Effect of retinol dehydrogenase gene transfer in a novel rat model of Stargardt disease.

Dysfunction of the ATPase-binding Cassette Transporter protein (ABCA4) can lead to early onset macular degeneration, in particular to Stargardt disease. To enable translational research into this form of blindness, we evaluated the effect of Cas9-induced disruptions of the ABCA4 gene to potentially generate new transgenic rat models of the disease. We show that deletion of the short exon preceding the second nucleotide-binding domain is sufficient to drastically knock down protein levels and results in accumulation of retinoid dimers similar to that associated with Stargardt disease. Overexpression of the retinol dehydrogenase enzymes RDH8 and RDH12 can to a limited extent offset the increase in the bisretinoid levels in the Abca4Ex42-/ - KO rats possibly by restricting the time window in which retinal can dimerize before being reduced to retinol. However, in vivo imaging shows that overexpression of RDH8 can induce retinal degeneration. This may be due to the depletion in the outer segment of the cofactor NADPH, needed for RDH function. The translational potential of RDH therapy as well as other Stargardt disease therapies can be tested using the Abca4 knockdown rat model.

Measurement of Serum Immunoglobulins: A National Survey of Practice.

Aim To survey how serum immunoglobulins (IgG, IgA, IgM) are measured and reported across public hospitals in Ireland. Methods We developed a seven-point questionnaire to elicit the methodology, reporting and reference intervals of serum immunoglobulins. It was distributed electronically by email to sixteen laboratory managers of Irish public hospitals. Results A total of twelve questionnaires were completed. The test method was the same in each laboratory, whilst the analyser and source of the reference intervals varied. In some institutions, the reference interval differed within the adult age population and for sex. The IgG parameter contained the highest spread of results. The lower reference limit ranged 5.4-8.0 g/L, with a standard deviation of 0.81, and the upper reference limit ranged 14.9-18.2 g/L (SD 1.04). Conclusion Considerable variation exists in the reference intervals of serum immunoglobulins within different Irish public hospitals. This has important implications for users of the test, patients and referral patterns.

Long-term expression of melanopsin and channelrhodopsin causes no gross alterations in the dystrophic dog retina.

Several preclinical studies have investigated the potential of algal channelrhodopsin and human melanopsin as optogenetic tools for vision restoration. In the present study, we assessed the potentially deleterious effects of long-term expression of these optogenes on the diseased retina in a large animal model of retinal degeneration, the RPE65-deficient Briard dog model of Leber congenital amaurosis. Intravitreal injection of adeno-associated virus vectors expressing channelrhodopsin and melanopsin had no effect on retinal thickness over a 16-month period post injection. Our data support the safety of the optogenetic approach for the treatment of blindness.

Hiding opaque eyes in transparent organisms: a potential role for larval eyeshine in stomatopod crustaceans.

Opaque screening pigments are a fundamental requisite for preserving resolution in image-forming eyes. Possession of any type of image-forming eye in a transparent, pelagic animal will thus undermine the ability of that animal to be invisible in the water column. Transparent, pelagic animals must therefore deal with the trade-off between the ability to see and the ability of other animals to see them. Stomatopod larvae, like many transparent crustaceans, possess specialized optics in their compound eyes that minimize the volume of the opaque retina. Though the volumes of these retinas are reduced, their opacity remains conspicuous to an observer. The light reflected from structures overlying the retinas of stomatopod crustacean larval eyes, referred to here as eyeshine, is hypothesized to further reduce the visibility of opaque retinas. Blue or green wavelengths of light are most strongly reflected in stomatopod larval eyeshine, suggesting a putative spectral matching to the light environment against which the larval eyes are viewed. We tested the efficacy of stomatopod crustacean larval eyeshine as an ocular camouflaging mechanism by photographing larvae in their natural light environment and analysing the contrast of eyes with the background light. To test for spectral matching between stomatopod larval eyeshine and the background light environment, we characterized the spectrum of eyeshine and calculated its performance using radiometric measurements collected at the time of each photographic series. These results are the first to demonstrate an operative mirror camouflage matched in both spectrum and radiance to the pelagic background light environment.

Management of maple syrup urine disease in the peri-operative period.

Maple syrup urine disease (MSUD) has an incidence of 1:125,000 newborns in Ireland. Patients, when fasting, or in a catabolic state build up toxic metabolites leading to progressive neurological dysfunction. We describe the necessary peri-operative management of a patient with MSUD who developed symptomatic gallstones requiring a laparoscopic cholecystectomy.

The molecular genetics and evolution of colour and polarization vision in stomatopod crustaceans.

Stomatopod crustaceans have the most complex assemblage of visual receptor classes known; retinas of many species are thought to express up to 16 different visual pigments. Physiological studies indicate that stomatopods contain up to six distinct middle-wavelength-sensitive (MWS) photoreceptor classes, suggesting that no more than six different MWS opsin gene copies exist per species. However, we previously reported the unexpected expression of 6-15 different MWS genes in retinas of each of five stomatopod species (Visual Neurosci 26: 255-266, 2009). Here, we present a review of the results reported in this publication, plus new results that shed light on the origins of the diverse colour and polarization visual capabilities of stomatopod crustaceans. Using in situ hybridization of opsins in photoreceptor cells, we obtained new results that support the hypothesis of an ancient functional division separating spatial and polarizational vision from colour vision in the stomatopods. Since evolutionary trace analysis indicates that stomatopod MWS opsins have diverged both with respect to spectral tuning and to cytoplasmic interactions, we have now further analyzed these data in an attempt to uncover the origins, diversity and potential specializations among clades for specific visual functions. The presence of many clusters of highly similar transcripts suggests exuberant opsin gene duplication has occurred in the stomatopods, together with more conservative, ancient gene duplication events within the stem crustacean lineage. Phylogenetic analysis of opsin relatedness suggests that opsins specialized for colour vision have diverged from those devoted to polarization vision, and possibly motion and spatial vision.

Speciation and stasis in marine ostracoda: climatic modulation of evolution.

Morphologic and paleozoogeographic analysis of Cenozoic marine Ostracoda from the Atlantic, Caribbean, and Pacific indicates that climatic change modulates evolution by disrupting long-term stasis and catalyzing speciation during sustained, unidirectional climatic transitions and, conversely, by maintaining morphologic stasis during rapid, high-frequency climatic oscillations. In the middle Pliocene, 4 to 3 million years ago, at least six new species of Puriana suddenly appeared as the Isthmus of Panama closed, changing oceanographic circulation and global climate. Since then morphologic stasis has characterized ancestral and descendant species during many glacial-interglacial cycles. The frequency and duration of climatic events have more impact on ostracode evolution than the magnitude of climatic changes.

Tidal vertical migration: an endogenous rhythm in estuarine crab larvae.

Field-caught larvae of the estuarine crab Rhithropanopeus harrisii have a tidal rhythm of vertical migration when maintained in constant conditions. Laboratory-reared larvae do not show this rhythm. Endogenous tidal vertical migrations aid the retention of these planktonic larvae in estuaries near the parent populations.

Crystal structure of the repetitive segments of spectrin.

The elongated proteins of the spectrin family (dystrophin, alpha-actinin, and spectrin) contain tandemly repeated segments and form resilient cellular meshworks by cross-linking actin filaments. The structure of one of the repetitive segments of alpha-spectrin was determined at a 1.8 angstrom resolution. A segment consists of a three-helix bundle. A model of the interface between two tandem segments suggests that hydrophobic interactions between segments may constrain intersegment flexibility. The helix side chain interactions explain how mutations that are known to produce hemolytic anemias disrupt spectrin associations that sustain the integrity of the erythrocyte membrane.

Micropaleontological evidence for increased meridional heat transport in the north atlantic ocean during the pliocene.

The Middle Pliocene ( approximately 3 million years ago) has been identified as the last time the Earth was significantly warmer than it was during the Last Interglacial and Holocene. A quantitative micropaleontological paleotemperature transect from equator to high latitudes in the North Atlantic indicates that Middle Pliocene warmth involved increased meridional oceanic heat transport.

Quaternary climates and sea levels of the u.s. Atlantic coastal plain.

Uranium-series dating of corals from marine deposits of the U.S. Atlantic Coastal Plain coupled with paleoclimatic reconstructions based on ostracode (marine) and pollen (continent) data document at least five relatively warm intervals during the last 500,000 years. On the basis of multiple paleoenvironmental criteria, we determined relative sea level positions during the warm intervals, relative to present mean sea level, were 7 +/- 5 meters at 188,000 years ago, 7.5 +/- 1.5 meters at 120,000 years ago, 6.5 +/- 3.5 meters at 94,000 years ago, and 7 +/- 3 meters at 72,000 years ago. The composite sea level chronology for the Atlantic Coastal Plain is inconsistent with independent estimates of eustatic sea level positions during interglacial intervals of the last 200,000 years. Hydroisostatic adjustment from glacial-interglacial sea level fluctuations, lithospheric flexure, and isostatic uplift from sediment unloading due to erosion provide possible mechanisms to account for the discrepancies. Alternatively, current eustatic sea level estimates for the middle and late Quaternary may require revision.

Behavioural evidence for polarisation vision in stomatopods reveals a potential channel for communication.

Polarisation sensitivity (PS) - the ability to detect the orientation of polarised light - occurs in a wide variety of invertebrates [1] [2] and vertebrates [3] [4] [5], many of which are marine species [1]. Of these, the crustacea are particularly well documented in terms of their structural [6] and neural [7] [8] adaptations for PS. The few behavioural studies conducted on crustaceans demonstrate orientation to, or local navigation with, polarised sky patterns [9]. Aside from this, the function of PS in crustaceans, and indeed in most animals, remains obscure. Where PS can be shown to allow perception of polarised light as a 'special sensory quality' [1], separate from intensity or colour, it has been termed polarisation vision (PV). Here, within the remarkable visual system of the stomatopod crustaceans (mantis shrimps) [10], we provide the first demonstration of PV in the crustacea and the first convincing evidence for learning the orientation of polarised light in any animal. Using new polarimetric [11] and photographic methods to examine stomatopods, we found striking patterns of polarisation on their antennae and telson, suggesting that one function of PV in stomatopods may be communication [12]. PV may also be used for tasks such as navigation [5] [9] [13], location of reflective water surfaces [14] and contrast enhancement [1] [15] [16] [17] [18]. It is possible that the stomatopod PV system also contributes to some of these functions.

A comparison of mental health legislation in five developed countries: a narrative review.

OBJECTIVES: To describe similarities and differences in mental health legislation between five jurisdictions: the Republic of Ireland, England and Wales, Scotland, Ontario (Canada), and Victoria (Australia). METHODS: An in-depth examination was undertaken focussing on the process of involuntary admission, review of Admission Orders and the legal processes in relation to treatment in the absence of patient consent in each of the five jurisdictions of interest. RESULTS: All jurisdictions permit the detention of a patient if they have a mental disorder although the definition of mental disorder varies between jurisdictions. Several additional differences exist between the five jurisdictions, including the duration of admission prior to independent review of involuntary detention and the role of supported decision making. CONCLUSIONS: Across the five jurisdictions examined, largely similar procedures for admission, detention and treatment of involuntary patients are employed, reflecting adherence with international standards and incorporation of human rights-based principles. Differences exist in relation to the criteria to define mental disorder, the occurrence of automatic review hearings in a timely fashion after a patient is involuntarily admitted and the role for supported decision making under mental health legislation.

Enhanced Arctic Amplification Began at the Mid-Brunhes Event ~400,000 years ago.

Arctic Ocean temperatures influence ecosystems, sea ice, species diversity, biogeochemical cycling, seafloor methane stability, deep-sea circulation, and CO2 cycling. Today's Arctic Ocean and surrounding regions are undergoing climatic changes often attributed to "Arctic amplification" - that is, amplified warming in Arctic regions due to sea-ice loss and other processes, relative to global mean temperature. However, the long-term evolution of Arctic amplification is poorly constrained due to lack of continuous sediment proxy records of Arctic Ocean temperature, sea ice cover and circulation. Here we present reconstructions of Arctic Ocean intermediate depth water (AIW) temperatures and sea-ice cover spanning the last ~ 1.5 million years (Ma) of orbitally-paced glacial/interglacial cycles (GIC). Using Mg/Ca paleothermometry of the ostracode Krithe and sea-ice planktic and benthic indicator species, we suggest that the Mid-Brunhes Event (MBE), a major climate transition ~ 400-350 ka, involved fundamental changes in AIW temperature and sea-ice variability. Enhanced Arctic amplification at the MBE suggests a major climate threshold was reached at ~ 400 ka involving Atlantic Meridional Overturning Circulation (AMOC), inflowing warm Atlantic Layer water, ice sheet, sea-ice and ice-shelf feedbacks, and sensitivity to higher post-MBE interglacial CO2 concentrations.

Dark regeneration of rhodopsin in crayfish photoreceptors.

The eyes of crayfish were exposed to lights of known spectral composition, and the course of regeneration was followed in the dark by measuring the content of rhodopsin and metarhodopsin in single rhabdoms isolated at various times after the adaptation, using an assay that is based on the fluorescence of metarhodopsin. Complete recovery requires several days in the dark after intense adaptation to orange light, but requires less than 2 d after blue light exposure. Following an orange light exposure with blue produces recovery kinetics characteristic of the blue light exposure alone. This quickening of recovery occurs whether the receptors are exposed to blue light either immediately or many hours after the original exposure to orange. Conversely, following blue light adaptation with orange leads to slow recovery, which is characteristic of orange alone. Recovery from long-wavelength adaptation is slower principally because many rhabdoms seem to delay the onset of regeneration. We suggest that the regeneration system is itself photosensitive, and after orange light adaptation the supply of active chromophore (presumably 11-cis retinal) limits the rate of recovery. Once started, recovery proceeds slowly and continuously, and the total pigment concentration (rhodopsin plus metarhodopsin) in the rhabdomeric membrane remains approximately constant. Within hours after intense adapting exposures, the rhabdoms become altered in appearance, the surfaces become coated with accessory pigment, and the bands of microvilli are less distinct. These changes persist until recovery of rhodopsin proceeds, which suggests that visual pigment regeneration results from addition of newly synthesized rhodopsin associated with membrane turn-over.

Photosensitivity spectrum of crayfish rhodopsin measured using fluorescence of metarhodopsin.

Discrepancies exist among spectral measurements of sensitivity of crayfish photoreceptors, their absorption in situ, and the number and absorption spectra of crayfish photopigments that are extracted by digitonin solutions. We have determined the photosensitivity spectrum of crayfish rhodopsin in isolated rhabdoms using long wavelength fluorescence emission from crayfish metarhodopsin as an intrinsic probe. There is no measurable metarhodopsin in the dark-adapted receptor, so changes in the emission level are directly proportional to metarhodopsin concentration. We therefore used changes in metarhodopsin fluorescence to construct relaxation and saturation ("photoequilibrium") spectra, from which the photosensitivity spectrum of crayfish rhodopsin was calculated. This spectrum peaks at or approximately 530 nm and closely resembles the previously measured difference spectrum for total bleaches of dark-adapted rhabdoms. Measurements of the kinetics of changes in rhabdom fluorescence and in transmittance at 580 nm were compared with predictions derived from several model systems containing one or two photopigments. The comparison shows that only a single rhodopsin and its metarhodopsin are present in the main rhabdom of crayfish, and that other explanations must be sought for the multiple pigments seen in digitonin solution. The same analysis shows that there is no detectable formation of isorhodopsin in the rhabdom.

Redundant proteolytic activation of a viral superantigen.

Proteolytic activation of viral superantigens (vSAgs)4 expressed in Chinese hamster ovary (CHO) cells is required for T cell stimulation, and is mediated primarily by the protein convertase (PC) furin. Three PC recognition sites are highly conserved in vSAgs, but it was not known which sites are required for PC dependent vSAg activation. Moreover, because the PC recognition sites are not conserved in all functional vSAgs it was possible that activation could occur by processing at any of several sites. To identify the location(s) where processing of vSAg7 generates an active superantigen, each of two PC recognition sites, and a third related site were altered by in vitro mutagenesis, and the mutant proteins were tested for their abilities to activate T cells. Mutation of the PC recognition site at position 68-71 in vSAg7 had no effect on its ability to activate T cells. Mutation of the processing site at position 169-172 completely abolished T cell activation, and indicated that cleavage at this position was obligatory for proteolytic activation of vSAg7. However, introduction of a PC recognition site at position 192-195, a position that in many other vSAgs encodes a PC recognition site, restored activity to a vSAg7 protein that lacked a recognition site at position 169-172. The data revealed that processing of vSAgs at either position 169-172 or 192-195 was sufficient for vSAg7 activation, and explain how vSAgs that lack some PC recognition sites can be activated by proteolytic processing.

Visual mate choice in poison frogs.

We investigated female mate choice on the basis of visual cues in two populations of Dendrobates pumilio, the strawberry poison frog, from the Bocas del Toro Archipelago in Panama, Central America. Mate choice experiments were carried out by presenting subject females of each of two morphs of this species (orange and green) from two different island populations (Nancy Key and Pope Island) with object frogs (one of each morph) under glass at one end of a terrarium. Recorded calls were played simultaneously from behind both object frogs. The experiments were carried out under two light regimes: (i) white light, and (ii) relatively monochromatic filtered blue light. Subject females from each population displayed a significant preference for their own morph under white light, but not under blue light. These results indicate that female D. pumilio use visual cues in mate choice, and suggest that colour may be the visual cue they use.

bcl-2 prolongs neuronal survival during hypoxia-induced apoptosis.

In vivo models of hypoxic-ischemic brain injury have shown altered expression of a number of genes that are important in regulating neuronal survival. However, it is not clear as to whether hypoxia alone can alter the expression of genes regulating neuronal survival. We hypothesized that (1) hypoxia alone alters the expression of bcl-2 in neurons, (2) the severity and duration of hypoxia influence bcl-2 expression, and (3) the alteration of bcl-2 expression has an important role in regulating neuronal survival during hypoxia. Embryonic rat neocortical neurons cultured for 7-10 days were exposed to 0.1, 1, or 3% oxygen for various durations and were removed for analyses at 24-h intervals. Under all hypoxic conditions, neurons exhibited morphologic changes, as assessed by electron microscopy and Annexin V staining, consistent with apoptosis. Immunoblot and immunofluorescence analyses revealed an increase in neuronal bcl-2 protein during hypoxic exposure. Quantitative immunofluorescence analyses of bcl-2 immunostained neurons indicated that expression of bcl-2 was altered by the duration and severity of hypoxia. Attenuation of bcl-2 expression by antisense oligonucleotides decreased the proportion of surviving neurons by approximately 50% after 48 h of exposure to 0.1% oxygen. We conclude that observed increase in bcl-2, in part, plays an important role in neuronal survival during exposure to hypoxia.

Single lung transplantation for primary pulmonary hypertension.

Single lung transplantation has become a therapeutic option for end-stage interstitial lung disease and obstructive lung disease. Our group recently extended this treatment to three patients with primary pulmonary hypertension. All patients had marked decreases in pulmonary artery pressures and pulmonary vascular resistance and increases in cardiac output following single lung transplantation. Spirometry, lung volumes, and diffusion capacity were not different in comparison to preoperative studies. Quantitative ventilation-perfusion scans revealed the majority of perfusion distributed to the transplanted lung, with ventilation approximately equally divided between the native and the transplanted lung. Despite ventilation-perfusion imbalance, there was no resting hypoxemia and there was no arterial oxygen desaturation with exercise. One patient expired on the 30th postoperative day due to cytomegalovirus infection of the lungs. In the remaining two patients, maximum exercise capacity following transplantation was near normal in one recipient and reduced in the second recipient. Of note, there was no evidence of ventilatory limitation or impaired oxygenation during exercise in these two recipients. Although an exaggerated exercise ventilatory response was present, this did not limit exercise performance. This report supports the use of single lung transplantation for the treatment of primary pulmonary hypertension.