Ligand gated receptor interactions: A key to the power of neuronal networks.

The discovery of the chemical synapse was a seminal finding in Neurobiology but the large body of microscopic interactions involved in synaptic transmission could hardly have been foreseen at the time of these first discoveries. Characterization of the molecular players at work at synapses and the increased granularity at which we can now analyze electrical and chemical signal processing that occur in even the simplest neuronal system are shining a new light on receptor interactions. The aim of this review is to discuss the complexity of some representative interactions between excitatory and inhibitory ligand-gated ion channels and/or G protein coupled receptors, as well as other key machinery that can impact neurotransmission and to explain how such mechanisms can be an important determinant of nervous system function.

Deficits across multiple behavioral domains align with susceptibility to stress in 129S1/SvImJ mice.

Acute physical or psychological stress can elicit adaptive behaviors that allow an organism maintain homeostasis. However, intense and/or prolonged stressors often have the opposite effect, resulting in maladaptive behaviors and curbing goal-directed action; in the extreme, this may contribute to the development of psychiatric conditions like generalized anxiety disorder, major depressive disorder, or post-traumatic stress disorder. While treatment of these disorders generally focuses on reducing reactivity to potentially threatening stimuli, there are in fact impairments across multiple domains including valence, arousal, and cognition. Here, we use the genetically stress-susceptible 129S1 mouse strain to explore the effects of stress across multiple domains. We find that 129S1 mice exhibit a potentiated neuroendocrine response across many environments and paradigms, and that this is associated with reduced exploration, neophobia, decreased novelty- and reward-seeking, and spatial learning and memory impairments. Taken together, our results suggest that the 129S1 strain may provide a useful model for elucidating mechanisms underlying myriad aspects of stress-linked psychiatric disorders as well as potential treatments that may ameliorate symptoms.

Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor.

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

The mutational spectrum of FOXA2 in endometrioid endometrial cancer points to a tumor suppressor role.

BACKGROUND: Forkhead box protein A2 (FOXA2) plays an important in development, cellular metabolism and tumorigenesis. The Cancer Genome Atlas (TCGA) identified a modest frequency of FOXA2 mutations in endometrioid endometrial cancers (EEC). The current study sought to determine the relationship between FOXA2 mutation and clinicopathologic features in EEC and FOXA2 expression. METHODS: Polymerase chain reaction (PCR) amplification and sequencing were used to identify mutations in 542 EEC. Western blot, quantitative reverse transcriptase PCR (qRT-PCR) and immunohistochemistry (IHC) were used to assess expression. Methylation analysis was performed using combined bisulfite restriction analysis (COBRA) and sequencing. Chi-squared, Fisher's exact, Student's t- and log-rank tests were performed. RESULTS: Fifty-one mutations were identified in 49 tumors (9.4% mutation rate). The majority of mutations were novel, loss of function (LOF) (78.4%) mutations, and most disrupted the DNA-binding domain (58.8%). Six recurrent mutations were identified. Only two tumors had two mutations and there was no evidence for FOXA2 allelic loss. Mutation status was associated with tumor grade and not associated with survival outcomes. Methylation of the FOXA2 promoter region was highly variable. Most tumors expressed FOXA2 at both the mRNA and protein level. In those tumors with mutations, the majority of cases expressed both alleles. CONCLUSION: FOXA2 is frequently mutated in EEC. The pattern of FOXA2 mutations and expression in tumors suggests complex regulation and a haploinsufficient or dominant-negative tumor suppressor function. In vitro studies may shed light on how mutations in FOXA2 affect FOXA2 pioneer and/or transcription factor functions in EEC.

Retransplantation after BK virus nephropathy in prior kidney transplant: an OPTN database analysis.

BK virus (BKV) has emerged as a major complication of kidney transplantation. Since June 30, 2004, the OPTN in the USA collects BKV as a primary or secondary cause of graft loss and also if treatment for BK virus (TBKV) is administered. In this study, we determined characteristics of those recipients of repeat kidney transplants from the OPTN database, where either (a) a graft loss occurred between June 30, 2004 and December 31, 2008 and database recorded prior TBKV in that allograft or (b) a graft loss between June 30, 2004 and December 31, 2008 was attributed primarily or secondarily due to BKV. In the study time period, 823 graft losses have occurred where TBKV or graft failure attributable to BKV was reported in prior transplant; of these, 126 have received a retransplant as of June 5, 2009. Induction and maintenance immunosuppression usage mirrored current trends. As of June 5, 2009, 118/126 grafts are still functioning, one graft failure attributed to BKV. TBKV was reported in 17.5% of the retransplants. In the retransplants performed through December 31, 2007, 1-year acute rejection rate was 7%, 1-year and 3-year Kaplan-Meier graft survival rates and median GFR were 98.5%, 93.6%, 65.5 and 68.4 mL/min, respectively. Retransplantation after BKV appears to be associated with good results.

Association between depression and nonadherence in recipients of kidney transplants: analysis of the United States renal data system.

INTRODUCTION: In our previous prospective single-center study, using validated self-administered instruments, we demonstrated correlation between depression and nonadherence in recipients of kidney transplants. The purpose of this study was to confirm our finding that depression was associated with nonadherence in a large database of transplant recipients for which we used the United States Renal Data System (USRDS). METHODS: We conducted a retrospective cohort study of 32,757 Medicare primary renal transplant recipients in the USRDS who underwent transplantation from January 1, 2000 to July 31, 2004 and were followed up through December 31, 2004, assessing Medicare claims showing depression and nonadherence based on codes of the International Classification of Diseases, 9th Revision. RESULTS: Logistic regression analysis (adjusted hazards ratio 1.69 with 95% confidence interval, 1.48-1.92) and log rank test (P < .0005) showed that there was a strong association of depression and nonadherence. Depression was associated with nonadherence, irrespective of the time of depression, whether it was pretransplantation (P < .001) or posttransplantation (P < .001). Nonadherence was also associated with black race (P < .001), younger age (P < .001), less HLA mismatch (P < .005), recipients of living kidneys and patients who underwent transplantation a longer time ago (P < .001). Furthermore, patients with 12 or less years of education were more nonadherent (P < .001). Among the transplant donor factors we investigated, donor black race (P < .001) and expanded criteria donor kidneys were strongly associated with nonadherence (P < .001). However, donor age and delayed graft function were not significantly associated with nonadherence. CONCLUSIONS: Future clinical trials of immunosuppressive therapy should assess the impact of depression on graft survival.

Acute kidney injury requiring hemodialysis in patients with anicteric leptospirosis.

BACKGROUND: Leptospirosis is an infrequent disease in the US, with most cases reported in the state of Hawaii. Renal involvement is common (44 - 67%), ranging from a mild prerenal azotemia in anicteric disease to renal failure requiring dialysis in Weil's syndrome (severe leptospirosis with jaundice, renal failure, and hemorrhage). METHODS: To describe the pattern of leptospiral renal disease at our institution, we performed a retrospective analysis (1992 - 2004) of all hospitalized cases of laboratory confirmed leptospirosis presenting with acute kidney injury (AKI), defined as a presenting serum creatinine > 1.5 mg/dl. RESULTS: During this time period, 18 patients were hospitalized with laboratory confirmed leptospirosis. Among these patients, 12 had AKI on presentation, and hemodialysis was required in 3 patients. Renal biopsies were performed in 2 of these patients, revealing acute tubulointerstitial nephritis. Interestingly, the patients who required dialysis did not have Weil's syndrome. They did not exhibit jaundice or hemorrhage, and serum AST (mean 51.7 U/l (range 36 - 60)), ALT (mean 51.0 U/l (range 38 - 64)), and total bilirubin (mean 1.2 mg/dl (range 0.8 - 1.8)) were either within normal limits or only slightly elevated, despite having the worst renal disease. CONCLUSIONS: This series adds to other evidence that severe AKI (requiring dialysis) can complicate anicteric leptospirosis in contrast to the notion that the AKI in anicteric disease is typically mild and prerenal. Leptospirosis should be considered in all patients who present with fever and AKI, especially if associated with thrombocytopenia or travel to an endemic area.

Hypoxia recruits a respiratory-related excitatory pathway to brainstem premotor cardiac vagal neurons in animals exposed to prenatal nicotine.

The most ubiquitous form of arrhythmia is respiratory sinus arrhythmia in which the heart beat slows during expiration and heart rate increases during inspiration. Whereas respiratory sinus arrhythmia benefits pulmonary gas exchange respiratory dysfunction presents a major challenge to the cardiorespiratory system. Hypoxia evokes a pronounced bradycardia mediated by increases in parasympathetic cardiac activity. It has been hypothesized that the fatal events in sudden infant death syndrome (SIDS) are exaggerated cardiorespiratory responses to hypoxia. This study tests whether premotor cardiac vagal neurons receive rhythmic respiratory-related excitatory synaptic inputs during normoxia and hypoxia, and if animals exposed to nicotine in the prenatal period have exaggerated responses to hypoxia. Premotor cardiac vagal neurons in the nucleus ambiguus were identified in rats by the presence of a fluorescent tracer in medullary slices that generate rhythmic inspiratory-related motor discharge. Respiratory activity was recorded from the hypoglossal nerve and excitatory synaptic events in cardiac vagal neurons were isolated using patch clamp techniques. Adult female rats were implanted with osmotic minipumps that delivered nicotine at a level approximately equivalent to those that occur in moderate to heavy smokers. During normal eupneic respiration, as well as during hypoxia, premotor cardiac vagal neurons from control animals did not receive any rhythmic respiratory-related excitatory inputs. However in animals exposed to nicotine throughout the prenatal period respiratory bursts during hypoxia dramatically increased the frequency of excitatory synaptic events in cardiac vagal neurons. In summary, in animals exposed to nicotine throughout the prenatal period, but not in unexposed animals, respiratory bursts that occur during hypoxia dramatically increase the frequency of excitatory synaptic events in cardiac vagal neurons. This study establishes a likely neurochemical mechanism for the heart rate responses to hypoxia and a link between prenatal nicotine exposure and exaggerated bradycardia responses during hypoxia that may contribute to sudden infant death syndrome.

Method for detecting the signature of noise-induced structures in spatiotemporal data sets.

Spatiotemporal stochastic resonance (STSR) is a phenomenon, where the stability of spatial patterns in an extended dynamical system displays a resonance-type dependence on the noise amplitude with the patterns being optimal at intermediate noise level. This dynamical behavior has been found in theoretical systems as well as in biochemical processes, where the noise level has been controlled externally. However, it is an open question how to identify the signature of a spatiotemporal stochastic resonance in a natural system, e.g., in ecology, when the noise amplitude is not known. This question is addressed in the present paper. We provide analysis tools, which allow to reconstruct the noise intensity in a spatiotemporal data set from the data alone. These tools are based on nearest-neighbor considerations inspired by cellular automata and are an appropriate method for detecting STSR, when combined with some measure of spatial order. As a test of our analysis tools, we apply them to sample data generated by four theoretical model systems. We show explicitly that without knowledge of the theoretical value of the noise amplitude for those systems displaying STSR the corresponding resonance curve can be reconstructed from the data alone. In addition, the other (nonresonant) cases are properly identified by our method with no resonance curve being found.

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant.

In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pHcyt) were monitored in mesophyll protoplasts from the CAM plant Kalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pHcyt indicator. At the beginning of the light phase, pHcyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pHcyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring delta pHcyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H(+)-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pHcyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pHcyt. A diurnal oscillation in pHcyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.

Cerebral vascular endothelial heme oxygenase: expression, localization, and activation by glutamate.

Endogenous carbon monoxide (CO) contributes to vasodilator responses of cerebral microvessels in newborn pigs. We investigated the expression, intracellular localization, and activity of heme oxygenase (HO), the key enzyme in CO production, in quiescent cerebral microvascular endothelial cells (CMVEC) from newborn pigs. HO-1 and HO-2 isoforms were detected by RT-PCR, immunoblotting, and immunofluorescence. HO-1 and HO-2 are membrane-bound proteins that have a strong preference for the nuclear envelope and perinuclear area of the cytoplasm. Betamethasone (10(-6) to 10(-4) M for 48 h) was associated with upregulation of HO-2 protein by approximately 50% and inhibition of Cox-2 but did not alter HO-1 or endothelial nitric oxide synthase expression in CMVEC. In vivo betamethasone treatment of newborn pigs (0.2 and 5.0 mg/kg im for 48 h) upregulated HO-2 in cerebral microvessels by 30-60%. HO activity as (14)CO production from [(14)C]glycine-labeled endogenous heme was inhibited by chromium mesoporphyrin (10(-6) to 10(-4) M). L-Glutamate (0.3-1.0 mM) stimulated HO activity 1.5-fold. High-affinity specific binding sites for L-[(3)H]glutamate suggestive of the glutamate receptors were detected in CMVEC. Altogether, these data suggest that, in cerebral circulation of newborn pigs, endothelium-derived CO may contribute to basal vascular tone and to responses that involve glutamate receptor activation.

Reconstruction of the extensor mechanism after proximal tibia endoprosthetic replacement.

The proximal tibia is a difficult area in which to perform a wide resection of a bone tumor. This difficulty is due to the intimate relationship of tumor in this location to the nerves and blood vessels of the leg, inadequate soft tissue coverage after endoprosthetic reconstruction, and the need to reconstruct the extensor mechanism. Competence of the extensor mechanism is the major determinant of functional outcome of these patients. Between 1980 and 1997, 55 patients underwent proximal tibia resection with endoprosthetic reconstruction for a variety of malignant and benign-aggressive tumors. Reconstruction of the extensor mechanism included reattachment of the patellar tendon to the prosthesis with a Dacron tape, reinforcement with autologous bone-graft, and attachment of an overlying gastrocnemius flap. All patients were followed for a minimum of 2 years; 6 patients (11%) had a transient peroneal nerve palsy, 4 patients (7.2%) had a fasciocutaneous flap necrosis, and 2 patients (3.6%) had a deep wound infection. Full extension to extension lag of 20 degrees was achieved in 44 patients, and 8 patients required secondary reinforcement of the patellar tendon. Function was estimated to be good to excellent in 48 patients (87%). Reattachment of the patellar tendon to the prosthesis and reinforcement with an autologous bone-graft and a gastrocnemius flap are reliable means to restore extension after proximal tibia endoprosthetic reconstruction.

Synaptic and neurotransmitter activation of cardiac vagal neurons in the nucleus ambiguus.

Cardiac vagal neurons play a critical role in the control of heart rate and cardiac function. These neurons, which are primarily located in the nucleus ambiguus (NA) and the dorsal motor nucleus of the vagus (DMNX), dominate the neural control of heart rate under normal conditions. Cardiac vagal activity is diminished and unresponsive in many disease states, while restoration of parasympathetic activity to the heart lessens ischemia and arrhythmias and decreases the risk of sudden death. Recent work has demonstrated that cardiac vagal neurons are intrinsically silent and therefore rely on synaptic input to control their firing. To date, three major synaptic inputs to cardiac vagal neurons have been identified. Stimulation of the nucleus tractus solitarius evokes a glutamatergic pathway that activates both NMDA and non-NMDA glutamatergic postsynaptic currents in cardiac vagal neurons. Acetylcholine excites cardiac vagal neurons via three mechanisms, activating a direct ligand-gated postsynaptic nicotinic receptor, enhancing postsynaptic non-NMDA currents, and presynaptically by facilitating transmitter release. This enhancement by nicotine is dependent upon activation of pre- and postsynaptic P-type voltage-gated calcium channels. Additionally, there is a GABAergic innervation of cardiac vagal neurons. The transsynaptic pseudorabies virus that expresses GFP (PRV-GFP) has been used to identify, for subsequent electrophysiologic study, neurons that project to cardiac vagal neurons. Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties, and the labeled baroreflex pathways that control heart rate are unaltered by the virus.

Stochastic noise interferes coherently with a model biological clock and produces specific dynamic behaviour.

The influence of noise is unavoidable in all living systems. Its impact on a model of a biological clock, normally running in regular oscillating modes, is examined. It is shown that in a specific system in which endogenous rhythmicity is produced by a beat oscillator acting on a feedback coupled metabolic pool system, noise can act coherently to produce unexpected dynamic behaviour, running from regular over pseudo-regular to irregular time-structures. If the biological system consists of a set of identical weakly coupled cells, stochasticity may lead to phase decoupling producing irregular spatio-temporal patterns. Synchronization via phase resetting can be achieved by external short-time temperature pulses. Explicit results are obtained for the well-studied circadian photosynthesis oscillations in plants performing crassulacean acid metabolism. Because of the generic structure of the underlying nonlinear dynamics they can, however, be regarded as a general property of the influence of noise on nonlinear excitable systems with fixed points occuring close to limit cycles.

Angiography and interventional radiology: percutaneous approaches to benign pancreatic disorders.

Interventional radiology contributes several avenues for the diagnosis and treatment of benign pancreatic disorders. Biopsy can provide supporting evidence for benign or equivocal lesions. Aspiration of collections suspected of infection can yield specific bacteriologic diagnosis. Bleeding complications of pancreatitis can be treated directly by transcatheter embolization.

Pancreatic pseudocysts and fluid collections: percutaneous approaches.

Radiological imaging and intervention play important roles in the management of pancreatic fluid collections and pseudocysts. Computed tomography evaluation of the severity of pancreatitis and assessment of its course are now routine. Percutaneous drainage of pancreatic pseudocysts and abscesses is commonly performed as an adjunct to surgical treatment and is frequently definitive therapy. Percutaneous débridement of pancreatic necrosis has recently emerged as a viable alternative to open surgical treatment.

The use of etomidate for rapid-sequence intubation in the air medical setting.

OBJECTIVE: To describe the use of etomidate for rapid-sequence intubation (RSI) in the air medical environment. METHODS: This was a retrospective review of a consecutive series of patients receiving etomidate for RSI by a university hospital-based air medical program. Records of all patients more than 10 years of age requiring intubation during a 13-month period were reviewed. Data collected included demographics, site of intubation, person performing intubation, indication, diagnosis, medications administered, complications, and pre- and post-

Activity of cardiorespiratory networks revealed by transsynaptic virus expressing GFP.

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alpha herpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

Susceptibility of ATP-sensitive K+ channels to cell stress through mediation of phosphoinositides as examined by photoirradiation.

Cell stress is implicated in a number of pathological states of metabolism, such as ischaemia, reperfusion and apoptosis in heart, neurons and other tissues. While it is known that the ATP-sensitive K+ (KATP) channel plays a role during metabolic abnormality, little information is available about the direct response of this channel to cell stress. Using photoirradiation stimulation, we studied the effects of cell stress on both native and cloned KATP channels. Single KATP channel currents were recorded from cell-attached and inside-out patches of rat ventricular myocytes and COS-1 cells coexpressing SUR2 and Kir6.2. KATP channel activity increased within < 1 min upon irradiation. The activity resulted from increased maximal open probability and decreased ATP inhibition. The effects remained after the irradiation was stopped. Irradiation also affected the channels formed only by Kir6.2DeltaC35. The irradiation-induced activation was comparable to that induced by phosphoinositides. Analysis of phosphatidylinositol composition revealed an elevated phosphatidylinositol bisphosphate level with irradiation. Wortmannin, an inhibitor of phosphatidylinositol kinases, decreased both the irradiation-induced channel activity and the production of phosphatidylinositol bisphosphates. Radical scavengers also reduced the irradiation-induced activation, suggesting a role for free radicals, an immediate product of photoirradiation. We conclude that photoirradiation can modify the single-channel properties of KATP, which appears to be mediated by phosphoinositides. Our study suggests that cellular stress may be linked with KATP channels, and we offer a putative mechanism for such a linkage.

Biopsy of musculoskeletal tumors. Current concepts.

Biopsy is a key step in the diagnosis of bone and soft tissue tumors. An inadequately performed biopsy may fail to allow proper diagnosis, have a negative impact on survival, and ultimately necessitate an amputation to accomplish adequate margins of resection. Poorly performed biopsy remains a common finding in patients with musculoskeletal tumors who are referred to orthopaedic oncology centers. The principles by which an adequate and safe biopsy of musculoskeletal tumors should be planned and performed are reviewed, and the surgical approach to different anatomic locations is emphasized.