Exploiting nanobodies and Affimers for superresolution imaging in light microscopy

Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibody provides several advantages for superresolution imaging

A novel ruthenium(II)–cobaloxime supramolecular complex for photocatalytic H_2 evolution: synthesis, characterisation and mechanistic studies

We report the synthesis and characterization of novel mixed-metal binuclear ruthenium(II)–cobalt(II) photocatalysts for hydrogen evolution in acidic acetonitrile. First, 2-(2′-pyridyl)benzothiazole (pbt), 1, was reacted with RuCl_(3)·xH_(2)O to produce [Ru(pbt)_(2)Cl_2]·0.25CH_(3)COCH_3, 2, which was then reacted with 1,10-phenanthroline-5,6-dione (phendione), 3, in order to produce [Ru(pbt)_(2)(phendione)](PF_(6))_2·4H_(2)O, 4. Compound 4 was then reacted with 4-pyridinecarboxaldehyde in order to produce [Ru(pbt)_(2)(L-pyr)](PF_6)_(2)·9.5H_(2)O, 5 (where L-pyr = (4-pyridine)oxazolo[4,5-f]phenanthroline). Compound 5 was then reacted with [Co(dmgBF_2)_(2)(H_(2)O)_2] (where dmgBF_(2) = difluoroboryldimethylglyoximato) in order to produce the mixed-metal binuclear complex, [Ru(pbt)_(2)(L-pyr)Co(dmgBF_(2))_(2)(H_(2)O)](PF_(6))_2·11H_(2)O·1.5CH_(3)COCH_3, 6. [Ru(Me_(2)bpy)_2(L-pyr)Co(dmgBF_2)_(2)(OH_2)](PF_6)_(2), 7 (where Me_(2)bpy = 1,10-phenanthroline, 4,4′-dimethyl-2,2′-bipyridine) and [Ru(phen)_(2)(L-pyr)Co(dmgBF_2)_(2)(OH_2)](PF_(6))_2, 8 were also synthesised. All complexes were characterized by elemental analysis, ESI MS, HRMS, UV-visible absorption, ^(11)B, ^(19)F, and ^(59)Co NMR, ESR spectroscopy, and cyclic voltammetry, where appropriate. Photocatalytic studies carried out in acidified acetonitrile demonstrated constant hydrogen generation longer than a 42 hour period as detected by gas chromatography. Time resolved spectroscopic measurements were performed on compound 6, which proved an intramolecular electron transfer from an excited Ru(II) metal centre to the Co(II) metal centre via the bridging L-pyr ligand. This resulted in the formation of a cobalt(I)-containing species that is essential for the production of H_2 gas in the presence of H^+ ions. A proposed mechanism for the generation of hydrogen is presented

<i>Gaia</i> Data Release 1. Summary of the astrometric, photometric, and survey properties

Context. At about 1000 days after the launch of Gaia we present the first Gaia data release, Gaia DR1, consisting of astrometry and photometry for over 1 billion sources brighter than magnitude 20.7. Aims. A summary of Gaia DR1 is presented along with illustrations of the scientific quality of the data, followed by a discussion of the limitations due to the preliminary nature of this release. Methods. The raw data collected by Gaia during the first 14 months of the mission have been processed by the Gaia Data Processing and Analysis Consortium (DPAC) and turned into an astrometric and photometric catalogue. Results. Gaia DR1 consists of three components: a primary astrometric data set which contains the positions, parallaxes, and mean proper motions for about 2 million of the brightest stars in common with the HIPPARCOS and Tycho-2 catalogues – a realisation of the Tycho-Gaia Astrometric Solution (TGAS) – and a secondary astrometric data set containing the positions for an additional 1.1 billion sources. The second component is the photometric data set, consisting of mean G-band magnitudes for all sources. The G-band light curves and the characteristics of ∼3000 Cepheid and RR-Lyrae stars, observed at high cadence around the south ecliptic pole, form the third component. For the primary astrometric data set the typical uncertainty is about 0.3 mas for the positions and parallaxes, and about 1 mas yr−1 for the proper motions. A systematic component of ∼0.3 mas should be added to the parallax uncertainties. For the subset of ∼94 000 HIPPARCOS stars in the primary data set, the proper motions are much more precise at about 0.06 mas yr−1. For the secondary astrometric data set, the typical uncertainty of the positions is ∼10 mas. The median uncertainties on the mean G-band magnitudes range from the mmag level to ∼0.03 mag over the magnitude range 5 to 20.7. Conclusions. Gaia DR1 is an important milestone ahead of the next Gaia data release, which will feature five-parameter astrometry for all sources. Extensive validation shows that Gaia DR1 represents a major advance in the mapping of the heavens and the availability of basic stellar data that underpin observational astrophysics. Nevertheless, the very preliminary nature of this first Gaia data release does lead to a number of important limitations to the data quality which should be carefully considered before drawing conclusions from the data

Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications

We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications

A qualitative evidence synthesis of employees' views of workplace smoking reduction or cessation interventions

Background The need to reduce smoking rates is a recognised public health policy issue in many countries. The workplace offers a potential context for offering smokers’ programmes and interventions to assist smoking cessation or reduction. A qualitative evidence synthesis of employees’ views about such programmes might explain why some interventions appear effective and others not, and can be used to develop evidence-based interventions for this population and setting. Methods A qualitative evidence synthesis of primary research exploring employees’ views about workplace interventions to encourage smoking cessation, including both voluntary programmes and passive interventions, such as restrictions or bans. The method used was theory-based “best fit” framework synthesis. Results Five relevant theories on workplace smoking cessation were identified and used as the basis for an a priori framework. A comprehensive literature search, including interrogation of eight databases, retrieved 747 unique citations for the review. Fifteen primary research studies of qualitative evidence were found to satisfy the inclusion criteria. The synthesis produced an evidence-based conceptual model explaining employees’ experiences of, and preferences regarding, workplace smoking interventions. Conclusion The synthesis suggests that workplace interventions should employ a range of different elements if they are to prove effective in reducing smoking among employees. This is because an employee who feels ready and able to change their behaviour has different needs and preferences from an employee who is not at that stage. Only a multi-faceted intervention can satisfy the requirements of all employees

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

ILC3 function as a double-edged sword in inflammatory bowel diseases

Inflammatory bowel diseases (IBD), composed mainly of Crohn’s disease (CD) and ulcerative colitis (UC), are strongly implicated in the development of intestinal inflammation lesions. Its exact etiology and pathogenesis are still undetermined. Recently accumulating evidence supports that group 3 innate lymphoid cells (ILC3) are responsible for gastrointestinal mucosal homeostasis through moderate generation of IL-22, IL-17, and GM-CSF in the physiological state. ILC3 contribute to the progression and aggravation of IBD while both IL-22 and IL-17, along with IFN-γ, are overexpressed by the dysregulation of NCR− ILC3 or NCR+ ILC3 function and the bias of NCR+ ILC3 towards ILC1 as well as regulatory ILC dysfunction in the pathological state. Herein, we feature the group 3 innate lymphoid cells’ development, biological function, maintenance of gut homeostasis, mediation of IBD occurrence, and potential application to IBD therapy