Nuclear physics and astronomical observations of compact objects / Física nuclear y observaciones astronómicas de objetos compactos

Abstract We discuss our predictions of two astrophysics observations: neutrino emission and element abundances. We studied the emission and possible detection of neutrinos from past black hole accretion disks. We find neutrinos are copiously emitted from these sites and encourage the development of large facilities for detection. We also studied changes in the synthesis of neutron-rich elements due to the suppression of key nuclear processes. We find important changes in the element abundances due to the, previously overlooked, alpha decay.

Resumen Discutimos nuestras predicciones de dos observaciones astrofísicas: la emisión de neutrinos y las abundancias de elementos. Hemos estudiado la emisión y posible detección de neutrinos emitidos por discos de acreción alrededor de agujeros negros en el pasado. Encontramos que los neutrinos son emitidos en abundancia por discos de acreción y sugerimos el desarrollo de detectores de gran escala para mejorar su detección. También hemos estudiado los cambios en la síntesis de elementos ricos en neutrones, debido a la supresión de procesos nucleares claves. Encontramos que hay cambios importantes en la abundancia de elementos debido al decaimiento alfa.

Activation of nuclear factor-kappa B signalling promotes cellular senescence.

Cellular senescence is a programme of irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress. The underlying signalling pathways, of major clinicopathological relevance, are unknown. We combined genome-wide expression profiling with genetic complementation to identify genes that are differentially expressed when conditionally immortalised human fibroblasts undergo senescence upon activation of the p16-pRB and p53-p21 tumour suppressor pathways. This identified 816 up and 961 downregulated genes whose expression was reversed when senescence was bypassed. Overlay of this data set with the meta-signatures of genes upregulated in cancer showed that nearly 50% of them were downregulated upon senescence showing that even though overcoming senescence may only be one of the events required for malignant transformation, nearly half of the genes upregulated in cancer are related to it. Moreover 65 of the up and 26 of the downregulated genes are known downstream targets of nuclear factor (NF)-κB suggesting that senescence was associated with activation of the NF-κB pathway. Direct perturbation of this pathway bypasses growth arrest indicating that activation of NF-κB signalling has a causal role in promoting senescence.

Thermal conductivity and phase separation of the crust of accreting neutron stars.

Recently, crust cooling times have been measured for neutron stars after extended outbursts. These observations are very sensitive to the thermal conductivity kappa of the crust and strongly suggest that kappa is large. We perform molecular dynamics simulations of the structure of the crust of an accreting neutron star using a complex composition that includes many impurities. The composition comes from simulations of rapid proton capture nucleosynthesis followed by electron captures. We find that the thermal conductivity is reduced by impurity scattering. In addition, we find phase separation. Some impurities with low atomic number Z are concentrated in a subregion of the simulation volume. For our composition, the solid crust must separate into regions of different compositions. This could lead to an asymmetric star with a quadrupole deformation. Observations of crust cooling can constrain impurity concentrations.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Association of CYP3A4 genotype with detection of Vy/JB trans-rearrangements in the peripheral blood leukocytes of pediatric cancer patients undergoing chemotherapy for all

Cancer patients receiving chemotherapy are exposed to high doses of cytotoxic and genotoxic drugs which, in some cases, can lead to treatment related leukemia. Since this only occurs in a minority of patients, however, it is possible some individuals are predisposed due to genetic polymorphisms in genes for enzymes that mediate drug metabolism. To address this possibility we measured the genotoxicity of chemotherapeutic agents in patients receiving treatment for ALL by the frequency of the Vã/Jâ trans-rearrangement in their peripheral blood leukocytes and compared this with CYP3A4 genotype. CYP3A4 is the most abundant of the cytochrome P450 (CYP) enzyme in the liver and intestine which contains a common −392A>G substitution in the promoter region (CYP3A4*1B allele). We found a significant increase in the frequency of rearrangements during chemotherapy only in patients homozygous for the wild type CYP3A4*1A allele. This provides a direct link between CYP3A4 genotype and susceptibility to drug genotoxicity thus strengthening the possibility that predisposition to treatment related leukemia may be measurable by simple genetic testing.

Endothelin B receptor gene hypermethylation in prostate adenocarcinoma.

BACKGROUND: Alterations in the methylation patterns of promoter CpG islands have been associated with the transcriptional inhibition of genes in many human cancers. These epigenetic alterations could be used as molecular markers for the early detection of cancer-that is, while potentially curable according to current therapeutic strategies. In prostate cancer, GSTP1 hypermethylation is the most common epigenetic alteration, and can be detected in up to 90% of cases. Thus, screening for methylation of other loci would probably increase the number of primary tumours amenable to screening. Moreover, previous studies have shown that the endothelin B receptor (EDNRB) gene is abnormally methylated in a high proportion of prostate tumours ( approximately 70%). AIMS: To investigate the potential use of EDNRB gene hypermethylation as a prostate cancer specific marker. METHODS: Methylation specific polymerase chain reaction (MSP) for the promoter region of EDNRB was performed on prospectively collected tissue samples from 48 patients harbouring clinically localised prostate cancer, and in a group of 23 patients with benign prostatic hyperplasia (BPH). Genomic DNA was isolated from the samples and the methylation status was examined in a blinded manner. RESULTS: EDNRB methylation was found in 40 of 48 of the adenocarcinomas. However, the same alteration was found in the paired normal tissue, and 21 of 23 of the BPH samples were found to harbour EDNRB hypermethylation. CONCLUSIONS: EDNRB hypermethylation at CpG sites upstream of the transcription start site can be detected in a high proportion of prostate adenocarcinomas. However, because this same alteration is also present in normal and hyperplastic tissue, it does not distinguish normal from neoplastic prostate cells, thus precluding its use as a prostate cancer marker.

Alternative spliced transcripts as cancer markers.

Eukaryotic mRNAs are transcribed as precursors containing their intronic sequences. These are subsequently excised and the exons are spliced together to form mature mRNAs. This process can lead to transcript diversification through the phenomenon of alternative splicing. Alternative splicing can take the form of one or more skipped exons, variable position of intron splicing or intron retention. The effect of alternative splicing in expanding protein repertoire might partially underlie the apparent discrepancy between gene number and the complexity of higher eukaryotes. It is likely that more than 50% form. Many cancer-associated genes, such as CD44 and WT1 are alternatively spliced. Variation of the splicing process occurs during tumor progression and may play a major role in tumorigenesis. Furthermore, alternatively spliced transcripts may be extremely useful as cancer markers, since it appears likely that there may be striking contrasts in usage of alternatively spliced transcript variants between normal and tumor tissue than in alterations in the general levels of gene expression.

Significant overexpression of oligophrenin-1 in colorectal tumors detected by cDNA microarray analysis.

The human oligophrenin-1 gene is ubiquitously expressed at low levels and expressed at high levels in the developing neuroepithelium of the neural tube. Mutations in this gene have been related to the X-linked mental retardation. Using cDNA microarrays, we found evidence that oligophrenin-1 is strongly up-regulated in colorectal tumors. Semiquantitative reverse transcriptase polymerase chain reaction confirmed this finding. Thus, a well-known nervous system-associated human gene transcript may also be an important colorectal tumor marker and potential therapeutic target.

Loss of chromosome arms 3p and 9p and inactivation of P16 (INK4a) in normal epithelium of patients with primary lung cancer.

The accumulation of genetic alterations in the respiratory epithelium may give rise to cancer and often is accompanied by a series of histologic alterations over a period of several years. Recent studies have identified some molecular alterations in histologically normal-appearing epithelium among patients with lung cancer. To extend these observations, we investigated clonal genetic alterations by using fluorescence in situ hybridization (FISH) analysis and immunohistochemistry in 69 biopsy samples of histologically normal-appearing bronchial epithelium from 22 patients with or without lung cancer. Thirty-seven biopsy specimens from 13 patients were examined for loss of 3p14, and 48 biopsy specimens from 18 patients were examined for loss at 9p21 by FISH. P16(INK4a) expression was analyzed in 54 biopsy samples from 19 patients. In at least one biopsy specimen from five of the 13 patients with primary lung cancer, FISH or immunohistochemistry detected loss of the 3p14 or 9p21 region. In contrast, no alterations were detected for the same regions in the nine patients without primary lung cancer. Our results support the concept that the normal epithelial surface of large bronchi of patients with lung cancer has molecular changes suggestive of the outgrowth of numerous clonal foci.

Mitochondrial mutations in early stage prostate cancer and bodily fluids.

We recently demonstrated the existence of specific patterns of somatic mitochondrial DNA (mtDNA) mutations in several cancers. Here we sought to identify the presence of mtDNA mutations in prostate cancer and their paired PIN lesions. The D-loop region, 16S rRNA, and the NADH subunits of complex I were sequenced to identify mtDNA mutations in 16 matched PIN lesions and primary prostate cancers. Twenty mtDNA mutations were detected in the tumor tissue of three patients. Identical mutations were also identified in the PIN lesion from one patient. This patient with multiple point mutations also harbored a high frequency of microsatellite instability (MSI-H) in nuclear mononucleotide repeat markers. Remarkably, identical mutations were also detected in all (3/3) matched urine and plasma samples obtained from these patients. Although mitochondrial mutations are less common in prostate adenocarcinoma, they occur early in cancer progression and they can be detected in bodily fluids of early stage disease patients. The identification of MtDNA mutations may complement other early detection approaches for prostate cancer.

Methylation status in the promoter region of the human PGP9.5 gene in cancer and normal tissues.

PGP 9.5 is a neurospecific peptide that functions to remove ubiquitin from ubiquitinated cellular proteins, thereby preventing them from targeted degradation by the proteasome-dependent pathway or regulating their localization, activity or structure. Using the serial analysis of gene expression method (SAGE), we initially found that the PGP9.5 transcript and protein was highly expressed in more than 50% of primary lung cancers and nearly all lung cancer cell lines but was not detectable in the normal lung. This increased expression could be the result of transcriptional regulation accompanied by methylation changes at the CpG island of the promoter region. We studied the methylation status of the cytosines at the promoter region of human PGP9.5 using sodium bisulfite genomic sequencing in normal and neoplastic cells. Although no methylation of PGP9.5 promoter was observed in the normal lung, normal cervical tissue, and lung cancer cell lines, this region was densely methylated in the HeLa cell line. Exposure to HeLa cells to the demethylating agent, 5-aza-2'-deoxycytidine, led to re-expression of PGP9.5. This data suggested that while other mechanisms may be involved in the frequent overexpression of PGP9.5 gene in lung tumors and lung cancer cell lines, promoter methylation may play a role in the transcriptional suppression of PGP9.5 gene expression in the cervical tissue-derived HeLa cell line.

Detection of c-myc amplification in uveal melanoma by fluorescent in situ hybridization.

PURPOSE: Genetic abnormalities of chromosomal arm 8q have been reported by many studies in uveal melanoma. To better understand the role of 8q abnormalities in uveal melanoma development, copy number anomalies of the c-myc oncogene (located on 8q24.1) have been investigated. METHODS: Forty-three uveal melanomas were analyzed by fluorescent in situ hybridization (FISH) with probes for c-myc and the chromosome 8 centromere. Results of the FISH analysis were compared with genetic changes previously detected by microsatellite analysis on chromosomes 3 and 6p. RESULTS: Thirty uveal melanomas (70%) had extra copies of c-myc, 2 tumors (5%) had loss of c-myc, and 11 tumors (25%) had no abnormalities in c-myc copy number. Of those with extra copies of c-myc, 13 tumors (43%) had amplification of the c-myc gene, 14 tumors (47%) had an intermediate relative increase in the c-myc copy number, and 3 tumors (10%) had a simple gain of chromosome 8. An association between larger tumor size and c-myc amplification was found (P < 0.01). Although extra copies of c-myc were seen in tumors with retention of chromosome 3, remarkably only tumors with monosomy 3 showed amplification of c-myc (P = 0.03). CONCLUSIONS: The specific amplification of the c-myc oncogene detected in at least 30% of primary uveal melanomas cannot be explained by the simple 8q abnormalities observed in cytogenetic studies. The striking association between c-myc amplification and monosomy 3 suggests a unique pathway of genetic progression in a subset of uveal melanomas.

Microsatellite instability as a tool for the classification of gastric cancer.

Microsatellite instability (MSI) is a common feature of gastric cancers that reflects underlying mismatch-repair deficiency in the tumor, caused most frequently by methylation of the hMLH1 promoter. Tumors with MSI have been found to inactivate certain target genes by permitting an increased frequency of mutations in mononucleotide runs in their coding regions. Gastric tumors with MSI have a distinct clinicopathological profile with a relatively good prognosis. Using the simple and robust methodologies available, MSI detection in gastrointestinal tumors promises to be one of the first widely used molecular prognostic tests for human cancer. Here, we review the molecular context of this exciting prospect with respect to one of the world's most prevalent cancers, that of the stomach.

A putative oncogenic role for MPP11 in head and neck squamous cell cancer.

Genetic alterations of chromosome 7 are common in human cancer. Furthermore, previous studies have supported the presence of a gene important in a broad range of cancers at 7q22-31.1. There is evidence that supports an oncogenic function for this putative gene, as well as evidence that supports a tumor suppressive role. In this study, we used a cross-species candidate gene approach in combination with physical mapping to identify MPP11 as a candidate for the putative cancer-related activity at 7q22-31.1. We then analyzed primary head and neck squamous cell tumors (HNSCCs) for loss of heterozygosity/allelic imbalance (LOH/AI) at the MPP11 genomic locus. Thirty-eight percent of tumors examined displayed LOH/AI involving the MPP11 genomic locus. Mutation analysis of MPP11 in the latter samples did not identify any inactivating mutations. However, immunohistochemical staining of primary tumor sections and Western blot analysis of HNSCC cell lines revealed a tumor-specific high level of expression of MPP11p. Fluorescence in situ hybridization analysis done on the cell lines identified increased chromosome 7 copy number with a concomitant increase in MPP11 copy number. These results suggest an oncogenic role for MPP11 in HNSCC.

AIS is an oncogene amplified in squamous cell carcinoma.

We and others recently isolated a human p53 homologue (p40/p51/p63/p73L) and localized the gene to the distal long arm of chromosome 3. Here we sought to examine the role of p40/p73L, two variants lacking the N-terminal transactivation domain, in cancer. Fluorescent in situ hybridization (FISH) analysis revealed frequent amplification of this gene locus in primary squamous cell carcinoma of the lung and head and neck cancer cell lines. (We named this locus AIS for amplified in squamous cell carcinoma.) Furthermore, amplification of the AIS locus was accompanied by RNA and protein overexpression of a variant p68(AIS) lacking the terminal transactivation domain. Protein overexpression in primary lung tumors was limited to squamous cell carcinoma and tumors known to harbor a high frequency of p53 mutations. Overexpression of p40(AIS) in Rat 1a cells led to an increase in soft agar growth and tumor size in mice. Our results support the idea that AIS plays an oncogenic role in human cancer.

Frequent gain of the p40/p51/p63 gene locus in primary head and neck squamous cell carcinoma.

We have identified a new human p53 homologue, p40 (p51/p63). This gene was mapped to the distal arm of 3q and was found to be essential for normal epithelial development. We used microsatellite and FISH analyses to search for genetic alterations of p40 in primary HNSCC. A more precise localization of p40 was completed using 6 known markers on 3q and a newly isolated microsatellite marker within the p40 gene. We also determined the genomic organization of the p40 gene using human YAC and BAC clones. Microsatellite analysis revealed that 14 of 26 (54%) primary HNSCC had allelic imbalance in at least 1 of the 7 microsatellite loci. However, FISH analysis with a p40 probe showed that a majority of HNSCC had an increased copy number of the locus regardless of allelic status. Thus, overrepresentation of the p40 locus may play an important role in the development of HNSCC.

Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.

Examination of human bladder, head and neck, and lung primary tumors revealed a high frequency of mitochondrial DNA (mtDNA) mutations. The majority of these somatic mutations were homoplasmic in nature, indicating that the mutant mtDNA became dominant in tumor cells. The mutated mtDNA was readily detectable in paired bodily fluids from each type of cancer and was 19 to 220 times as abundant as mutated nuclear p53 DNA. By virtue of their clonal nature and high copy number, mitochondrial mutations may provide a powerful molecular marker for noninvasive detection of cancer.

[Monitoring AIDS patients for the development of cytomegalovirus (CMV) disease using multiplex PCR]. / Monitoramento de pacientes com AIDS para o desenvolvimento de doença por citomegalovirus (CMV) usando-se PCR multiplex.

The human cytomegalovirus is an important pathogen in patients infected with the human immunodeficiency virus (HIV). The CMV viral load seems to be predictor of the development of the CMV disease in these patients. We used a multiplex PCR protocol that also provides quantitative information in those samples from which a single band is amplified and contains fewer viral genomes than those from which both targets are amplified. Monthly blood samples were collected from 270 AIDS patients. From twenty patients, two CMV targets were amplified three or more consecutive times and these patients developed CMV related disease during the study. In contrast, patients who did not result positive for both viral targets, for three or more consecutive times, or who had alternating positive and negative samples during the follow up did not present CMV related disease. The results suggest that the PCR multiplex can be used for the identification of HIV positive patients with higher risk of development of CMV disease.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay.

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 10(6) leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 10(6) leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.