Identifying rhesus macaque gene orthologs using heterospecific human CNV probes

AbstractWe used the Affymetrix® Genome-Wide Human SNP Array 6.0 to identify heterospecific markers and compare copy number and structural genomic variation between humans and rhesus macaques. Over 200,000 human copy number variation (CNV) probes were mapped to a Chinese and an Indian rhesus macaque sample. Observed genomic rearrangements and synteny were in agreement with the results of a previously published genomic comparison between humans and rhesus macaques. Comparisons between each of the two rhesus macaques and humans yielded 206 regions with copy numbers that differed by at least two fold in the Indian rhesus macaque and human, 32 in the Chinese rhesus macaque and human, and 147 in both rhesus macaques. The detailed genomic map and preliminary CNV data are useful for better understanding genetic variation in rhesus macaques, identifying derived changes in human CNVs that may have evolved by selection, and determining the suitability of rhesus macaques as human models for particular biomedical studies

Tales from the EMR: Does a 21st-Century Data Warehouse Facilitate Clinical Research for Pancreatic Cancer?

Background: The importance of an electronic medical record has been highlighted for both clinical care and research. In the current era, data warehouses and repositories have been established to serve the dual function of patient care and investigation. Purpose: The aim of this study was to compare a newly developed institutional clinical data warehouse, linked with the hospital information system (HIS), to a prospectively-maintained departmental database. Methods: A novel HIS-linked institutional clinical data warehouse was queried for 9 primary and secondary ICD-9-CM discharge diagnosis codes for pancreatic cancer. The database captured inpatient and outpatient clinical and billing information from a pool of over 2 million patients evaluated at an academic medical institution and its affiliates since 1995. A cohort was identified; following Institutional Review Board approval, demographic and clinical data was obtained. This data was compared to a manually-entered and prospectively-maintained surgical oncology database of the same institution, tracking 394 patients since 1999. Duplicated patients, and those unique to either dataset, were flagged. Patients with diagnosis dates prior to 1999 were excluded to allow comparison over the same time period. For validation purposes, a 10% random sample of remaining patients unique to each dataset underwent manual review of medical records including clinic notes, admission/discharge notes, diagnostic imaging, and pathology reports. Results: 1107 patients were identified from the HIS-linked dataset with pancreatic neoplasm-associated diagnosis codes dating from 1999 to 2009. Of these, 254 (22.9%) were captured in both datasets, while 853 (77.1%) were only in the HIS-linked dataset. Manual review of the 10% subset of the HIS-only group demonstrated that 55.6% of patients were without identifiable pancreatic pathology, suggesting miscoding, while 31.7% had diagnoses consistent with pancreatic neoplasm, and 12.7% had pseudocyst or pancreatitis. Of the 394 patients tracked by surgical oncology, 254 (64.5%) were captured in both datasets, while 140 (35.5%) had not been captured in the HIS-linked dataset. Manual review of the 10% subset of the non-captured patients demonstrated 93.3% with pancreatic neoplasm and 6.7% with pseudocyst or pancreatitis. Lastly, a review of the 10% subset of the 254 patient overlap demonstrated that 87.5% of patients were with pancreatic neoplasm, 8.3% with pseudocyst or pancreatitis, and 4.2% without pancreatic pathology. Conclusions: While technological advances provide a powerful means to automate institutional-level cohort identification and data collection, a high degree of misclassification may be present if queries are based solely on ICD-9-CM discharge codes. For that reason, careful validation and data cleaning are critical steps prior to research use. These results also suggest cautious interpretation of national-level administrative data utilizing ICD-9-CM diagnosis codes. Our findings suggest that the current state-of-the-art data warehouses continue to require clinical correlation and validation through traditional retrospective mechanisms

Is pancreatic cancer palliatable? A national study

Background: Pancreatic cancer is frequently diagnosed at advanced stages where potentially curative resection is no longer possible. Palliative procedures can be performed; however, results on a national level are unknown. This study examines pancreatic cancer patients who underwent potentially palliative procedures including gastric bypass, biliary bypass surgery, celiac block, biliary stent, gastrostomy or jejunostomy, and examines post-intervention complications and 30-day mortality. Methods: SEER-Medicare 1991-2005 was used to identify patients with Stage 3-4 pancreatic cancer. Complication rates were calculated including post-op infection, myocardial infarction, aspiration pneumonia, DVT/PE, pulmonary compromise, gastric bleed, acute renal failure, and reoperation. Kaplan-Meier survival analysis was performed. Finally, Cox proportional hazards modeling was used to control for the effects of age, sex, race, stage, and resection. Results: Of 22,314 pancreatic cancer patients, 858 (3.9%) patients were Stage 3, and 11,149 (50.0%) stage 4. Post-procedure median survival for all patients is approximately two months, with longest survival for biliary bypass patients (3.2mo, 95% CI(2.9-3.7), and lowest survival for jejunostomy 1.3 mo (1.2-1.5) and gastrostomy 1.5 mo (1.4-1.8). Post-procedure 30-day mortality was highest for gastrostomy patients at 41.5%; followed by jejunostomy (39.1%), celiac plexus block (30.0%), gastric bypass (23.8%), biliary bypass (17.8%), and biliary stent (21.2%). The rate of complications averaged 40%, with highest rate for gastrostomy (47.4%) and gastric bypass (45.3%) and lowest for celiac plexus block (29.3%). Stage 4 disease was an independent predictor of death for patients undergoing five out of six procedures. Conclusion: We found that morbidity and mortality of palliative procedures in unresectable pancreatic cancer is high, especially in stage 4 patients. Further studies need to be conducted to identify patients who will have sufficient expected post-procedure survival to benefit from these palliative interventions

Genetic diversity and population structure of long-tailed macaque (Macaca fascicularis) populations in Peninsular Malaysia

Background: The genetic diversity and structure of long-tailed macaques (Macaca fascicularis) in Peninsular Malaysia, a widely used non-human primate species in biomedical research, have not been thoroughly characterized. Methods: Thirteen sites of wild populations of long-tailed macaques representing six states were sampled and analyzed with 18 STR markers. Results: The Sunggala and Penang Island populations showed the highest genetic diversity estimates, while the Jerejak Island population was the most genetically discrete due to isolation from the mainland shelf. Concordant with pairwise Fst estimates, STRUCTURE analyses of the seven PCA-correlated clusters revealed low to moderate differentiation among the sampling sites. No association between geographic and genetic distances exists, suggesting that the study sites, including island study sites, are genetically if not geographically contiguous. Conclusions: The status of the genetic structure and composition of long-tailed macaque populations require further scrutiny to develop this species as an important animal model in biomedical research

Ancestry, Plasmodium cynomolgi prevalence and rhesus macaque admixture in cynomolgus macaques (Macaca fascicularis) bred for export in Chinese breeding farms

Background: Most cynomolgus macaques (Macaca fascicularis) used in the United States as animal models are imported from Chinese breeding farms without documented ancestry. Cynomolgus macaques with varying rhesus macaque ancestry proportions may exhibit differences, such as susceptibility to malaria, that affect their suitability as a research model. Methods: DNA of 400 cynomolgus macaques from 10 Chinese breeding farms was genotyped to characterize their regional origin and rhesus ancestry proportion. A nested PCR assay was used to detect Plasmodium cynomolgi infection in sampled individuals. Results: All populations exhibited high levels of genetic heterogeneity and low levels of inbreeding and genetic subdivision. Almost all individuals exhibited an Indochinese origin and a rhesus ancestry proportion of 5%-48%. The incidence of P. cynomolgi infection in cynomolgus macaques is strongly associated with proportion of rhesus ancestry. Conclusions: The varying amount of rhesus ancestry in cynomolgus macaques underscores the importance of monitoring their genetic similarity in malaria research

EssC:domain structures inform on the elusive translocation channels in the Type VII secretion system.

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein

Arsenic resistance in the archaeon "Ferroplasma acidarmanus" : new insights into the structure and evolution of the ars genes

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus " was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42444/1/s00792-002-0303-6.pd

hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

<p>Abstract</p> <p>Background</p> <p>Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.</p> <p>Methods</p> <p>Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.</p> <p>Results</p> <p>We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.</p> <p>By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.</p> <p>Conclusions</p> <p>Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.</p

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists