?The Infectious Disease Society of America In-Training Examination (IDSA ITE) is a feedback tool used to help fellows track their knowledge acquisition during fellowship training. We determined whether the scores on the IDSA ITE and from other major medical knowledge assessments predict performance on the American Board of Internal Medicine (ABIM) Infectious Disease Certification Examination.
Recently the decision-making committee of the compulsory Swiss accident insurance scheme needed to make a basic decision as to whether to fund hand transplantation under that scheme or not. A Health Technology Assessment was commissioned to inform decision-making and gain experience with applicability of the method. The following were main findings from various domains. Compared with prosthesis fitting, the outcome of hand transplantation is satisfactory for function and sensibility. Complications due to immunosuppression are frequent, sometimes severe and potentially life-shortening. The direct medical costs over the entire life span calculated for a 35-year-old unilaterally amputated base case patient were CHF 528,600 (EUR 438,500) higher than for a prosthesis. There are challenging ethical, legal and organizational issues. The committee decided not to reimburse hand transplantation for ethical reasons. The Health Technology Assessment has been shown to be a useful tool for decision-making in the context of Swiss accident insurance. Level of evidence: IV.
The ability of human participants to integrate fragmented stimulus elements into perceived coherent contours (amidst a field of distracter elements) has been intensively studied across a large number of contour element parameters, ranging from luminance contrast and chromaticity to motion and stereo. The evidence suggests that contour integration performance depends on the low-level Fourier properties of the stimuli. Thus, to understand contour integration, it would be advantageous to understand the properties of the low-level filters that the visual system uses to process contour stimuli. We addressed this issue by examining the role of stimulus element orientation bandwidth in contour integration, a previously unexplored area. We carried out three psychophysical experiments, and then simulated all of the experiments using a recently developed two-stage filter-overlap model whereby the contour grouping occurs by virtue of the overlap between the filter responses to different elements. The first stage of the model responds to the elements, while the second stage integrates the responses along the contour. We found that the first stage had to be fairly broadly tuned for orientation to account for our results. The model showed a very good fit to a large data set with relatively few free parameters, suggesting that this class of model may have an important role to play in helping us to better understand the mechanisms of contour integration.
Experienced clinicians derive many diagnoses intuitively, because most new problems they see closely resemble problems they've seen before. The majority of these diagnoses, but not all, will be correct. This study determined whether further reflection regarding initial diagnoses improves diagnostic accuracy during a high-stakes board exam, a model for studying clinical decision making.
We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (3), AZA34 (4), and AZA35, respectively) in mussels, seawater, and Azadinium spinosum culture. Approximately 700 ?g of 3 and 250 ?g of 4 were isolated from a bulk culture of A. spinosum, and their structures determined by MS and NMR spectroscopy. These compounds differ significantly at the carboxyl end of the molecule from known AZA analogues and therefore provide valuable information on structure-activity relationships. Initial toxicological assessment was performed using an in vitro model system based on Jurkat T lymphocyte cytotoxicity, and the potencies of 3 and 4 were found to be 0.22- and 5.5-fold that of AZA1 (1), respectively. Thus, major changes in the carboxyl end of 1 resulted in significant changes in toxicity. In mussel extracts, 3 was detected at low levels, whereas 4 and AZA35 were detected only at extremely low levels or not at all. The structures of 3 and 4 are consistent with AZAs being biosynthetically assembled from the amino end.
Purpose: To assess whether partial occlusion of the non-amblyopic eye with Bangerter filters can immediately reduce suppression and promote binocular summation of contrast in observers with amblyopia. Methods: In Experiment 1, suppression was measured for 22 observers (mean age 20 years, range 14-32 years) with strabismic or anisometropic amblyopia and 10 controls using our previously established "balance point" protocol. Measurements were made at baseline and with 0.6, 0.4 and 0.2 strength Bangerter filters placed over the non-amblyopic/dominant eye. In Experiment 2, measurements of contrast sensitivity were made under binocular and monocular viewing conditions for 25 observers with anisometropic amblyopia (mean age 17 years, range 11-28 years) and 22 controls (mean age 24 years, range 22 - 27 years). Measurements were made at baseline and with 0.4 and 0.2 strength Bangerter filters placed over the non-amblyopic/dominant eye. Binocular summation ratios (BSRs) were calculated at baseline and with Bangerter filters. Results: Experiment 1: Bangerter filters reduced suppression in observers with amblyopia and induced suppression in controls (p = 0.025). The 0.2 strength filter eliminated suppression in observers with amblyopia and this was not a visual acuity effect. Experiment 2: Bangerter filters were able to induce normal levels of binocular contrast summation in the group of observers with anisometropic amblyopia for a stimulus with a spatial frequency of 3 cycles per degree (cpd) (p = 0.006). The filters reduced binocular summation in controls. Conclusions: Bangerter filters can immediately reduce suppression and promote binocular summation for mid/low spatial frequencies in observers with amblyopia.
Occlusion therapy for amblyopia is predicated on the idea that amblyopia is primarily a disorder of monocular vision; however, there is growing evidence that patients with amblyopia have a structurally intact binocular visual system that is rendered functionally monocular due to suppression. Furthermore, we have found that a dichoptic treatment intervention designed to directly target suppression can result in clinically significant improvement in both binocular and monocular visual function in adult patients with amblyopia. The fact that monocular improvement occurs in the absence of any fellow eye occlusion suggests that amblyopia is, in part, due to chronic suppression. Previously the treatment has been administered as a psychophysical task and more recently as a video game that can be played on video goggles or an iPod device equipped with a lenticular screen. The aim of this case-series study of 14 amblyopes (six strabismics, six anisometropes and two mixed) ages 13 to 50 years was to investigate: 1. whether the portable video game treatment is suitable for at-home use and 2. whether an anaglyphic version of the iPod-based video game, which is more convenient for at-home use, has comparable effects to the lenticular version.
While the contrast sensitivity approach has been successful in evaluating the processing of first-order stimuli, there is a need to develop comparable ways of assessing second-order vision. Our purpose here is to establish normative data on second-order contrast-, orientation-, and motion-modulation sensitivity in humans. We propose a unified framework, applying the quick contrast sensitivity function (qCSF) method, which was recently developed for the rapid measurement of contrast sensitivity across the full spatial-frequency range (Lesmes, Lu, Baek, & Albright, 2010), to measure both first- and second-order sensitivity functions. We first show that the qCSF methodology can be successfully adapted to different kinds of first- and second-order measurements. We provide a normative dataset for both first- and second-order sensitivity, and we show that the sensitivity to all these stimuli is equal in the two eyes. Our results confirm some strong differences between first- and second-order processing, in accordance with the classical filter-rectify-filter model. They suggest a common contrast detection mechanism but different second-order mechanisms.
?-N-methylamino-l-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 µg/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 µg/g DW), while only several samples contained quantifiable free BMAA.
Embryonic development, adult tissue repair and cancer share a number of common regulating pathways. The basic processes that govern the events that induce mesenchymal properties in epithelial cells - a process known as epithelial-mesenchymal transition - are central for embryonic development, and can be resumed in adults either during wound healing or tissue regeneration. A misregulation of these pathways is involved in pathological situations, such as tissue fibrosis and cancer. Proteomic approaches have emerged as promising tools to better understand the signaling pathways that govern these complex biological processes. This review focuses on the recent proteomic-based contributions to better understand the modulation of Transforming growth factor-beta (TGF-?), wingless-type MMTV integration site family (Wnt), Notch and Receptor tyrosine kinase (RTK) signaling pathways. New advances include the description of new protein interactions, the formation of new protein complexes or the description on how some post-translational modifications are regulating these pathways. Understanding protein interactions and the tempo-spatial modulation of these pathways might lead us to interesting research quests in cancer, embryonic development or even on improving adult tissue regeneration capabilities. This article is protected by copyright. All rights reserved.
Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.
In this work, both experimental data and a model are presented on the coupling between living cells and graphene solution-gated field-effect transistors. Modified HEK 293 cells are successfully cultured on graphene transistor arrays and electrically accessed by the patch clamp method. Transistor recordings are presented, showing the opening and closing of voltage-gated potassium ion channels in the cell membrane. The experimental data is compared with the broadly used standard point-contact model. The ion dynamics in the cell-transistor cleft are analyzed to account for the differences between the model and the experimental data revealing a significant increase in the total ionic strength in the cleft. In order to describe the influence of the ion concentration resulting from the cell activity, the ion-sensitivity of graphene solution-gated field-effect transistors is investigated experimentally and modelled by considering the screening effect of the ions on the surface potential at the graphene/electrolyte interface. Finally, the model of the cell-transistor coupling is extended to include the effect of ion accumulation and ion sensitivity. The experimental data shows a very good agreement with this extended model, emphasizing the importance of considering the ion concentration in the cleft to properly understand the cell-transistor coupling.
The genus Amanita encompasses both symbiotic, ectomycorrhizal fungi and asymbiotic litter decomposers; all species are derived from asymbiotic ancestors. Symbiotic species are no longer able to degrade plant cell walls. The carbohydrate esterases family 1 (CE1s) is a diverse group of enzymes involved in carbon metabolism, including decomposition and carbon storage. CE1 genes of the ectomycorrhizal A. muscaria appear diverged from all other fungal homologues, and more similar to CE1s of bacteria, suggesting a horizontal gene transfer (HGT) event. In order to test whether AmanitaCE1s were acquired horizontally, we built a phylogeny of CE1s collected from across the tree of life, and describe the evolution of CE1 genes among Amanita and relevant lineages of bacteria. CE1s of symbiotic Amanita were very different from CE1s of asymbiotic Amanita, and are more similar to bacterial CE1s. The protein structure of one CE1 gene of A. muscaria matched a depolymerase that degrades the carbon storage molecule poly((R)-3-hydroxybutyrate) (PHB). Asymbiotic Amanita do not carry sequence or structural homologues of these genes. The CE1s acquired through HGT may enable novel metabolisms, or play roles in signaling or defense. This is the first evidence for the horizontal transfer of carbohydrate metabolism genes into ectomycorrhizal fungi.
Ubiquitin is a small 8.5 kDa protein that is conjugated to a target protein in a concerted three step enzymatic process. Ubiquitin addition can drastically affect function or target the modified protein for degradation. Ubiquitin modifications have important regulatory roles in disease progression, such as in cancer and neurodegenerative diseases to name a few. As a consequence, it is imperative to identify important ubiquitin targets to elucidate the role of the modification. Proteomic studies have sought to understand this role by identifying proteome-wide ubiquitylated proteins. Two central ideas have developed to characterize the ubiquitylome: affinity purification of ubiquitylated proteins and optimization of GG-peptide enrichment. In this review, we will discuss recent advances in both approaches and discuss how these studies are essential to pharmacoproteomics.
The S100A8/A9 heterodimer (calprotectin) acts as a danger signal when secreted into the extracellular space during inflammation and tissue damage. It promotes proinflammatory responses and drives tumor development in different models of inflammation-driven carcinogenesis. S100A8/A9 is strongly expressed in several human tumors, including hepatocellular carcinoma (HCC). Apart from this evidence, the role of calprotectin in hepatocyte transformation and tumor microenvironment is still unknown. The aim of this study was to define the function of S100A8/A9 in inflammation-driven HCC. Mice lacking S100a9 were crossed with the Mdr2(-) (/) (-) model, a prototype of inflammation-induced HCC formation. S100a9(-) (/) (-) Mdr2(-) (/) (-) (dKO) mice displayed no significant differences in tumor incidence or multiplicity compared to Mdr2(-) (/) (-) animals. Chronic liver inflammation, fibrosis and oval cell activation were not affected upon S100a9 deletion. Our data demonstrate that, although highly upregulated, calprotectin is dispensable in the onset and development of HCC, and in the maintenance of liver inflammation.
Transposable elements (TEs) are ubiquitous inhabitants of eukaryotic genomes and their proliferation and dispersal shape genome architectures and diversity. Nevertheless, TE dynamics are often explored for one species at a time and are rarely considered in ecological contexts. Recent work with plant pathogens suggests a link between symbiosis and TE abundance. The genomes of pathogenic fungi appear to house an increased abundance of TEs, and TEs are frequently associated with the genes involved in symbiosis. To investigate whether this pattern is general, and relevant to mutualistic plant-fungal symbioses, we sequenced the genomes of related asymbiotic (AS) and ectomycorrhizal (ECM) Amanita fungi. Using methods developed to interrogate both assembled and unassembled sequences, we characterized and quantified TEs across three AS and three ECM species, including the AS outgroup Volvariella volvacea. The ECM genomes are characterized by abundant numbers of TEs, an especially prominent feature of unassembled sequencing libraries. Increased TE activity in ECM species is also supported by phylogenetic analysis of the three most abundant TE superfamilies; phylogenies revealed many radiations within contemporary ECM species. However, the AS species Amanita thiersii also houses extensive amplifications of elements, highlighting the influence of additional evolutionary parameters on TE abundance. Our analyses provide further evidence for a link between symbiotic associations among plants and fungi, and increased TE activity, while highlighting the importance individual species' natural histories may have in shaping genome architecture.
Exhibiting a combination of exceptional structural and electronic properties, graphene has a great potential for the development of highly sensitive sensors. To date, many challenging chemical, biochemical, and biologic sensing tasks have been realized based on graphene. However, many of these sensors are rather unspecific. To overcome this problem, for instance, the sensor surface can be modified with analyte-specific transducers such as enzymes. One problem associated with the covalent attachment of such biomolecular systems is the introduction of crystal defects that have a deleterious impact on the electronic properties of the sensor. In this work, we present a versatile platform for biosensing applications based on polymer-modified CVD-grown graphene transistors. The functionalization method of graphene presented here allows one to integrate several functional groups within surface-bound polymer brushes without the introduction of additional defects. To demonstrate the potential of this polymer brush functionalization scaffold, we modified solution-gated graphene field-effect transistors with the enzyme acetylcholinesterase and a transducing group, allowing the detection of the neurotransmitter acetylcholine. Taking advantage of the transducing capability of graphene transistors and the versatility of polymer chemistry and enzyme biochemistry, this study presents a novel route for the fabrication of highly sensitive, multipurpose transistor sensors that can find application for a multitude of biologically relevant analytes.
Neurons in the visual cortex process a local region of visual space, but in order to adequately analyze natural images, neurons need to interact. The notion of an ‘‘association field’’ proposes that neurons interact to extract extended contours. Here, we identify the site and properties of contour integration mechanisms. We used functional magnetic resonance imaging (fMRI) and population receptive field (pRF) analyses. We devised pRF mapping stimuli consisting of contours. We isolated the contribution of contour integration mechanisms to the pRF by manipulating the contour content. This stimulus manipulation led to systematic changes in pRF size. Whereas a bank of Gabor filters quantitatively explains pRF size changes in V1, only V2/V3 pRF sizes match the predictions of the association field. pRF size changes in later visual field maps, hV4, LO-1, and LO-2 do not follow either prediction and are probably driven by distinct classical receptive field properties or other extraclassical integration mechanisms. These pRF changes do not follow conventional fMRI signal strength measures. Therefore, analyses of pRF changes provide a novel computational neuroimaging approach to investigating neural interactions. We interpreted these results as evidence for neural interactions along cooriented, cocircular receptive fields in the early extrastriate visual cortex (V2/V3), consistent with the notion of a contour association field.
Because the deubiquitinating enzyme USP33 is involved in several important cellular processes (?-adrenergic receptor recycling, centrosome amplification, RalB signaling, and cancer cell migration), its levels must be carefully regulated. Using quantitative mass spectrometry, we found that the intracellular level of USP33 is highly sensitive to the activity of p97. Knockdown or chemical inhibition of p97 causes robust accumulation of USP33 due to inhibition of its degradation. The p97 adaptor complex involved in this function is the Ufd1-Npl4 heterodimer. Furthermore, we identified HERC2, a HECT domain-containing E3 ligase, as being responsible for polyubiquitination of USP33. Inhibition of p97 causes accumulation of polyubiquitinated USP33, suggesting that p97 is required for postubiquitination processing. Thus, our study has identified several key molecules that control USP33 degradation within the ubiquitin-proteasome system.
S100/calgranulins (S100A8, S100A9 and S100A12) are key players of innate immune function and elevated levels are a characteristic feature of acute and chronic inflammation, and inflammation-associated carcinogenesis. However, reduced S100A8 and S100A9 expression has been detected for squamous cell carcinoma, including the head and neck region (HNSCC), which originate from mucosal epithelia with abundant expression of both proteins under physiological conditions. In contrast to S100A8 and S100A9, only sparse information is available for S100A12 and a comparative study of all three S100/calgranulins in HNSCC is still missing. We analyzed S100/calgranulin protein levels in a retrospective patient cohort (n?=?131) of oropharyngeal squamous cell carcinoma (OPSCC) by immunohistochemical staining of tissue microarrays. Common characteristics of all three S100/calgranulins were: (i) abundant expression in supra-basal keratinocytes of normal mucosa with predominant nuclear staining, (ii) low expression in 30.4-51.9% of primary OPSCCs and (iii) variable accumulation of S100/calgranulin-positive immune cells in the tumor stroma. These features were associated with histopathological characteristics, such as tumor grade, lymph node metastasis and tumor stage. Furthermore, univariate and multivariate analysis revealed worse overall survival of OPSCC patients with simultaneous reduction of S100A8 and S100A12 expression, while expression of S100A9 or presence of the S100A8/S100A9 heterodimer had no impact, suggesting distinct regulation and function of individual S100/calgranulins in the pathogenesis of HNSCCs.
To better understand the relationship between dichoptic masking in normal vision and suppression in amblyopia we address three questions: First, what is the time course of dichoptic masking in normals and amblyopes? Second, is interocular suppression low-pass or band-pass in its spatial dependence? And third, in the above two regards, is dichoptic masking in normals different from amblyopic suppression?
The Polo-like Kinase 1 (PLK1) protein regulates cell cycle progression and is overexpressed in many malignant tissues. Overexpression is associated with poor prognosis in several cancer entities, whereby expression of PLK1 shows high inter-individual variability. Although PLK1 is extensively studied, not much is known about the genetic variability of the PLK1 gene. The function of PLK1 and the expression of the corresponding gene could be influenced by genomic variations. Hence, we investigated the gene for functional polymorphisms. Such polymorphisms could be useful to investigate whether PLK1 alters the risk for and the course of cancer and they could have an impact on the response to PLK1 inhibitors.
Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs.
Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 ?g·kg-1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 ?g·kg-1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell-1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.
An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30 min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods.
Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy). Specifically, we explore genetic delivery of a small immunoRNase consisting of an EGFR-binding scFv antibody fragment fused to the RNase Onconase (ONCEGFR ) that induces tumor cell death by RNA degradation after cellular internalization. Onconase is a frog RNase that combines lack of immunogenicity and excellent safety in patients with high tumor killing potency due to its resistance to the human cytosolic RNase inhibitor. We show that ONCEGFR expression by oncolytic adenoviruses is feasible with an optimized, replication-dependent gene expression strategy. Virus-encoded ONCEGFR induces potent and EGFR-dependent bystander killing of tumor cells. Importantly, the ONCEGFR -encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR-positive tumor cells in vitro and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The latter demonstrates that ONCEGFR is expressed at levels sufficient to trigger tumor cell killing in vivo. The established ONCEGFR -encoding oncolytic adenovirus represents a novel agent for treatment of EGFR-positive tumors. This viro-antibody therapy platform can be further developed for targeted/personalized cancer therapy by exploiting antibody diversity to target further established or emerging tumor markers or combinations thereof.
To test the hypothesis that fixational stability of the amblyopic eye in strabismics will improve when viewing provides both bifoveal fixation and reduced inter-ocular suppression by reducing the contrast to the fellow eye.
The visual system pools information from local samples to calculate textural properties. We used a novel stimulus to investigate how signals are combined to improve estimates of global orientation. Stimuli were 29 × 29 element arrays of 4 c/deg log Gabors, spaced 1° apart. A proportion of these elements had a coherent orientation (horizontal/vertical) with the remainder assigned random orientations. The observer's task was to identify the global orientation. The spatial configuration of the signal was modulated by a checkerboard pattern of square checks containing potential signal elements. The other locations contained either randomly oriented elements ("noise check") or were blank ("blank check"). The distribution of signal elements was manipulated by varying the size and location of the checks within a fixed-diameter stimulus. An ideal detector would only pool responses from potential signal elements. Humans did this for medium check sizes and for large check sizes when a signal was presented in the fovea. For small check sizes, however, the pooling occurred indiscriminately over relevant and irrelevant locations. For these check sizes, thresholds for the noise check and blank check conditions were similar, suggesting that the limiting noise is not induced by the response to the noise elements. The results are described by a model that filters the stimulus at the potential target orientations and then combines the signals over space in two stages. The first is a mandatory integration of local signals over a fixed area, limited by internal noise at each location. The second is a task-dependent combination of the outputs from the first stage.
Marine biotoxins are algal metabolites that can accumulate in fish or shellfish and render these foodstuffs unfit for human consumption. These toxins, released into seawater during algal occurrences, can be monitored through passive sampling. Acetone, methanol and isopropanol were evaluated for their efficiency in extracting toxins from algal biomass. Isopropanol was chosen for further experiments thanks to a slightly higher recovery and no artifact formation. Comparison of Oasis HLB, Strata-X, BondElut C18 and HP-20 sorbent materials in SPE-mode led to the choice of Oasis HLB, HP-20 and Strata-X. These three sorbents were separately exposed as passive samplers for 24 h to seawater spiked with algal extracts containing known amounts of okadaic acid (OA), azaspiracids (AZAs), pinnatoxin-G (PnTX-G), 13-desmethyl spirolide-C (SPX1) and palytoxins (PlTXs). Low density polyethylene (LDPE) and silicone rubber (PDMS) strips were tested in parallel on similar mixtures of spiked natural seawater for 24 h. These strips gave significantly lower recoveries than the polymeric sorbents. Irrespective of the toxin group, the adsorption rate of toxins on HP-20 was slower than on Oasis HLB and Strata-X. However, HP-20 and Strata-X gave somewhat higher recoveries after 24 h exposure. Irrespective of the sorbent tested, recoveries were generally highest for cyclic imines and OA group toxins, slightly lower for AZAs, and the lowest for palytoxins. Trials in re-circulated closed tanks with mussels exposed to Vulcanodinium rugosum or Prorocentrum lima allowed for further evaluation of passive samplers. In these experiments with different sorbent materials competing for toxins in the same container, Strata-X accumulated toxins faster than Oasis HLB, and HP-20, and to higher levels. The deployment of these three sorbents at Ingril French Mediterranean lagoon to detect PnTX-G in the water column showed accumulation of higher levels on HP-20 and Oasis HLB compared to Strata-X. This study has significantly extended the range of sorbents for passive sampling of marine toxins. In particular, sorbents were included that had previously been evaluated for polyhalogenated contaminants, pharmaceuticals, phytochemicals or veterinary residues. Moreover, this study has for the first time demonstrated the usefulness of the polymeric Oasis HLB and Strata-X sorbents in laboratory and field studies for various microalgal toxins.
The amblyopic visual system was once considered to be structurally monocular. However, it now evident that the capacity for binocular vision is present in many observers with amblyopia. This has led to new techniques for quantifying suppression that have provided insights into the relationship between suppression and the monocular and binocular visual deficits experienced by amblyopes. Furthermore, new treatments are emerging that directly target suppressive interactions within the visual cortex and, on the basis of initial data, appear to improve both binocular and monocular visual function, even in adults with amblyopia. The aim of this review is to provide an overview of recent studies that have investigated the structure, measurement and treatment of binocular vision in observers with strabismic, anisometropic and mixed amblyopia.
The programmed formation of specific tissues from embryonic stem cells is a major goal of regenerative medicine. To identify points of intervention in cardiac tissue formation, we performed an siRNA screen in murine embryonic stem cells to identify ubiquitin system genes that repress cardiovascular tissue formation. Our screen uncovered an F-box protein, Fbxl16, as a repressor of one of the earliest steps in the cardiogenic lineage: FLK1+ progenitor formation. Whereas F-box proteins typically form SCF ubiquitin ligases, shotgun mass spectrometry revealed that FBXL16 instead binds protein phosphatase 2A (PP2A) containing a B55 specificity subunit (PP2A(B55)). Phosphoproteomic analyses indicate that FBXL16 negatively regulates phosphorylation of the established PP2A(B55) substrate, vimentin. We suggest that FBXL16 negatively regulates the activity of B55?-PP2A to modulate the genesis of FLK1+ progenitor cells.
Human T-cell leukemia virus type-1 (HTLV-1) is estimated to infect 15-25 million people worldwide, with several areas including southern Japan and the Caribbean basin being endemic. The virus is the etiological agent of debilitating and fatal diseases, for which there is currently no long-term cure. In the majority of cases of leukemia caused by HTLV-1, only a single viral gene, hbz, and its cognate protein, HBZ, are expressed and their importance is increasingly being recognized in the development of HTLV-1-associated disease. We hypothesized that HBZ, like other HTLV-1 proteins, has properties and functions regulated by post-translational modifications (PTMs) that affect specific signaling pathways important for disease development. To date, PTM of HBZ has not been described. We used an affinity-tagged protein and mass spectrometry method to identify seven modifications of HBZ for the first time. We examined how these PTMs affected the ability of HBZ to modulate several pathways, as measured using luciferase reporter assays. Herein, we report that none of the identified PTMs affected HBZ stability or its regulation of tested pathways.
Our aim was to compare sensitivity for horizontal and vertical disparity corrugations and to resolve whether these stimuli are processed by similar or radically different underlying mechanisms. We measure global disparity sensitivity as a function of carrier spatial frequency for equi-detectable carriers and found a similar optimal carrier relationship for vertical and horizontal stimuli. Sensitivity as a function of corrugation spatial frequency for stimuli of comparable spatial summation and composed of optimal, equi-detectable narrowband carriers did not significantly differ for vertical and horizontal stimuli. A small anisotropy was revealed when fixed, high contrast broadband carriers were used. In a separate discrimination-at-threshold experiment, multiple mechanisms of similar tuning were revealed to underlie the detection of both vertical and horizontal disparity corrugations. We conclude that the processing of the horizontal and vertical disparity corrugations occurs along similar lines.
Phase information is a fundamental aspect of visual stimuli. However, the nature of the binocular combination of stimuli defined by modulations in contrast, so-called second-order stimuli, is presently not clear. To address this issue, we measured binocular combination for first- (luminance modulated) and second-order (contrast modulated) stimuli using a binocular phase combination paradigm in seven normal adults. We found that the binocular perceived phase of second-order gratings depends on the interocular signal ratio as has been previously shown for their first order counterparts; the interocular signal ratios when the two eyes were balanced was close to 1 in both first- and second-order phase combinations. However, second-order combination is more linear than previously found for first-order combination. Furthermore, binocular combination of second-order stimuli was similar regardless of whether the carriers in the two eyes were correlated, anti-correlated, or uncorrelated. This suggests that, in normal adults, the binocular phase combination of second-order stimuli occurs after the monocular extracting of the second-order modulations. The sensory balance associated with this second-order combination can be obtained from binocular phase combination measurements.
The American Board of Medical Specialties (ABMS) certification and maintenance of certification (MOC) programs strive to provide the public with guidance about a physicians competence. This study summarizes the literature on the effectiveness of these programs.
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
Practice-based learning and improvement is a core competency that all medical residents must demonstrate. Because confidence is important in translating competence into action, effective quality improvement (QI) curricula should evaluate trainees knowledge and confidence to perform QI. Past efforts to assess educational outcomes in QI have not adequately evaluated trainees confidence from a multidimensional perspective.
Tumor suppressor microRNA-126 (miR-126) is often down-regulated in cancer cells, and its overexpression is found to inhibit cancer metastasis. To elucidate the mechanism of tumor suppression by miR-126, we analyzed the proteomic response to miR-126 overexpression in the human metastatic breast cancer cell line MDA-MB-231. To acquire quantitative, time-resolved information, we combined two complementary proteomic methods, BONCAT and SILAC. We discovered a new direct target of miR-126: CD97, a pro-metastatic G-protein-coupled receptor (GPCR) that has been reported to promote tumor cell invasion, endothelial cell migration, and tumor angiogenesis. This discovery establishes a link between down-regulation of miR-126 and overexpression of CD97 in cancer and provides new mechanistic insight into the role of miR-126 in inhibiting both cell-autonomous and non-cell-autonomous cancer progression.
Evidence is accumulating that suppression may be the cause of amblyopia rather than a secondary consequence of mismatched retinal images. For example, treatment interventions that target suppression may lead to better binocular and monocular outcomes. Furthermore, it has recently been demonstrated that the measurement of suppression may have prognostic value for patching therapy. For these reasons, the measurement of suppression in the clinic needs to be improved beyond the methods that are currently available, which provide a binary outcome.
Stereopsis is dependent on the average level of illumination, especially if it differs between the two eyes. We manipulate the mean luminance seen by both eyes or the interocular difference in mean luminance by using neutral density (ND) filters placed in front of both eyes or just one eye respectively. Stereo acuity was measured using a one temporal interval forced choice task for detecting the sign of a Gaussian depth perturbation in a noise field with a comparable spectrum to that found in natural images. We show that the effect of changing mean luminance is spatial scale independent within the range of 0.5 to 4 cpd, certainly not larger at higher spatial scales. To investigate its origin we manipulate two factors, the temporal synchrony between the two eyes and the interocular contrast. Both factors are implicated in the loss of stereo performance when the mean luminance is different between the eyes, suggesting an underlying explanation in terms of temporal low-pass filtering resulting in the combination of a luminance-dependent temporal delay and a luminance-dependent change in contrast gain.
Previous studies demonstrated that fibroblast-derived and JUN-dependent soluble factors play a crucial role on keratinocyte proliferation and differentiation during cutaneous wound healing. Furthermore, mice with a deficiency in Jun N-terminal kinases, JNK1 or JNK2, showed impaired skin development and delayed wound closure. To decipher the role of dermal JNK in keratinocyte behavior during these processes we employed a heterologous co-culture model combining primary human keratinocytes and murine fibroblasts. While co-cultured JNK1/JNK2-deficient fibroblasts did not affect keratinocyte proliferation, temporal monitoring of the transcriptome of differentiating keratinocytes revealed that efficient keratinocyte differentiation not only requires the support by fibroblast-derived soluble factors but critically depends on JNK1 and JNK2 signaling in these cells. Moreover, we showed that the repertoire of fibroblast transcripts encoding secreted proteins is severely disarranged upon loss of JNK under the co-culture conditions applied. Finally, our data demonstrate that efficient keratinocyte terminal differentiation requires constant presence of JNK-dependent and fibroblast-derived soluble factors. Taken together, our results imply that mesenchymal JNK plays a pivotal role in the paracrine crosstalk between dermal fibroblasts and epidermal keratinocytes during wound healing.Journal of Investigative Dermatology accepted article preview online, 16 December 2013. doi:10.1038/jid.2013.535.
Amblyopia is a neurodevelopmental disorder of vision caused by abnormal visual experience during early childhood that is often considered to be untreatable in adulthood. Recently, it has been shown that a novel dichoptic videogame-based treatment for amblyopia can improve visual function in adult patients, at least in part, by reducing inhibition of inputs from the amblyopic eye to the visual cortex. Non-invasive anodal transcranial direct current stimulation has been shown to reduce the activity of inhibitory cortical interneurons when applied to the primary motor or visual cortex. In this double-blind, sham-controlled cross-over study we tested the hypothesis that anodal transcranial direct current stimulation of the visual cortex would enhance the therapeutic effects of dichoptic videogame-based treatment. A homogeneous group of 16 young adults (mean age 22.1?±?1.1 years) with amblyopia were studied to compare the effect of dichoptic treatment alone and dichoptic treatment combined with visual cortex direct current stimulation on measures of binocular (stereopsis) and monocular (visual acuity) visual function. The combined treatment led to greater improvements in stereoacuity than dichoptic treatment alone, indicating that direct current stimulation of the visual cortex boosts the efficacy of dichoptic videogame-based treatment. This intervention warrants further evaluation as a novel therapeutic approach for adults with amblyopia.
Epigenetic regulation of gene expression is, at least in part, mediated by histone modifications. PTMs of histones change chromatin structure and regulate gene transcription, DNA damage repair, and DNA replication. Thus, studying histone variants and their modifications not only elucidates their functional mechanisms in chromatin regulation, but also provides insights into phenotypes and diseases. A challenge in this field is to determine the best approach(es) to identify histone variants and their PTMs using a robust high-throughput analysis. The large number of histone variants and the enormous diversity that can be generated through combinatorial modifications, also known as histone code, makes identification of histone PTMs a laborious task. MS has been proven to be a powerful tool in this regard. Here, we focus on bottom-up, middle-down, and top-down MS approaches, including CID and electron-capture dissociation/electron-transfer dissociation based techniques for characterization of histones and their PTMs. In addition, we discuss advances in chromatographic separation that take advantage of the chemical properties of the specific histone modifications. This review is also unique in its discussion of current bioinformatic strategies for comprehensive histone code analysis.
Yeast Cdc48 (p97/VCP in human cells) is a hexameric AAA ATPase that is thought to use ATP hydrolysis to power the segregation of ubiquitin-conjugated proteins from tightly bound partners. Current models posit that Cdc48 is linked to its substrates through adaptor proteins, including a family of seven proteins (13 in human) that contain a Cdc48-binding UBX domain. However, few substrates for specific UBX proteins are known, and hence the generality of this hypothesis remains untested. Here, we use mass spectrometry to identify ubiquitin conjugates that accumulate in cdc48 and ubx mutants. Different ubx mutants exhibit unique patterns of conjugate accumulation that point to functional specialization of individual Ubx proteins. To validate our findings, we examined in detail the endoplasmic reticulum-bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. Mutant ubx2? cells are deficient in both cleaving the ubiquitinated 120 kDa precursor of Spt23 to form active p90 and in localizing p90 to the nucleus, resulting in reduced expression of the target gene OLE1, which encodes fatty acid desaturase. Our findings provide a resource for future investigations on Cdc48, illustrate the utility of proteomics to identify ligands for specific ubiquitin receptor pathways, and uncover Ubx2 as a key player in the regulation of membrane lipid biosynthesis.
Board certification has evolved from a "point-in-time" event to a process of periodic learning and reevaluation of medical competence through maintenance of certification (MOC). To better understand MOC participation, the transtheoretical model (TTM) was used to describe physicians perceptions of MOC as a sequence of attitudinal changes.
The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55? or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55? levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55? has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55? complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55? complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55?, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55? in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55? results in marked dephosphorylation of p107 while B55? knockdown results in hyperphosphorylation. More importantly, knockdown of B55? dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.
Amblyopia is a neurodevelopmental disorder of vision that is associated with abnormal patterns of neural inhibition within the visual cortex. This disorder is often considered to be untreatable in adulthood because of insufficient visual cortex plasticity. There is increasing evidence that interventions that target inhibitory interactions within the visual cortex, including certain types of noninvasive brain stimulation, can improve visual function in adults with amblyopia.
The emerging field of chemo- and pharmacoproteomics studies the mechanisms of action of bioactive molecules in a systems pharmacology context. In contrast to traditional drug discovery, pharmacoproteomics integrates the mechanism of a drugs action, its side effects including toxicity, and the discovery of new drug targets in a single approach. Thus, it determines early favorable (e.g. multiple kinase target in cancer drugs) and unfavorable (e.g. side effects) polypharmacology. Target profiling is accomplished using either active site-labeling probes or immobilized drugs. This strategy identifies direct targets and has in fact enabled even the determination of binding curves and half maximum inhibitory concentrations of these targets. In addition, the enrichment greatly reduces the complexity of the proteome to be analyzed by quantitative MS. Complementary to these approaches, global proteomics profiling studying drug treatement-induced changes in protein expression levels and/or post-translational modification status have started to become possible mostly due to significant improvements in instrumentation. Particularly, when using multidimensional separations, a considerable proteome depth of up to 10 000 proteins can be achieved with current state-of-the-art mass spectrometers and bioinformatics tools. In summary, chemo- and pharmacoproteomics has already contributed significantly to the identification of novel drug targets and their mechanisms of action(s). Aided by further technological advancements, this interdisciplinary approach will likely be used more broadly in the future.
Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of "fast-acting toxins", its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC?? values of 0.38 µg mL?¹ and 0.19 µg mL?¹ were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL?¹). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.
Mass-spectrometry-based proteomics has evolved as the preferred method for the analysis of complex proteomes. Undoubtedly, recent advances in mass spectrometry instrumentation have greatly enhanced proteomic analysis. A popular instrument platform in proteomics research is the LTQ-Orbitrap mass analyzer. In this tutorial, we discuss the significance of evaluating and optimizing mass spectrometric settings on the LTQ-Orbitrap during CID data-dependent acquisition (DDA) mode to improve protein and peptide identification rates. We focus on those MS and MS/MS parameters that have been systematically examined and evaluated by several researchers and are commonly used during DDA. More specifically, we discuss the effect of mass resolving power, preview mode for FTMS scan, monoisotopic precursor selection, signal threshold for triggering MS/MS events, number of microscans per MS/MS scan, number of MS/MS events, automatic gain control target value (ion population) for MS and MS/MS, maximum ion injection time for MS/MS, rapid and normal scan rate, and prediction of ion injection time. We furthermore present data from the latest generation LTQ-Orbitrap system, the Orbitrap Elite, along with recommended MS and MS/MS parameters. The Orbitrap Elite outperforms the Orbitrap Classic in terms of scan speed, sensitivity, dynamic range, and resolving power and results in higher identification rates. Several of the optimized MS parameters determined on the LTQ-Orbitrap Classic and XL were easily transferable to the Orbitrap Elite, whereas others needed to be reevaluated. Finally, the Q Exactive and HCD are briefly discussed, as well as sample preparation, LC-optimization, and bioinformatics analysis. We hope this tutorial will serve as guidance for researchers new to the field of proteomics and assist in achieving optimal results.
Aberrant expression of the receptor for advanced glycation end products (RAGE) and its ligands, such as S100/Calgranulins, has been demonstrated in squamous cell carcinomas of the upper aerodigestive tract. However, the question whether RAGE signaling is causally linked with neoplastic transformation of keratinocytes in mucosal epithelia has not been addressed so far. We used the well-established mouse model of 4-nitroquinoline-1-oxide (4-NQO) induced tumorigenesis to investigate tumor development in control and RAGE-deficient (Rage(-/-)) animals. Although 4-NQO induced lesions of the tongue and the esophagus showed strong induction of the RAGE ligands S100a8 and S100a9, we did not observe any significant difference in tumor incidence or multiplicity between control and Rage(-/-) mice. Furthermore, detailed analysis of tumor sections by histological and immunohistochemical staining revealed no difference in either the size or histological architecture of dysplastic lesions, tumor cell proliferation, or the number of inflammatory immune cells in the tumor microenvironment. Finally, we detected induced transcript and protein levels of the Toll-like receptor 4 (Tlr4) in 4-NQO induced lesions, suggesting that signaling via the S100-Tlr4 axis may compensate for the lack of RAGE in early stages of tumor development. Our data demonstrate that RAGE is dispensable in the onset of genotoxic induced oral and esophageal squamous cell carcinoma and provide evidence for an alternative pathway of S100-Calgranulin signaling via Tlr4.
The K shell excitation of H-like uranium (U(91+)) in relativistic collisions with different gaseous targets has been studied at the experimental storage ring at GSI Darmstadt. By performing measurements with different targets as well as with different collision energies, we were able to observe for the first time the effect of electron-impact excitation (EIE) process in the heaviest hydrogenlike ion. The large fine-structure splitting in H-like uranium allowed us to unambiguously resolve excitation into different L shell levels. State-of-the-art calculations performed within the relativistic framework which include excitation mechanisms due to both protons (nucleus) and electrons are in good agreement with the experimental findings. Moreover, our experimental data clearly demonstrate the importance of including the generalized Breit interaction in the treatment of the EIE process.
Amblyopia is a neurological disorder of binocular vision affecting up to 3% of the population resulting from a disrupted period of early visual development. Recently, it has been shown that vision can be partially restored by intensive monocular or dichoptic training (4-6 weeks). This can occur even in adults owing to a residual degree of brain plasticity initiated by repetitive and successive sensory stimulation. Here we show that the binocular imbalance that characterizes amblyopia can be reduced by occluding the amblyopic eye with a translucent patch for as little as 2.5?hours, suggesting a degree of rapid binocular plasticity in adults resulting from a lack of sensory stimulation. The integrated binocular benefit is larger in our amblyopic group than in our normal control group. We propose that this rapid improvement in function, as a result of reduced sensory stimulation, represents a new form of plasticity operating at a binocular site.
Adults with amblyopia, a common visual cortex disorder caused primarily by binocular disruption during an early critical period, do not respond to conventional therapy involving occlusion of one eye. But it is now clear that the adult human visual cortex has a significant degree of plasticity, suggesting that something must be actively preventing the adult brain from learning to see through the amblyopic eye. One possibility is an inhibitory signal from the contralateral eye that suppresses cortical inputs from the amblyopic eye. Such a gating mechanism could explain the apparent lack of plasticity within the adult amblyopic visual cortex. Here we provide direct evidence that alleviating suppression of the amblyopic eye through dichoptic stimulus presentation induces greater levels of plasticity than forced use of the amblyopic eye alone. This indicates that suppression is a key gating mechanism that prevents the amblyopic brain from learning to see.
We developed a dichoptic global orientation coherence paradigm to quantify interocular suppression in amblyopia. This task is biased towards ventral processing and allows comparison with two other techniques-global motion processing, which is more dorsally biased, and binocular phase combination, which most likely reflects striate function. We found a similar pattern for the relationship between coherence threshold and interocular contrast curves (thresholds vs. interocular contrast ratios or TvRs) in our new paradigm compared with those of the previous dichoptic global motion coherence paradigm. The effective contrast ratios at balance point (where the signals from the two eyes have equal weighting) in our new paradigm were larger than those of the dichoptic global motion coherence paradigm but less than those of the binocular phase combination paradigm. The measured effective contrast ratios in the three paradigms were also positively correlated with each other, with the two global coherence paradigms having the highest correlation. We concluded that: (a) The dichoptic global orientation coherence paradigm is effective in quantifying interocular suppression in amblyopia; and (b) Interocular suppression, while sharing a common suppression mechanism at the early stage in the pathway (e.g., striate cortex), may have additional extra-striate contributions that affect both dorsal and ventral streams differentially.
Binocularity is a fundamental property of primate vision. Ocular dominance describes the perceptual weight given to the inputs from the two eyes in their binocular combination. There is a distribution of sensory dominance within the normal binocular population with most subjects having balanced inputs while some are dominated by the left eye and some by the right eye. Using short-term monocular deprivation, the sensory dominance can be modulated as, under these conditions, the patched eyes contribution is strengthened. We address two questions: Is this strengthening a general effect such that it is seen for different types of sensory processing? And is the strengthening specific to pattern deprivation, or does it also occur for light deprivation? Our results show that the strengthening effect is a general finding involving a number of sensory functions, and it occurs as a result of both pattern and light deprivation.
Luminance plays a modulating role in the processes of several visual tasks, which in turn provides significant information for the understanding of visual processing. Here, using a binocular phase combination paradigm, we studied the effect of unilateral changes in mean luminance on binocular combination in both normal and amblyopic vision. We found, in normal observers, attenuation of one eyes stimulus luminance with neutral density filters produces binocular phase combination similar to those of amblyopic subjects. Correspondingly, in amblyopic observers, reduction of the fellow eyes stimulus luminance produces binocular phase combination similar to those of normal subjects. These phenomena could be explained by an attenuated contribution of the filtered eye to the binocular phase percept due to reduced gain-control. The findings have major implications both for the study of binocular combination and for amblyopia treatment.
To better understand the pooling properties underlying global stereopsis we examined the relationship between carrier luminance spatial frequency and modulator disparity spatial frequency. Thresholds for detecting global sinusoidal disparity corrugations of spatially band-pass noise were measured as a function of modulator disparity spatial frequency for both centrally and peripherally located stimuli using a standard 2-IFC task. We found a characteristic relationship that depended on modulator disparity spatial frequency. At high modulator disparity spatial frequencies (>1c/d), there is an optimal ratio of around 2.6, whereas at low modulator disparity spatial frequencies, there is an optimal absolute carrier luminance spatial frequency (i.e., 3c/d). In the periphery, vision is restricted to modulator disparity spatial frequencies below 1c/d and, as a consequence, following the above rule, there is an optimum absolute carrier luminance spatial frequency that reduces in spatial frequency with increasing eccentricity. This finding is consistent with there being more than one channel processing global stereo that is subsequently confirmed using a 2×2 AFC detection/discrimination paradigm. Furthermore, because of the different carrier/modulator relationships in central and peripheral vision, peripheral global stereo cannot be simply related to central global stereo by a scaling factor and thus cannot be simply due to cortical magnification, as originally thought.
Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication.
Mycobacterium tuberculosis (Mtb) causes tuberculosis, one of the leading causes of fatal infectious diseases worldwide. Cell-cell recognition between the pathogen Mtb and its host is mediated in part by glycosylated proteins. So far, glycoproteins in Mtb are understudied and for only very few glycoproteins glycosylation sites have been described, e.g., alanine and proline rich secreted protein apa, superoxide dismutase SODC, lipoprotein lpqH and MPB83/MPT83. In this study, glycosylated proteins in Mtb culture filtrate were investigated using liquid chromatography-mass spectrometry approaches and bioinformatic analyses. To validate the presence of glycoproteins, several strategies were pursued including collision induced dissociation, high energy collision dissociation and electron transfer dissociation techniques, and bioinformatics analyses involving a neutral loss search for glycosylated moieties. After extensive data curation, we report glycosylation sites for thirteen Mtb glycoproteins using a combination of mass spectrometry techniques on a dataset collected from culture filtrate proteins. This is the first glycoproteomics study identifying glycosylation sites on mycobacterial culture filtrate proteins (CFP) on a global scale.
It has been reported that a single session of 1 Hz or 10 Hz repetitive transcranial magnetic stimulation (rTMS) of the visual cortex can temporarily improve contrast sensitivity in adults with amblyopia. More recently, continuous theta burst stimulation (cTBS) of the visual cortex has been found to improve contrast sensitivity in observers with normal vision.
Changes in certification requirements and examinee characteristics are likely to influence the validity of the evidence associated with interpretations made based on test data. We examined whether changes in Educational Commission for Foreign Medical Graduates (ECFMG) certification requirements over time were associated with changes in internal medicine (IM) residency program director ratings and certification examination scores. Comparisons were made between physicians who were ECFMG-certified before and after the Clinical Skills Assessment (CSA) requirement. A multivariate analysis of covariance was conducted to examine the differences in program director ratings based on CSA cohort and whether the examinees emigrated for undergraduate medical education (national vs. international students). A univariate analysis of covariance was conducted to examine differences in scores from the American Board of Internal Medicine (ABIM) Internal Medicine Certification Examination. For both analyses, United States Medical Licensing Examination (USMLE) Step 1 and Step 2 scores were used as covariates. Results indicate that, of those certified by ECFMG between 1993 and 1997, 17 % (n = 1,775) left their country of citizenship for undergraduate medical education. In contrast, 38 % (n = 1,874) of those certified between 1999 and 2003 were international students. After adjustment by covariates, the main effect of cohort membership on the program director ratings was statistically significant (Wilks ? = 0.99, F 5, 15391 = 19.9, P < 0.001). However, the strength of the relationship between cohort group and the ratings was weak (? = 0.01). The main effect of migration status was statistically significant and weak (Wilks ? = 0.98, F 5,15391 = 45.3, P < 0.01; ? = 0.02). Differences in ABIM Internal Medicine Certification Examination scores based on whether or not CSA were required was statistically significant, although the magnitude of the association between these variables was very small. The findings suggest that the implementation of an additional evaluation of skills (e.g., history-taking, physical examination) as a prerequisite to postgraduate medical education (residency) provides some additional, relevant data to those who select ECFMG-certified residents.
Middle-down mass spectrometry (MS) combined with electron capture dissociation (ECD) represents an attractive method for characterization of proteins and their post-translational modifications (PTMs). Coupling online chromatographic separation with tandem mass spectrometry enables a high-throughput analysis, while improving sensitivity of the electrosprayed peptides and reducing sample amount requirements. However, middle-down ECD has not been thus far coupled with online chromatographic separation. In this work, we examine the feasibility of coupling middle-down ECD with online nanoflow-liqiud chromatography (nano-LC) for the analysis of large, >3 kDa, and highly modified polypeptides in a data-dependent acquisition mode. We evaluate the effectiveness of the method by analyzing peptides derived from Asp-N and Glu-C digestions of unfractionated histones from calf thymus and acid-extracted histones from HeLa, MCF-7, and Jurkat cells. Our results demonstrate that middle-down ECD is compatible with online chromatographic separation, providing high peptide and protein sequence coverage while allowing precise mapping of PTM sites. The high mass accuracy, obtained by the ICR mass analyzer, for both the precursor and product ions greatly increases confidence in peptide identification, particularly for modified peptides. Overall, for all samples examined, several histone variants were identified and modification sites were successfully localized, including single, multiple, and positional isomeric PTM sites. The vast majority of the identified peptides were in the mass range from 3 to 9 kDa. The data presented here highlight the feasibility and utility of nano-LC-ECD-MS/MS for high-throughput middle-down analysis.
High-risk types of human papilloma virus (HPV) are increasingly associated with oropharyngeal squamous cell carcinoma (OPSCC). Strikingly, patients with HPV-positive OPSCC are highly curable with ionizing radiation and have better survival compared with HPV-negative patients, but the underlying molecular mechanisms remain poorly understood. We applied an array-based approach to monitor global changes in CpG island hypermethylation between HPV-negative and HPV-positive OPSCCs and identified a specific pattern of differentially methylated regions that critically depends on the presence of viral transcripts. HPV-related alterations were confirmed for the majority of candidate gene promoters by mass spectrometric, quantitative methylation analysis. There was a significant inverse correlation between promoter hypermethylation of ALDH1A2, OSR2, GATA4, GRIA4, and IRX4 and transcript levels. Interestingly, Kaplan-Meier analysis revealed that a combined promoter methylation pattern of low methylation levels in ALDH1A2 and OSR2 promoters and high methylation levels in GATA4, GRIA4, and IRX4 promoters was significantly correlated with improved survival in 3 independent patient cohorts. ALDH1A2 protein levels, determined by immunohistochemistry on tissue microarrays, confirmed the association with clinical outcome. In summary, our study highlights specific alterations in global gene promoter methylation in HPV-driven OPSCCs and identifies a signature that predicts the clinical outcome in OPSCCs.
Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.
We set out to determine whether extra-striate ventral stream function was compromised in amblyopia and to compare any observed deficit with previous data on comparable dorsal stream function. We devised a multi-element orientation task where orientation coherence sensitivity could be measured in a comparable way to motion coherence. The use of spatial frequency narrowband elements allowed for accurate correction of any upstream contrast sensitivity influence and ensured that the orientation bandwidth of our elements did not covary with the measured coherence. Using a standard equivalent noise analysis, we varied both the local orientation bandwidth of individual elements as well as the global orientation bandwidth of the element array to obtain estimates of both local and global internal noise and efficiency. The results show that for this ventral stream task there is only a subtle amblyopic deficit in processing global orientation relative to control observers. This deficit is present for both amblyopic and fixing eyes, and appears to reflect poorer efficiency in processing local orientation, suggesting a subtle deficit at the input stage to extra-striate cortex where orientation coherence is processed.
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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.