The mitochondrial RNA granule modulates manganese-dependent cell toxicity.

Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and nonadaptive cellular processes targeted by manganese remains mostly unexplored. Here we study mechanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinson's gene PARK2. We found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule composition and function, resulting in disruption of mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. We conclude that impairment of the mitochondrial RNA granule function is a protective mechanism for acute manganese toxicity.

Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.

Painful, palpable and pathological: anomalous flexor digitorum superficialis brevis in the palm, comparative anatomical context, and an updated classification of anomalies of the flexor digitorum superficialis.

Anomalies of the flexor digitorum superficialis are rare and can present a diagnostic dilemma. Patients present with a painful or palpable mass, or symptoms of carpal tunnel syndrome. This review article summarizes previously reported anomalies of the flexor digitorum superficialis, reports a further case, and proposes a new classification.

Modelling catchment management impact on in-stream phosphorus loads in northern Victoria.

Phosphorus pollution severely impairs the water quality of rivers in Australia and worldwide. Conceptual models have proved useful to assess management impact on phosphorus loads, particularly in data-sparse environments. This paper develops and evaluates the coupling of a point-scale model (HowLeaky2008) to a catchment scale model (CatchMODS) to enhance modelling of farm management impacts on in-stream phosphorus loads. The model was tested in two adjacent catchments in northern Victoria (Avon-Richardson and Avoca), Australia. After calibration of the in-stream attenuation parameter against measurements at gauging stations, the model simulated specific annual phosphorus loads across the catchments well (Nash-Sutcliffe model efficiency of 0.52 in the Avon-Richardson and 0.83 for the Avoca catchment). Phosphorus loads at both catchment outlets under current conditions were estimated at 7 t y(-1) and were dominated by field exports. Changes to farm management practices, i.e. the use of perennial pastures in grazing systems and zero-tillage in cropping systems were estimated to reduce phosphorus load by 31% in the Avon-Richardson catchment and 19% in the Avoca catchment, relative to current practices (annual pasture and minimum tillage). The model afforded a major improvement in conceptual modelling by explicit simulation of the impacts of soil and climatic conditions on field-scale exports and by placing them in the context of landscape processes.

Interplay of endothelial and inducible nitric oxide synthases modulates the vascular response to ischaemia-reperfusion in the rabbit lung.

AIM: Lung ischaemia-reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia-reperfusion-induced pulmonary vasoconstriction. The aim was to determine whether ischaemia-reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion. METHODS: Ischaemia-reperfusion was induced for various times in anesthetized rabbits with ventilated lungs by reversibly occluding the right pulmonary artery and initiating reperfusion. Nitric oxide synthase activity/expression and phosphorylation, reactive nitrogen species generation and total nitrate/nitrite were determined in lung tissue. RESULTS: Inducible nitric oxide synthase expression and activity, and reactive nitrogen species formation coincided with increased pulmonary vascular resistance during reperfusion and increased with ischaemia duration, further increasing after 2-h reperfusion. Total nitrate/nitrite also increased with ischaemia but decreased after 2-h reperfusion. Pre-treatment with an inducible nitric oxide synthase inhibitor (1400W; Cayman Chemical Company, Ann Arbor, MI, USA) attenuated inducible nitric oxide synthase activity, reactive nitrogen species generation and pulmonary vascular resistance, but did not affect total nitrate/nitrite. Endothelial nitric oxide synthase expression was unchanged by ischaemia-reperfusion; however, its phosphorylation on serine 1177 and dephosphorylation on threonine 495 was uncoupled, suggesting decreased endothelial nitric oxide synthase activity. 1400W prevented uncoupling of endothelial nitric oxide synthase phosphorylation, maintaining its activity during reperfusion. CONCLUSION: Ischaemia-reperfusion up-regulates inducible nitric oxide synthesis and/activity, which coincides with reduced endothelial nitric oxide synthase activity as suggested by its uncoupling and may contribute to ischaemia-reperfusion-induced pulmonary vasoconstriction.

Hypertonicity activates pulmonary vagal afferents independently of vasoconstriction.

Injecting hypertonic saline into the lung periphery causes a vagally mediated neural hyperpnea and tachypnea (the excitatory lung reflex, ELR). In the present study, we tested the hypothesis that hypertonic saline activates lung afferents mainly by increasing fluid flux from pulmonary vessels into the alveoli. If our hypothesis is correct, reducing perfusion of the vagal sensory region will reduce the fluid flux and attenuate the ELR. In anesthetized, open chest and mechanically ventilated rabbits, using intravital video microscopy, we confirmed that topical KCl (100 mM) constricted sub-pleural blood vessels and limited blood flow significantly, as indicated by a 43.3±9% decrease in arteriolar diameters (p<0.005), sluggish microvascular flow and paleness of alveolar walls. Then, we compared respiratory responses (assessed from phrenic nerve activity) to injections of hypertonic saline (8.1%, 0.1 ml) into the lung periphery before and after locally injecting KCl to limit fluid flux. The respiratory responses were the same with or without vasoconstriction. However, the responses were significantly decreased (from 22±5% to 1±2% for phrenic amplitude and from 75±9% to 13±6% for phrenic burst rate; n=14, p<0.02) after local injection of 2% lidocaine to block sensory endings. Since the ELR was not attenuated by vasoconstriction, increased transvascular fluid flux does not appear to be a major mechanism for hypertonic saline induced ELR.

The use of µCT technology to identify skull fracture in a case involving blunt force trauma.

A 40-year-old man was admitted to hospital with a scalp wound but died 22 days later after unsuccessful treatment. Initial assessment of the cranial fragments removed during surgery revealed fine fracture lines on the endocranial surface, and a dark arcuate line on the ectocranial surface. To investigate the extent of the fractures a µCT scan of the fragments was taken, examined in 3D, and compared to plain radiographs. Some fractures were found to extend through the full thickness of the skull. This case presents a novel application of µCT technology to forensic radiology.

The mechanics of gravitaxis in Paramecium.

An analysis of swimming patterns in the ciliate Paramecium shows that the ability to swim preferentially upwards (negative gravitaxis) is primarily the result of upwardly curving trajectories. The trajectory characteristics are consistent with those produced by mechanical orientation. Cell profile measurements from microscope images suggest that the characteristic front-rear body asymmetry accounts for the observed orientation rates. Gravikinesis may result from interactions between the propelling cilia and the sedimentary flow around the cell, and it seems unlikely that an internal physiological gravity receptor exists in Paramecium.

Mechanisms of fibrinogen-induced microvascular dysfunction during cardiovascular disease.

Fibrinogen (Fg) is a high molecular weight plasma adhesion protein and a biomarker of inflammation. Many cardiovascular and cerebrovascular disorders are accompanied by increased blood content of Fg. Increased levels of Fg result in changes in blood rheological properties such as increases in plasma viscosity, erythrocyte aggregation, platelet thrombogenesis, alterations in vascular reactivity and compromises in endothelial layer integrity. These alterations exacerbate the complications in peripheral blood circulation during cardiovascular diseases such as hypertension, diabetes and stroke. In addition to affecting blood viscosity by altering plasma viscosity and erythrocyte aggregation, growing experimental evidence suggests that Fg alters vascular reactivity and impairs endothelial cell layer integrity by binding to its endothelial cell membrane receptors and activating signalling mechanisms. The purpose of this review is to discuss experimental data, which demonstrate the effects of Fg causing vascular dysfunction and to offer possible mechanisms for these effects, which could exacerbate microcirculatory complications during cardiovascular diseases accompanied by increased Fg content.

Lower-crustal intrusion on the North Atlantic continental margin.

When continents break apart, the rifting is sometimes accompanied by the production of large volumes of molten rock. The total melt volume, however, is uncertain, because only part of it has erupted at the surface. Furthermore, the cause of the magmatism is still disputed-specifically, whether or not it is due to increased mantle temperatures. We recorded deep-penetration normal-incidence and wide-angle seismic profiles across the Faroe and Hatton Bank volcanic margins in the northeast Atlantic. Here we show that near the Faroe Islands, for every 1 km along strike, 360-400 km(3) of basalt is extruded, while 540-600 km(3) is intruded into the continent-ocean transition. We find that lower-crustal intrusions are focused mainly into a narrow zone approximately 50 km wide on the transition, although extruded basalts flow more than 100 km from the rift. Seismic profiles show that the melt is intruded into the lower crust as sills, which cross-cut the continental fabric, rather than as an 'underplate' of 100 per cent melt, as has often been assumed. Evidence from the measured seismic velocities and from igneous thicknesses are consistent with the dominant control on melt production being increased mantle temperatures, with no requirement for either significant active small-scale mantle convection under the rift or the presence of fertile mantle at the time of continental break-up, as has previously been suggested for the North Atlantic Ocean.

The anatomy demonstrator of the future: an examination of the role of the medically-qualified anatomy demonstrator in the context of tomorrow's doctors and modernizing medical careers.

In 1993, the UK General Medical Council published Tomorrow's Doctors leading to a nationwide restructuring of undergraduate medical courses. Traditional courses with distinct pre-clinical and clinical phases gave way to a more integrated approach to undergraduate medical education, with an emphasis on the quality and variety of teaching provided. More than a decade after Tomorrow's Doctors, postgraduate medical training is being transformed. Modernising Medical Careers is leading to the introduction of a two-year Foundation Programme, with subsequent streamlined specialist training. In the context of these changes, we consider how the creation of posts for medically-qualified anatomy demonstrators would present an opportunity to fulfil needs in both undergraduate education and postgraduate training. We outline the threats facing established posts, and how these problems may be resolved. We hope that this overview of the challenges facing undergraduate and postgraduate education in the UK, with particular reference to anatomy, may offer some useful insight to teachers and learners in other countries. We conclude that the role of the medically-qualified anatomy demonstrator has proved valuable in the context of Tomorrow's Doctors, and that this role can evolve and expand as part of the 21st century "modern medical career."

Homotypic association between tumour-associated VHL proteins leads to the restoration of HIF pathway.

The von Hippel-Lindau (VHL) tumour suppressor gene encodes a substrate-specifying component of an E3 ubiquitin ligase that targets hypoxia-inducible factor (HIF) alpha subunits for degradation under normoxia. The VHL protein is composed of an N-terminal HIFalpha-binding beta domain and a C-terminal alpha domain, which is necessary and sufficient for the formation of the E3 multiprotein enzyme. A large number of disease-causing mutations in either the alpha or beta domain renders HIFalpha stable irrespective of oxygen tension, leading to the upregulation of numerous HIF-target genes, such as GLUT1 and VEGF. Here, we show that VHL forms a self-associated complex in vivo, but not in vitro, and demonstrate that coexpression of two different VHL missense mutants -- one in the alpha domain and the other in the beta domain -- restores HIF-mediated gene expression profile. These findings indicate that VHL homotypic complexes can function in vivo in a complementary fashion to target HIFalpha for ubiquitin-mediated proteolysis, and potentially explain why VHL-associated tumours with a missense mutation-carrying VHL allele is almost invariably accompanied by a second VHL allele harbouring a gross truncation or deletion.

Effect of frusemide on bodyweight loss and recovery in racing Standardbreds.

This study was conducted to compare bodyweight (bwt) loss and recovery in Standardbred horses receiving frusemide compared to controls. Thirty Standardbred horses from 7 training stables that were racing at the Red Mile in Lexington, Kentucky, during the spring 2001 pari-mutuel meeting were studied. Fourteen horses (FRU) received frusemide (250 mg/horse i.v.) 4 h prior to racing, while 16 horses (NFRU) did not received frusemide. Horses were weighed on the morning of race day, prior to warm-up, after racing and the next day. Changes in bwt were calculated as percentage increase or decrease from initial bwt. Prior to warm-up, FRU horses had a mean bwt reduction of 1.30 +/- 0.63%, (P<0.01), compared to a 0.19 +/- 0.63% increase for the NFRU horses. FRU horses had a greater (P<0.03) postrace bwt loss (3.55 +/- 0.55%) than the NFRU horses (2.71 +/- 0.66%). On the morning after racing, bwt was still below initial values by 0.90 +/- 0.55 and 1.24 +/- 0.35% for the FRU and the NFRU horses, respectively. While frusemide administration increased the postrace weight loss in racing Standardbreds, it did not affect short-term bwt recovery. Further research is required into management strategies required to allow horses to replenish fluids and electrolytes safely following strenuous exercise.

Bipartite interaction between neurofibromatosis type I protein (neurofibromin) and syndecan transmembrane heparan sulfate proteoglycans.

The neurofibromatosis type 1 (NF1) gene encodes a large tumor suppressor protein (neurofibromin). Although it is known to possess Ras GTPase-activating protein (GAP) activity, the cellular role of neurofibromin remains unclear. Here we used yeast two-hybrid screening to identify neurofibromin-interacting proteins. Syndecan-2, a transmembrane heparan sulfate proteoglycan (HSPG), was isolated as a binding partner for two distinct regions of the neurofibromin protein. We subsequently found that neurofibromin can bind all four mammalian syndecans. NF1 interaction requires the transmembrane domain and a membrane-proximal region of the cytoplasmic tail of syndecan, but not the C terminus of syndecan known to bind to CASK, a membrane-associated guanylate kinase (MAGUK). Neurofibromin, syndecans, and CASK have overlapping subcellular distributions in axons and synapses of neurons, as shown by biochemical fractionation and immunostaining. Moreover, neurofibromin exists in a complex with syndecan and CASK in vivo, as evidenced by their coimmunoprecipitation from rat brain. Our findings suggest that interaction with different members of the syndecan family may be a mechanism for localizing neurofibromin to specialized domains of the plasma membrane.

Chemoreflexes: an experimental study.

HYPOTHESIS: Transmyocardial laser revascularization (TMLR) will not denervate the heart, because it does not destroy all of the afferents. This study was designed to determine if stimulation of cardiac sympathetic and vagal afferents from an area of the left ventricle treated with TMLR could evoke reflex effects, and thus whether TMLR would denervate the heart. METHODS: The effect of TMLR on reflexes evoked by chemically stimulating cardiac afferents was examined in 9 dogs. Bradykinin and capsaicin were applied topically or injected into the left anterior descending coronary artery before and after TMLR and after bilateral vagotomy and sympathectomy. Aortic (AoP) and left ventricular pressures (LVP) and electrocardiography were monitored. The first derivatives of LVP (dP/dt) were calculated. RESULTS: Topical bradykinin elicited variable hemodynamic responses. Topical capsaicin evoked pressor responses, increasing mean (+/- SEM) AoP (105+/-9 to 115+/-9 mm Hg; P<.001) and positive dP/dt (+dP/dt) (1032+/-81 to 1159+/-10 mm Hg/s; P<.01) before TMLR. Intracoronary capsaicin evoked a depressor response before TMLR. Pressor responses remained intact after TMLR with increases in mean AoP and +dP/dt (115+/-6 to 128+/-5 mm Hg and 1039+/-98 to 1136+/-100 mm Hg/s, respectively; P<.01). Depressor responses also remained intact after TMLR (91+/-10 vs 101+/-11 mm Hg [P<.02], and 865+/-104 vs 931+/-104 mm Hg/s [P<.05], respectively). Hemodynamic responses were diminished after bilateral vagotomy and abolished after bilateral sympathectomy. CONCLUSION: Since activation of cardiac afferent nerves and reflex responses remained intact after TMLR, but changed after vagotomy or sympathectomy, TMLR does not denervate the heart sufficiently to be the cause of improved angina after TMLR.

Pulmonary rapidly adapting receptor stimulation does not increase airway resistance in anesthetized rabbits.

In open-chest artificially ventilated rabbits, removal followed by replacement of positive end-expiratory pressure (PEEP maneuver) favors stimulation of airway rapidly adapting receptors (RARs). The purpose of the present study was to determine whether activation of RARs can cause bronchoconstriction. We measured airway pressure, airflow, and tidal volume, and calculated dynamic lung compliance and total lung resistance. PEEP maneuver increased airway pressure swings (16.4 +/- 4% above control; p = 0.0016) and decreased compliance (to 84.8 +/- 2.8% of control; p = 0.0002) without changing resistance (108.0 +/- 4.4% of control; p = 0.85). On the other hand, the resistance increased greatly (93 +/- 13%, p < 0.01) after intravenous injection of acetylcholine or electrical stimulation of vagal efferents, indicating that our system could detect increases in the resistance. In a separate group, we stimulated RARs by stroking the trachea with a cotton tip (tickling), tickling produced cough, manifested by increased pressure and flow without resistance changing. These changes were abolished after paralysis with succinylcholine. Because we did not detect an increase in airflow resistance during activation of RARs by the PEEP maneuver and tickling, we conclude that increase in resistance may not be an important reflex component of airway RARs.

Pulmonary artery occlusion and reperfusion causes microvascular constriction in the rabbit lung.

INTRODUCTION: Reperfusion injury of the lung after ischemia is associated with altered alveolar blood flow and ventilation-perfusion mismatch, which is a significant cause of morbidity and mortality after lung transplantation. We examined the effect of ischemia and reperfusion on the tone of individual subpleural arterioles in the pulmonary circulation by using video microscopy with polarized epiillumination. METHODS: In 11 open-chested rabbits anesthetized with pentobarbital (2.3 to 2.5 kg), we ventilated the lungs through the lower trachea (air or 50% oxygen) and examined the response of subpleural arterioles (diameter 75 +/- 13 microm) to ischemia (76 +/- 32 min) of the right lung caused by occluding the right main pulmonary artery. Observations were made during baseline, occlusion, and during early (20 to 32 min) and late (48 to 63 min) reperfusion using a long working distance lens (550x) with a dipping cone held at the pleural surface while the lungs were statically inflated (10 cm H2O) with oxygen for brief periods. Data are expressed as mean +/- standard deviation. RESULTS: Arteriolar diameter was decreased 57% +/- 19% during early reperfusion. There was a decrease in blood flow and alveolar walls were pale, indicating reduced capillary perfusion. During late reperfusion, arteriolar diameter was diminished (19% +/- 26%); flow was still reduced. Overall pulmonary vascular resistance increased during early reperfusion but returned to baseline level during late reperfusion. Arterial partial pressure of oxygen averaged 200 mm Hg during ischemia and reperfusion. CONCLUSIONS: Constriction of small arterioles by ischemia and reperfusion can have a significant effect on the early phase of ventilation-perfusion mismatch and pulmonary dysfunction by altering alveolar perfusion. This response does not appear to be mediated by hypoxia because it was not prevented by ventilation with oxygen.