Climate and human stressors on global penguin hotspots: Current assessments for future conservation.

As charismatic and iconic species, penguins can act as "ambassadors" or flagship species to promote the conservation of marine habitats in the Southern Hemisphere. Unfortunately, there is a lack of reliable, comprehensive, and systematic analysis aimed at compiling spatially explicit assessments of the multiple impacts that the world's 18 species of penguin are facing. We provide such an assessment by combining the available penguin occurrence information from Global Biodiversity Information Facility (>800,000 occurrences) with three main stressors: climate-driven environmental changes at sea, industrial fisheries, and human disturbances on land. Our analyses provide a quantitative assessment of how these impacts are unevenly distributed spatially within species' distribution ranges. Consequently, contrasting pressures are expected among species, and populations within species. The areas coinciding with the greatest impacts for penguins are the coast of Perú, the Patagonian Shelf, the Benguela upwelling region, and the Australian and New Zealand coasts. When weighting these potential stressors with species-specific vulnerabilities, Humboldt (Spheniscus humboldti), African (Spheniscus demersus), and Chinstrap penguin (Pygoscelis antarcticus) emerge as the species under the most pressure. Our approach explicitly differentiates between climate and human stressors, since the more achievable management of local anthropogenic stressors (e.g., fisheries and land-based threats) may provide a suitable means for facilitating cumulative impacts on penguins, especially where they may remain resilient to global processes such as climate change. Moreover, our study highlights some poorly represented species such as the Northern Rockhopper (Eudyptes moseleyi), Snares (Eudyptes robustus), and Erect-crested penguin (Eudyptes sclateri) that need internationally coordinated efforts for data acquisition and data sharing to understand their spatial distribution properly.

Designer proteins: applications of genetic code expansion in cell biology.

Designer amino acids, beyond the canonical 20 that are normally used by cells, can now be site-specifically encoded into proteins in cells and organisms. This is achieved using 'orthogonal' aminoacyl-tRNA synthetase-tRNA pairs that direct amino acid incorporation in response to an amber stop codon (UAG) placed in a gene of interest. Using this approach, it is now possible to study biology in vitro and in vivo with an increased level of molecular precision. This has allowed new biological insights into protein conformational changes, protein interactions, elementary processes in signal transduction and the role of post-translational modifications.

Using a quadruplet codon to expand the genetic code of an animal.

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here, we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

Three-dimensional feedback-driven trapping of a single nanoparticle or molecule in aqueous solution with a confocal fluorescence microscope.

Control of electroosmotic flows in a two-layer microfluidic device with crossed channels is used to counteract Brownian diffusion in aqueous solution for three-dimensional trapping of a single nanoparticle or molecule within the probe volume of a confocal fluorescence microscope. A field programmable gate array sorts and counts photons into four channels synchronous with laser pulses in four beams focused to waists slightly offset from the center of the confocal volume and uses the counts to update voltages between the four fluidic inlets every 13.5 µs. Trapping is demonstrated for 40 nm nanoparticles for up to 240 s, 20 nm nanoparticles for up to 25 s, and single molecules of streptavidin-Alexa 647 for up to 1.2 s.

Early and long-term outcomes after manual and remote magnetic navigation-guided catheter ablation for ventricular tachycardia.

Aims: Remote magnetic navigation (RMN) is a safe and effective means of performing ventricular tachycardia (VT) ablation. It may have advantages over manual catheter ablation due to ease of manoeuvrability and catheter stability. We sought to compare the safety and efficacy of RMN vs. manual VT ablation. Methods and results: Retrospective study of procedural outcomes of 139 consecutive VT ablation procedures (69 RMN, 70 manual ablation) in 113 patients between 2009 and 2015 was performed. Remote magnetic navigation was associated with overall higher acute procedural success (80% vs. 60%, P = 0.01), with a trend to fewer major complications (3% vs. 9% P = 0.09). Seventy-nine patients were followed up for a median of 17.0 [interquartile range (IQR) 3.0-41.0] months for the RMN group and 15.5 (IQR 6.5-30.0) months for manual ablation group. In the ischaemic cardiomyopathy subgroup, RMN was associated with longer survival from the composite endpoint of VT recurrence leading to defibrillator shock, re-hospitalization or repeat catheter ablation and all-cause mortality; single-procedure adjusted hazard ratio (HR) 0.240 (95% CI 0.070-0.821) P = 0.023, multi-procedure HR 0.170 (95% CI 0.046-0.632) P = 0.002. In patients with implanted defibrillators, multi-procedure VT-free survival was superior with RMN, HR 0.199 (95% CI 0.060-0.657) P = 0.003. Conclusion: Remote magnetic navigation may improve clinical outcomes after catheter ablation of VT in patients with ischaemic cardiomyopathy. Further prospective clinical studies are required to confirm these findings.

Circuit Impedance Could Be a Crucial Factor Influencing Radiofrequency Ablation Efficacy and Safety: A Myocardial Phantom Study of the Problem and its Correction.

BACKGROUND: Circuit impedance could affect the safety and efficacy of radiofrequency (RF) ablation. AIM: To perform irrigated RF ablations with graded impedance to compare (1) lesion dimensions and overheated dimensions in fixed power ablations (2) and in power corrected ablations. METHODS: Ablations were performed with irrigated Navistar Thermocool catheter and Stockert EP shuttle generator at settings of 40 W power for 60 seconds, in a previously validated myocardial phantom. The impedance of the circuit was set at 60 Ω, 80 Ω, 100 Ω, 120 Ω, 140 Ω, and 160 Ω. The lesion and overheated dimensions were measured at 53 °C and 80 °C isotherms, respectively. In the second set of ablations, power was corrected according to circuit impedance. RESULTS: In total, 70 ablations were performed. The lesion volume was 72.0 ± 4.8% and 44.7 ± 4.6% higher at 80 Ω and 100 Ω, respectively, compared to that at 120 Ω and it was 15.4 ± 1.2%, 28.1 ± 2.0%, and 38.0 ± 1.8% lower at 140 Ω, 160 Ω, and 180 Ω, respectively. The overheated volume was four times larger when impedance was reduced to 80 Ω from 100 Ω. It was absent at 120 Ω and above. In the power corrected ablations, the lesion volumes were similar to that of 40 W/120 Ω ablations and there was no evidence of overheating. CONCLUSION: The lesion and overheated dimensions were significantly larger with lower circuit impedance during irrigated RF ablation and the lesion size was smaller in high impedance ablations. Power delivery adjusted to impedance using a simple equation improved the consistency of lesion formation and prevented overheating.

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.

The Wearable Cardioverter Defibrillator: an Early Single Centre Australian Experience. Some Pitfalls and Caveats for Use.

BACKGROUND: Wearable Cardioverter Defibrillators (WCD) have been effectively used for more than a decade in North America and Europe for prevention of sudden cardiac death (SCD) due to ventricular arrhythmias. This device has only recently been available in Australia. METHOD: At Westmead hospital, WCD has been used since 2013 as a bridging therapy to an implantable cardioverter defibrillator (ICD) for those at high risk, but are temporarily not suitable for an implantable device. Indications for use were explanted infected ICD, dilated cardiomyopathy, post partum cardiomyopathy, valvular heart disease and myocarditis. The default device settings were: ventricular tachycardia (VT) and ventricular fibrillation (VF) threshold of 150 bpm and 200 bpm respectively and response times were 60 secs for VT and 25 secs for VF. OUTCOME: WCD was used in eight patients. Duration of use ranged from five to 180 days with median of 77 days. Daily usage averaged 23.4±0.6hours. All except one were compliant with the device and none of our patients received shock or died during device usage. Four of the eight patients received ICD, two declined ICD, one was judged to no longer require ICD and one remains under assessment. CONCLUSION: WCD is easy to use, well tolerated and is effective for SCD prevention in patients who are temporarily not suitable for ICD. However patients need to be actively followed-up to reduce the duration of WCD usage and thereby be cost effective.

Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (ß-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

High-speed multiparameter photophysical analyses of fluorophore libraries.

There is a critical need for high-speed multiparameter photophysical measurements of large libraries of fluorescent probe variants for imaging and biosensor development. We present a microfluidic flow cytometer that rapidly assays 10(4)-10(5) member cell-based fluorophore libraries, simultaneously measuring fluorescence lifetime and photobleaching. Together, these photophysical characteristics determine imaging performance. We demonstrate the ability to resolve the diverse photophysical characteristics of different library types and the ability to identify rare populations.

Pollution, habitat loss, fishing, and climate change as critical threats to penguins.

Cumulative human impacts across the world's oceans are considerable. We therefore examined a single model taxonomic group, the penguins (Spheniscidae), to explore how marine species and communities might be at risk of decline or extinction in the southern hemisphere. We sought to determine the most important threats to penguins and to suggest means to mitigate these threats. Our review has relevance to other taxonomic groups in the southern hemisphere and in northern latitudes, where human impacts are greater. Our review was based on an expert assessment and literature review of all 18 penguin species; 49 scientists contributed to the process. For each penguin species, we considered their range and distribution, population trends, and main anthropogenic threats over the past approximately 250 years. These threats were harvesting adults for oil, skin, and feathers and as bait for crab and rock lobster fisheries; harvesting of eggs; terrestrial habitat degradation; marine pollution; fisheries bycatch and resource competition; environmental variability and climate change; and toxic algal poisoning and disease. Habitat loss, pollution, and fishing, all factors humans can readily mitigate, remain the primary threats for penguin species. Their future resilience to further climate change impacts will almost certainly depend on addressing current threats to existing habitat degradation on land and at sea. We suggest protection of breeding habitat, linked to the designation of appropriately scaled marine reserves, including in the High Seas, will be critical for the future conservation of penguins. However, large-scale conservation zones are not always practical or politically feasible and other ecosystem-based management methods that include spatial zoning, bycatch mitigation, and robust harvest control must be developed to maintain marine biodiversity and ensure that ecosystem functioning is maintained across a variety of scales.

Three-dimensional tracking of a single fluorescent nanoparticle using four-focus excitation in a confocal microscope.

We report high sensitivity detection and tracking of a single fluorescent nanoparticle in solution by use of four alternately pulsed laser diodes for fluorescence excitation in a confocal microscope. Slight offsets between the centers of the overlapping laser foci together with time-resolved photon counting enable sub-micron precision position measurements. Real-time correction for diffusional motion with a xyz-piezo stage then enables tracking of a nanoparticle with diffusivity up to ~12 µm(2) s(-1). Fluorescence correlation spectroscopy and calibration measurements indicate a net fluorescence photon detection efficiency of ~6-9%, comparable to that of an optimized single-molecule microscope.

Ants of the Florida Keys: species accounts, biogeography, and conservation (Hymenoptera: Formicidae).

As a tropical archipelago, the Florida Keys provide an ideal environment to examine the historic and short-term processes that structure and influence biological diversity. Through a new survey of the ants of the Florida Keys, we increase our knowledge of the number of species to 94 representing 34 genera and 8 subfamilies. Through detailed collection information, we provide an in depth picture of the distribution of each species across the Keys. On the basis of these data and information on the native and known distributions of each species, we confirm the historical trend toward continued immigration of nonnative species into the Florida Keys and present these findings in the context of the proportion of native to nonnative species. We find a similar number of species introduced from the Old World and Neotropical mainland and discuss the probable immigration of mainland Florida species during the exposure of the Florida Shelf during the last glacial episode and the subsequent isolation of some populations as sea level rose following the last glaciation. Lastly, we discuss the possible threats to these populations due to rapid climate change and other human influences.

Rapid, single-molecule assays in nano/micro-fluidic chips with arrays of closely spaced parallel channels fabricated by femtosecond laser machining.

Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

Percutaneous transcatheter left atrial appendage closure devices: role in the long-term management of atrial fibrillation.

OBJECTIVE: Atrial fibrillation remains the most common cardiac rhythmic disorder worldwide and is associated with significant health hazards, most notably an increase in the rate of cerebrovascular events. Stroke prevention in atrial fibrillation has traditionally been managed with warfarin therapy which is encumbered by risk of bleeding, drug administration logistics and interactions as well as issues of non-compliance. Occlusion of the left atrial appendage has recently been explored as an alternative method of stroke prevention. The aim of this article is to evaluate the history, efficacy and draw-backs associated with percutaneous left atrial appendage occlusion devices in the management of atrial fibrillation. METHODS: The current literature and clinical experience was used to summarise the history and evaluate the efficacy of percutaneous left atrial appendage occlusion devices. RESULTS: Percutaneous left atrial appendage occlusion devices are effective novel therapies for stroke prevention in atrial fibrillation, with proven reductions in thromboembolic events in comparison with placebo and non-inferiority with warfarin therapy. Pericardial effusions and embolic strokes are primary peri-procedural adverse reactions. The rates of adverse reactions reduce with operator experience. CONCLUSIONS: Percutaneous left atrial appendage occlusion is an exciting and novel therapy of stroke prevention in atrial fibrillation. Whilst further trials and long-term data are required prior to widespread implementation of this procedure, trials so far have highlighted the clinical efficacy of the procedure.

Genetic Encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.

Cardiac implantable electronic device therapy for bradyarrhythmias and tachyarrhythmias.

Pacemakers originally were developed for patients with profound bradycardia and complete heart block who, without them, usually suffered from syncope, heart failure and an early demise. Since that time, devices have evolved to include pacing and shock therapies for the management of tachyarrhythmias and heart failure with the aim of improving quality, and if possible, length of life. Whether to insert a device depends on a balance between the potential benefits of device therapy and its risks, which are not inconsiderable. We discuss current agreed indications for pacemakers and implantable defibrillators and some current controversies surrounding their use.

Towards the protection of ammunition headstamps during fingermark enhancement processing; a preliminary study.

Forensic recovery from fired ammunition casings remains one of the most challenging tasks during high-profile investigations. Often, the decision must be made between screening for DNA or fingerprints, and, in doing so, the impact these processes will have on the examination of ballistic markings imparted on the ammunition from the firearm itself. Traditionally, fingermark enhancement processes have yielded low success rates in their efforts to identify suspects by enhancing friction ridge detail left on the cartridge casings. Moreover, the enhancement methods utilised may often induce detrimental physical changes to the casing(s), rendering them unsuitable for subsequent ballistics (marking) examination. Recently, new technology has been shown to increase the success rate of fingermark recovery from fired ammunition, and the growing adoption of such innovation means that new challenges are encountered to maximise evidence recovery and streamline forensic workflows. One such example arises from the potential obscuration of the ammunition headstamp area during such treatments. Accordingly, this study outlines the preliminary investigations and developments of a polymer mask substrate that serves to protect the headstamp of fired ammunition casings during relevant fingermark enhancement processes. The technique also has the capacity to be used as a surface protection device to eliminate unwanted chemical deposition across other areas of interest and evidence types.