Intraretinal Hyper-Reflective Foci Are Almost Universally Present and Co-Localize With Intraretinal Fluid in Diabetic Macular Edema.

Purpose: In diabetic macular edema (DME), hyper-reflective foci (HRF) has been linked to disease severity and progression. Using an automated approach, we aimed to investigate the baseline distribution of HRF in DME and their co-localization with cystoid intraretinal fluid (IRF). Methods: Baseline spectral-domain optical coherence tomography (SD-OCT) volume scans (N = 1527) from phase III clinical trials YOSEMITE (NCT03622580) and RHINE (NCT03622593) were segmented using a deep-learning-based algorithm (developed using B-scans from BOULEVARD NCT02699450) to detect HRF. The HRF count and volume were assessed. HRF distributions were analyzed in relation to best-corrected visual acuity (BCVA), central subfield thickness (CST), and IRF volume in quartiles, and Diabetic Retinopathy Severity Scores (DRSS) in groups. Co-localization of HRF with IRF was calculated in the central 3-mm diameter using the en face projection. Results: HRF were present in most patients (up to 99.7%). Median (interquartile range [IQR]) HRF volume within the 3-mm diameter Early Treatment Diabetic Retinopathy Study ring was 1964.3 (3325.2) pL, and median count was 64.0 (IQR = 96.0). Median HRF volumes were greater with decreasing BCVA (nominal P = 0.0109), and increasing CST (nominal P < 0.0001), IRF (nominal P < 0.0001), and DRSS up to very severe nonproliferative diabetic retinopathy (nominal P < 0.0001). HRF co-localized with IRF in the en face projection. Conclusions: Using automated HRF segmentation of full SD-OCT volumes, we observed that HRF are a ubiquitous feature in DME and exhibit relationships with BCVA, CST, IRF, and DRSS, supporting a potential link to disease severity. The spatial distribution of HRF closely followed that of IRF.

Crested Auklets.

Ian Jones and Bruce Lyon introduce the Crested Auklet, a seabird with mutual sexual selection.

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics.

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer's disease, or vision impairment caused by ganglion cell degeneration in the retina.

Drug-sensitized zebrafish screen identifies multiple genes, including GINS3, as regulators of myocardial repolarization.

BACKGROUND: Cardiac repolarization, the process by which cardiomyocytes return to their resting potential after each beat, is a highly regulated process that is critical for heart rhythm stability. Perturbations of cardiac repolarization increase the risk for life-threatening arrhythmias and sudden cardiac death. Although genetic studies of familial long-QT syndromes have uncovered several key genes in cardiac repolarization, the major heritable contribution to this trait remains unexplained. Identification of additional genes may lead to a better understanding of the underlying biology, aid in identification of patients at risk for sudden death, and potentially enable new treatments for susceptible individuals. METHODS AND RESULTS: We extended and refined a zebrafish model of cardiac repolarization by using fluorescent reporters of transmembrane potential. We then conducted a drug-sensitized genetic screen in zebrafish, identifying 15 genes, including GINS3, that affect cardiac repolarization. Testing these genes for human relevance in 2 concurrently completed genome-wide association studies revealed that the human GINS3 ortholog is located in the 16q21 locus, which is strongly associated with QT interval. CONCLUSIONS: This sensitized zebrafish screen identified 15 novel myocardial repolarization genes. Among these genes is GINS3, the human ortholog of which is a major locus in 2 concurrent human genome-wide association studies of QT interval. These results reveal a novel network of genes that regulate cardiac repolarization.

Relationships between mercury burden, sex, and sexually selected feather ornaments in crested auklet (Aethia cristatella).

Individuals with higher contaminant burdens are expected to be in poorer physical health and be of lower individual body condition and energetic status, potentially resulting in reduced ornamentation or increased asymmetry in bilateral features. The degree and magnitude of this effect also would be expected to vary by sex, as female birds depurate contaminants into eggs. We tested for relationships among mercury in feathers, sex, and elaborate feather ornaments that relate to individual quality in crested auklets (Aethia cristatella), small planktivorous seabirds in the North Pacific Ocean. We found no relationships between mercury and the size of individuals' forehead crest or degree of measurement asymmetry in auricular plumes, both of which are favoured by intersexual selection. Females had significantly greater mercury concentrations than males (females. 1.02 ± 0.39 µg/g; males, 0.75 ± 0.32 µg/g); but concentrations were below that known to have physiological effects, as expected for a secondary consumer. Sex differences in overwintering area for this long-distance migrant species (more females in the Kuroshio Current Large Marine Ecosystem than males) could be the reason for this seemingly counterintuitive result between sexes. Further research relating mercury burden to overwintering ecology and diet contents would build on our results and further elucidate interrelationships between sex, sexually selected feather ornaments and contaminant burden.

Quantifying and addressing the prevalence and bias of study designs in the environmental and social sciences.

Building trust in science and evidence-based decision-making depends heavily on the credibility of studies and their findings. Researchers employ many different study designs that vary in their risk of bias to evaluate the true effect of interventions or impacts. Here, we empirically quantify, on a large scale, the prevalence of different study designs and the magnitude of bias in their estimates. Randomised designs and controlled observational designs with pre-intervention sampling were used by just 23% of intervention studies in biodiversity conservation, and 36% of intervention studies in social science. We demonstrate, through pairwise within-study comparisons across 49 environmental datasets, that these types of designs usually give less biased estimates than simpler observational designs. We propose a model-based approach to combine study estimates that may suffer from different levels of study design bias, discuss the implications for evidence synthesis, and how to facilitate the use of more credible study designs.

Cystatin C as a filtration marker--haemodialysis patients expose its strengths and limitations.

OBJECTIVE: The reliability of serum cystatin C (s-Cys) as a filtration marker depends on the intra- and inter-individual variation and influence of non-renal factors of its production rate (Cys(pr)), non-renal clearance (CL(nr)) and sieving coefficient (S). Haemodialysis patients with no residual renal function would be the best population in which to investigate these variables, which otherwise require reliable GFR measurements. MATERIAL AND METHODS: Seventy-nine haemodialysis (HD) patients with negligible residual renal function (Group 1) were investigated and compared with 55 HD patients with varying degrees of residual renal function (Group 2) and 923 non-dialysis patients (Group 3). The equation eGFR = Cys(pr)/s-Cys-CL(nr) was used to analyse the turnover and variation of cystatin C. RESULTS: A formula for estimating GFR, eGFR = 99/s-Cys-14.1, calculated from Group 3, was shown to fit the HD patients. The measured s-Cys in Group 1 was 6.9+/-0.9 and 6.4+/-1.1 mg/L in Group 2. The calculated 95% confidence interval of eGFR of +/-(30-40) % increased sharply below GFR 20 mL/min/1.73 m(2), which means that s-Cys cannot be used for calculating low GFR, including the residual GFR of dialysis patients. CONCLUSIONS: Nicotine users in Group 1 had significantly higher s-Cys than non-users (7.5+/-0.9 mg/L compared to 6.7+/-0.8; p = 0.0008), which may be a factor to include in the eGFR formulae. However, s-Cys was independent of non-renal factors such as sex, age, LBM, body weight, malnutrition and CRP and also of changes in CRP.

Activity periods and questing behavior of the seabird tick Ixodes uriae (Acari: Ixodidae) on Gull Island, Newfoundland: the role of puffin chicks.

Questing behavior of Ixodes uriae and their associated seasonal, host-feeding patterns are crucial to our understanding of tick life history strategies and the ecology of diseases that they transmit. Consequently, we quantified questing behavior of nymphs and adult female I. uriae ticks at Gull Island, a seabird colony in Newfoundland, Canada, to examine seasonal variation of off-host and on-host tick activity. We sampled a total of 133 adult Atlantic puffins (Fratercula arctica), 152 puffin chicks, and 145 herring gull (Larus argentatus) chicks for ticks during the breeding seasons of 2004 and 2005. Questing ticks were sampled by dragging a white flannel cloth across the grassy breeding areas during the mo of May, June, July, and August. Nymph questing activity reached a peak during mid-July (79 and 110 individuals/hr in 2004 and 2005, respectively). The prevalence of nymphs and adult female ticks on different seabird hosts varied between years and during the seasons. Puffin chicks had the highest prevalence (above 70% in July) of nymphs in both years and this was correlated with questing activity. Female ticks rarely fed on puffin chicks, but were prevalent on adult puffins and gulls, although prevalence and questing of ticks were not correlated in these hosts. These patterns of off-host and on-host tick activity suggests that I. uriae ticks likely use a combination of questing and passive waiting, e.g., in puffin burrows, to detect hosts, depending on the tick stage and the host species.

A study of wrecked Dovekies (Alle alle) in the western North Atlantic highlights the importance of using standardized methods to quantify plastic ingestion.

Quantification of plastic ingestion across a range of seabirds is required to assess the prevalence of plastics in marine food webs. We quantified plastic ingestion in beached Dovekies (Alle alle), following a wreck in Newfoundland, Canada. Of 171 birds, 30.4% had ingested plastic (mean 0.81±0.30 SE pieces per bird, mass 0.005±0.002 SE g per bird). Most plastics were fragments of polyethylene and polypropylene. Surprisingly, 37% were burned or melted, indicating a previously unreported source of ingested plastics (incinerated waste). We found no relationship between plastic ingestion and age, sex or body condition. By comparing our results with a similar nearby study, we illustrate the need for researchers to adopt standardized methods for plastic ingestion studies. We underline the importance of using histological techniques to reliably identify gastric pathologies, and advise caution when inferring population level trends in plastic ingestion from studies of emaciated, wrecked birds.

A method for electrophysiological characterization of hamster retinal ganglion cells using a high-density CMOS microelectrode array.

Knowledge of neuronal cell types in the mammalian retina is important for the understanding of human retinal disease and the advancement of sight-restoring technology, such as retinal prosthetic devices. A somewhat less utilized animal model for retinal research is the hamster, which has a visual system that is characterized by an area centralis and a wide visual field with a broad binocular component. The hamster retina is optimally suited for recording on the microelectrode array (MEA), because it intrinsically lies flat on the MEA surface and yields robust, large-amplitude signals. However, information in the literature about hamster retinal ganglion cell functional types is scarce. The goal of our work is to develop a method featuring a high-density (HD) complementary metal-oxide-semiconductor (CMOS) MEA technology along with a sequence of standardized visual stimuli in order to categorize ganglion cells in isolated Syrian Hamster (Mesocricetus auratus) retina. Since the HD-MEA is capable of recording at a higher spatial resolution than most MEA systems (17.5 µm electrode pitch), we were able to record from a large proportion of RGCs within a selected region. Secondly, we chose our stimuli so that they could be run during the experiment without intervention or computation steps. The visual stimulus set was designed to activate the receptive fields of most ganglion cells in parallel and to incorporate various visual features to which different cell types respond uniquely. Based on the ganglion cell responses, basic cell properties were determined: direction selectivity, speed tuning, width tuning, transience, and latency. These properties were clustered to identify ganglion cell types in the hamster retina. Ultimately, we recorded up to a cell density of 2780 cells/mm(2) at 2 mm (42°) from the optic nerve head. Using five parameters extracted from the responses to visual stimuli, we obtained seven ganglion cell types.

High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels.

Studies on information processing and learning properties of neuronal networks would benefit from simultaneous and parallel access to the activity of a large fraction of all neurons in such networks. Here, we present a CMOS-based device, capable of simultaneously recording the electrical activity of over a thousand cells in in vitro neuronal networks. The device provides sufficiently high spatiotemporal resolution to enable, at the same time, access to neuronal preparations on subcellular, cellular, and network level. The key feature is a rapidly reconfigurable array of 26 400 microelectrodes arranged at low pitch (17.5 µm) within a large overall sensing area (3.85 × 2.10 mm(2)). An arbitrary subset of the electrodes can be simultaneously connected to 1024 low-noise readout channels as well as 32 stimulation units. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any neuron on the array. We demonstrate the applicability and potential of this device for various different experimental paradigms: large-scale recordings from whole networks of neurons as well as investigations of axonal properties of individual neurons.

A tangerine-scented social odour in a monogamous seabird.

Social odours, conspecific chemical signals, have been demonstrated in every class of vertebrate except birds. The apparent absence is surprising, as every bird examined has a functional olfactory system and many produce odours. The crested auklet (Aethia cristatella), a monogamous seabird, exhibits a distinctive tangerine-like scent closely associated with courtship. Using T-maze experiments, we tested whether auklets preferred conspecific odours and whether they distinguished between different types of scent, two prerequisites of chemical communication. Crested auklets exhibited: (i) an attraction to conspecific feather odour; (ii) a preference for two chemical components of feather scent (cis-4-decenal and octanal), which we identified as seasonally elevated; and (iii) differential responses to odours, as indicated by a preference for auklet odour, an aversion to mammalian musk, but no significant response to banana essence (amyl acetate). Our results suggest that crested auklets detect plumage odour and preferentially orientate towards this stimulus. The striking and well-described courtship display that involves the scented neck region, the 'ruff sniff', provides a conspicuous behavioural mechanism for odour transmission and the potential for scent assessment during sexual selection. Although the importance and full social function of chemical signals are just beginning to be understood in birds, including crested auklets, social odours promise to reveal a largely unexplored and possibly widespread means of avian communication.

Evaluation of seabirds in Newfoundland and Labrador, Canada, as hosts of influenza A viruses.

Influenza A viruses infect a wide range of hosts, including many species of birds. Avian influenza A virus (AIV) infection appears to be most common in Anseriformes (ducks, geese, and swans) and some Charadriiformes (shorebirds and gulls), but many other birds also serve as hosts of AIV. Here, we evaluated the role of seabirds as hosts for AIV. We tested 3,160 swab samples from 13 seabird species between May 2008 and December 2011 in Newfoundland and Labrador, Canada. We also tested 156 serum samples for evidence of previous infection of AIV in Common Murres (Uria aalge) and Atlantic Puffins (Fratercula arctica). Avian influenza A virus was detected in breeding Common Murres and nonbreeding Thick-billed Murres (Uria lomvia), and Common Murres also had high antibody prevalence (44%). From these findings, combined with other studies showing AIV infection in murres, we conclude that murres are important for the ecology of AIV. For other species (Razorbill, Alca torda; Leach's Storm-Petrel, Oceanodroma leucorhoa; Black-legged Kittiwake, Rissa tridactyla; Atlantic Puffin) with good coverage (>100 samples) we did not detect AIV. However, serology indicates infection does occur in Atlantic Puffins, with 22% antibody prevalence found. The possibility of virus spread through dense breeding colonies and the long distance movements of these hosts make a more thorough evaluation of the role for seabirds as hosts of AIV important.

Ingestion of plastic marine debris by Common and Thick-billed Murres in the northwestern Atlantic from 1985 to 2012.

Plastic ingestion by seabirds is a growing conservation issue, but there are few time series of plastic ingestion with large sample sizes for which one can assess temporal trends. Common and Thick-billed Murres (Uria aalge and U. lomvia) are pursuit-diving auks that are legally harvested in Newfoundland and Labrador, Canada. Here, we combined previously unpublished data on plastic ingestion (from the 1980s to the 1990s) with contemporary samples (2011-2012) to evaluate changes in murres' plastic ingestion. Approximately 7% of murres had ingested plastic, with no significant change in the frequency of ingestion among species or periods. The number of pieces of plastic/bird, and mass of plastic/bird were highest in the 1980s, lowest in the late 1990s, and intermediate in contemporary samples. Studying plastic ingestion in harvested seabird populations links harvesters to conservation and health-related issues and is a useful source of large samples for diet and plastic ingestion studies.

Recording from defined populations of retinal ganglion cells using a high-density CMOS-integrated microelectrode array with real-time switchable electrode selection.

In order to understand how retinal circuits encode visual scenes, the neural activity of defined populations of retinal ganglion cells (RGCs) has to be investigated. Here we report on a method for stimulating, detecting, and subsequently targeting defined populations of RGCs. The possibility to select a distinct population of RGCs for extracellular recording enables the design of experiments that can increase our understanding of how these neurons extract precise spatio-temporal features from the visual scene, and how the brain interprets retinal signals. We used light stimulation to elicit a response from physiologically distinct types of RGCs and then utilized the dynamic-configurability capabilities of a microelectronics-based high-density microelectrode array (MEA) to record their synchronous action potentials. The layout characteristics of the MEA made it possible to stimulate and record from multiple, highly overlapping RGCs simultaneously without light-induced artifacts. The high-density of electrodes and the high signal-to-noise ratio of the MEA circuitry allowed for recording of the activity of each RGC on 14±7 electrodes. The spatial features of the electrical activity of each RGC greatly facilitated spike sorting. We were thus able to localize, identify and record from defined RGCs within a region of mouse retina. In addition, we stimulated and recorded from genetically modified RGCs to demonstrate the applicability of optogenetic methods, which introduces an additional feature to target a defined cell type. The developed methodologies can likewise be applied to other neuronal preparations including brain slices or cultured neurons.

Auklet (Charadriiformes: Alcidae, Aethia spp.) chick meals from the Aleutian Islands, Alaska, have a very low incidence of plastic marine debris.

The ingestion of plastic marine debris is a chronic problem for some of the world's seabird species, contributing to reduced chick survival, population declines, and deposition of contaminants via absorption in birds' gastrointestinal tract. We analysed the frequency of ingested plastic in chick meals delivered by adults in four species of auklet - Crested (Aethia cristatella), Least (A. pusilla), Parakeet (A. psittacula), and Whiskered (A. pygmaea) - from three breeding colonies in the Aleutian Islands, Alaska, USA over a 14-year period from 1993 to 2006. Among 2541 chick meals, we found plastic in only one - from a Whiskered Auklet on Buldir Island in 1993. While adult Parakeet Auklets have a high frequency of plastic ingestion (over 90%), no chick meals contained plastic. Unlike other seabirds, the planktivorous auklets do not appear to offload plastic to their chicks, and we conclude that auklet chicks are probably at a low risk of contamination from plastic debris.

Efficient formal synthesis of oseltamivir phosphate (Tamiflu) with inexpensive D-ribose as the starting material.

An efficient formal synthesis of oseltamivir phosphate (Tamiflu) has been achieved in 12 steps with use of the inexpensive and highly abundant D-ribose as the starting material. This concise alternative route does not utilize protecting groups and features the introduction of 3-pentylidene ketal as the latent 3-pentyl ether, the use of a highly efficient RCM reaction to form the Tamiflu skeleton, and selective functional group manipulations.

Total synthesis of (+/-)-cordypyridones A and B and related epimers.

Efficient racemic synthesis of two antibacterial and antimalarial natural products, cordypyridones A and B, was achieved from inexpensive, commercially available starting materials in an overall yield of 15% (8% and 7%, respectively). This convergent synthesis utilizes a key coupling step of two fragments and the subsequent functional group transformations lead to the target compounds and their 8-epi-analogues.

In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation.

In the last decade the zebrafish has become a major model organism for the study of development and organogenesis. To maximize the experimental utility of this organism, it will be important to establish methods for adult phenotyping. We previously proposed that the embryonic zebrafish may be useful in high-throughput screening for drug-induced cardiotoxicity. We now describe a method for the reproducible recording of the adult zebrafish ECG and illustrate its application in the investigation of QT-prolonging drugs. Zebrafish ECGs were obtained by inserting two needle electrodes through the ventral epidermis. Fish were perfused orally, and motion artifacts were eliminated with a paralytic dose of mu-conotoxin GIIIB. Test compounds were delivered via the perfusion system. Without a means of hydration and oxygenation, the fish succumb rapidly. The use of a perfusion system allowed stable recording for > 6 h. Baseline conduction intervals were as follows: PR, 66 ms (SD 14); QRS, 34 ms (SD 11); QT, 242 ms (SD 54); and R-R, 398 ms (SD 77). The known QT-prolonging agents astemizole, haloperidol, pimozide, and terfenadine caused corrected QT increases of 18% (SD 9), 16% (SD 11), 17% (SD 9), and 11% (SD 6), respectively. The control drugs clonidine, penicillin and propranolol did not prolong the corrected QT interval. In conclusion, perfusion and muscular paralysis allows stable, low-noise recording of zebrafish ECGs. Agents known to cause QT prolongation in humans caused QT prolongation in fish in each case. The development of rigorous tools for the phenotyping of adult zebrafish will complement the high-throughput assays currently under development for embryonic and larval fish.