Germline variants in IL4, MGMT and AKT1 are associated with prostate cancer-specific mortality: An analysis of 12,082 prostate cancer cases.

BACKGROUND: Prostate cancer (PCa) is a leading cause of mortality and genetic factors can influence tumour aggressiveness. Several germline variants have been associated with PCa-specific mortality (PCSM), but further replication evidence is needed. METHODS: Twenty-two previously identified PCSM-associated genetic variants were genotyped in seven PCa cohorts (12,082 patients; 1544 PCa deaths). For each cohort, Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals for risk of PCSM associated with each variant. Data were then combined using a meta-analysis approach. RESULTS: Fifteen SNPs were associated with PCSM in at least one of the seven cohorts. In the meta-analysis, after adjustment for clinicopathological factors, variants in the MGMT (rs2308327; HR 0.90; p-value = 3.5 × 10-2) and IL4 (rs2070874; HR 1.22; p-value = 1.1 × 10-3) genes were confirmed to be associated with risk of PCSM. In analyses limited to men diagnosed with local or regional stage disease, a variant in AKT1, rs2494750, was also confirmed to be associated with PCSM risk (HR 0.81; p-value = 3.6 × 10-2). CONCLUSIONS: This meta-analysis confirms the association of three genetic variants with risk of PCSM, providing further evidence that genetic background plays a role in PCa-specific survival. While these variants alone are not sufficient as prognostic biomarkers, these results may provide insights into the biological pathways modulating tumour aggressiveness.

Novel associations between blood DNA methylation and body mass index in middle-aged and older adults.

BACKGROUND/OBJECTIVES: There is increasing evidence of a relationship between blood DNA methylation and body mass index (BMI). We aimed to assess associations of BMI with individual methylation measures (CpGs) through a cross-sectional genome-wide DNA methylation association study and a longitudinal analysis of repeated measurements over time. SUBJECTS/METHODS: Using the Illumina Infinium HumanMethylation450 BeadChip, DNA methylation measures were determined in baseline peripheral blood samples from 5361 adults recruited to the Melbourne Collaborative Cohort Study (MCCS) and selected for nested case-control studies, 2586 because they were subsequently diagnosed with cancer (cases) and 2775 as controls. For a subset of 1088 controls, these measures were repeated using blood samples collected at wave 2 follow-up, a median of 11 years later; weight was measured at both time points. Associations between BMI and blood DNA methylation were assessed using linear mixed-effects regression models adjusted for batch effects and potential confounders. These were applied to cases and controls separately, with results combined through fixed-effects meta-analysis. RESULTS: Cross-sectional analysis identified 310 CpGs associated with BMI with P<1.0 × 10-7, 225 of which had not been reported previously. Of these 225 novel associations, 172 were replicated (P<0.05) using the Atherosclerosis Risk in Communities (ARIC) study. We also replicated using MCCS data (P<0.05) 335 of 392 associations previously reported with P<1.0 × 10-7, including 60 that had not been replicated before. Associations between change in BMI and change in methylation were observed for 34 of the 310 strongest signals in our cross-sectional analysis, including 7 that had not been replicated using the ARIC study. CONCLUSIONS: Together, these findings suggest that BMI is associated with blood DNA methylation at a large number of CpGs across the genome, several of which are located in or near genes involved in ATP-binding cassette transportation, tumour necrosis factor signalling, insulin resistance and lipid metabolism.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer.

BACKGROUND: Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH). METHODS: Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines. RESULTS: Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10(-11)). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10(-5) and 10(-29)), and identified breast and pancreatic cell lines with BRCA defects. CONCLUSION: The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Hepatotoxicosis in dogs consuming a diet of camel meat contaminated with indospicine.

BACKGROUND: Four dogs presented with clinical signs of severe hepatic disease after consuming a commercial camel meat diet. METHODS: Laboratory investigation revealed evidence of severe liver disease, including markedly increased serum alanine aminotransferase (ALT) activity and total bilirubin concentration, and prolonged clotting times. RESULTS: Two dogs deteriorated despite supportive therapy and were euthanased. Histologically, both livers appeared similar, with the main lesion being extensive periacinar necrosis and haemorrhage. Indospicine, a toxic amino acid of plant origin, was detected in the serum and/or plasma from all four dogs, as well as in tissues of a dog that was necropsied and in a sample of the camel meat fed to this animal. Serum biochemistry tests using blood samples collected from 15 additional dogs identified as having eaten the diet detected indospicine was in the serum of 14 and 3 had increased ALT activity. One of the latter dogs subsequently developed clinical signs of severe liver disease and was euthanased. CONCLUSION: To the authors' knowledge, this is the first published report of the detection of indospicine residues in camel meat and the occurrence of severe, sometimes fatal, liver disease in dogs that consumed this contaminated meat.

Association of FGFR4 genetic polymorphisms with prostate cancer risk and prognosis.

The fibroblast growth factor receptor 4 (FGFR4) is thought to be involved in many critical cellular processes and has been associated with prostate cancer risk. Four single nucleotide polymorphisms (SNPs) within or near FGFR4 were analyzed in a population-based study of 1458 prostate cancer patients and 1352 age-matched controls. We found no evidence to suggest that any of the FGFR4 SNP genotypes were associated with prostate cancer risk or with disease aggressiveness, Gleason score or stage. A weak association was seen between rs351855 and prostate cancer-specific mortality. Subset analysis of cases that had undergone radical prostatectomy revealed an association between rs351855 and prostate cancer risk. Although our results confirm an association between FGFR4 and prostate cancer risk in radical prostatectomy cases, they suggest that the role of FGFR4 in disease risk and outcomes at a population-based level appears to be minor.

High sensitivity, specificity and predictive value of the Connective Tissue Disease Screening Questionnaire among urban African-American women.

The Connective Tissue Disease Screening Questionnaire (CSQ), developed to screen populations for SLE and other CTDs, has been validated in a predominantly Caucasian population with hospital-based controls. We aimed to test the performance characteristics of the CSQ in an urban, predominantly African-American population. The CSQ was administered by interview to women recruited for a study of environmental risk factors and SLE, including 99 cases with SLE validated by medical record review and 202 healthy controls recruited from the community. Overall, 88% of subjects had African heritage, 6% were Hispanic and 4% were non-Hispanic Caucasian. Controls were more likely to report African heritage than cases (91% versus 82%, P = 0.001). Sensitivity for detecting SLE was 88% and specificity was 91%. In this study, where the prevalence of SLE was 33%, predictive value of a positive CSQ was 82% and predictive value of a negative CSQ was 94%. The CSQ has slightly lower sensitivity but greater specificity for SLE in an urban, predominantly African-American population with community-based controls compared with a Caucasian population with hospital-based controls. These results suggest that the CSQ has adequate sensitivity and specificity and could be used in population studies to screen African-American women for SLE.

Preimplantation genetic diagnosis of chromosome balance in embryos from a patient with a balanced reciprocal translocation.

Duplications or deletions are present in a high percentage of the gametes produced by individuals carrying balanced translocations. Preimplantation genetic diagnosis was used to examine chromosome balance in embryos from a patient having a reciprocal translocation within the short arms of chromosomes 5 and 8 (46,XX,t(5;8)(p13;p23)). This woman has two sisters with the translocation unbalanced, resulting in a partial trisomy for chromosome 5 and partial monosomy for chromosome 8 (46,XX,-8, +der(8)t(5;8)(p13;p23)) with associated mental retardation and physical abnormalities. The patient and her husband desired to have children without the abnormal chromosome balance and wished to reduce the likelihood of spontaneous abortion or need for therapeutic abortion. Fluorescence in-situ hybridization (FISH) probes for the alpha-satellite region of chromosome 8 and for a region on the short arm of chromosome 5 (5p15.2) were tested initially on lymphocytes from the patient and her sisters. The hybridization signal for chromosome 5 was detected in the expected two copies for the patient and three copies for the sisters in 87% of the cells. Two hybridization signals for chromosome 8 were detected in 96% of the cells from all individuals. Additional probe testing was done using blastomeres from polyspermic embryos. The couple then proceeded with a stimulated in-vitro fertilization (IVF) cycle and biopsies were done on 13 embryos at the 7-10-cell stage using a method of zona drilling and fluid displacement. Diagnosis was possible on at least one blastomere for nine embryos. Three embryos had nuclei with three hybridization signals for chromosome 5, three had fewer than two signals for one or both chromosomes, one was mosaic, and two had two signals for each chromosome. The latter were transferred to the patient, but pregnancy was not achieved. The results demonstrate that preimplantation genetic diagnosis for patients with reciprocal translocations can be used to identify embryos having normal chromosome balance. The potential advantages and limitations of this approach are discussed.

Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir.

The reservoir of human immunodeficiency virus (HIV) in semen is unknown. Several lines of evidence suggest that semen HIV may not arise from the same reservoir of infection as peripheral blood. If true, the viral burden in the two compartments could be qualitatively and quantitatively different, a scenario of potentially profound significance for the design of effective strategies of treatment, disease monitoring, and infection containment. We report here that the ratio of infected to uninfected leukocytes in ejaculated semen specimens is highly discordant with paired blood samples, demonstrating that they derive from distinct populations of infected cells. In addition, infectious HIV was isolated from semen cells, but not from blood cells, of an individual on triple antiretroviral therapy; the absence of major resistance-conferring mutations in the semen virus indicates that it was replicating in isolation from the antiviral agents. The compartmentalization of blood and semen infection was further supported by genetic analysis of several infectious HIV clones isolated from semen cells and peripheral blood cells of another donor not on antiretroviral therapy. Protease gene sequence analyses revealed significant divergence of the two viral populations. These findings confirm the distinct compartmentalization of HIV in the semen of this study cohort, and support the concept that semen HIV arises from an isolated reservoir of infection that may function independently in the pathobiology of HIV disease.

Biosynthesis of 'essential' amino acids by scleractinian corals.

Animals rely on their diet for amino acids that they are incapable either of synthesizing or of synthesizing in sufficient quantities to meet metabolic needs. These are the so-called 'essential amino acids'. This set of amino acids is similar among the vertebrates and many of the invertebrates. Previously, no information was available for amino acid synthesis by the most primitive invertebrates, the Cnidaria. The purpose of this study was to examine amino acid synthesis by representative cnidarians within the Order Scleractinia. Three species of zooxanthellate reef coral, Montastraea faveolata, Acropora cervicornis and Porites divaricata, and two species of non-zooxanthellate coral, Tubastrea coccinea and Astrangia poculata, were incubated with 14C-labelled glucose or with the 14C-labelled amino acids glutamic acid, lysine or valine. Radiolabel tracer was followed into protein amino acids. A total of 17 amino acids, including hydroxyproline, were distinguishable by the techniques used. Of these, only threonine was not found radiolabelled in any of the samples. We could not detect tryptophan or cysteine, nor distinguish between the amino acid pairs glutamic acid and glutamine, or aspartic acid and asparagine. Eight amino acids normally considered essential for animals were made by the five corals tested, although some of them were made only in small quantities. These eight amino acids are valine, isoleucine, leucine, tyrosine, phenylalanine histidine, methionine and lysine. The ability of cnidarians to synthesize these amino acids could be yet another indicator of a separate evolutionary history of the cnidarians from the rest of the Metazoa.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

PCR amplification of HIV and cellular DNA sequences in formaldehyde-fixed, immunoreactive white blood cells.

Full utilization of the diagnostic power of PCR amplification of specific DNA sequences has been hampered by the logistical difficulties in handling potentially infectious human DNAs and by the polymerization of nonspecific, competing DNA products in PCR assays of low-frequency DNA sequences. To overcome these difficulties, we have devised a sensitive, specific PCR strategy that readily detects 5 copies of HIV provirus in a background of 10(5) disinfected white blood cells collected from formaldehyde-fixed peripheral blood.

Codon usage in bony fishes.

Bony fishes are excellent experimental models that have been used extensively in biochemical and molecular genetic studies. As proteins are isolated and characterized from these organisms, information on codon usage by bony fishes can be used for subsequent recombinant DNA studies. Codon usage and nucleotide bias within codons from three species of bony fishes and two composites of 14 and 15 bony-fish species were analyzed. Although differences in codon usage increased from seven amino acids between fish species to eight amino acids between fish genera, the small number of differences (3 amino acids) between a single species and a fish composite minus that species suggests that codon usage tables constructed from large numbers of fish species are representative of bony fish in general. Furthermore, we found few differences in codon usage between two vertebrate phyla (fish and rat). Codons in fish DNA sequences end predominantly in G or C, even though the coding sequences are not enriched in these nucleotides. This positional base bias can be used to locate putative protein coding regions in fish DNA sequences.

A just alternative. Church tribunals may be the best forum for settling wrongful discharge disputes.

The threat of a wrongful discharge lawsuit should be a concern for every employer. However, Church employers can minimize their risk if they follow Church teaching in their employment practices and policies and if they use the Church tribunal system to settle employer-employee disputes. As it originally developed in common law, the "employment-at-will" concept stipulated that an employer could discharge an employee at any time, for any reason, or for no reason at all. Most states have adopted this common law concept, although courts and legislatures have created a number of exceptions to it. Despite these exceptions, employment-at-will has proven to be a powerful tool in the hands of employers. However, employees of Church institutions who feel they have been discharged wrongfully can turn to Church tribunals, which are governed by canon law. A Church tribunal can do virtually anything a civil court can do, with the exception of ordering someone to jail. Moreover, because the standards of justice and equity applied by Church tribunals are stricter than those applied by American courts, employees of Catholic institutions in the United States may make increasing use of them in the future. Disputes that come before a Church tribunal will be settled by either arbitration or mediation. Arbitration is a kind of informal litigation. Mediation, however, is preferable because it forces the parties involved to examine themselves, their motives, and their effects on each other and the Church as a whole.

Adverse effects of gossypol analogs on TM4 cell mitochondrial function.

Three aqueous-soluble gossypol Schiff's bases, SP562: bis-8,8'-[(N-(2-(dimethylamino)ethyl]-iminomethylene]- 1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2'-binaphthalene dihydrochloride; SP563: bis-8,8'-[(N-(2-(diethylamino)ethyl]-iminomethylene]-1,1',6,6',7,7'- hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2'-binaphthalene dihydrochloride; and SP564: bis-8,8'-[(N-(2-(diethylamino)propyl]-iminomethylene]-1,1',6,6',7,7'- [hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-2,2'-binaphthalene dihydrochloride, were examined for their effects on TM4 cell mitochondrial function and proliferation. Among the three gossypol analogs, SP562 had the most severe effect on decreasing TM4 cell population that accumulated rhodamine 123 into their mitochondria. This adverse effect exerted by SP562, however, was not as strong as that caused by (+/-) gossypol acetic acid of the same molarity. On the other hand, SP564 had the most severe effect on proliferation of TM4 cells. This severe effect was even more so than that caused by an equimolarity of (+/-) gossypol acetic acid.

Incorporation of gossypol and formation of its protein conjugates in mouse transformed Sertoli (TM4) cells.

Mitochondria of both mouse transformed Sertoli (TM4) cells and primary-cultured rat and mouse Sertoli cells were shown to be preferentially affected by gossypol (Tanphaichitr et al, 1984). To investigate whether this selective effect was due to a greater of TM4 cells to accumulate gossypol, TM4 and other somatic cell lines, including dog (MDCK) and kangaroo (PtK2) kidney epithelial cells, rat embryo fibroblasts (Rat-1) and mouse BALB/c 3T3 fibroblasts, were incubated with [14C]gossypol and the incorporated specific activity of the drug was assessed. The results indicate that TM4 cells accumulated [14C]gossypol at the highest level. Incorporated [14C]gossypol appeared to bind to TM4 cell macromolecules and remained in the dialysis tubing after extensive dialysis. Characterization of these gossypol-conjugated proteins by SDS-polyacrylamide gel electro-proteins had apparent Mr's of 92,500, 70,000, 63,200, 60,000, 58,100, 54,000, 52,000, 50,000, 47,500, 40,000 37,000, 35,000, 30,000, 20,000, and 14,500 daltons. Conjugation of these proteins with gossypol may result in macromolecular dysfunction and abnormal structure.

Differential effects of (+) and (-) gossypol enantiomers on mitochondrial function and proliferation of cultured TM4 cells.

The in vitro effects of (+) and (-)gossypol enantiomers on the mitochondrial functions of mouse transformed Sertoli, TM4 cells were investigated by monitoring mitochondrial rhodamine 123 accumulation. When TM4 cells were cultured in medium without fetal calf serum, 5 micrograms/ml of both enantiomers caused similar declines in mitochondrial rhodamine 123 staining. By contrast, (-)gossypol had a greater adverse action than did (+)gossypol on the mitochondrial of TM4 cells that were cultured in medium supplemented with 2% fetal calf serum. Construction of dose response curves for the effects of the two enantiomers on rhodamine 123 accumulation by TM4 cells after 5 hr of drug treatment gave an EC50 of 7.5 micrograms/ml for the (-)gossypol isomer compared with 18 micrograms/ml for the (+)gossypol isomer. Similarly, TM4 cell proliferation was also more disturbed by (-)gossypol in medium supplemented with other concentrations of fetal calf serum. The results suggest that the lower effect of (+)gossypol on TM4 cells may be attributed to its higher affinity to serum components, which may impede its entrance into TM4 cells.

Egg-penetration ability and structural properties of human sperm prepared by Percoll-gradient centrifugation.

Human sperm with a high zona-free hamster egg-penetration ability were obtained by centrifuging freshly ejaculated sperm through a discontinuous two-step (47% and 90%) Percoll gradient at 600g at room temperature for 30 min. Highly motile sperm with good penetration ability were recovered in the pellet fraction (Per-sperm), whereas those with low penetration ability were in the gradient interface. The increased penetration ability of Per-sperm, as compared to sperm capacitated by other methods such as a single-tube swim-up or multiple-tube swim-up preparation, was not due to an increased proportion of acrosome reacted sperm. Rather, transmission electron microscopy indicated that Per-sperm were devoid of coating envelopes, which were present around both the head and tail regions of noncapacitated and single-tube swim-up sperm. Changes to the surface of Per-sperm were demonstrated by their decreased interaction with UEA I lectin, which binds specifically to fucose residues. Removal of the coating envelopes as well as other changes on the sperm surface may lead to an enhanced binding of Per-sperm to the oocyte. In addition, 99% of Per-sperm contained chromatin that was fully condensed. By contrast, about 15% of swim-up sperm still possessed incompletely condensed chromatin. With a higher penetration ability, "clean" appearance, and homogeneity of condensed chromatin, Per-sperm are recommended for insemination and studies of human sperm capacitation.