Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells.

Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO's role in modulating OVCA cells' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs for OVCAs.

An improved method for quantification of Vibrio vulnificus in oysters.

Chromogenic agar was compared with the FDA recommended cellobiose-colistin agar for assessment of Vibrio vulnificus in oysters. A two-step culture confirmation method was also evaluated. The inclusion of CA gave a 33% increase in the detection rate and the two-step culture confirmation eliminated 62.5% of false positives.

Effector biology of plant-associated organisms: concepts and perspectives.

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

Clinicopathologic study of 85 cases of melanoma of the female genitalia.

BACKGROUND: Melanoma of the female genitalia has poor overall prognosis. OBJECTIVE AND METHODS: To examine prognostic factors influencing survival, the Duke Melanoma and Tumor Registry Databases were queried for patients who had received their clinical care at Duke University Medical Center, with a diagnosis of melanoma of the female genitalia, including vulva, vagina, and cervix, between 1970 and 2009. From this group, any available histopathologic specimens were procured for further review. RESULTS: Eighty-five patients were identified. The median follow-up time was 8.8 years with 60% of the patients experiencing melanoma-related mortality at last follow-up. Survival rates at 1, 5, and 10 years were 85%, 51%, and 30%, respectively. The available histopathologic specimens from 36 cases were reviewed by a dermatopathologist (M.A.S.). Fifteen of 36 cases were notable for the presence of atypical melanocytic hyperplasia adjacent to the primary melanoma. Breslow depth, lymph node status, systemic therapy, and surgery were also examined for differences in survival distributions using the log-rank test. In general, survival was inversely correlated with Breslow depth, extent of nodal involvement, and provision of systemic therapy. A higher survival rate was observed among those who received wide local excision. Log-rank test demonstrated that survival between different decades of diagnosis was not significantly different. LIMITATIONS: Because of its small sample size, this study may be underpowered. CONCLUSION: Despite new treatments developed and attempted, there is no evidence that survival has improved over the past 40 years. In summary, patients with thinner melanomas amenable to surgical resection had a better prognosis than those with more extensive, metastatic disease at presentation.

Evaluation of 39 cases of pediatric cutaneous head and neck melanoma.

BACKGROUND: Studies examining head and neck (H&N) melanoma in the pediatric population are scarce. OBJECTIVE: The goal of this study is to describe pediatric H&N melanoma with the intent of increasing understanding of the course of disease. METHODS: The Duke Melanoma Database and Duke Tumor Registry Database were searched for patients with a diagnosis of melanoma occurring on the H&N before age 18 years, with exclusion of ocular/mucosal/aerodigestive melanomas. RESULTS: Queries yielded 39 Caucasian pediatric patients, 24 (61.5%) of them male. The mean age at diagnosis was 14.2 years (15 years, median). The primary sites were represented as follows: cutaneous auricular (1/39, 2.6%), facial (15/39, 38.5%), and scalp/neck (23/39, 59%). The follow-up time ranged from 2 months to 23 years with a median of 9.9 years (95% confidence interval: 6.2-13 years). At the time of follow-up, there were 12 (12/39, 30.8%) melanoma-associated deaths. The anatomic distribution of primary melanoma for these 12 patients follows: 4 (33.3%) facial and 8 (66.7%) scalp/neck. Histologic data revealed 24 (61.5%) tumors classified as superficial spreading melanoma with nodular melanoma (12.8%) a distant second. The mean Breslow depth for patients with melanoma-related mortality was 2.4 mm, compared with 1.8 mm for those who were alive at last follow-up. LIMITATIONS: Small sample size limited this study. CONCLUSION: This study found that the majority (59%) of H&N melanomas presented as scalp or neck lesions with a predilection for adolescents and boys. Those who experienced melanoma-related mortality had thicker lesions. Superficial spreading melanoma was the most common subtype.

Treating thermally injured children suffering symptoms of acute stress with imipramine and fluoxetine: a randomized, double-blind study.

INTRODUCTION: For pediatric burn patients with the symptoms of acute stress disorder (ASD) a first line medication is not widely agreed upon. A prospective, randomized, placebo controlled, double-blind design was used to test the efficacy of imipramine and fluoxetine. METHOD: Patients 4-18 years of age with symptoms of ASD were randomized to 1 of 3 groups: imipramine, fluoxetine, or placebo for 1 week. Daily imipramine dose was 1mg/kg, with the maximum dose being 100mg. Daily fluoxetine dose was 5mg for children weighing >or=40 kg; 10mg for those weighing between 40 and 60 kg; 20mg for those weighing >60 kg. RESULTS: Sixty participants, 16 females and 44 males, had an average body surface area burn of 53% (S.D.=18) and average age of 11 years (S.D.=4). Imipramine subjects received an average daily dose of 1.00+/-0.29 mg/kg. Fluoxetine subjects received an average daily dose of 0.29+/-0.16 mg/kg. Between group differences were not detected. Fifty-five percent responded positively to placebo; 60% responded positively to imipramine; and 72% responded positively to fluoxetine. CONCLUSION: Within the parameters of this study design and sample, placebo was statistically as effective as either drug in treating symptoms of ASD.

A faculty development program to train tutors to be discussion leaders rather than facilitators.

PURPOSE: During 2003, 2004, and 2005, the role of 70 tutors was changed from that of facilitator to discussion leader, in a preclinical PBL learning course, Gastrointestinal Pathophysiology, by use of three key business school teaching strategies: questions, summaries, and schematics. The purpose of this study was to learn what difference this new approach made. METHOD: During each of the three study years, 171 (2003), 167 (2004), and 170 (2005) students were given Likert-scale attitudinal questionnaires to rate whether their tutors encouraged student direction of the tutorials and whether the summaries and closure schematics benefited their learning. Students' overall course evaluations and mean USMLE scores were quantitatively analyzed, pre- and postintervention. A variety of statistical tests were used to assess the statistical significance of means at the confidence level of .05. RESULTS: In the third year of the program, student ratings indicated that their tutors were significantly better at encouraging student direction of the tutorials than in the first year (P < .05). The students reported that the tutorial made a more important contribution to their learning (P < .05), and the course objectives were better stated (P = .038) and better met (P = .007). Overall satisfaction with the course also improved significantly (P = .006). Part I gastrointestinal system mean scores of the USMLE showed a statistically significant increase in 2005 compared with 2001 or 2002. CONCLUSIONS: The tutor as a discussion leader who questions, summarizes, and uses schematics to illustrate concepts had a significant and positive impact on learning in tutorials, achieving course objectives, improving overall course satisfaction, and increasing a standardized national exam's mean score.

Early treatment of acute stress disorder in children with major burn injury.

OBJECTIVE: This study examines retrospectively the response rate of pediatric burn survivors with acute stress disorder to either imipramine or fluoxetine. METHODS: On retrospective chart review, 128 intensive care unit patients (85 boys, 43 girls) with 52%+/- 20% total body surface area burn, length of stay of 32.8+/- 25.2 days, mean age of 9.1+/- 4.7 yrs, and age range of 13 months to 19 yrs met criteria for acute stress disorder after >or=2 days of symptoms and were treated with either imipramine or fluoxetine. If significant improvement did not occur within 7 days, the medication was either increased or switched to the other class. RESULTS: Initially, 104 patients were treated with imipramine and 24 with fluoxetine. A total of 84 patients responded to imipramine: seven of these patients required a higher dose. A total of 18 patients responded to initial fluoxetine treatment. Of 26 nonresponders to the initial medication, 13 imipramine failures and one fluoxetine failure refused further treatment. The other 12 responded to the second medication. Therefore, 114 of 128 treated patients (89%) responded to either fluoxetine (mean dose, 0.30+/- 0.14 mg/kg) or imipramine (mean dose, 1.30+/- 0.55 mg/kg). Response was independent of sex and age but was less for those with burns of >60% total body surface area. The side effects of each medication were not significant. Most patients continued treatment for >or=3 months; some required 6 months of treatment before successful discontinuation. CONCLUSIONS: Early treatment of acute stress disorder with either imipramine or fluoxetine is often able to reduce its symptoms. This is a review of a single hospital's experience in managing psychiatric distress in this very high-risk group of burned children. Additional clinical studies are needed before generalizing these findings.

Infliximab: Use in Inflammatory Bowel Disease.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic and often debilitating inflammatory bowel diseases (IBD) without medical cures. Despite the existence of multiple therapies, the medical treatment of these diseases often has proven insufficient and surgery is frequently required. However, as our understanding of the pathogenesis of these disorders and other immune-mediated inflammatory diseases (eg, rheumatoid arthritis and psoriasis) has grown, new and more specific biologic therapies have been developed that are proving more effective than traditional agents. Infliximab is a genetically engineered monoclonal antibody that targets the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and represents the first effective biologic therapy for IBD and has largely revolutionized treatment. Infliximab initially was developed to be used in patients with moderate to severe luminal or fistulizing CD who are refractory to standard medical therapy. More and more practitioners now are using infliximab as first-line therapy because of its superior efficacy. Infliximab rapidly induces remission in CD, but when given chronically, it can provide long-term maintenance of remission. In addition, there are some data to support its use as a steroid-sparing agent and treatment for various extraintestinal manifestations of IBD and, although used predominantly to treat CD, recent data suggest that infliximab also may have a role in the management of UC. Overall, infliximab represents a clinically useful, cost-effective therapy that works well, even though careful patient monitoring is required to avoid rare but significant toxicities. The hope is that infliximab, together with other biologic agents that currently are in development, will allow us to modify the course of IBD, avoid complications such as strictures and abscesses, and reduce the need for surgery.

The Role of Antibiotics in Inflammatory Bowel Disease.

Broad-spectrum antibiotics are the mainstay of therapy for patients with Crohn's disease (CD) who present with localized peritonitis due to a microperforation bacterial overgrowth secondary to chronic strictures. They are essential adjuncts to drainage therapy for CD-associated abscesses and for complicated perineal disease. The lack of well-designed, placebo-controlled trials has led to much skepticism about the efficacy of antibiotics as primary therapy for CD. However, a careful review of the experience with antibiotics, including clinical observations and controlled trials, leads to the conclusion that antibiotics have a role as primary therapy in active uncomplicated CD. The efficacy of their response must be considered in well-defined subsets of patients. Ciprofloxacin and metronidazole, the two most widely studied antibiotics, are effective therapy for patients with active ileocolonic and colonic disease and have been shown to reduce recurrence rates after ileocolonic resection. The benefits of these drugs are less clear for patients with uncomplicated ileal disease. Additionally, ciprofloxacin and metronidazole may also serve as an adjunct to immunomodulator therapy. The role of antimycobacterial therapy in treatment of CD is an attractive alternative, and hopefully this therapy will be further clarified when results of ongoing trials become available. In toxic patients with fulminant ulcerative colitis (UC), with or without megacolon, broad-spectrum antibiotics should be a part of the treatment program. In less severely ill patients requiring hospitalization, antibiotics may be given to cover for the potential of a superimposed infection until the workup for infection, including Clostridium difficile is completed. There may be a subset of patients with severe nontoxic colitis with persistent fever and bandemia after steroid therapy who respond to antibiotics, but to date controlled trials have not shown efficacy in this group. Antibiotics should not be routinely used for mild to moderately ill patients with UC, although a trial of ciprofloxacin is not unreasonable prior to colectomy for otherwise refractory patients. The use of rifaximin in UC requires further evaluation in larger studies.

Detection of mRNA species in bulbospinal neurons isolated from the rostral ventrolateral medulla using single-cell RT-PCR.

The rostral ventrolateral medulla (RVL) contains neurons which are critically involved in the tonic and reflex control of blood pressure. Some of these neurons project to the intermediolateral cell column of the thoracolumbar spinal cord and excite preganglionic sympathetic neurons. In order to gain a better understanding of the properties of the RVL neurons at the cellular and molecular level, a protocol was developed utilizing acute dissociation and the reverse transcription-polymerase chain reaction (RT-PCR) to study the expression of several genes in single RVL neurons. Neurons were dissociated from the RVL region of young rats, and classified as spinally projecting or non-spinal by the presence or absence of retrogradely transported fluorescent beads injected into the upper thoracic segments of the spinal cord. Individual neurons were collected by aspiration into a glass micropipette and analysed by RT-PCR. The presence of either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or neuron-specific enolase (NSE) mRNA was used as the criterion for selecting cells for further analysis. A subpopulation (50%) of spinally projecting, GAPDH- or NSE-positive neurons expressed mRNA for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT), indicative of catecholaminergic or C1 adrenergic neurons, respectively. Some bulbospinal RVL neurons, including those that were TH- or PNMT-positive, were also found to express mRNA for the mineralocorticoid receptor (MR), the glucocorticoid receptor (GR), noradrenaline transporter (NET), and neuronal glutamate transporter (EAAC1). The glial glutamate transporter (GLT), glycine transporter (GLYT2), glutamic acid decarboxylase (GAD67) and gamma-amino butyric acid (GABA) transporter (GAT-1) were not expressed. The single-cell RT-PCR protocol is a powerful, yet simple and relatively rapid method for analysis of mRNA expression in a defined neuronal population. It can be combined with whole-cell patch-clamp recording prior to RT-PCR analysis, allowing linkage of the molecular analysis of mRNA expression to the electrophysiological and pharmacological properties of single neurons. The method is very sensitive, enabling mRNA transcripts in low abundance to be detected, and its application in our recent studies provided novel information about neurons involved in blood-pressure regulation at the molecular and cellular level.

Effects of cyanide and hypoxia on membrane currents in neurones acutely dissociated from the rostral ventrolateral medulla of the rat.

Previous reports suggested that some neurones located in the rostral ventrolateral medulla (RVL) can act as fast oxygen sensors which enhance the sympathetic activity and blood pressure independent of peripheral chemoreceptors. The aim of this study was to compare hypoxic responses of different subpopulations of RVL neurones to ascertain whether the hypoxic sensitivity is restricted to one group of these neurones. Whole-cell patch-clamp recordings were made from acutely dissociated neurones obtained from RVL of P13-P19 rats. Short-lasting hypoxia (1-2 min) was evoked by pressure injection of NaCN or lowering pO2. Cells projecting to the upper thoracic segments were retrogradely labelled with fluorescent beads. Catecholaminergic (CA) or non-catecholaminergic (non-CA) neurones were identified using single-cell reverse-transcription polymerase chain reaction (RT-PCR) or immunocytochemistry. Recordings were made from 38 neurones (26 spinally-projecting, 12 non-spinal) using Cs+/TEA or K+-containing pipettes. In most of the cells tested with slow depolarising ramp commands (78%; including spinally-projecting and non-spinal neurones, as well as CA and non-CA neurones), NaCN or hypoxia evoked a reversible increase of the sustained inward current. Extracellular application of 1 mM Co2+ or 25 nM TTX revealed three components of the hypoxia-sensitive inward current which resembled the persistent sodium (INaP), low threshold calcium (LVA Ca2+) and high threshold calcium (HVA Ca2+) currents. The NaCN or hypoxia induced increase of the current could also be observed during step commands. Recordings with K+-containing pipettes during similar depolarising ramps revealed, in addition, a reversible increase of IK in 78% of tested cells (in all four types of examined neurones). These results are consistent with the concepts that RVL neurones can act as a central oxygen sensor. However, in contrast to the previously published data demonstrating that in pentobarbital anaesthetised rats only the barosensitive and spinally projecting cells were affected by a short-lasting hypoxia, our findings obtained with dissociated RVL neurones indicate that sensitivity to hypoxia is widely distributed within this part of the medulla oblongata.

Whole cell patch-clamp study of putative vasomotor neurons isolated from the rostral ventrolateral medulla.

A distinct subpopulation of neurons in the rostral and ventrolateral part of the medulla oblongata (RVL) plays a key role in controlling sympathetic vasomotor tone. To characterize these neurons under conditions in which all cell-to-cell interactions are eliminated, RVL neurons were acutely dissociated from 13- to 19-day old rats. Cells projecting to the upper thoracic segments were retrogradely labeled with fluorescent beads. Fifty-two percent (17/33) of examined spinally projecting neurons were catecholaminergic, as demonstrated by single-cell reverse transcription-polymerase chain reaction or immunocytochemistry. No spontaneous (capacitive) spikes were revealed in the tight seal cell-attached configuration. Whole cell recordings were made from 54 spinally projecting neurons using Cs(+)- or K(+)-containing pipettes. No spontaneous firing was observed in current-clamp mode with K(+)-based pipettes (membrane potential, -61.5 +/- 2.3 mV). Step depolarizations (300- or 400-ms pulses, up to 100 pA) evoked regular firing or one to four spikes. Several voltage-gated currents, resembling the transient and persistent Na+, delayed rectifier and low- and high-threshold Ca2+, were revealed in voltage-clamp mode. These results show that isolated spinally projecting RVL neurons display no pacemaker-like activity. Because data from the literature indicate that these neurons are capable of generating such activity under different experimental conditions, the factors responsible for different behavior need to be determined. Dissociated RVL neurons provide a useful new model for studying biophysical and other properties of neurons involved in blood pressure control.

O2-sensitive K+ current in undifferentiated and NGF-treated PC12 cell variants.

It has been reported that cultured PC12 cells can be used as a model for studying mechanisms of O2 sensitivity, previously examined in peripheral chemoreceptors and some neurons. This study compared the hypoxic depression of K+ currents in two PC12 variants, before and after differentiation into neurone-like cells induced by nerve growth factor (NGF). The results show that interaction of O2 and K+ channels is strongly-voltage dependent in the PC12/TM but not the PC12/ES subline. In PC12/TM cells an effect of hypoxia on the K+ current was appreciable only at moderately depolarized voltages, with a loss of sensitivity at +40 to +50 mV. NGF-induced transformation did not affect the responses seen in undifferentiated cells. These results emphasize the importance of screening PC12 cells before selecting a variant for studying O2 sensitivity. In view of evidence cited in the literature that hypoxia may effect membrane channels directly, further molecular and biophysical studies of the differences among PC12 variants are required.

[Recurrent hemorrhage from the small intestine in a patient with type 1 neurofibromatosis]. / Rezividierende Dünndarmblutungen bei einer Patientin mit Neurofibromatose Typ-1 (NF-1).

Patients with NF-1 may exhibit visceral neurofibromas, Schwannomas and other tumors of ectodermal origin beside the classical cutaneous and neuronal manifestations of neurofibromatosis. In a 46-year-old woman with a previous diagnosis of NF-1 and a history of recurrent intestinal bleeding of obscure origin a well vascularized tumor in a branch of the A. messenterica superior was found by use of scintigraphy and angiography. Intraoperatively two inhomogeneons tumors were found in the proximal jejunum. Histologically these tumors were classified as neurofibromas. After partial resection of the jejunum no further intestinal bleeding occurred in this patient. Therefore the intestinal neurofibromas were presumably the source of the recurrent bleeding.

[Radiodiagnosis of glucagonoma and vipoma, 2 rare endocrine pancreatic tumors]. / Radiodiagnostik des Glukagonom und Vipom, zwei seltenen endokrinen Pankreastumoren.

From the non-B-cell tumors of the pancreas the gastrinoma (Zollinger-Ellison-syndrome) is the best known. The glucagonoma and the vipoma are less known and less common. The diagnosis of these syndroms, produced by overproduction of hormones, is a clinical one. The radiodiagnost has to localize the primary tumor and to exclude metastases. The x-ray findings of the glucagonoma in 3 patients and of the vipoma in 1 patient are described.