The road to precision oncology

The ultimate goal of precision medicine is to use population-based molecular, clinical and other data to make individually tailored clinical decisions for patients, although the path to achieving this goal is not entirely clear. A new study shows how knowledge banks of patient data can be used to make individual treatment decisions in acute myeloid leukemia

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy

Understanding the Role of Host Hemocytes in a Squid/Vibrio Symbiosis Using Transcriptomics and Proteomics

The symbiosis between the squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, serves as a model for understanding interactions between beneficial bacteria and animal hosts. The establishment and maintenance of the association is highly specific and depends on the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environment. Current evidence suggests that the host’s cellular innate immune system, in the form of macrophage-like hemocytes, helps to mediate host tolerance of V. fischeri. To begin to understand the role of hemocytes in this association, we analyzed these cells by high-throughput 454 transcriptomic and liquid chromatography/tandem mass spectrometry (LC-MS/MS) proteomic analyses. 454 high-throughput sequencing produced 650, 686 reads totaling 279.9 Mb while LC-MS/MS analyses of circulating hemocytes putatively identified 702 unique proteins. Several receptors involved with the recognition of microbial-associated molecular patterns were identified. Among these was a complete open reading frame to a putative peptidoglycan recognition protein (EsPGRP5) with conserved residues for amidase activity. Assembly of the hemocyte transcriptome showed EsPGRP5 had high coverage, suggesting it is among the 5% most abundant transcripts in circulating hemocytes. Other transcripts and proteins identified included members of the conserved NF-κB signaling pathway, putative members of the complement pathway, the carbohydrate binding protein galectin, and cephalotoxin. Quantitative Real-Time PCR of complement-like genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes from adult squid with colonized light organs compared to those isolated from hosts where the symbionts were removed. These data suggest that the presence of the symbiont influences gene expression of the cellular innate immune system of E. scolopes

The repeatability and reproducibility of four techniques for measuring horizontal heterophoria: Implications for clinical practice

PURPOSE: Convergence insufficiency, the most common binocular vision anomaly, is characterised by a receded near point of convergence and an exophoria which is at least 4 prism dioptres (Δ) larger at near than at distance. However, the repeatability of standard heterophoria measures are poorly understood. This study assessed the ability of four common heterophoria tests to detect differences of 4Δ by evaluating the inter- and intra-examiner variability of the selected techniques. METHODS: Distance and near horizontal heterophorias of 20 visually-normal adults were measured with the alternating prism cover test, von Graefe prism dissociation, Howell Card and Maddox Rod by two examiners at two separate visits using standardised instructions and techniques. We investigated inter- and intra-examiner variability using repeatability and reproducibility indices, as well as Bland-Altman analysis with acceptable limits of agreement defined as ±2Δ. RESULTS: The Howell card test had the lowest intra-examiner variability at both distance and near, as well as the best 95% limits of agreement (±1.6Δ for distance and ±3.7Δ for near). Inter-examiner reproducibility results were similar, although at near the alternating prism cover test had better repeatability (1.1Δ, 95% confidence intervals −1.1Δ to 4.0Δ) than the Howell card (1.4Δ, 95% confidence intervals −1.9Δ to 5.9Δ). CONCLUSION: The low repeatability of many standard clinical heterophoria tests limits the ability to reliably detect a 4Δ difference. The Howell Card provided the most repeatable and reproducible results indicating that this technique should be used to detect small changes in heterophoria magnitude and direction

Exploring the Origin and Fate of the Magellanic Stream with Ultraviolet and Optical Absorption

(Abridged) We present an analysis of ionization and metal enrichment in the Magellanic Stream (MS), the nearest gaseous tidal stream, using HST/STIS and FUSE ultraviolet spectroscopy of two background AGN, NGC 7469 and Mrk 335. For NGC 7469, we include optical spectroscopy from VLT/UVES. In both sightlines the MS is detected in low-ion and high-ion absorption. Toward NGC 7469, we measure a MS oxygen abundance [O/H]_MS=[OI/HI]=-1.00+/-0.05(stat)+/-0.08(syst), supporting the view that the Stream originates in the SMC rather than the LMC. We use CLOUDY to model the low-ion phase of the Stream as a photoionized plasma using the observed Si III/Si II and C III/C II ratios. Toward Mrk 335 this yields an ionization parameter log U between -3.45 and -3.15 and a gas density log (n_H/cm^-3) between -2.51 and -2.21. Toward NGC 7469 we derive sub-solar abundance ratios for [Si/O], [Fe/O], and [Al/O], indicating the presence of dust in the MS. The high-ion column densities are too large to be explained by photoionization, but also cannot be explained by a single-temperature collisional-ionization model (equilibrium or non-equilibrium). This suggests the high-ion plasma is multi-phase. Summing over the low-ion and high-ion phases, we derive conservative lower limits on the ratio N(total H II)/N(H I) of >19 toward NGC 7469 and >330 toward Mrk 335, showing that along these two directions the vast majority of the Stream has been ionized. The presence of warm-hot plasma together with the small-scale structure observed at 21 cm provides evidence for an evaporative interaction with the hot Galactic corona. This scenario, predicted by hydrodynamical simulations, suggests that the fate of the MS will be to replenish the Galactic corona with new plasma, rather than to bring neutral fuel to the disk.Comment: Accepted for publication in ApJ. 18 pages, 7 figures, all in colo

Neurotransmitters Drive Combinatorial Multistate Postsynaptic Density Networks

The mammalian postsynaptic density (PSD) comprises a complex collection of ~1100 proteins. Despite extensive knowledge of individual proteins, the overall organization of the PSD is poorly understood. Here, we define maps of molecular circuitry within the PSD based on phosphorylation of postsynaptic proteins. Activation of a single neurotransmitter receptor, the N-methyl-D-aspartate receptor (NMDAR), changed the phosphorylation status of 127 proteins. Stimulation of ionotropic and metabotropic glutamate receptors and dopamine receptors activated overlapping networks with distinct combinatorial phosphorylation signatures. Using peptide array technology, we identified specific phosphorylation motifs and switching mechanisms responsible for the integration of neurotransmitter receptor pathways and their coordination of multiple substrates in these networks. These combinatorial networks confer high information-processing capacity and functional diversity on synapses, and their elucidation may provide new insights into disease mechanisms and new opportunities for drug discover

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales

Association of type XI collagen genes with chronic Achilles tendinopathy in independent populations from South Africa and Australia

BACKGROUND: Type XI collagen, which is expressed in developing tendons and is encoded by the COL11A1, COL11A2 and COL2A1 genes, shares structural and functional homology with type V collagen, which plays an important role in collagen fibril assembly. We investigated the association of these three polymorphisms with Achilles tendinopathy (AT) and whether these polymorphisms interact with COL5A1 to modulate the risk of AT. METHODS: 184 participants diagnosed with chronic AT (TEN) and 338 appropriately matched asymptomatic controls (CON) were genotyped for the three polymorphisms. RESULTS: Although there were no independent associations with AT, the TCT pseudohaplotype constructed from rs3753841 (T/C), rs1676486 (C/T) and rs1799907 (T/A) was significantly over-represented (p=0.006) in the TEN (25.9%) compared with the CON (17.1%) group. The TCT(AGGG) pseudohaplotypes constructed using these type XI collagen polymorphisms and the functional COL5A1 rs71746744 (-/AGGG) polymorphism were also significantly over-represented (p<0.001) in the TEN (25.2%) compared with the CON (9.1%) group. DISCUSSION: The genes encoding structural and functionally related type XI (COL11A1 and COL11A2) and type V (COL5A1) collagens interact with one another to collectively modulate the risk for AT. Although there are no immediate clinical applications, the results of this study provide additional evidence that interindividual variations in collagen fibril assembly might be an important molecular mechanism in the aetiology of chronic AT.This study was supported in part by funds from the National Research Foundation of South Africa (grant number: CPR20110712000020673), University of Cape Town, and the South African Medical Research Council.http://bjsm.bmj.com/hb201

Estimating Historical Forest Density From Land‐Survey Data: A Response to Baker and Williams (2018)

In the Western United States, historical forest conditions are used to inform land management and ecosystem restoration goals (North et al. 2009, Stephens et al. 2016). This interest is based on the premise that historical forests were resilient to ecological disturbances (Keane et al. 2018). Researchers throughout the United States have used the General Land Office (GLO) surveys of the late 19th and early 20th centuries to estimate historical forest conditions (Bourdo 1956, Schulte and Mladenoff 2001, Cogbill et al. 2002, Paciorek et al. 2016). These surveys were conducted throughout the United States and represent a systematic, historical sample of trees across a broad geographic area. A challenge of using GLO survey data is the accurate estimation of tree density from sparse witness tree data. Levine et al. (2017) tested the accuracy and precision of four plotless density estimators that can be applied to GLO survey sample data, including the Cottam (Cottam and Curtis 1956), Pollard (Pollard 1971), Morisita (Morisita 1957), and mean harmonic Voronoi density (MHVD; Williams and Baker 2011) estimators. The Cottam, Pollard, and Morisita are count‐based plotless density estimators (PDE) and have a history of being applied to GLO data (e.g., Kronenfeld and Wang 2007, Rhemtulla et al. 2009, Hanberry et al. 2012, Maxwell et al. 2014, Goring et al. 2016). The MHVD estimator is an area‐based PDE that has been applied by the study\u27s authors to sites in the western United States (Baker 2012, 2014), but had not been independently evaluated. Levine et al. (2017) found that the Morisita estimator was the least biased and most precise estimator for estimating density from GLO survey data, with a relative root mean square error ranging from 0.11 to 0.78 for the six study sites. Levine et al. (2017) also demonstrated the MHVD approach consistently overestimated density from 16% to 258% in all six study areas that were analyzed