Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling.

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor-mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine-induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada -/- and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder.

Neuronal and axonal degeneration in experimental spinal cord injury: in vivo proton magnetic resonance spectroscopy and histology.

Longitudinal in vivo proton magnetic resonance spectroscopy (1H-MRS) and immunohistochemistry were performed to investigate the tissue degeneration in traumatically injured rat spinal cord rostral and caudal to the lesion epicenter. On 1H-MRS significant decreases in N-acetyl aspartate (NAA) and total creatine (Cr) levels in the rostral, epicenter, and caudal segments were observed by 14 days, and levels remained depressed up to 56 days post-injury (PI). In contrast, the total choline (Cho) levels increased significantly in all three segments by 14 days PI, but recovered in the epicenter and caudal, but not the rostral region, at 56 days PI. Immunohistochemistry demonstrated neuronal cell death in the gray matter, and reactive astrocytes and axonal degeneration in the dorsal, lateral, and ventral white-matter columns. These results suggest delayed tissue degeneration in regions both rostrally and caudally from the epicenter in the injured spinal cord tissue. A rostral-caudal asymmetry in tissue recovery was seen both on MRI-observed hyperintense lesion volume and the Cho, but not NAA and Cr, levels at 56 days PI. These studies suggest that dynamic metabolic changes take place in regions away from the epicenter in injured spinal cord.

Outcomes of safety and effectiveness in a multicenter randomized, controlled trial of whole-body hypothermia for neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: Whole-body hypothermia reduced the frequency of death or moderate/severe disabilities in neonates with hypoxic-ischemic encephalopathy in a randomized, controlled multicenter trial. OBJECTIVE: Our goal was to evaluate outcomes of safety and effectiveness of hypothermia in infants up to 18 to 22 months of age. DESIGN/METHODS: A priori outcomes were evaluated between hypothermia (n = 102) and control (n = 106) groups. RESULTS: Encephalopathy attributable to causes other than hypoxia-ischemia at birth was not noted. Inotropic support (hypothermia, 59% of infants; control, 56% of infants) was similar during the 72-hour study intervention period in both groups. Need for blood transfusions (hypothermia, 24%; control, 24%), platelet transfusions (hypothermia, 20%; control, 12%), and volume expanders (hypothermia, 54%; control, 49%) was similar in the 2 groups. Among infants with persistent pulmonary hypertension (hypothermia, 25%; control, 22%), nitric-oxide use (hypothermia, 68%; control, 57%) and placement on extracorporeal membrane oxygenation (hypothermia, 4%; control, 9%) was similar between the 2 groups. Non-central nervous system organ dysfunctions occurred with similar frequency in the hypothermia (74%) and control (73%) groups. Rehospitalization occurred among 27% of the infants in the hypothermia group and 42% of infants in the control group. At 18 months, the hypothermia group had 24 deaths, 19 severe disabilities, and 2 moderate disabilities, whereas the control group had 38 deaths, 25 severe disabilities, and 1 moderate disability. Growth parameters were similar between survivors. No adverse outcomes were noted among infants receiving hypothermia with transient reduction of temperature below a target of 33.5 degrees C at initiation of cooling. There was a trend in reduction of frequency of all outcomes in the hypothermia group compared with the control group in both moderate and severe encephalopathy categories. CONCLUSIONS: Although not powered to test these secondary outcomes, whole-body hypothermia in infants with encephalopathy was safe and was associated with a consistent trend for decreasing frequency of each of the components of disability.

Timely follow-up of abnormal diagnostic imaging test results in an outpatient setting: are electronic medical records achieving their potential?

BACKGROUND: Given the fragmentation of outpatient care, timely follow-up of abnormal diagnostic imaging results remains a challenge. We hypothesized that an electronic medical record (EMR) that facilitates the transmission and availability of critical imaging results through either automated notification (alerting) or direct access to the primary report would eliminate this problem. METHODS: We studied critical imaging alert notifications in the outpatient setting of a tertiary care Department of Veterans Affairs facility from November 2007 to June 2008. Tracking software determined whether the alert was acknowledged (ie, health care practitioner/provider [HCP] opened the message for viewing) within 2 weeks of transmission; acknowledged alerts were considered read. We reviewed medical records and contacted HCPs to determine timely follow-up actions (eg, ordering a follow-up test or consultation) within 4 weeks of transmission. Multivariable logistic regression models accounting for clustering effect by HCPs analyzed predictors for 2 outcomes: lack of acknowledgment and lack of timely follow-up. RESULTS: Of 123 638 studies (including radiographs, computed tomographic scans, ultrasonograms, magnetic resonance images, and mammograms), 1196 images (0.97%) generated alerts; 217 (18.1%) of these were unacknowledged. Alerts had a higher risk of being unacknowledged when the ordering HCPs were trainees (odds ratio [OR], 5.58; 95% confidence interval [CI], 2.86-10.89) and when dual-alert (>1 HCP alerted) as opposed to single-alert communication was used (OR, 2.02; 95% CI, 1.22-3.36). Timely follow-up was lacking in 92 (7.7% of all alerts) and was similar for acknowledged and unacknowledged alerts (7.3% vs 9.7%; P = .22). Risk for lack of timely follow-up was higher with dual-alert communication (OR, 1.99; 95% CI, 1.06-3.48) but lower when additional verbal communication was used by the radiologist (OR, 0.12; 95% CI, 0.04-0.38). Nearly all abnormal results lacking timely follow-up at 4 weeks were eventually found to have measurable clinical impact in terms of further diagnostic testing or treatment. CONCLUSIONS: Critical imaging results may not receive timely follow-up actions even when HCPs receive and read results in an advanced, integrated electronic medical record system. A multidisciplinary approach is needed to improve patient safety in this area.

Postoperative renal function preservation with nonischemic femoral arterial cannulation for thoracoabdominal aortic repair.

BACKGROUND: Renal failure after thoracoabdominal aortic repair is a significant clinical problem. Distal aortic perfusion for organ and spinal cord protection requires cannulation of the left femoral artery. In 2006, we reported the finding that direct cannulation led to leg ischemia in some patients and was associated with increased renal failure. After this finding, we modified our perfusion technique to eliminate leg ischemia from cannulation. In this article, we present the effects of this change on postoperative renal function. METHODS: Between February 1991 and July 2008, we repaired 1464 thoracoabdominal aortic aneurysms. Distal aortic perfusion was used in 1088, and these were studied. Median patient age was 68 years, and 378 (35%) were women. In September 2006, we began to adopt a sidearm femoral cannulation technique that provides distal aortic perfusion while maintaining downstream flow to the leg. This was used in 167 patients (15%). We measured the joint effects of preoperative glomerular filtration rate (GFR) and cannulation technique on the highest postoperative creatinine level, postoperative renal failure, and death. Analysis was by multiple linear or logistic regression with interaction. RESULTS: The preoperative GFR was the strongest predictor of postoperative renal dysfunction and death. No significant main effects of sidearm cannulation were noted. For peak creatinine level and postoperative renal failure, however, strong interactions between preoperative GFR and sidearm cannulation were present, resulting in reductions of postoperative renal complications of 15% to 20% when GFR was <60 mL>/min/1.73 m(2). For normal GFR, the effect was negated or even reversed at very high levels of GFR. Mortality, although not significantly affected by sidearm cannulation, showed a similar trend to the renal outcomes. CONCLUSION: Use of sidearm cannulation is associated with a clinically important and highly statistically significant reduction in postoperative renal complications in patients with a low GFR. Reduced renal effect of skeletal muscle ischemia is the proposed mechanism. Effects among patients with good preoperative renal function are less clear. A randomized trial is needed.

Sulforaphane improves cognitive function administered following traumatic brain injury.

Recent studies have shown that sulforaphane, a naturally occurring compound that is found in cruciferous vegetables, offers cellular protection in several models of brain injury. When administered following traumatic brain injury (TBI), sulforaphane has been demonstrated to attenuate blood-brain barrier permeability and reduce cerebral edema. These beneficial effects of sulforaphane have been shown to involve induction of a group of cytoprotective, Nrf2-driven genes, whose protein products include free radical scavenging and detoxifying enzymes. However, the influence of sulforaphane on post-injury cognitive deficits has not been examined. In this study, we examined if sulforaphane, when administered following cortical impact injury, can improve the performance of rats tested in hippocampal- and prefrontal cortex-dependent tasks. Our results indicate that sulforaphane treatment improves performance in the Morris water maze task (as indicated by decreased latencies during learning and platform localization during a probe trial) and reduces working memory dysfunction (tested using the delayed match-to-place task). These behavioral improvements were only observed when the treatment was initiated 1h, but not 6h, post-injury. These studies support the use of sulforaphane in the treatment of TBI, and extend the previously observed protective effects to include enhanced cognition.

Long-lasting hyperexcitability induced by depolarization in the absence of detectable Ca2+ signals.

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

Synaptic vesicle dynamics in mouse rod bipolar cells.

To better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of 7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.

Rationale for reading fluconazole MICs at 24 hours rather than 48 hours when testing Candida spp. by the CLSI M27-A2 standard method.

We investigated if CLSI M27-A2 Candida species breakpoints for fluconazole MIC are valid when read at 24 h. Analysis of a data set showed good correlation between 48- and 24-h MICs, as well as similar outcomes and pharmacodynamic efficacy parameters, except for isolates in the susceptible dose-dependent category, such as Candida glabrata.

Treatment evolution in high-risk congenital diaphragmatic hernia: ten years' experience with diaphragmatic agenesis.

OBJECTIVE: The objective of this study was to evaluate the impact of newer therapies on the highest risk patients with congenital diaphragmatic hernia (CDH), those with agenesis of the diaphragm. SUMMARY BACKGROUND DATA: CDH remains a significant cause of neonatal mortality. Many novel therapeutic interventions have been used in these infants. Those children with large defects or agenesis of the diaphragm have the highest mortality and morbidity. METHODS: Twenty centers from 5 countries collected data prospectively on all liveborn infants with CDH over a 10-year period. The treatment and outcomes in these patients were examined. Patients were followed until death or hospital discharge. RESULTS: A total of 1,569 patients with CDH were seen between January 1995 and December 2004 in 20 centers. A total of 218 patients (14%) had diaphragmatic agenesis and underwent repair. The overall survival for all patients was 68%, while survival was 54% in patients with agenesis. When patients with diaphragmatic agenesis from the first 2 years were compared with similar patients from the last 2 years, there was significantly less use of ECMO (75% vs. 52%) and an increased use of inhaled nitric oxide (iNO) (30% vs. 80%). There was a trend toward improved survival in patients with agenesis from 47% in the first 2 years to 59% in the last 2 years. The survivors with diaphragmatic agenesis had prolonged hospital stays compared with patients without agenesis (median, 68 vs. 30 days). For the last 2 years of the study, 36% of the patients with agenesis were discharged on tube feedings and 22% on oxygen therapy. CONCLUSIONS: There has been a change in the management of infants with CDH with less frequent use of ECMO and a greater use of iNO in high-risk patients with a potential improvement in survival. However, the mortality, hospital length of stay, and morbidity in agenesis patients remain significant.

Temporal Control of Leaf Complexity by miRNA-Regulated Licensing of Protein Complexes

The tremendous diversity of leaf shapes has caught the attention of naturalists for centuries. In addition to interspecific and intraspecific differences, leaf morphologies may differ in single plants according to age, a phenomenon known as heteroblasty. In Arabidopsis thaliana, the progression from the juvenile to the adult phase is characterized by increased leaf serration. A similar trend is seen in species with more complex leaves, such as the A. thaliana relative Cardamine hirsuta, in which the number of leaflets per leaf increases with age. Although the genetic changes that led to the overall simpler leaf architecture in A. thaliana are increasingly well understood, less is known about the events underlying age-dependent changes within single plants, in either A. thaliana or C. hirsuta. Here, we describe a conserved miRNA transcription factor regulon responsible for an age-dependent increase in leaf complexity. In early leaves, miR319-targeted TCP transcription factors interfere with the function of miR164-dependent and miR164-independent CUC proteins, preventing the formation of serrations in A. thaliana and of leaflets in C. hirsuta. As plants age, accumulation of miR156-regulated SPLs acts as a timing cue that destabilizes TCP-CUC interactions. The destabilization licenses activation of CUC protein complexes and thereby the gradual increase of leaf complexity in the newly formed organs. These findings point to posttranslational interaction between unrelated miRNA-targeted transcription factors as a core feature of these regulatory circuits.

Whey protein isolate decreases murine stomach weight and intestinal length and alters the expression of Wnt signalling-associated genes

Acknowledgements K. N. N. was supported by the Teagasc Vision Programme on Obesity (RMIS5974). L. M. was supported by the Teagasc Walsh Fellowship. J. R. S. was supported by a 1000-talents professorship from the Chinese government. The funding bodies had no input on the design of the study or in the interpretation of the data. The authors’ contributions are as follows: L. M., J. R. S., J. F. C. and K. N. N. designed the study; K. N. N. and J. F. C. obtained ethical approval for the study; L. M. performed the experiments; L. M. and J. R. S. analysed the data; L. M. generated the figures. All authors contributed to the drafting of the manuscript. All authors approved the final version for submission. The authors declare that there is no competing interest.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.

Temporomandibular joint disorder (TMJD) is known for its mastication-associated pain. TMJD is medically relevant because of its prevalence, severity, chronicity, the therapy-refractoriness of its pain, and its largely elusive pathogenesis. Against this background, we sought to investigate the pathogenetic contributions of the calcium-permeable TRPV4 ion channel, robustly expressed in the trigeminal ganglion sensory neurons, to TMJ inflammation and pain behavior. We demonstrate here that TRPV4 is critical for TMJ-inflammation-evoked pain behavior in mice and that trigeminal ganglion pronociceptive changes are TRPV4-dependent. As a quantitative metric, bite force was recorded as evidence of masticatory sensitization, in keeping with human translational studies. In Trpv4(-/-) mice with TMJ inflammation, attenuation of bite force was significantly less than in wildtype (WT) mice. Similar effects were seen with systemic application of a specific TRPV4 inhibitor. TMJ inflammation and mandibular bony changes were apparent after injections of complete Freund adjuvant but were remarkably independent of the Trpv4 genotype. It was intriguing that, as a result of TMJ inflammation, WT mice exhibited significant upregulation of TRPV4 and phosphorylated extracellular-signal-regulated kinase (ERK) in TMJ-innervating trigeminal sensory neurons, which were absent in Trpv4(-/-) mice. Mice with genetically-impaired MEK/ERK phosphorylation in neurons showed resistance to reduction of bite force similar to that of Trpv4(-/-) mice. Thus, TRPV4 is necessary for masticatory sensitization in TMJ inflammation and probably functions upstream of MEK/ERK phosphorylation in trigeminal ganglion sensory neurons in vivo. TRPV4 therefore represents a novel pronociceptive target in TMJ inflammation and should be considered a target of interest in human TMJD.