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1.  Rag GTPase-mediated regulation of mTORC1 by nutrients is necessary for neonatal autophagy and survival 
Nature  2012;493(7434):679-683.
The mTOR Complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors1. Cell-based studies showed that mTORC1 senses amino acids through the Rag family of GTPases 2,3, but their importance in mammalian physiology is unknown. Here, we generated knock-in mice that express a constitutively active form of RagA (RagAGTP) from its endogenous promoter. RagAGTP/GTP mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarian-section, fasted RagAGTP/GTP neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a drop in plasma amino acid levels. In contrast, mTORC1 inhibition does not occur in RagAGTP/GTP neonates, despite identical reductions in blood nutrient levels. With prolonged fasting, wild-type neonates recover their plasma glucose levels, but RagAGTP/GTP mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagAGTP/GTP neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagAGTP/GTP neonates, we hypothesized that the Rag pathway signals glucose as well as amino acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagAGTP/GTP fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino acid levels to mTORC1, and play an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.
PMCID: PMC4000705  PMID: 23263183
2.  Signaling Regulated Endocytosis and Exocytosis Lead to Mating Pheromone Concentration Dependent Morphologies in Yeast 
FEBS letters  2012;586(23):4208-4214.
Polarized cell morphogenesis requires actin cytoskeleton rearrangement for polarized transport of proteins, organelles and secretory vesicles, which fundamentally underlies cell differentiation and behavior. During yeast mating, S. cerevisiae responds to extracellular pheromone gradients by extending polarized projections, which are likely maintained through vesicle transport to (exocytosis) and from (endocytosis) the membrane. We experimentally demonstrate that the projection morphology is pheromone concentration-dependent, and propose the underlying mechanism through mathematical modeling. The inclusion of membrane flux and dynamically evolving cell boundary into our yeast mating signaling model shows good agreement with experimental measurements, and provides a plausible explanation for pheromone-induced cell morphology.
PMCID: PMC3921116  PMID: 23108052
3.  Contemporary Ventilator Management in Patients With and at Risk of ALI/ARDS 
Respiratory care  2013;58(4):10.4187/respcare.01755.
Ventilator practices in patients at risk for acute lung injury (ALI) and ARDS are unclear. We examined factors associated with choice of set tidal volumes (VT), and whether VT < 8 mL/kg predicted body weight (PBW) relates to the development of ALI/ARDS.
We performed a secondary analysis of a multicenter cohort of adult subjects at risk of lung injury with and without ALI/ARDS at onset of invasive ventilation. Descriptive statistics were used to describe ventilator practices in specific settings and ALI/ARDS risk groups. Logistic regression analysis was used to determine the factors associated with the use of VT < 8 mL/kg PBW and the relationship of VT to ALI/ARDS development and outcome.
Of 829 mechanically ventilated patients, 107 met the criteria for ALI/ARDS at time of intubation, and 161 developed ALI/ARDS after intubation (post-intubation ALI/ARDS). There was significant intercenter variability in initial ventilator settings, and in the incidence of ALI/ARDS and post-intubation ALI/ARDS. The median VT was 7.96 (IQR 7.14–8.94) mL/kg PBW in ALI/ARDS subjects, and 8.45 (IQR 7.50–9.55) mL/kg PBW in subjects without ALI/ARDS (P = .004). VT decreased from 8.40 (IQR 7.38–9.37) mL/kg PBW to 7.97 (IQR 6.90–9.23) mL/kg PBW (P < .001) in those developing post-intubation ALI/ARDS. Among subjects without ALI/ARDS, VT ≥ 8 mL/kg PBW was associated with shorter height and higher body mass index, while subjects with pneumonia were less likely to get ≥ 8 mL/kg PBW. Initial VT ≥ 8 mL/kg PBW was not associated with the post-intubation ALI/ARDS (adjusted odds ratio 1.30, 95% CI 0.74–2.29) or worse outcomes. Post-intubation ALI/ARDS subjects had mortality similar to subjects intubated with ALI/ARDS.
Clinicians seem to respond to ALI/ARDS with lower initial VT. Initial VT, however, was not associated with the development of post-intubation ALI/ARDS or other outcomes.
PMCID: PMC3840036  PMID: 22906363
acute lung injury; ARDS; mechanical ventilation; mortality; tidal volume
4.  The sea lamprey has a primordial accessory olfactory system 
A dual olfactory system, represented by two anatomically distinct but spatially proximate chemosensory epithelia that project to separate areas of the forebrain, is known in several classes of tetrapods. Lungfish are the earliest evolving vertebrates known to have this dual system, comprising a main olfactory and a vomeronasal system (VNO). Lampreys, a group of jawless vertebrates, have a single nasal capsule containing two anatomically distinct epithelia, the main (MOE) and the accessory olfactory epithelia (AOE). We speculated that lamprey AOE projects to specific telencephalic regions as a precursor to the tetrapod vomeronasal system.
To test this hypothesis, we characterized the neural circuits and molecular profiles of the accessory olfactory epithelium in the sea lamprey (Petromyzon marinus). Neural tract-tracing revealed direct and reciprocal connections with the dorsomedial telencephalic neuropil (DTN) which in turn projects directly to the dorsal pallium and the rostral hypothalamus. High-throughput sequencing demonstrated that the main and the accessory olfactory epithelia have virtually identical profiles of expressed genes. Real time quantitative PCR confirmed expression of representatives of all 3 chemoreceptor gene families identified in the sea lamprey genome.
Anatomical and molecular evidence shows that the sea lamprey has a primordial accessory olfactory system that may serve a chemosensory function.
PMCID: PMC3765145  PMID: 23957559
5.  Small Cell Carcinoma ex-Pleomorphic Adenoma of the Parotid Gland 
Head and Neck Pathology  2012;6(4):502-506.
Small cell carcinoma (SCC) is a high-grade malignancy usually encountered in the lungs but also seen in almost any site including the salivary glands. SCC is important to recognize because it often metastasizes widely and is treated with systemic chemotherapy. Carcinoma ex pleomorphic adenoma is a malignant epithelial neoplasm arising in a pre-existing benign mixed tumor (i.e., pleomorphic adenoma, PA). While virtually any salivary carcinoma can arise from a PA, to our knowledge SCC ex-PA has not been described. We report a case of a woman presenting with fullness of the right neck and a parotid gland mass. The tumor was resected and evaluated grossly, by light microscopy, and by immunohistochemistry. Grossly, a 1.6 cm well-circumscribed nodule was identified within the parotid. Microscopic examination revealed foci of SCC associated with high-grade adenocarcinoma, in the background of a PA. The SCC was immunoreactive for cytokeratin in a dot-like pattern and neuroendocrine markers synaptophysin and CD56. Despite the focal nature of the SCC in the parotid, a pure SCC metastasis was present in one neck level II lymph node. The patient was free of disease with 8 months of follow-up. Our case illustrates that: (1) although rare, in the salivary glands SCC may arise from lower grade neoplasms including PAs; (2) SCC ex PA may metastasize as pure SCC even if the primary SCC component was focal; (3) adequate sampling of PAs is crucial to prevent missing a rare SCC that must be treated with chemotherapy.
PMCID: PMC3500895  PMID: 22736150
Carcinoma ex pleomorphic adenoma; Carcinoma ex mixed tumor; Malignant mixed tumor; Small cell carcinoma; Parotid gland; Salivary glands
6.  Protocols in the management of critical illness 
Critical Care  2012;16(2):306.
Care of the critically ill patient is becoming increasingly complex. Protocols, which standardize care of patients with similar diseases, represent a potential solution to managing multiple simultaneous problems in critically ill patients. In this article, we examine the advantages and disadvantages to care protocolization, and posit that careful and thoughtful implementation of protocols is likely to benefit patients. We also discuss the potential for unintended consequences, and even harm, with protocolization in critically ill patients using the Critical Illness Outcomes Study as a model to examine the effects of protocolization in large populations of intensive care patients.
PMCID: PMC3584719  PMID: 22424130
7.  Gene expression profiling of blood in brain arteriovenous malformation patients 
Translational stroke research  2011;2(4):575-587.
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. Gene expression profiling of blood has led to the identification of stroke biomarkers, and may help identify BAVM biomarkers and illuminate BAVM pathogenesis. It is unknown whether blood gene expression profiles differ between 1) BAVM patients and healthy controls, or 2) unruptured and ruptured BAVM patients at presentation. We characterized blood transcriptional profiles in 60 subjects (20 unruptured BAVM, 20 ruptured BAVM, and 20 healthy controls) using Affymetrix whole genome expression arrays. Expression differences between groups were tested by ANOVA, adjusting for potential confounders. Genes with absolute fold change ≥ 1.2 (false discovery rate corrected p ≤ 0.1) were selected as differentially expressed and evaluated for over-representation in KEGG biological pathways (p ≤ 0.05). Twenty-nine genes were differentially expressed between unruptured BAVM patients and controls, including 13 which may be predictive of BAVM. Patients with ruptured BAVM compared to unruptured BAVM differed in expression of 1490 genes, with over-representation of genes in 8 pathways including MAPK, VEGF, Wnt signaling and several inflammatory pathways. These results suggest clues to the pathogenesis of BAVM and/or BAVM rupture and point to potential biomarkers or new treatment targets.
PMCID: PMC3241209  PMID: 22184505
arteriovenous malformation; blood; gene expression; intracranial hemorrhage; microarray analysis
8.  Spontaneous intracranial hypotension secondary to anterior thoracic osteophyte: Resolution after primary dural repair via posterior approach 
Spontaneous intracranial hypotension (SIH) is an uncommon syndrome widely attributed to CSF hypovolemia, typically secondary to spontaneous CSF leak. Although commonly associated with postural headache and variable neurological symptoms, one of the most severe consequences of SIH is bilateral subdural hematomas with resultant neurological deterioration.
We present the case of a patient diagnosed with SIH secondary to an anteriorly positioned thoracic osteophyte with resultant dural disruption, who after multiple attempts at nonsurgical management developed bilateral subdural hematomas necessitating emergent surgical intervention. The patient underwent a unilateral posterior repair of his osteophyte with successful anterior decompression. At 36 months follow up, the patient reported completely resolved headaches with no focal neurological deficits.
We outline our posterior approach to repair of the dural defect and review the management algorithm for the treatment of patients with SIH. We also examine the current hypotheses as to the origin, pathophysiology, diagnosis and treatment of this syndrome.
A posterior approach was utilized to repair the dural defect caused by an anterior thoracic osteophyte in a patient with severe SIH complicated by bilateral subdural hematomas. This approach minimizes morbidity compared to an anterior approach and allowed for removal of the osteophyte and repair of the dural defect.
PMCID: PMC3537944  PMID: 23108168
Spontaneous intracranial hypotension; CSF leak; Thoracic osteophyte; Posterior repair
9.  Detection of Promoter Hypermethylation in Salivary Rinses as a Biomarker for Head and Neck Squamous Cell Carcinoma Surveillance 
Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP).
Experimental Design
Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1.
We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8–80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2–6.5; P = 0.016).
We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.
PMCID: PMC3215270  PMID: 21628494
10.  Intramedullary spinal cord metastasis from prostate carcinoma: a case report 
Although vertebral and epidural metastases are common, intradural metastases and intramedullary spinal cord metastases are rare. The indications for the treatment of intramedullary spinal cord metastases remain controversial. We present the first biopsy-proven case of an intramedullary spinal cord metastasis from adenocarcinoma of the prostate.
Case presentation
Our patient was a 68-year-old right-handed Caucasian man with a Gleason grade 4 + 3 prostate adenocarcinoma who had previously undergone a prostatectomy, androgen blockade and transurethral debulking. He presented with new-onset saddle anesthesia and fecal incontinence. Magnetic resonance imaging demonstrated a spindle-shaped intramedullary lesion of the conus medullaris. Our patient underwent decompression and an excisional biopsy; the lesion’s pathology was consistent with metastatic adenocarcinoma of the prostate. Postoperatively, our patient received CyberKnife® radiosurgery to the resection cavity at a marginal dose of 27Gy to the 85% isodose line. At three months follow-up, our patient remains neurologically stable with no new deficits or lesions.
We review the literature and discuss the indications for surgery and radiosurgery for intramedullary spinal cord metastases. We also report the novel use of stereotactic radiosurgery to sterilize the resection cavity following an excisional biopsy of the metastasis.
PMCID: PMC3419088  PMID: 22657386
11.  Carcinoma ex Pleomorphic Adenoma of the Nasal Cavity 
Head and Neck Pathology  2011;5(4):405-409.
Carcinoma ex pleomorphic adenoma (CXPA) is a malignant epithelial neoplasm arising in a benign mixed tumor (i.e., pleomorphic adenoma or PA); it accounts for approximately 3–4% of all salivary gland neoplasms. CXPA is exceedingly rare in the nasal cavity, with only three cases previously documented.
We report two cases of women presenting with masses of the nasal cavity. The tumors were resected and evaluated grossly and by routine microscopy.
Both patients presented with nasal obstruction, and one patient reported epistaxis. Grossly, the tumors were 2.5 and 3 cm in greatest dimension, respectively. In each case, microscopic examination of the tumor revealed an infiltrating carcinoma arising in association with foci of benign PA. The carcinoma types were adenoid cystic carcinoma and high grade adenocarcinoma, not otherwise specified. Both cases were widely (i.e., >1.5 mm) invasive beyond the preexisting PA. The findings were diagnostic of CXPA.
Carcinomatous transformation of a PA in the nasal cavity is extremely rare, but does occur. Accordingly, PAs of this site should be thoroughly sampled and closely examined to exclude the possibility of malignant transformation.
PMCID: PMC3210223  PMID: 21505850
Carcinoma ex pleomorphic adenoma; Carcinoma ex mixed tumor; Malignant mixed tumor; Nasal cavity; Nasal septum
12.  Potential application of hydrogen in traumatic and surgical brain injury, stroke and neonatal hypoxia-ischemia 
Medical Gas Research  2012;2:11.
This article summarized findings of current preclinical studies that implemented hydrogen administration, either in the gas or liquid form, as treatment application for neurological disorders including traumatic brain injury (TBI), surgically induced brain injury (SBI), stroke, and neonatal hypoxic-ischemic brain insult (HI). Most reviewed studies demonstrated neuroprotective effects of hydrogen administration. Even though anti-oxidative potentials have been reported in several studies, further neuroprotective mechanisms of hydrogen therapy remain to be elucidated. Hydrogen may serve as an adjunct treatment for neurological disorders.
PMCID: PMC3353846  PMID: 22515516
Hydrogen; Neuroprotection; Oxidative stress; Reactive oxygen species
13.  The Role of MAGEA2 in Head and Neck Cancer 
To examine the role of MAGEA2 in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC).
Primary tissue microarray data and quantitative reverse transcription–polymerase chain reaction (RT-PCR) showed that MAGEA2 is differentially over-expressed in HNSCC. Functional analyses were then performed using MAGEA2 transfections and small-interfering RNA knockdowns with subsequent anchorage-dependent growth studies and cell cycle analyses. Quantitative RT-PCR was used to evaluate expression changes in p53 downstream targets after transfection of MAGEA2 into normal upper aerodigestive cell lines.
MAGEA2 is differentially overexpressed in HNSCC. In addition, MAGEA2 promotes growth in normal oral keratinocytes, whereas knockdown of MAGEA2 in HNSCC cells decreases growth. Using the HCT116 p53 wt and null cell line system, transfection of MAGEA2 induced growth in the p53 wt cell line while providing no growth advantage in the p53 mutant cells. Subsequently, transfection of MAGEA2 induced a decrease in messenger RNA expression of the p53 downstream targets CDKN1A and BAX and decreased G1 arrest in cells allowed to remain confluent for longer than 48 hours.
These data suggest that MAGEA2 is differentially expressed in HNSCC and functions, in part, through the p53 pathway by increasing cellular proliferation and abrogating cell cycle arrest. This improved understanding of MAGEA2 function and expression patterns will potentially allow for the improved ability to use MAGEA2 for detection, surveillance, and targeted therapeutics.
PMCID: PMC3309612  PMID: 21422315
14.  Early Identification of Patients at Risk of Acute Lung Injury 
Rationale: Accurate, early identification of patients at risk for developing acute lung injury (ALI) provides the opportunity to test and implement secondary prevention strategies.
Objectives: To determine the frequency and outcome of ALI development in patients at risk and validate a lung injury prediction score (LIPS).
Methods: In this prospective multicenter observational cohort study, predisposing conditions and risk modifiers predictive of ALI development were identified from routine clinical data available during initial evaluation. The discrimination of the model was assessed with area under receiver operating curve (AUC). The risk of death from ALI was determined after adjustment for severity of illness and predisposing conditions.
Measurements and Main Results: Twenty-two hospitals enrolled 5,584 patients at risk. ALI developed a median of 2 (interquartile range 1–4) days after initial evaluation in 377 (6.8%; 148 ALI-only, 229 adult respiratory distress syndrome) patients. The frequency of ALI varied according to predisposing conditions (from 3% in pancreatitis to 26% after smoke inhalation). LIPS discriminated patients who developed ALI from those who did not with an AUC of 0.80 (95% confidence interval, 0.78–0.82). When adjusted for severity of illness and predisposing conditions, development of ALI increased the risk of in-hospital death (odds ratio, 4.1; 95% confidence interval, 2.9–5.7).
Conclusions: ALI occurrence varies according to predisposing conditions and carries an independently poor prognosis. Using routinely available clinical data, LIPS identifies patients at high risk for ALI early in the course of their illness. This model will alert clinicians about the risk of ALI and facilitate testing and implementation of ALI prevention strategies.
Clinical trial registered with (NCT00889772).
PMCID: PMC3056224  PMID: 20802164
respiratory distress syndrome, adult; prevention; prediction model; acute respiratory failure
15.  A rare case of an aldosterone secreting metastatic adrenocortical carcinoma and papillary thyroid carcinoma in a 31-year-old male 
Rare Tumors  2011;3(4):e45.
We report a rare synchronous presentation of adrenocortical carcinoma (ACC) and papillary thyroid carcinoma (PTC). A 31-year-old male first presented with a large left adrenal mass that was identified during the workup for refractory hypertension due to hyperaldosteronism. The mass was removed surgically with pathology showing ACC. The patient was then treated with adjuvant radiation therapy and mitotane chemotherapy. Four months post ACC resection, metastatic ACC to the right upper lung and PTC in the left lobe of the thyroid were found in surveillance imaging. He subsequently developed pulmonary, contralateral adrenal and brain metastases from his ACC. Li Fraumeni syndrome and Multiple Endocrine Neoplasia Type I (MEN I) were considered, but testing of both P53 and menin genes showed no mutation. We also performed a review of the literature and found three similar cases, however gene mutation analysis was not performed..
PMCID: PMC3282450  PMID: 22355500
adrenocortical carcinoma; papillary thyroid carcinoma; hereditary cancer syndrome.
16.  Presence of HPV DNA in convalescent salivary rinses is an adverse prognostic marker in head and neck squamous cell carcinoma 
Oral oncology  2008;44(10):915-919.
Human papillomavirus (HPV) 16 is present in up to 60% of patients with head and neck squamous cell carcinoma (HNSCC) and confers a favorable prognosis in terms of recurrence and mortality. Previous reports demonstrated that HPV-16 DNA can be detected in the initial salivary rinses from these patients. In this study, we assessed the feasibility of post-treatment HPV DNA shed from the oral mucosa as a prognostic marker for persistent/recurrent head and neck cancer. Fresh tumor samples and pre- and post-treatment salivary rinses were collected from 59 patients with HNSCC. HPV-16 E6 and E7 DNA copy number in these samples were quantified by real time PCR. Twenty of 59 patients (33.9%) were HPV-16 positive in their tumors before treatment. Four of 20 HPV tumor positive patients ultimately developed recurrence, and 2 of these 4 patients were HPV-16 positive in surveillance salivary rinses (sensitivity = 50%). Of the 39 (66.1%) HPV-16 negative patients on initial clinical presentation and the 16 HPV-16 positive patients who did not recur, none were HPV-16 positive in salivary rinses after treatment (specificity = 100%). HPV-16 presence in follow-up salivary rinses preceded clinical detection of disease recurrence by an average of 3.5 months. Patients with presence of HPV-16 DNA in surveillance salivary rinses are at significant risk for recurrence. Quantitative measurement of salivary HPV-16 DNA has promise for surveillance and early detection of recurrence.
PMCID: PMC3215237  PMID: 18329326
Human Papilloma Virus (HPV); Head and Neck Squamous Cell Carcinoma (HNSCC); quantitative PCR
17.  MicroRNA alterations in Head and Neck Squamous Cell Carcinoma 
MicroRNAs (mirs) are small non-coding RNA molecules (~22 nucleotides) that regulate post-transcriptional gene expression. Currently, there has not been a comprehensive study of their role in primary HNSCC. To determine the role of mirs in head and neck squamous cell carcinoma (HNSCC), we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs.
An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines were analyzed for mature microRNA expression by microarray. Significance was determined using Significance Analysis of Microarrays (SAM). Nine microRNAs were found by SAM to be up-regulated or down-regulated in tumor tissue including mir-21,let-7,18,29c,142-3p,155,146b(over-expressed) and 494(under-expressed). Mir-21 was validated by qRT-PCR.
Functional validation by growth assays was performed, further validating mir-21. Transfection of mir-21 into JHU-011 and JHU-012 cell lines showed a 39% increase in cell growth at 72 hrs relative to controls (p<.05). Transfection of the inhibitor into JHU-O12 cell lines showed a 92% decrease in cell growth relative to controls at 72hrs (p<.05). In addition, flow cytometry analysis of JHU-012 cells 48 hrs after mir-21 inhibitor transfection showed a statistically significantly increase in cytochrome c release and increased apoptosis.
These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir-21 is a putative oncogenic microRNA in head and neck cancer.
PMCID: PMC3184846  PMID: 18798260
MicroRNA; HNSCC; Mir-21; Mir-494; Mir; Squamous Cell Cancer
18.  EIF2C Is Overexpressed and Amplified in Head and Neck Squamous Cell Carcinoma 
To discover putative oncogenes in head and neck squamous cell carcinoma (HNSCC) by integrating data from whole-genome comparison of array-based comparative genomic hybridization (CGH) and expression microarray analysis of HNSCC.
We integrated published data defining regions of loss/gain identified from the profiling of 21 HNSCC using high-resolution (<1 Mb) CGH arrays and data from an mRNA expression microarray (approx. 12,000 genes) comparing 6 normal tissues and 8 HNSCC tumor tissues. Eukaryotic translation initiation factor 2C subunit 2 (EIF2C2) was found to be the most significantly overexpressed gene by mRNA expression array, and corresponded to the most common region of amplification found by the CGH array described by Sparano et al. We validated EIF2C2 overexpression in primary tissue, overexpression and amplification in HNSCC lines (JHU-011, JHU-012, FADU) relative to a minimally transformed oral keratinocyte cell line (OKF6) and performed knockdown experiments.
The tumor tissues had an average mRNA expression level of 123 (SD = 49) compared to the normal tissues (18.6, SD = 10) (p = 0.0005) by expression array. Quantitative RT-PCR validation of our expression arrays found that normal tissues had an average expression of 0.76 (SE = 0.08) and tumor tissues of 2.1 (SE = 0.35) (p = 0.0008). EIF2C2 was found to be amplified and overexpressed in 3 HNSCC cell lines. Knockdown of EIF2C2 in cell lines (JHU-012 and JHU-011) inhibited proliferation.
EIF2C2 is amplified and overexpressed in HNSCC cell lines and primary tumors and functionally significant in cell lines.
PMCID: PMC2975733  PMID: 20924207
Head and neck squamous cell carcinoma; EIF2C2
19.  Impact of Common Medications on Serum Total Prostate-Specific Antigen Levels: Analysis of the National Health and Nutrition Examination Survey 
Journal of Clinical Oncology  2010;28(25):3951-3957.
Previous studies suggest that some common medications alter prostate-specific antigen (PSA) levels. It remains unclear whether these reported medication effects are due to clinicodemographic factors or concurrent use of other medications. We investigated the impact of individual and combinations of common medications on PSA in a large cross-sectional study of the United States population.
Patients and Methods
The study included men ≥ 40 years old without prostate cancer from the 2003 to 2004 and 2005 to 2006 cycles of the National Health and Nutrition Examination Survey (NHANES). Men with recent prostate manipulation, prostatitis, and those on hormone therapy were excluded. Weighted multivariate linear regression was performed on log-transformed total PSA to determine the effect of the 10 most commonly prescribed medication classes, adjusting for potential confounders including demographics, clinical characteristics, physical examination, laboratory studies, and duration of medication use.
In total, 1,864 men met inclusion criteria. Nonsteroidal anti-inflammatory drug (NSAID; P = .03), statin (P = .01), and thiazide diuretic (P = .025) intake was inversely related to PSA levels. Five years of NSAID, statin, and thiazide diuretic use was associated with PSA levels lower by 6%, 13%, and 26%, respectively. The combination of statins and thiazide diuretics showed the greatest reduction in PSA levels: 36% after 5 years. Concurrent calcium channel blocker use minimizes or negates the inverse relationship of statin use and PSA level.
We found that men using NSAIDs, statins, and thiazide diuretics have reduced PSA levels by clinically relevant amounts. The impact of regularly consuming these common medications on prostate cancer screening is unknown.
PMCID: PMC2940393  PMID: 20679596
20.  CyberKnife Stereotactic Radiosurgery for Recurrent, Metastatic, and Residual Hemangiopericytomas 
Hemangiopericytoma is a rare and aggressive meningeal tumor. Although surgical resection is the standard treatment, hemangiopericytomas often recur with high incidences of metastasis. The purpose of this study was to evaluate the role of CyberKnife stereotactic radiosurgery (CK) in the management of recurrent, metastatic, and residual hemangiopericytomas.
In a review of the Stanford radiosurgery database between 2002 and 2009, the authors found 14 patients who underwent CK therapy for recurrent, metastatic, and residual hemangiopericytomas. A total of 24 tumors were treated and the median patient age was 52 years (range 29-70 years) at the time of initial CK therapy. The median follow-up period was 37 months (10-73 months) and all patients had been previously treated with surgical resection. Mean tumor volume was 9.16 cm3 and the mean marginal and maximum radiosurgical doses to the tumors were 21.2 Gy and 26.8 Gy, respectively.
Of the 24 tumors treated, 22 have clinical follow-up data at this time. Of those 22 tumors, 12 decreased in size (54.5%), 6 remained unchanged (27.3%), and 4 showed recurrence (18.2%) after CK therapy. Progression-free survival rate was 95%, 71.5%, and 71.5% at 1, 3, and 5 years after multiple CK treatments. The 5-year survival rate after CK was 81%.
CK is an effective and safe management option for hemangiopericytomas. The current series demonstrates a tumor control of 81.8%. Other institutions have demonstrated similar outcomes with stereotactic radiosurgery, with tumor control ranging from 46.4% to 100%.
PMCID: PMC3118387  PMID: 21645367
21.  Inactivation of the Tumor Suppressor Genes Causing the Hereditary Syndromes Predisposing to Head and Neck Cancer via Promoter Hypermethylation in Sporadic Head and Neck Cancers 
Fanconi anemia (FA) and dyskeratosis congenita (DC) are rare inherited syndromes that cause head and neck squamous cell cancer (HNSCC). Prior studies of inherited forms of cancer have been extremely important in elucidating tumor suppressor genes inactivated in sporadic tumors. Here, we studied whether sporadic tumors have epigenetic silencing of the genes causing the inherited forms of HNSCC. Using bisulfite sequencing, we investigated the incidence of promoter hypermethylation of the 17 Fanconi- and DC-associated genes in sporadic HNSCC. Genes that only showed methylation in the tumor patients were chosen for quantitative methylation-specific PCR (qMSP) in a set of 45 tumor and 16 normal patients. Three gene promoters showed differences in methylation: FancB (FAAP95, FA core complex), FancJ (BRIP1, DNA Helicase/ATPase), and DKC1 (dyskeratin). Bisulfite sequencing revealed that only FancB and DKC1 showed no methylation in normal patients, yet the presence of promoter hypermethylation in tumor patients. On qMSP, 1/16 (6.25%) of the normal mucosal samples from non-cancer patients and 14/45 (31.1%) of the tumor patients demonstrated hypermethylation of the FancB locus (p < 0.05). These results suggest that inactivation of FancB may play a role in the pathogenesis of sporadic HNSCC.
PMCID: PMC2881891  PMID: 20332657
Hypermethylation; Fanconi anemia; Dyskeratosis congenita; Leukoplakia; Head and neck cancer
22.  Identification of Hypermethylated Genes Associated with Cisplatin Resistance in Human Cancers 
Cancer research  2010;70(7):2870-2879.
Cisplatin is among the most widely used cytotoxic anti-cancer agents in solid tumors, however, the development of secondary resistance remains a major obstacle to clinical efficacy. Treatment-related DNA hypermethylation may play a role in creating drug resistant phenotypes by inactivating genes that are required for cytotoxicity. We applied a pharmacologic unmasking approach to detect hypermethylated genes whose inactivation contributes to cisplatin resistance. Utilizing three pairs of isogeneic, cisplatin-sensitive and -resistant cell lines derived from two parental cell lines (KB-3-1 and SCC25), we identified several hundred genes that were down-regulated in each resistant cell line and re-activated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC). Among them, 30 genes were common to ≥ 2 cell lines, and/or reported to be down-regulated in previous studies. Bisulfite sequencing confirmed that 14 genes were hypermethylated in resistant cell lines, but not in the sensitive parental cell lines. Six of 14 genes (SAT, C8orf4, LAMB3, TUBB, G0S2, MCAM) were cisplatin-inducible in sensitive, but not in resistant cell lines. siRNA knockdown of two genes, SAT and S100P, increased cell viability with cisplatin treatment in sensitive parental cell lines. S100P knockdown significantly decreased the S-phase fraction (SPF) of parental sensitive cell lines and slowed cell proliferation, which was associated with decreased sensitivity to cisplatin. Based on these findings, we conclude that DNA methylation is a frequent event in cells that are chronically exposed to cisplatin, and that methylation-induced gene silencing may play a role in the development of resistance to cytotoxic chemotherapeutic agents.
PMCID: PMC2849007  PMID: 20215521
cisplatin; drug resistance; S100P; methylation; epigenetics
23.  Chronic CSE Treatment Induces the Growth of Normal Oral Keratinocytes via PDK2 Upregulation, Increased Glycolysis and HIF1α Stabilization 
PLoS ONE  2011;6(1):e16207.
Exposure to cigarette smoke is a major risk factor for head and neck squamous cell carcinoma (HNSCC). We have previously established a chronic cigarette smoke extract (CSE)-treated human oral normal keratinocyte model, demonstrating an elevated frequency of mitochondrial mutations in CSE treated cells. Using this model we further characterized the mechanism by which chronic CSE treatment induces increased cellular proliferation.
Methodology/Principal Findings
We demonstrate that chronic CSE treatment upregulates PDK2 expression, decreases PDH activity and thereby increases the glycolytic metabolites pyruvate and lactate. We also found that the chronic CSE treatment enhanced HIF1α accumulation through increased pyruvate and lactate production in a manner selectively reversible by ascorbate. Use of a HIF1α small molecule inhibitor blocked the growth induced by chronic CSE treatment in OKF6 cells. Furthermore, chronic CSE treatment was found to increase ROS (reactive oxygen species) production, and application of the ROS scavengers N-acetylcysteine abrogated the expression of PDK2 and HIF1α. Notably, treatment with dichloroacetate, a PDK2 inhibitor, also decreased the HIF1α expression as well as cell proliferation in chronic CSE treated OKF6 cells.
Our findings suggest that chronic CSE treatment contribute to cell growth via increased ROS production through mitochondrial mutations, upregulation of PDK2, attenuating PDH activity thereby increasing glycolytic metabolites, resulting in HIF1α stabilization. This study suggests a role for chronic tobacco exposure in the development of aerobic glycolysis and normoxic HIFα activation as a part of HNSCC initiation. These data may provide insights into development of chemopreventive strategies for smoking related cancers.
PMCID: PMC3023770  PMID: 21283817
24.  Chronic Cigarette Smoke Extract Treatment Selects For Apoptotic Dysfunction and Mitochondrial mutations in Minimally Transformed Oral Keratinocytes 
Cigarette smoke demonstrates a carcinogenic effect through chronic exposure, not acute exposures. However, current cell line models study only the acute effects of cigarette smoke. Using a cell line model, we compared the effects of acute versus chronic cigarette-smoke-extract (CSE) on mitochondria in minimally-transformed oral keratinocytes (OKF6).
OKF6 cells were treated with varying concentrations of CSE for 6-months. Cells were analyzed monthly by flow cytometry for mitochondrial-membrane-potential (MMP), cytochrome-c release, caspase-3 activation and viability after CSE-exposure. At each time point the same assays were performed after 24hrs of valinomycin (MMP depolarizing agent) treatment. The mitochondrial-DNA of chronically CSE-treated cells was sequenced.
After 6-months of CSE-treatment, the cells were increasingly resistant to CSE-mediated and valinomycin induced cell death. In addition, chronic CSE-treatment caused chronic depolarization of MMP, cytochrome c release, and caspase activation. Cells grown in the presence of only CSE vapor also exhibited the same resistance and chronic baseline apoptotic activation. Mitochondrial DNA sequencing found that chronic CSE treated cells had more amino acid changing mitochondrial mutations than acutely treated cells.
CSE treatment of normal cells select for apoptotic dysfunction as well as mitochondrial mutations. These findings suggest that chronic tobacco exposure induce carcinogenesis via selection of apoptosis resistance and mitochondrial mutation in addition to previously known genotoxic effects that were found by acute treatments. Chronic models of tobacco exposure on upper aerodigestive epithelia may be more insightful than models of acute exposure in studying head and neck carcinogenesis
PMCID: PMC2818069  PMID: 19634139
Cigarette Smoke Extract; CSE; Apoptosis; Mitochondria; Keratinocytes
25.  A Novel Neural Substrate for the Transformation of Olfactory Inputs into Motor Output 
PLoS Biology  2010;8(12):e1000567.
Anatomical and physiological experiments in the lamprey reveal the neural circuit involved in transforming olfactory inputs into motor outputs, which was previously unknown in a vertebrate.
It is widely recognized that animals respond to odors by generating or modulating specific motor behaviors. These reactions are important for daily activities, reproduction, and survival. In the sea lamprey, mating occurs after ovulated females are attracted to spawning sites by male sex pheromones. The ubiquity and reliability of olfactory-motor behavioral responses in vertebrates suggest tight coupling between the olfactory system and brain areas controlling movements. However, the circuitry and the underlying cellular neural mechanisms remain largely unknown. Using lamprey brain preparations, and electrophysiology, calcium imaging, and tract tracing experiments, we describe the neural substrate responsible for transforming an olfactory input into a locomotor output. We found that olfactory stimulation with naturally occurring odors and pheromones induced large excitatory responses in reticulospinal cells, the command neurons for locomotion. We have also identified the anatomy and physiology of this circuit. The olfactory input was relayed in the medial part of the olfactory bulb, in the posterior tuberculum, in the mesencephalic locomotor region, to finally reach reticulospinal cells in the hindbrain. Activation of this olfactory-motor pathway generated rhythmic ventral root discharges and swimming movements. Our study bridges the gap between behavior and cellular neural mechanisms in vertebrates, identifying a specific subsystem within the CNS, dedicated to producing motor responses to olfactory inputs.
Author Summary
Animal behaviors, including locomotion, can be driven by olfactory cues, such as pheromones or food sources. The neural substrate (neuroanatomical connections and physiological signals) that permits the transformation of olfactory inputs into locomotor responses is still unknown in vertebrates. In the present study, we identify such a neural substrate in the lamprey. Here, olfactory signals from the outside world are transmitted to the reticulospinal neurons in the lower brainstem, which provide the descending locomotor command to the spinal cord. We found that this circuit originates in the medial portion of the olfactory bulb and that connections are made in the posterior tuberculum, a ventral diencephalic structure. These inputs are then conveyed to the mesencephalic locomotor region, known to project extensively to brainstem reticulospinal neurons and thereby activate locomotion. Our results illuminate a specific dedicated neural substrate in the brain of lampreys that underlies olfactory-motor responses, which is activated by both food-related or pheromonal olfactory cues. It will be of interest to determine whether such a pathway is preserved in all vertebrates.
PMCID: PMC3006349  PMID: 21203583

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