A gap isolation method to investigate electrical and mechanical properties of fully contracting skeletal muscle fibers.

In the green alga Chlamydomonas chlamyrhodopsin fulfills its role as a light sensor by absorbing light and activating photoreceptor channels within the eyespot area. At intense light stimuli, the photoreceptor (P) current triggers a fast and a slow flagellar current that finally leads to backward swimming (stop response). Here we report about probing the photoreceptor current directly at the eyespot. This allows the detection of the whole P current with a size of above 50 pA. The P current appears with a delay of less than 50 microseconds, suggesting that rhodopsin and the P channel are closely coupled or form one ion channel complex. The Ca2+ dependence of the P current has been demonstrated with the established suction technique in a capacitive mode. The P current shows the maximum amplitude at only 300 nM Ca2+, and it gradually declines at higher Ca2+. In addition to Ca2+, the photoreceptor and the fast flagellar current can be carried by Sr2+ and Ba2+. Mg2+ is conducted less efficiently and at high concentrations blocks the photoreceptor channel. A motion analysis of the cells shows that only Ca2+ and Sr2+ can induce physiological stop responses, whereas the large Ba2+ currents cause abnormal long-lasting cell spiraling.     

Gone with the forest: Assessing global woodpecker conservation from land use patterns

Aim

As a result of their ecological traits, woodpeckers (Picidae, Aves) are highly sensitive to forest cover change. We explored the current land cover in areas of high species richness of woodpeckers to determinate regions where urgent conservation actions are needed. In addition, we identified woodpecker species that are sensitive to forest loss and that have high levels of human habitat modification and low levels of protection (through protected areas) in their distribution ranges.

Location

Global.

Methods

We joined available range maps for all extant 254 woodpecker species with information of their conservation status and tolerances to human habitat modifications and generated a richness map of woodpecker species worldwide. Then, we associated this information (the richness pattern and individual species’ maps) with land cover and protected areas (PAs) maps.

Result

We found that the foremost woodpecker species richness hotspot is in Southeast Asia and is highly modified. At the second species richness hotspot in the eastern Andes, we observed a front of deforestation at its southern extreme and a greater deforested area in its northern extreme but most of its area remains with forest coverage. At the species level, 17 species that are sensitive to forest modification experience extensive deforestation and have low extents of PAs in their ranges.

Main conclusions

The most diverse woodpecker hotspots are mostly occupied by human‐modified landscapes, and a large portion of the species there avoids anthropogenic environments. The level of representation of woodpecker species in PAs is low as a global general pattern, although slightly better in Asia. Our global analysis of threats to woodpecker from land use patterns reiterates the urgent conservation needs for Southeast Asian forests. Finally, based on our results, we recommend a re‐evaluation for inclusion in the Red List of five woodpecker species.

     

Seed vigour contributes to yield improvement in dry direct‐seeded rainfed lowland rice

Dry direct‐seeded rice (DSR) cultivation is widely spreading in tropical Asia, but drought and nutrient deficiency stresses often cause crop failure in rainfed lowlands. The objective of this study was to dissect the physio‐morphological characteristics associated with crop establishment and early vigour of DSR under drought and P deficiency conditions in the Philippines. It was found that new drought‐resistant cultivars bred for DSR (Rc348 and Rc192) had faster germination and sprout growth than popular irrigated rice cultivars (Rc222 and Rc10) under soil water deficit due to rapid moisture acquisition by the germinating seeds from drying soils. There was a significant correlation between seed moisture content and the reduction in seed dry weight, and between reduction in seed dry weight and shoot elongation under both control and drought stress treatments at the germination stage. At the seedling stage, the root growth of Rc348 under drought tended to be more vigorous with its higher root‐to‐shoot ratio compared to Rc222 and Rc10. The seedling vigour of Rc348 under P deficiency was also greater than that of Rc222 due to its greater root growth and P uptake. The yields of Rc348 and Rc192 grown under rainfed condition at the target drought‐prone site where a dry spell of 13 days occurred during crop establishment were higher (4.0–4.1 t ha^?1) than the yield of Rc10 (3.0 t ha^?1). These results suggest that quick germination and seedling vigour with quick root anchorage and great nutrient uptake capacity, even with limitations of soil moisture and nutrients, would be important traits for DSR in rainfed lowlands.     

The Efficiency of Informality: Quantifying Greenhouse Gas Reductions from Informal Recycling in Bogotá, Colombia

The dual challenges of increasing urbanization and consumption are centered in cities in the Global South, where growing waste production threatens public and environmental health. Reuse and recycling are widely recognized to provide broad environmental benefits. Although most industrialized cities replaced their informal recycling sectors with municipally run recycling schemes and have had to build their recycling rates anew, most industrializing cities in the Global South remain centers of recycling and reuse through the work of informal workers. Bogotá, Colombia, is emblematic of many cities in the Global South seeking to modernize their city, in part by formalizing their recycling system. This article asks: What are the greenhouse gas (GHG) emission implications of this modernization? Using interviews and observation in combination with life cycle assessment, we compare GHG emissions resulting from the baseline case (1,200 tonnes per day [t/d] recycled through informal channels; 5,700 t/d landfilled) to three alternative scenarios that formalize the recycling sector: the prohibition of informal recycling; a reduction in informal recycling coupled with a scale‐up of formalized recycling; and the replacement of informal recycling with formal recycling. We find that the baseline recycling scenario, dependent on the informal sector only, emits far fewer GHGs than do all formalization scenarios. Three processes drive the results, in order of magnitude: informal textile reuse (largest GHG savings); landfilling (largest emitter of GHGs); and metal recycling (GHG savings). A hybrid model could combine the incentives and efficiency of the informal system with the better working conditions of the municipal one.     

A Novel Admixture-Based Pharmacogenetic Approach to Refine Warfarin Dosing in Caribbean Hispanics

Aim

This study is aimed at developing a novel admixture-adjusted pharmacogenomic approach to individually refine warfarin dosing in Caribbean Hispanic patients.

Patients & Methods

A multiple linear regression analysis of effective warfarin doses versus relevant genotypes, admixture, clinical and demographic factors was performed in 255 patients and further validated externally in another cohort of 55 individuals.

Results

The admixture-adjusted, genotype-guided warfarin dosing refinement algorithm developed in Caribbean Hispanics showed better predictability (R² = 0.70, MAE = 0.72mg/day) than a clinical algorithm that excluded genotypes and admixture (R² = 0.60, MAE = 0.99mg/day), and outperformed two prior pharmacogenetic algorithms in predicting effective dose in this population. For patients at the highest risk of adverse events, 45.5% of the dose predictions using the developed pharmacogenetic model resulted in ideal dose as compared with only 29% when using the clinical non-genetic algorithm (p<0.001). The admixture-driven pharmacogenetic algorithm predicted 58% of warfarin dose variance when externally validated in 55 individuals from an independent validation cohort (MAE = 0.89 mg/day, 24% mean bias).

Conclusions

Results supported our rationale to incorporate individual’s genotypes and unique admixture metrics into pharmacogenetic refinement models in order to increase predictability when expanding them to admixed populations like Caribbean Hispanics.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01318057","term_id":"NCT01318057"}}NCT01318057     

Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers

Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.     

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols¹. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.     

<Emphasis Type="Italic">Trichoderma</Emphasis> Modulates Stomatal Aperture and Leaf Transpiration Through an Abscisic Acid-Dependent Mechanism in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Trichoderma species are widespread phytostimulant fungi that act through biocontrol of root pathogens, modulation of root architecture, and improving plant adaptation to biotic and abiotic stress. With the major challenge to better understand the contribution of Trichoderma symbionts to plant adaptation to climate changes and confer stress tolerance, we investigated the potential of Trichoderma virens and Trichoderma atroviride in modulating stomatal aperture and plant transpiration. Arabidopsis wild-type (WT) seedlings and ABA-insensitive mutants, abi1-1 and abi2-1, were co-cultivated with either T. virens or T. atroviride, and stomatal aperture and water loss were determined in leaves. Arabidopsis WT seedlings inoculated with these fungal species showed both decreased stomatal aperture and reduced water loss when compared with uninoculated seedlings. This effect was absent in abi1-1 and abi2-1 mutants. T. virens and T. atroviride induced the abscisic acid (ABA) inducible marker abi4:uidA and produced ABA under standard or saline growth conditions. These results show a novel facet of Trichoderma-produced metabolites in stomatic aperture and water-use efficiency of plants.     

The failed invasion of Harmonia axyridis in the Azores,Portugal: Climatic restriction or wrong population origin?

We tested two questions:(i)whether the climatic conditions of the Azorean Islands in Portugal may have restricted the invasion of Harmonia axyridis across this archipelago and(ii)determine what population of this species could have a higher probability of invading the islands.We used MaxEnt to project the climate requirements of different H.axyridis populations from three regions of the world,and the potential global niche of the species in the Azorean islands.Then we assessed the suitability of the islands for each of the three H.axyridis populations and global potential niche through histograms analysis,Principal Component Analysis(PCA)of climate variables,and a variable-by-variable assessment of the suitability response curves compared with the climatic conditions of the Azores.Climatic conditions of the Azores are less suitable for the U.S.and native Asian populations of H.axyridis,and more suitable for European populations and the global potential niche.The PCA showed that the climatic conditions of the islands differed from the climatic requirements of H.axyridis.This difference is mainly explained by precipitation of the wettest month,isothermality,and the minimum temperature of the coldest month.We concluded that the climatic conditions of the Azores could have influenced the establishment and spread of H.axyridis on these islands from Europe.Our results showed that abiotic resistance represented by the climate of the potentially colonizable zones could hinder the establishment of invasive insects,but it could vary depending of the origin of the colonizing population.