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author:("soubry, Ethan")
1.  Expression Microarray Analysis Reveals Alternative Splicing of LAMA3 and DST Genes in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2014;9(3):e91263.
Purpose
Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors.
Experimental Design
We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score—the Maximum-Minimum Exon Score (MMES) – designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort.
Results
After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001).
Conclusion
Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.
doi:10.1371/journal.pone.0091263
PMCID: PMC3967989  PMID: 24675808
2.  Mitochondrial DNA Mutations in Respiratory Complex-I in Never-Smoker Lung Cancer Patients Contribute to Lung Cancer Progression and associated with EGFR gene mutation 
Journal of cellular physiology  2012;227(6):2451-2460.
Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with EGFR and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients’ samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P=0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P=0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients’ population. MtDNA mutation was significantly (P=0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P=0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
doi:10.1002/jcp.22980
PMCID: PMC3256258  PMID: 21830212
Lung cancer; never-smokers; MtDNA mutation; Respiratory Complex-I; EGFR mutation
3.  Trends in incidence and susceptibility among methicillin-resistant Staphylococcus aureus isolated from intranasal cultures associated with rhinosinusitis 
Background:
Reports regarding the incidence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in rhinosinusitis (RS) are limited. This study was designed to identify epidemiology and trends of MRSA incidence and antimicrobial resistance in the sinonasal cavities.
Methods:
This is a retrospective case series. All intranasal/sinus cultures obtained by otolaryngologists at Stanford over a 20-year period (1990–2010) were retrospectively reviewed by mining the microbiology database. Nested searches were then made for all S. aureus and MRSA cultures. Patterns of incidence and changes in antibiotic susceptibilities were tabulated and statistical analysis was performed.
Results:
Our search retrieved 10,387 positive intranasal culture samples, with S. aureus found in 800 (7.7%), and MRSA comprising 110 (1.06%) of this subset. Between the years of 1990 and 1999, only 2/112 (1.7%) of S. aureus–positive nasal cultures were positive for MRSA, with a sharp rise in incidence to 86/606 (14.2%) from 2000 to 2005, and to 22/82, 26.8% from 2006 to 2010. On a percent basis, using logistic regression modeling, this represents a statistically significant increasing trend (p < 0.0001) for MRSA sinusitis. However, over the 20-year interval studied, the patterns of antibiotic resistance among MRSA remained unaltered, especially with regard to trimethoprim-sulfamethoxazole and vancomycin.
Conclusion:
S. aureus and MRSA isolates from intranasal cultures, which were essentially absent before the year 2000, became significantly more common earlier this decade. These data show the increased role of MRSA in sinusitis. MRSA antibiotic susceptibilities have remained, however, largely stable during this time period.
doi:10.2500/ajra.2013.27.3858
PMCID: PMC3901439  PMID: 23562203
Antibiotic resistance; antibiotic susceptibility; intranasal/sinus culture; methicillin-resistant S. aureus; MRSA; Staphylococcus aureus
4.  Clinical and public health research using methylated DNA Immunoprecipitation (MeDIP): A comparison of commercially available kits to examine differential DNA methylation across the genome 
Epigenetics  2012;7(1):106-112.
The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies.
doi:10.4161/epi.7.1.18647
PMCID: PMC3337832  PMID: 22207357
differential methylation; Methylated DNA Immunoprecipitation (MeDIP); translational research genome-wide promoter methylation
5.  MAGEB2 is Activated by Promoter Demethylation in Head and Neck Squamous Cell Carcinoma 
PLoS ONE  2012;7(9):e45534.
Purpose
Although promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented.
Experimental Design
In this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene.
Results
From the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines.
Conclusion
In conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2's exact role in HNSCC.
doi:10.1371/journal.pone.0045534
PMCID: PMC3454438  PMID: 23029077

Results 1-5 (5)