific homologous regions. A total of 768 genes were placed on the RH map in addition to 319 microsatellites used as anchor markers. Of these, 638 had human orthologs with mapping data, thus permitting construction of an ordered comparative map. The large number of ordered loci revealed > or =105 conserved segments between the two genomes. The comparative map suggests that 41 translocation events, a minimum of 54 internal rearrangements, and repositioning of all but one centromere can account for the observed organizations of the cattle and human genomes. In addition, the COMPASS in silico mapping tool was shown to be 95% accurate in its ability to predict cattle chromosome location from random sequence data, demonstrating this tool to be valuable for efficient targeting of specific regions for detailed mapping. The comparative map generated will be a cornerstone for elucidating mammalian chromosome phylogeny and the identification of genes of agricultural importance."Ought we, for instance, to begin by discussing each separate species-in virtue of some common element of their nature, and proceed from this as a basis for the consideration of them separately?" from Aristotle, On the Parts of Animals, 350 B.C.E.

Comparative organization of cattle chromosome 5 revealed by comparative mapping by annotation and sequence similarity and radiation hybrid mapping.

A whole genome cattle-hamster radiation hybrid cell panel was used to construct a map of 54 markers located on bovine chromosome 5 (BTA5). Of the 54 markers, 34 are microsatellites selected from the cattle linkage map and 20 are genes. Among the 20 mapped genes, 10 are new assignments that were made by using the comparative mapping by annotation and sequence similarity strategy. A LOD-3 radiation hybrid framework map consisting of 21 markers was constructed. The relatively low retention frequency of markers on this chromosome (19%) prevented unambiguous ordering of the other 33 markers. The length of the map is 398.7 cR, corresponding to a ratio of approximately 2.8 cR(5,000)/cM. Type I genes were binned for comparison of gene order among cattle, humans, and mice. Multiple internal rearrangements within conserved syntenic groups were apparent upon comparison of gene order on BTA5 and HSA12 and HSA22. A similarly high number of rearrangements were observed between BTA5 and MMU6, MMU10, and MMU15. The detailed comparative map of BTA5 should facilitate identification of genes affecting economically important traits that have been mapped to this chromosome and should contribute to our understanding of mammalian chromosome evolution.

A genome scan for QTL influencing milk production and health traits in dairy cattle.

A genome scan was conducted in the North American Holstein-Friesian population for quantitative trait loci (QTL) affecting production and health traits using the granddaughter design. Resource families consisted of 1,068 sons of eight elite sires. Genome coverage was estimated to be 2,551 cM (85%) for 174 genotyped markers. Each marker was tested for effects on milk yield, fat yield, protein yield, fat percentage, protein percentage, somatic cell score, and productive herd life using analysis of variance. Joint analysis of all families identified marker effects on 11 chromosomes that exceeded the genomewide, suggestive, or nominal significance threshold for QTL effects. Large marker effects on fat percentage were found on chromosomes 3 and 14, and multimarker regression analysis was used to refine the position of these QTL. Half-sibling families from Israeli Holstein dairy herds were used in a daughter design to confirm the presence of the QTL for fat percentage on chromosome 14. The QTL identified in this study may be useful for marker-assisted selection and for selection of a refined set of candidate genes affecting these traits.

A radiation hybrid map of BTA23: identification of a chromosomal rearrangement leading to separation of the cattle MHC class II subregions.

Bovine chromosome 23 (BTA23) contains the bovine major histocompatibility complex (MHC) and is thus of particular interest because of the role of MHC genes in immunity. Previous studies have shown cattle MHC class II genes to be subdivided into two distinct subregions separated by a variable genetic distance of 15-30 cM. To elucidate the genetic events that resulted in the present organization of the class II and other MHC genes, a framework radiation hybrid (RH) map of BTA23 was developed by testing DNA samples from a 5000 rad whole genome RH panel. Twenty-six markers were screened with an average retention frequency of 0.27, ranging from 0.14 to 0.42. Total length of the chromosome was 220 cR5000, with 4.1 cR5000/cM when compared to linkage data. Gene orders for the markers common to both the RH framework map and the consensus framework linkage map are identical. Large centiray intervals, D23S23-D23S7, DYA-D23S24 and CYP21-D23S31, were observed compared to linkage distances. These data may indicate a much larger physical distance or suppression of recombination in the interval separating the class II subregions and also within the class I region than previously estimated. Comparison of 13 Type I genes conserved between BTA23 and the human homolog HSA6p suggests the occurrence of an inversion encompassing the centromeric half of the bovine chromosome, thus explaining the large distance between the bovine class IIa and IIb clusters. These results exemplify the power of RH mapping in solving problems in comparative genomics and evolution. Furthermore, noncongruence of the genetic and physical RH map distances indicates that caution must be observed in using either resource alone in searching for candidate genes controlling traits of economic importance.

Determination of allele frequency from DNA pools using bovine trinucleotide microsatellites.

Three sets of fluorescent labelled primers were used to amplify bovine trinucleotide microsatellites from DNA pools. DNA from 20 individuals were collected to create 3 pools differing in allele frequencies. Replicate mixes from each pool were used as template for PCR reactions. PCR products were separated and quantified on an automated DNA sequencer. Allele frequency estimates from pooled samples corrected for overlapping shadow peaks were calculated. Rare alleles representing only 2.5% of the total pool were accurately detected. Standard error of allele frequency estimates expressed as percent of the total 40 chromosomes per pool ranged between 0.8%-4.6% for different microsatellite-pool combinations as compared to 8.0% binomial sampling error. Regression coefficients of actual allele frequencies, determined by individual genotyping, on estimated frequencies ranged from 0.96-1.06. As regression slopes were close to unity it can be deduced that corrected peak height values from a DNA pool are unbiased estimates of actual allele frequencies. With standard error of the y-intercept of 0.21, the 95% confidence interval of allele frequency is 0.42 alleles or 1% in a pool of 40 chromosomes. Thus, it would be possible to detect an allele with a frequency of greater than 1% within the pool.

Isolation of microsatellites from a bovine YAC clone harbouring the SOD1 gene.

A bovine yeast artificial chromosome (YAC) clone containing the superoxide dismutase 1 (SOD1) gene was used as a template for polymerase chain reaction (PCR) amplification using a conserved short interspersed nuclear element (SINE) primer. Two highly polymorphic microsatellites with nine and eight alleles were isolated and mapped by linkage analysis to the centromeric region of BTA1. These microsatellites will be used in the construction of a genetic and physical map of the SOD1 region towards positional cloning of the polled gene.

A medium-density genetic linkage map of the bovine genome.

A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density. Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees. Seven hundred and three of the loci are linked to at least one other locus. All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere. There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes. One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny. This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project.

Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome.

Trinucleotide (AGC)n microsatellites are found as 3' tails of the artiodactyl short insterspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)n microsatellites with 2-4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)n microsatellites. Enrichment for (AGC)n microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.

Misidentification rate in the Israeli dairy cattle population and its implications for genetic improvement.

The DNA microsatellites can be efficiently used to determine incorrect paternity attribution of cattle without genotyping of dams. Allelic frequencies of the population were determined for 12 microsatellites using the maternal alleles of 102 AI sires. The frequency of the most common microsatellite allele ranged from 0.27 to 0.58. Most loci had at least one allele that was present in only a single individual. Paternity of 9 of 173 cows (5.2%) and 3 of 102 bulls (2.9%) was excluded because putative paternal alleles were not present in progeny for at least one locus. For 4 of the 9 cows and all 3 bulls, exclusion was based on at least two loci. Mean probability of exclusion was 0.85 for cows and 0.99 for bulls. With an assumed cost of US $5 per genotype, a misidentification rate of 5%, and a discount rate of 0.05, additional profit for the Israeli-Holstein breeding program from genotyping 100 test daughters of each young sire becomes positive within 10 yr and reaches nearly US $2.4 million after 20 yr.