Altered parvalbumin basket cell inputs in the dorsolateral prefrontal cortex of schizophrenia subjects.

Cortical circuitry dysfunction in schizophrenia has been studied at many different levels of resolution, but not at the most basic unit of network organization--synaptic inputs. Multi-label electron or confocal light microscopy is required to examine specific types of synaptic inputs, and application of these methods to quantitatively study disease-related changes in human postmortem tissue has not been feasible for technical reasons. We recently developed a multi-label confocal light microscopic approach that makes possible the systematic identification and quantification of synaptic inputs, and of the relative levels of proteins localized to these inputs, in human postmortem tissue. We applied this approach to quantify parvalbumin basket cell (PVBC) inputs in area 9 of the dorsolateral prefrontal cortex from schizophrenia and matched comparison subjects. Tissue sections were triple-labeled for the 65 kD isoform of glutamic acid decarboxylase (GAD65), PV and the GABA(A) receptor α1 subunit. PVBC axonal boutons were defined as PV/GAD65 dual-labeled puncta, and PVBC inputs were defined as a PVBC bouton that overlapped a GABA(A) receptor α1 subunit punctum. The density of PVBC inputs was unchanged in subjects with schizophrenia, but levels of PV protein were lower in PVBC boutons. In concert with prior reports, these findings indicate that PVBC dysfunction in schizophrenia reflects molecular and not structural alterations in these cells and their axon terminals.

Cardiac gene therapy in large animals: bridge from bench to bedside.

Several clinical trials are evaluating gene transfer as a therapeutic approach to treat cardiac diseases. Although it has just started on the path to clinical application, recent advances in gene delivery technologies with increasing knowledge of underlying mechanisms raise great expectations for the cardiac gene therapy. Although in vivo experiments using small animals provide the therapeutic potential of gene transfer, there exist many fundamental differences between the small animal and the human hearts. Before applying the therapy to clinical patients, large animal studies are a prerequisite to validate the efficacy in an animal model more relevant to the human heart. Several key factors including vector type, injected dose, delivery method and targeted cardiac disease are all important factors that determine the therapeutic efficacy. Selecting the most optimal combination of these factors is essential for successful gene therapy. In addition to the efficacy, safety profiles need to be addressed as well. In this regard, large animal studies are best suited for comprehensive evaluation at the preclinical stages of therapeutic development to ensure safe and effective gene transfer. As the cardiac gene therapy expands its potential, large animal studies will become more important to bridge the bench side knowledge to the clinical arena.

Evidence that dynamin-2 functions as a signal-transducing GTPase.

The role of dynamin GTPases in the regulation of receptor-mediated endocytosis is well established. Here, we present new evidence that the ubiquitously expressed isoform dynamin-2 (dyn2) can also function in a signal transduction pathway(s). A 200-fold overexpression of dyn1, the 70% identical neuronal isoform, has no effect. Our data suggest that dyn2 can act as a signal transducing GTPase affecting transcriptional regulation.

The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly.

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

Succession of bacterial and fungal communities within biofilms of a chlorinated drinking water distribution system.

Understanding the temporal dynamics of multi-species biofilms in Drinking Water Distribution Systems (DWDS) is essential to ensure safe, high quality water reaches consumers after it passes through these high surface area reactors. This research studied the succession characteristics of fungal and bacterial communities under controlled environmental conditions fully representative of operational DWDS. Microbial communities were observed to increase in complexity after one month of biofilm development but they did not reach stability after three months. Changes in cell numbers were faster at the start of biofilm formation and tended to decrease over time, despite the continuing changes in bacterial community composition. Fungal diversity was markedly less than bacterial diversity and had a lag in responding to temporal dynamics. A core-mixed community of bacteria including Pseudomonas, Massillia and Sphingomonas and the fungi Acremonium and Neocosmopora were present constantly and consistently in the biofilms over time and conditions studied. Monitoring and managing biofilms and such ubiquitous core microbial communities are key control strategies to ensuring the delivery of safe drinking water via the current ageing DWDS infrastructure.

Interferon-gamma and tumor necrosis factor-alpha specifically induce formation of cytomegalovirus-permissive monocyte-derived macrophages that are refractory to the antiviral activity of these cytokines.

Monocytes/macrophages are key cells in the pathogenesis of human cytomegalovirus (HCMV). Although HCMV infection in monocytes is restricted to early events of gene expression, productive infection has been demonstrated in differentiated macrophages in vitro. We examined the cellular and cytokine components that are essential for HCMV replication in Concanavalin A-stimulated monocyte-derived macrophages (MDM). By negative selection, depletion of CD8+ T lymphocytes, but not CD4+ T lymphocytes, CD19+ B cells, or CD56+ NK cells, resulted in a 60-70% reduction in the number of HCMV-infected MDM, and a 4 log decrease in virus production. Neutralization of IFN-gamma and TNF-alpha, but not IL-1, IL-2, or TGF-beta, decreased production of virus by 4 logs and 2 logs, respectively. Subsequently, addition of recombinant IFN-gamma or TNF-alpha to purified monocyte cultures was sufficient to produce HCMV-permissive MDM. While IFN-gamma and TNF-alpha possess antiviral properties, addition of these cytokines to permissive MDM cultures did not affect production of HCMV. Thus, rather than inhibiting replication of HCMV, IFN-gamma and TNF-alpha specifically induce differentiation of monocytes into HCMV-permissive MDM, which are resistant to the antiviral effects of these cytokines.

Biofilm structures (EPS and bacterial communities) in drinking water distribution systems are conditioned by hydraulics and influence discolouration.

High-quality drinking water from treatment works is degraded during transport to customer taps through the Drinking Water Distribution System (DWDS). Interactions occurring at the pipe wall-water interface are central to this degradation and are often dominated by complex microbial biofilms that are not well understood. This study uses novel application of confocal microscopy techniques to quantify the composition of extracellular polymeric substances (EPS) and cells of DWDS biofilms together with concurrent evaluation of the bacterial community. An internationally unique, full-scale, experimental DWDS facility was used to investigate the impact of three different hydraulic patterns upon biofilms and subsequently assess their response to increases in shear stress, linking biofilms to water quality impacts such as discolouration. Greater flow variation during growth was associated with increased cell quantity but was inversely related to EPS-to-cell volume ratios and bacterial diversity. Discolouration was caused and EPS was mobilised during flushing of all conditions. Ultimately, biofilms developed under low-varied flow conditions had lowest amounts of biomass, the greatest EPS volumes per cell and the lowest discolouration response. This research shows that the interactions between hydraulics and biofilm physical and community structures are complex but critical to managing biofilms within ageing DWDS infrastructure to limit water quality degradation and protect public health.

Genetic and morphological features of human iPSC-derived neurons with chromosome 15q11.2 (BP1-BP2) deletions.

BACKGROUND: Copy number variation on chromosome 15q11.2 (BP1-BP2) causes deletion of CYFIP1, NIPA1, NIPA2 and TUBGCP5; it also affects brain structure and elevates risk for several neurodevelopmental disorders that are associated with dendritic spine abnormalities. In rodents, altered cyfip1 expression changes dendritic spine morphology, motivating analyses of human neuronal cells derived from iPSCs (iPSC-neurons). METHODS: iPSCs were generated from a mother and her offspring, both carrying the 15q11.2 (BP1-BP2) deletion, and a non-deletion control. Gene expression in the deletion region was estimated using quantitative real-time PCR assays. Neural progenitor cells (NPCs) and iPSC-neurons were characterized using immunocytochemistry. RESULTS: CYFIP1, NIPA1, NIPA2 and TUBGCP5 gene expression was lower in iPSCs, NPCs and iPSC-neurons from the mother and her offspring in relation to control cells. CYFIP1 and PSD95 protein levels were lower in iPSC-neurons derived from the CNV bearing individuals using Western blot analysis. At 10 weeks post-differentiation, iPSC-neurons appeared to show dendritic spines and qualitative analysis suggested that dendritic morphology was altered in 15q11.2 deletion subjects compared with control cells. CONCLUSIONS: The 15q11.2 (BP1-BP2) deletion is associated with reduced expression of four genes in iPSC-derived neuronal cells; it may also be associated altered iPSC-neuron dendritic morphology.

A prospective, randomized study of the effects of prostacyclin on platelets and blood loss during coronary bypass operations.

A randomized, double-blind study was designed to evaluate the therapeutic effect and safety of prostacyclin (epoprostenol) in patients undergoing cardiopulmonary bypass. One hundred patients having isolated coronary bypass grafting received 300 units/kg of heparin and then either prostacyclin (12.5 ng/kg/min from heparinization until cardiopulmonary bypass, 25 ng/kg/min during bypass) or buffer/diluent in a similar manner. Standardized anesthetic, perfusion, and surgical techniques were used. Drug and placebo groups were similar in demographic data and bypass times, and there were no deaths. Activated coagulation time and platelet count were significantly higher during cardiopulmonary bypass in patients receiving prostacyclin. Platelet count remained significantly higher 24 hours after bypass in the active drug group. Immediately after operation, there was significantly less prolongation of bleeding time (1.3 versus 2.9 minutes; p = 0.009) in the patients receiving prostacyclin. Blood loss was significantly reduced during the first 4 hours postoperatively in the prostacyclin group (261 +/- 159 versus 347 +/- 197 ml; p = 0.02). There was no significant difference between the groups when total blood loss was compared (710 +/- 351 versus 869 +/- 498 ml; p = 0.07). Patients receiving prostacyclin required an average of 257 ml less blood transfused in the intensive care unit (p = 0.02). We conclude that the clinical impact of prostacyclin in patients undergoing coronary artery operations was demonstrable, but small. Prostacyclin may provide clinical benefits in patients undergoing cardiopulmonary bypass when there are contraindications to or other difficulties with blood transfusion. With prostacyclin, reduced heparin dose is possible and therefore reduced protamine requirement would offer a potential benefit of less cardiovascular depression immediately after bypass. However, the advantages offered by prostacyclin are not sufficient to recommend its routine use during cardiopulmonary bypass.

A prospective, randomized study of the effects of prostacyclin on neuropsychologic dysfunction after coronary artery operation.

This randomized, double-blind study was designed to evaluate the effect of prostacyclin (epoprostenol) on the incidence and severity of postoperative neuropsychologic dysfunction in patients undergoing coronary artery operation. Four days before operation and 1 week after operation, 100 patients having coronary artery bypass grafting underwent detailed neurologic and psychologic examinations and computed tomographic scans of the brain. The psychologic examination was repeated 2 months after operation. During cardiopulmonary bypass, all patients received 300 U/kg of heparin and then either buffer-diluent or prostacyclin (12.5 ng/kg/min from the time of heparinization until onset of cardiopulmonary bypass and 25 ng/kg/min during cardiopulmonary bypass). No deaths or major neurologic complications occurred in this series. Ninety-six patients completed the psychologic and neurologic evaluations 1 week after operation; 74 of these patients were evaluated psychologically 2 months after operation. Psychologic testing demonstrated similar declines in postoperative performance in both the prostacyclin-treated and the control groups; these changes were no longer present in either group 2 months after operation. Results of neurologic examinations and computed tomographic scans of the brain were unchanged. We conclude that the administration of prostacyclin during cardiopulmonary bypass in patients undergoing routine coronary artery operation has no effect on perioperative cognitive changes.

A cluster of severe postoperative bleeding following open heart surgery.

OBJECTIVE: To investigate a cluster of postoperative bleeding following open heart surgery. DESIGN: A cohort and case/control study. SETTING: Palo Alto Veterans Administration Medical Center, Palo Alto, California. PARTICIPANTS: Six (21.4%) of 28 patients undergoing open heart surgery who developed severe, nonsurgical, postoperative bleeding from July 1 through August 30, 1988 (outbreak period). All case-patients had chest tube drainage of greater than or equal to 1000 ml within 4 hours of surgery but did not have identifiable bleeding vessel(s) on exploration. RESULTS: Upon comparison of the pre-outbreak (January 1986 through June 1988) and the outbreak period, a significant increase was found in the incidence of postoperative nonsurgical bleeding (5/440 versus 6/28, p = .0006), but not of postoperative surgical bleeding (8/440 versus 0/28, p = 1.0). Of all patients undergoing open heart surgery during the outbreak period, case patients were found to be older (67.8 versus 60.6, p = .02) and to have received a larger volume of hetastarch (HES), a synthetic colloidal plasma-volume expander (mean = 19.4 ml/kg versus 14.1 ml/kg, p = .02). CONCLUSIONS: We conclude that the use of large volumes of HES during surgery in the elderly open heart surgery patient may increase the risk for severe, nonsurgical postoperative bleeding, probably caused by alterations of the coagulation system. As the incidence of open heart surgery increases among the elderly, surgeons and anesthesiologists should be alert to possible adverse reactions from exposures not associated with adverse reactions in younger patients.

Blood glucose profiles in surgically prepared flaxedilized and anesthetized rats.

Blood glucose profiles were monitored over an extended period of time in surgical rat preparations which included femoral artery and vein cannulations, tracheotomy, carotid artery cannulation and a craniotomy to expose the brain. Rats were under pentobarbital anesthesia, urethane anesthesia or Flaxedil neuromuscular blockage and local anesthesia, Fasted and unfasted rats were compared. These profiles are a foundation for studies of the central nervous system control of feeding behavior and metabolic homeostasis and of hypothalamic glucoreceptors, for which knowledge of the baseline blood glucose profile is particularly important. Such studies utilize the surgical preparations examined here. The importance of rapid "on the spot" "moment to moment" monitoring of blood glucose during the course of an experiment and adjustment of the experiment accordingly is demonstrated. Methods for such monitoring are evaluated in comparison with the standard enzymatic fluorometric assay for glucose. The YSI Glucose Analyzer seems to be a suitable one and is feasible for physiological and psychological laboratories without biochemical expertise.

Effects of bipiperidyl mustard (BPM) lesions on insulin hypoglycemic convulsions.

Systemic gold thioglucose (GTG) is well known to produce hyperphagia, resulting in obesity, and histological damage focused relatively selectively in the ventromedial hypothalamus (VMH). Although structurally very different, bipiperidyl mustard (BPM) produces apparently similar effects. However, a proposed mechanism for concentration and hence localization of GTG toxicity depends on its structural similarity to glucose, binding it to glucoreceptors and focusing the cytotoxicity of the gold thio-portion. We recently showed that GTG treatment also produces an early decrease and a later increase in sensitivity to insulin hypoglycemic convulsions. We report here that BPM also produces a similar biphasic change in sensitivity to insulin hypoglucemic convulsions. For both, the differences are in the brain's convulsive response to hypoglycemia, rather than in the degree of hypoglycemia in response to insulin. Thus, GTG and BPM cytotoxic lesions appear similar in this regard as well. BPM is another way of producing a relatively discrete brain lesion which alters the brain's functional adjustment to hypoglycemia. The significance of this control center and its relationship to the control(s) of feeding and systemic metabolism are discussed.

Anesthesia crisis resource management training: teaching anesthesiologists to handle critical incidents.

The authors have developed a course in Anesthesia Crisis Resource Management (ACRM) analogous to courses in Crew (Cock-pit) Resource Management (CRM) conducted in commercial and military aviation. Anesthesiologists do not typically receive formal training in crisis management although they are called upon to manage life-threatening crises at a moment's notice. Two model demonstration courses in ACRM were conducted using a realistic anesthesia simulation system to test the feasibility and acceptance of this kind of training. Anesthesiologists received didactic instruction in dynamic decision-making, human performance issues in anesthesia, and in the principles of anesthesia crisis resource management. After familiarization with the host institution's operating rooms and with the simulation environment, they underwent a 2-h simulation session followed by a debriefing session which used a videotape of their simulator performance. Participants rated the course as intense, helpful to their practice of anesthesiology, and highly enjoyable. Several aspects of the course were highly rated, including: videotapes of actual anesthetic mishaps, simulation sessions, and debriefing sessions. Scores on written tests of knowledge about anesthesia crisis management showed a significant improvement following the first course (residents) but not the second course (experienced anesthesiologists). Although the ultimate utility of this training for anesthesiologists cannot easily be determined, the course appeared to be a useful method for addressing important issues of anesthesiologist performance which have previously been dealt with haphazardly. The authors believe that ACRM training should become a regular part of the initial and continuing education of anesthesiologists.

Suicide awareness at the elementary school level.

1. Suicidal behavior is a real and growing problem in elementary school-age children. 2. Childhood suicides often are mistaken for accidents. 3. Knowledge is an effective tool in preventing suicides. It is imperative that suicide awareness and orientation for all school staff and parents be initiated at the elementary school level.

JNK modulates FOXO3a for the expression of the mitochondrial death and mitophagy marker BNIP3 in pathological hypertrophy and in heart failure.

Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) is a mitochondrial death and mitophagy marker, which is involved in inducing cardiac remodeling post myocardial infarction. In this study, we show that BNIP3 expression increases in stressed cardiomyocytes in vitro and in response to pressure overload in vivo, and that its transcription is directly related to JNK activity. BNIP3 expression gradually increased in the first weeks after pressure overload and peaked at the heart failure stage. Ultrastructurally, the mitochondrial area was inversely proportional to BNIP3 expression. Both JNK and AKT activities increased with pressure overload; however, JNK signaling dominated over AKT signaling for the activation of the transcription factor FOXO3a and for the transcription of its effector, BNIP3. 3-methyladenine attenuated JNK signaling and significantly decreased BNIP3 expression and reversed cardiac remodeling in heart failure. Ultrastructurally, the mitochondrial area was significantly increased in the 3-methyladenine group compared with placebo. Moreover, adenoviral gene delivery of dominant negative JNK in a rat model of pressure overload hypertrophy abolished the increase in BNIP3 expression in response to pressure overload. These results suggest that JNK signaling is a critical modulator of the transcription factor FOXO3a driving the expression of its effector, BNIP3, in heart failure and that JNK, through BNIP3, induces mitochondrial apoptosis and mitophagy.

Relationship of cannabinoid CB1 receptor and cholecystokinin immunoreactivity in monkey dorsolateral prefrontal cortex.

Exposure to cannabis impairs cognitive functions reliant on the circuitry of the dorsolateral prefrontal cortex (DLPFC) and increases the risk of schizophrenia. The actions of cannabis are mediated via the brain cannabinoid 1 receptor (CB1R), which in rodents is heavily localized to the axon terminals of cortical GABA basket neurons that contain cholecystokinin (CCK). Differences in the laminar distribution of CB1R-immunoreactive (IR) axons have been reported between rodent and monkey neocortex, suggesting that the cell type(s) containing CB1Rs, and the synaptic targets of CB1R-IR axon terminals, may differ across species; however, neither the relationship of CB1Rs to CCK-containing interneurons, nor the postsynaptic targets of CB1R and CCK axon terminals, have been examined in primate DLPFC. Consequently, we compared the distribution patterns of CB1R- and CCK-IR structures, determined the proportions of CB1R and CCK neurons that were dual-labeled, and identified the synaptic types and postsynaptic targets of CB1R- and CCK-IR axon terminals in macaque monkey DLPFC. By light microscopy, CB1R- and CCK-IR axons exhibited a similar laminar distribution, with their greatest densities in layer 4. Dual-label fluorescence experiments demonstrated that 91% of CB1R-IR neurons were immunopositive for CCK, whereas only 51% of CCK-IR neurons were immunopositive for CB1R. By electron microscopy, all synapses formed by CB1R-IR axon terminals were symmetric, whereas CCK-IR axon terminals formed both symmetric (88%) and asymmetric (12%) synapses. The primary postsynaptic target of both CB1R- and CCK-IR axon terminals forming symmetric synapses was dendritic shafts (81-88%), with the remainder targeting cell bodies or dendritic spines. Thus, despite species differences in laminar distribution, CB1Rs are principally localized to CCK basket neuron axons in both rodent neocortex and monkey DLPFC. These axons target the perisomatic region of pyramidal neurons, providing a potential anatomical substrate for the impaired function of the DLPFC associated with cannabis use and schizophrenia.

Effect of hydration state on testosterone and cortisol responses to training-intensity exercise in collegiate runners.

Exercise intensity powerfully influences testosterone, cortisol, and testosterone : cortisol ratio (T:C) responses to endurance exercise. Hydration state may also modulate these hormones, and therefore may alter the anabolic/catabolic balance in response to endurance exercise and training. This study examined the effect of running intensity on testosterone, cortisol, and T : C when exercise was initiated in a hypohydrated state. Nine male collegiate runners (age = 20 +/- 0 y, height = 178 +/- 2 cm, mass = 67.0 +/- 1.8 kg, body fat % = 9.8 +/- 0.7 %, V.O2max = 65.7 +/- 1.1 ml.kg (-1).min (-1)) completed four 10-min treadmill runs differing in pre-exercise hydration status (euhydrated, or hypohydrated by 5 % of body mass) and exercise intensity (70 % or 85 % V.O2max). Body mass, urine osmolality, and urine-specific gravity documented fluid balance; blood samples drawn pre-, immediately post-, and 20 min post-exercise were analyzed for testosterone, cortisol, and T : C. Except for heart rate measured during the 70 % V.O2max trials, heart rate, V.O2, and plasma lactate were similar between euhydrated and hypohydrated conditions for a given intensity, suggesting hypohydration did not measurably increase the physiological stress of the exercise bouts. Furthermore, hydration state had no measurable effect on testosterone concentrations before, during, or after exercise at either intensity. Regardless of exercise intensity, cortisol concentrations were greater during hypohydration than euhydration pre-exercise and 20 min post-exercise. Additionally, T : C was significantly lower 20 min post-exercise at 70 % V.O2max when subjects were initially hypohydrated (T : C = 0.055) versus euhydrated (T : C = 0.072). These findings suggest that depending on exercise intensity, T : C may be altered by hydration state, therefore influencing the balance between anabolism and catabolism in response to running exercise performed at typical training intensities.

Influence of aroclor 1242 concentration on polychlorinated biphenyl biotransformations in hudson river test tube microcosms.

When 93.3 to 933 (mu)mol of Aroclor 1242 per kg was added to Hudson River sediment test tube microcosms, the rates of polychlorinated biphenyl biotransformations increased with increasing Aroclor 1242 concentration after a 4- to 8-week acclimation period. In contrast, when 37.3 (mu)mol of Aroclor 1242 per kg was added, polychlorinated biphenyl biotransformations occurred at slow constant rates.