Evaluation of mass spectrometric methods for detection of the anti-protozoal drug imidocarb

Imidocarb [N,N\u27-bis[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]urea, C19H20N6O1, m.w. 348.41] is a symmetrical carbanilide derivative used to treat disease caused by protozoans of the Babesia genus. Imidocarb, however, is also considered capable of suppressing Babesia-specific immune responses, allowing Babesia-positive horses to pass a complement fixation test (CFT) without eliminating the infection. This scenario could enable Babesia-infected horses to pass CFT-based importation tests. It is imperative to unequivocally identify and quantify equine tissue residues of imidocarb by mass spectrometry to address this issue. As a pretext to development of sensitive tissue assays, we have investigated possibilities of mass spectrometric (MS) detection of imidocarb. Our analyses disclosed that an unequivocal mass spectral analysis of imidocarb is challenging because of its rapid fragmentation under standard gas chromatography (GC)-MS conditions. In contrast, solution chemistry of imidocarb is more stable but involves distribution into mono- and dicationic species, m/z 349 and 175, respectively, in acid owing to the compound\u27s inherent symmetrical nature. Dicationic imidocarb was the preferred complex as viewed by either direct infusion-electrospray-MS or by liquid chromatography (LC)-MS. Dicationic imidocarb multiple reaction monitoring (MRM: m/z 175 → 162, 145, and 188) therefore offer the greatest opportunities for sensitive detection and LC-MS is more likely than GC-MS to yield a useful quantitative forensic analytical method for detecting imidocarb in horses

Zebrafish: A See-Through Host and a Fluorescent Toolbox to Probe Host–Pathogen Interaction

In many ways, the zebrafish represents a hybrid between mouse and invertebrate infection models. Powerful forwardgenetic tools that have made invertebrates justifiably famous are not only relatively accessible in the zebrafish, but have been exploited to yield new insights into human infectious diseases, including leprosy and tuberculosis [1]. Transgenic technologies have enabled detailed, non-invasive in vivo visualization of macrophages and neutrophils in pitched battle with bacteria and fungi [2,3]. Reverse genetics with morpholinos, vivo-morpholinos, and zinc-finger nucleases (but unfortunately not homologous recombination, which for the moment remains out of reach in this organism) enable examination of the roles of specific genes during infection. Flexible genetic systems such as Gal4-UAS and Cre-Lox permit tissue-specific transformation and ablation ([3]; Figure 1)

OMEGA: A NEW COLD X-RAY SIMULATION FACILITY FOR THE EVALUATION OF OPTICAL COATINGS

We report on recent progress for the development of a new cold X-ray optical test capability using the Omega Facility located at the Laboratory for Laser Energetics (LLE) at the University of Rochester. These tests were done on the 30 kJ OMEGA laser at the Laboratory for Laser Energetics (LLE) at the University of Rochester, Rochester, NY. We conducted a six-shot series called OMEGA II on 14 July 2006 in one eight-hour day (supported by the Defense Threat Reduction Agency). The initial testing was performed using simple protected gold optical coatings on fused silica substrates. PUFFTFT analyses were completed and the specimen's thermal lateral stress and transverse stress conditions were calculated and interpreted. No major anomalies were detected. Comparison of the pre- and posttest reflective measurements coupled with the TFCALC analyses proved invaluable in guiding the analyses and interpreting the observed damage. The Omega facility is a high quality facility for performing evaluation of optical coatings and coupons and provides experience for the development of future National Ignition Facility (NIF) testing

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

The importance of context: an exploration of factors influencing the adoption of student-centered teaching among chemistry, biology, and physics faculty

Background: Research at the secondary and postsecondary levels has clearly demonstrated the critical role that individual and contextual characteristics play in instructors’ decision to adopt educational innovations. Although recent research has shed light on factors influencing the teaching practices of science, technology, engineering, and mathematics (STEM) faculty, it is still not well understood how unique departmental environments impact faculty adoption of evidence-based instructional practices (EBIPs) within the context of a single institution. In this study, we sought to characterize the communication channels utilized by STEM faculty, as well as the contextual and individual factors that influence the teaching practices of STEM faculty at the departmental level. Accordingly, we collected survey and observational data from the chemistry, biology, and physics faculty at a single large research-intensive university in the USA. We then compared the influencing factors experienced by faculty in these different departments to their instructional practices. Results: Analyses of the survey data reveal disciplinary differences in the factors influencing adoption of EBIPs. In particular, the physics faculty (n = 15) had primarily student-centered views about teaching and experienced the most positive contextual factors toward adoption of EBIPs. At the other end of the spectrum, the chemistry faculty (n = 20) had primarily teacher-centered views and experienced contextual factors that hindered the adoption of student-centered practices. Biology faculty (n = 25) fell between these two groups. Classroom observational data reflected these differences: The physics classrooms were significantly more student-centered than the chemistry classrooms. Conclusions: This study demonstrates that disciplinary differences exist in the contextual factors teaching conceptions that STEM faculty experience and hold, even among faculty within the same institution. Moreover, it shows that these differences are associated to the level of adoption of student-centered teaching practices. This work has thus identified the critical need to carefully characterize STEM faculty’s departmental environment and conceptions about teaching before engaging in instructional reform efforts, and to adapt reform activities to account for these factors. The results of this study also caution the over generalization of findings from a study focused on one type of STEM faculty in one environment to all STEM faculty in any environment

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human β-adrenoceptors

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of -9.53 and -8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]-butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol. 1983, 5, 430-437.

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands

The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

Multimessenger Search for Sources of Gravitational Waves and High-Energy Neutrinos: Results for Initial LIGO-Virgo and IceCube

We report the results of a multimessenger search for coincident signals from the LIGO and Virgo gravitational-wave observatories and the partially completed IceCube high-energy neutrino detector, including periods of joint operation between 2007-2010. These include parts of the 2005-2007 run and the 2009-2010 run for LIGO-Virgo, and IceCube's observation periods with 22, 59 and 79 strings. We find no significant coincident events, and use the search results to derive upper limits on the rate of joint sources for a range of source emission parameters. For the optimistic assumption of gravitational-wave emission energy of $10^{-2}$\,M$_\odot$c$^2$ at $\sim 150$\,Hz with $\sim 60$\,ms duration, and high-energy neutrino emission of $10^{51}$\,erg comparable to the isotropic gamma-ray energy of gamma-ray bursts, we limit the source rate below $1.6 \times 10^{-2}$\,Mpc$^{-3}$yr$^{-1}$. We also examine how combining information from gravitational waves and neutrinos will aid discovery in the advanced gravitational-wave detector era