Widefield multifrequency fluorescence lifetime imaging using a two‐tap complementary metal‐oxide semiconductor camera with lateral electric field charge modulators

Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD‐FLIM system based on a complementary metal‐oxide semiconductor camera equipped with two‐tap true correlated double sampling lock‐in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31‐harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean‐Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD‐FLIM system is a valuable tool in the biomedical field.      

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.     

Meta‐analysis reveals the importance of matrix composition for animals in fragmented habitat

Aim Connectivity is a key determinant of the distribution and abundance of organisms and is greatly influenced by anthropogenic landscape modification, yet we lack a synthetic perspective on the magnitude and extent of matrix effects on connectivity. We synthesize results from published studies to understand the importance of matrix effects on fragmented animal populations. Location Global. Methods We conduct a meta‐analysis of 283 fragmented populations representing 184 terrestrial animal taxa to determine the strength of matrix composition effects on the occurrence and abundance of animals in fragmented habitat. Results Studies that use data on matrix composition report greater effects on abundance and occupancy of fragmented populations than studies that define connectivity without regard to the surrounding matrix (i.e. ‘binary’ studies that describe only characteristics of patch habitat). Main conclusions Our findings underscore that conservation strategies must consider the importance of matrix habitat, have important implications for metapopulation and metacommunity paradigms, and provide direct large‐scale, multi‐taxa evidence that matrix habitat is an important driver of ecological dynamics in heterogeneous landscapes.     

The potential of methane‐oxidizing bacteria for applications in environmental biotechnology

Methanotrophic bacteria possess a unique set of enzymes enabling them to oxidize, degrade and transform organic molecules and synthesize new compounds. Therefore, they have great potential in environmental biotechnology. The application of these unique properties was demonstrated in three case studies: (i) Methane escaping from leaky gas pipes may lead to massive mortality of trees in urban areas. Lack of oxygen within the soil surrounding tree roots caused by methanotrophic activity was identified as one of the reasons for this phenomenon. The similarity between metabolic reactions performed by the key enzymes of methanotrophs (methane monooxygenase) and ammonium oxidizers (ammonium monooxygenase) might offer a solution to this problem by applying commercially available nitrification and urease inhibitors. (ii) Methanotrophs are able to co‐metabolically degrade contaminants such as low‐molecular‐weight‐chlorinated hydrocarbons in soil and water in the presence of methane. Batch and continuous trichloroethylene degradation experiments in laboratory‐scale reactors using Methylocystis sp. GB 14 were performed, partly with cells entrapped in a polymer matrix. (iii) Using a short, two‐stage pilot‐scale process, the intracellular polymer accumulation of poly‐β‐hydroxybutyrate (PHB) in methanotrophs reached a maximum of 52%. Interestingly, an ultra‐high‐molecular‐weight PHB of 3.1 MDa was accumulated under potassium deficiency. Under strictly controlled conditions (temperature, pH and methane supply) this process can be nonsterile because of the establishment of a stable microbial community (dominant species Methylocystis sp. GB 25 ≥86% by biomass). The possibility to substitute methane with biogas from renewable sources facilitates the development of a methane‐based PHB production process that yields a high‐quality biopolymer at competitive costs.     

Host specificity testing and examination for plant pathogens reveals that the gall‐inducing psyllid Calophya latiforceps is safe to release for biological control of Brazilian peppertree

Brazilian peppertree, Schinus terebinthifolia Raddi (Anacardiaceae), is one of the worst upland exotic weeds in Florida, USA. Foreign exploration for natural enemies led to the discovery of a pit‐galling psyllid, Calophya latiforceps Burckhardt (Hemiptera: Calophyidae), in the state of Bahia, Brazil, in 2010. Crawlers of C. latiforceps stimulate the formation of galls on the leaves of S. terebinthifolia resulting in leaf discoloration and in some cases leaf abscission. To determine whether C. latiforceps is a safe candidate for biological control of S. terebinthifolia, host specificity and the presence of selected plant pathogens were examined. Adult oviposition, gall formation, and adult survival of C. latiforceps were examined on 89 plant species under no‐choice and choice conditions. We found that C. latiforceps laid eggs on plants in seven families; however, crawlers stimulated gall formation and completed development to adult only on S. terebinthifolia. All crawlers on non‐target plants died, likely due to starvation caused either by the absence of a feeding stimulus or by a hypersensitive plant response. Under no‐choice conditions, 10% of adults lived for 19 days on the target weed, but adult survival was reduced to <3 days on non‐target plants. Choice testing revealed that females preferred to oviposit on S. terebinthifolia compared to non‐target plants. Molecular methods and indicator host inoculations did not detect the presence of ‘Candidatus Liberibacter solanacearum’, ‘Ca. L. asiaticus’, ‘Ca. L. americanus’, ‘Ca. L. africanus’, or plant viruses in adult C. latiforceps. We conclude that releasing C. latiforceps in the USA will have extremely low risk to non‐target plants, and provides another tool for the management of S. terebinthifolia.     

Proteomic characterization of EL4 lymphoma‐derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase‐6 during tumor cell death in vivo

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in‐depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label‐free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase‐3 and an upregulation of caspase‐6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor.     

The secretion of the bacterial phytase PHY‐US417 by Arabidopsis roots reveals its potential for increasing phosphate acquisition and biomass production during co‐growth

Phytic acid (PA) is a major source of inorganic phosphate (Pi) in the soil; however, the plant lacks the capacity to utilize it for Pi nutrition and growth. Microbial phytases constitute a group of enzymes that are able to remobilize Pi from PA. Thus, the use of these phytases to increase the capacity of higher plants to remobilize Pi from PA is of agronomical interest. In the current study, we generate transgenic Arabidopsis lines (ePHY) overexpressing an extracellular form of the phytase PHY‐US417 of Bacillus subtilis, which are characterized by high levels of secreted phytase activity. In the presence of PA as sole source of Pi, while the wild‐type plants show hallmark of Pi deficiency phenotypes, including the induction of the expression of Pi starvation‐induced genes (PSI, e.g. PHT1;4) and the inhibition of growth capacity, the ePHY overexpressing lines show a higher biomass production and no PSI induction. Interestingly, when co‐cultured with ePHY overexpressors, wild‐type Arabidopsis plants (or tobacco) show repression of the PSI genes, improvement of Pi content and increases in biomass production. In line with these results, mutants in the high‐affinity Pi transporters, namely pht1;1 and pht1;1‐1;4, both fail to accumulate Pi and to grow when co‐cultured with ePHY overexpressors. Taken together, these data demonstrate the potential of secreted phytases in improving the Pi content and enhancing growth of not only the transgenic lines but also the neighbouring plants.     

Water‐compatible silica sol–gel molecularly imprinted polymer as a potential delivery system for the controlled release of salicylic acid

Molecularly imprinted polymers (MIPs) for salicylic acid were synthesized and evaluated in aqueous environments in the aim to apply them as drug delivery carriers. One organic MIP and one inorganic MIP based on the sol–gel process were synthesized. The organic MIP was prepared by radical polymerization using the stoichiometric functional monomer, 1‐(4‐vinylphenyl)‐3‐(3,5‐bis(trifluoromethyl)phenyl)urea, which can establish strong electrostatic interactions with the –COOH of salicylic acid. The sol–gel MIP was prepared with 3‐(aminopropyl)triethoxysilane and trimethoxyphenylsilane, as functional monomers and tetraethyl orthosilicate as the crosslinker. While the organic MIPs bound the target specifically in acetonitrile, they exhibited lower binding in the presence of water, although the imprinting factor increased under these conditions, due to reduced non‐specific binding. The sol–gel MIP has a high specificity and capacity for the drug in ethanol, a solvent compatible with drug formulation and biomedical applications. In vitro release profiles of the polymers in water were evaluated, and the results were modelled by Fick's law of diffusion and the power law. Analysis shows that the release mechanism was predominantly diffusion‐controlled. Copyright © 2014 John Wiley & Sons, Ltd.     

Genomic data reveals potential for hybridization,introgression, and incomplete lineage sorting to confound phylogenetic relationships in an adaptive radiation of narrow‐mouth frogs

The microhylid frog genus Kaloula is an adaptive radiation spanning the edge of the Asian mainland and multiple adjacent island archipelagos, with much of the clade's diversity associated with an endemic Philippine radiation. Relationships among clades from the Philippines, however, remain unresolved. With ultraconserved element (UCE) and mitogenomic data, we identified highly supported differences in topology and areas of poor resolution, for each marker set. Using the UCE data, we then identified possible instances of contemporary hybridization, past introgression, and incomplete lineage sorting (ILS) within the Philippine Kaloula. Using a simulation approach, and an estimate of the Philippine Kaloula clade origin (12.7—21.0 mya), we demonstrate that an evolutionary history including inferred instances of hybridization, introgression, and ILS leads to phylogenetic reconstructions that show concordance with results from the observed mitogenome and UCE data. In the process of validating a complex evolutionary scenario in the Philippine Kaloula, we provide the first demonstration of the efficacy of UCE data for phylogenomic studies of anuran amphibians.