Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging. Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin

2012 Market Report on U.S. Wind Technologies in Distributed Applications

At the end of 2012, U.S. wind turbines in distributed applications reached a 10-year cumulative installed capacity of more than 812 MW from more than 69,000 units across all 50 states. In 2012 alone, nearly 3,800 wind turbines totaling 175 MW of distributed wind capacity were documented in 40 states and in the U.S. Virgin Islands, with 138 MW using utility-scale turbines (i.e., greater than 1 MW in size), 19 MW using mid-size turbines (i.e., 101 kW to 1 MW in size), and 18.4 MW using small turbines (i.e., up to 100 kW in size). Distributed wind is defined in terms of technology application based on a wind project’s location relative to end-use and power-distribution infrastructure, rather than on technology size or project size. Distributed wind systems are either connected on the customer side of the meter (to meet the onsite load) or directly to distribution or micro grids (to support grid operations or offset large loads nearby). Estimated capacity-weighted average costs for 2012 U.S. distributed wind installations was $2,540/kW for utility-scale wind turbines,$2,810/kW for mid-sized wind turbines, and $6,960/kW for newly manufactured (domestic and imported) small wind turbines. An emerging trend observed in 2012 was an increased use of refurbished turbines. The estimated capacity-weighted average cost of refurbished small wind turbines installed in 2012 was$4,080/kW. As a result of multiple projects using utility-scale turbines, Iowa deployed the most new overall distributed wind capacity, 37 MW, in 2012. Nevada deployed the most small wind capacity in 2012, with nearly 8 MW of small wind turbines installed in distributed applications. In the case of mid-size turbines, Ohio led all states in 2012 with 4.9 MW installed in distributed applications. State and federal policies and incentives continued to play a substantial role in the development of distributed wind projects. In 2012, U.S. Treasury Section 1603 payments and grants and loans from the U.S. Department of Agriculture’s Rural Energy for America Program were the main sources of federal funding for distributed wind projects. State and local funding varied across the country, from rebates to loans, tax credits, and other incentives. Reducing utility bills and hedging against potentially rising electricity rates remain drivers of distributed wind installations. In 2012, other drivers included taking advantage of the expiring U.S. Treasury Section 1603 program and a prosperous year for farmers. While 2012 saw a large addition of distributed wind capacity, considerable barriers and challenges remain, such as a weak domestic economy, inconsistent state incentives, and very competitive solar photovoltaic and natural gas prices. The industry remains committed to improving the distributed wind marketplace by advancing the third-party certification process and introducing alternative financing models, such as third-party power purchase agreements and lease-to-own agreements more typical in the solar photovoltaic market. Continued growth is expected in 2013

The Current State of Performance Appraisal Research and Practice: Concerns, Directions, and Implications

On the surface, it is not readily apparent how some performance appraisal research issues inform performance appraisal practice. Because performance appraisal is an applied topic, it is useful to periodically consider the current state of performance research and its relation to performance appraisal practice. This review examines the performance appraisal literature published in both academic and practitioner outlets between 1985 and 1990, briefly discusses the current state of performance appraisal practice, highlights the juxtaposition of research and practice, and suggests directions for further research

Gliadin Nanoparticles Induce Immune Tolerance to Gliadin in Mouse Models of Celiac Disease

Peer reviewe

Ciprofloxacin Causes Persister Formation by Inducing the TisB toxin in Escherichia coli

Persisters are specialized survivor cells that arise in populations of E. coli after antibiotic-mediated DNA damage induces the production of a small membrane-acting peptide TisB, which causes reversible dormancy. The TisB-dependent persisters are tolerant to multiple antibiotics

Strategic and practical guidelines for successful structured illumination microscopy

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes

The role of released ATP in killing Candida albicans and other extracellular microbial pathogens by cationic peptides

A unifying theme common to the action of many cationic peptides that display lethal activities against microbial pathogens is their specific action at microbial membranes that results in selective loss of ions and small nucleotides chiefly ATP. One model cationic peptide that induces non-lytic release of ATP from the fungal pathogen Candida albicans is salivary histatin 5 (Hst 5). The major characteristic of Hst 5-induced ATP release is that it occurs rapidly while cells are still metabolically active and have polarized membranes, thus precluding cell lysis as the means of release of ATP. Other cationic peptides that induce selective release of ATP from target microbes are lactoferricin, human neutrophil defensins, bactenecin, and cathelicidin peptides. The role of released extracellular ATP induced by cationic peptides is not known, but localized increases in extracellular ATP concentration may serve to potentiate cell killing, facilitate further peptide uptake, or function as an additional signal to activate the host innate immune system at the site of infection