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

Determinants of pain interference and headache impact in patients who have chronic migraine with medication overuse: Results from the MOTS trial

OBJECTIVE: Pain interference and headache impact refer to negative consequences that pain and headache have on one\u27s life. This study investigated determinants of these negative impacts in a large patient cohort who have chronic migraine with medication overuse. METHODS: Six hundred and eleven adults were enrolled from 34 headache, neurology, and primary care clinics. Negative consequences of chronic migraine with medication overuse were determined using the Patient-Reported Outcomes Measurement Information System (PROMIS) Pain Interference 6b questionnaire and the Headache Impact Test 6. Relationships between PROMIS-6b and Headache Impact Test 6 scores with demographics, headache characteristics, medication use, anxiety symptoms, and depression symptoms were assessed with linear regression. Elastic Net regression was used to develop a multiple regression model. RESULTS: PROMIS-6b T-Scores averaged 65.2 (SD 5.4) and Headache Impact Test 6 scores averaged 65.0 (SD 5.3), indicating severe negative consequences of chronic migraine with medication overuse. Chronic migraine with medication overuse interfered with enjoyment of life, concentration, daily activities, doing tasks away from home, and socializing. Depression symptom severity had the strongest relationship with pain interference and headache impact. Moderate-to-severe headache frequency, headache intensity, and anxiety symptoms were also associated with pain interference and headache impact. CONCLUSIONS: Chronic migraine with medication overuse is associated with substantial negative consequences, the extent of which is most strongly related to depression symptoms

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

Single Nucleotide Polymorphisms That Increase Expression of the Guanosine Triphosphatase RAC1 Are Associated With Ulcerative Colitis

BACKGROUND & AIMS: RAC1 is a GTPase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases (IBD) are associated with dysregulation of immune defenses. We studied the role of RAC1 in IBD using human genetic and functional studies and animal models of colitis. METHODS: We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms (SNPs) in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 mRNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulphate sodium (DSS). RESULTS: We observed a genetic association between RAC1 with ulcerative colitis (UC) in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the SNPs rs10951982 (Pcombined UC = 3.3 × 10–8, odds ratio [OR]=1.43 [1.26–1.63]) and rs4720672 (Pcombined UC=4.7 × 10–6, OR=1.36 [1.19–1.58]). Patients with IBD who had the rs10951982 risk allele had increased expression of RAC1, compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected them against DSS-induced colitis. CONCLUSION: Studies of human tissue samples and knockout mice demonstrated a role for the GTPase RAC1 in the development of UC; increased expression of RAC1 was associated with susceptibility to colitis

The Photon Ring in M87*

We report measurements of the gravitationally lensed secondary image—the first in an infinite series of so-called “photon rings”—around the supermassive black hole M87* via simultaneous modeling and imaging of the 2017 Event Horizon Telescope (EHT) observations. The inferred ring size remains constant across the seven days of the 2017 EHT observing campaign and is consistent with theoretical expectations, providing clear evidence that such measurements probe spacetime and a striking confirmation of the models underlying the first set of EHT results. The residual diffuse emission evolves on timescales comparable to one week. We are able to detect with high significance a southwestern extension consistent with that expected from the base of a jet that is rapidly rotating in the clockwise direction. This result adds further support to the identification of the jet in M87* with a black hole spin-driven outflow, launched via the Blandford-Znajek process. We present three revised estimates for the mass of M87* based on identifying the modeled thin ring component with the bright ringlike features seen in simulated images, one of which is only weakly sensitive to the astrophysics of the emission region. All three estimates agree with each other and previously reported values. Our strongest mass constraint combines information from both the ring and the diffuse emission region, which together imply a mass-to-distance ratio of 4.20 − 0.06 + 0.12 μ as and a corresponding black hole mass of (7.13 \ub1 0.39) 7 109 M ⊙, where the error on the latter is now dominated by the systematic uncertainty arising from the uncertain distance to M87*

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