Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This does not occur in the western painted turtle, which overwinters for months without oxygen. Neurological damage is avoided through anoxia-mediated decreases in NMDA and AMPA receptor currents that are dependent upon a modest rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) originating from mitochondria. Anoxia also blocks mitochondrial reactive oxygen species (ROS) generation, which is another potential signaling mechanism to regulate glutamate receptors. To assess the effects of decreased intracellular [ROS] on NMDA and AMPA receptor currents, we scavenged ROS with N-2-mercaptopropionylglycine (MPG) or N-acetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100%, while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca(2+)]i concentrations were unaffected by ROS manipulation. Because decreases in [ROS] increased NMDA receptor currents, we next asked whether mitochondrial Ca(2+) release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATP-sensitive potassium (mKATP) channels with diazoxide decreased NMDA receptor currents and was unaffected by subsequent ROS scavenging. Diazoxide application following ROS scavenging did not rescue scavenger-mediated increases in NMDA receptor currents. Fluorescent measurement of [Ca(2+)]i and ROS levels demonstrated that [Ca(2+)]i increases before ROS decreases. We conclude that decreases in ROS concentration are not linked to anoxia-mediated decreases in NMDA/AMPA receptor currents but are rather associated with an increase in NMDA receptor currents that is prevented during anoxia by mitochondrial Ca(2+) release.
In the BRIM-3 trial, vemurafenib was associated with risk reduction versus dacarbazine of both death and progression in patients with advanced BRAF(V600) mutation-positive melanoma. We present an extended follow-up analysis of the total population and in the BRAF(V600E) and BRAF(V600K) mutation subgroups.
Food web ecologists have long sought to characterize the trophic niches of animals using stable isotopic analysis. However, distilling trophic position from isotopic composition has been difficult, largely because of the variability associated with trophic discrimination factors (inter-trophic isotopic fractionation and routing). We circumvented much of this variability using compound-specific isotopic analysis (CSIA). We examined the (15)N signatures of amino acids extracted from organisms reared in pure culture at four discrete trophic levels, across two model communities. We calculated the degree of enrichment at each trophic level and found there was a consistent trophic discrimination factor (~7.6‰). The constancy of the CSIA-derived discrimination factor permitted unprecedented accuracy in the measurement of animal trophic position. Conversely, trophic position estimates generated via bulk-(15)N analysis significantly underestimated trophic position, particularly among higher-order consumers. We then examined the trophic hierarchy of a free-roaming arthropod community, revealing the highest trophic position (5.07) and longest food chain ever reported using CSIA. High accuracy in trophic position estimation brings trophic function into sharper focus, providing greater resolution to the analysis of food webs.
Perception of scenes has typically been investigated by using static or simplified visual displays. How attention is used to perceive and evaluate dynamic, realistic scenes is more poorly understood, in part due to the problem of comparing eye fixations to moving stimuli across observers. When the task and stimulus is common across observers, consistent fixation location can indicate that that region has high goal-based relevance. Here we investigated these issues when an observer has a specific, and naturalistic, task: closed-circuit television (CCTV) monitoring. We concurrently recorded eye movements and ratings of perceived suspiciousness as different observers watched the same set of clips from real CCTV footage. Trained CCTV operators showed greater consistency in fixation location and greater consistency in suspiciousness judgements than untrained observers. Training appears to increase between-operators consistency by learning "knowing what to look for" in these scenes. We used a novel "Dynamic Area of Focus (DAF)" analysis to show that in CCTV monitoring there is a temporal relationship between eye movements and subsequent manual responses, as we have previously found for a sports video watching task. For trained CCTV operators and for untrained observers, manual responses were most highly related to between-observer eye position spread when a temporal lag was introduced between the fixation and response data. Several hundred milliseconds after between-observer eye positions became most similar, observers tended to push the joystick to indicate perceived suspiciousness. Conversely, several hundred milliseconds after between-observer eye positions became dissimilar, observers tended to rate suspiciousness as low. These data provide further support for this DAF method as an important tool for examining goal-directed fixation behavior when the stimulus is a real moving image.
Anoxic insults cause hyperexcitability and cell death in mammalian neurons. Conversely, in anoxia-tolerant turtle brain, spontaneous electrical activity is suppressed by anoxia (i.e., spike arrest; SA) and cell death does not occur. The mechanism(s) of SA is unknown but likely involves GABAergic synaptic transmission, because GABA concentration increases dramatically in anoxic turtle brain. We investigated this possibility in turtle cortical neurons exposed to anoxia and/or GABA(A/B) receptor (GABAR) modulators. Anoxia increased endogenous slow phasic GABAergic activity, and both anoxia and GABA reversibly induced SA by increasing GABA(A)R-mediated postsynaptic activity and Cl(-) conductance, which eliminated the Cl(-) driving force by depolarizing membrane potential (?8 mV) to GABA receptor reversal potential (?-81 mV), and dampened excitatory potentials via shunting inhibition. In addition, both anoxia and GABA decreased excitatory postsynaptic activity, likely via GABA(B)R-mediated inhibition of presynaptic glutamate release. In combination, these mechanisms increased the stimulation required to elicit an action potential >20-fold, and excitatory activity decreased >70% despite membrane potential depolarization. In contrast, anoxic neurons cotreated with GABA(A+B)R antagonists underwent seizure-like events, deleterious Ca(2+) influx, and cell death, a phenotype consistent with excitotoxic cell death in anoxic mammalian brain. We conclude that increased endogenous GABA release during anoxia mediates SA by activating an inhibitory postsynaptic shunt and inhibiting presynaptic glutamate release. This represents a natural adaptive mechanism in which to explore strategies to protect mammalian brain from low-oxygen insults.
Methyl salicylate, an herbivore-induced plant volatile, has been shown to attract natural enemies and affect herbivore behavior. In this study, methyl salicylate was examined for its attractiveness to natural enemies of the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), and for its direct effects on soybean aphid population growth rates. Methyl salicylate lures were deployed in plots within organic soybean [Glycine max (L.) Merr.] fields. Sticky card traps adjacent to and 1.5 m from the lure measured the relative abundance of natural enemies, and soybean aphid populations were monitored within treated and untreated plots. In addition, exclusion cage studies were conducted to determine methyl salicylates effect on soybean aphid population growth rates in the absence of natural enemies. Significantly greater numbers of syrphid flies (Diptera: Syrphidae) and green lacewings (Neuroptera: Chrysopidae) were caught on traps adjacent to the methyl salicylate lure, but no differences in abundance were found at traps 1.5 m from the lure. Furthermore, abundance of soybean aphids was significantly lower in methyl salicylate-treated plots. In exclusion cage studies, soybean aphid numbers were significantly reduced on treated soybean plants when all plants were open to natural enemies. When plants were caged, however, soybean aphid numbers and population growth rates did not differ between treated and untreated plants suggesting no effect of methyl salicylate on soybean aphid reproduction and implicating the role of natural enemies in depressing aphid populations. Although aphid populations were reduced locally around methyl salicylate lures, larger scale studies are needed to assess the technology at the whole-field scale.
Low-level stimulus salience and task relevance together determine the human fixation priority assigned to scene locations (Fecteau and Munoz in Trends Cogn Sci 10(8):382-390, 2006). However, surprisingly little is known about the contribution of task relevance to eye movements during real-world visual search where stimuli are in constant motion and where the target for the visual search is abstract and semantic in nature. Here, we investigate this issue when participants continuously search an array of four closed-circuit television (CCTV) screens for suspicious events. We recorded eye movements whilst participants watched real CCTV footage and moved a joystick to continuously indicate perceived suspiciousness. We find that when multiple areas of a display compete for attention, gaze is allocated according to relative levels of reported suspiciousness. Furthermore, this measure of task relevance accounted for twice the amount of variance in gaze likelihood as the amount of low-level visual changes over time in the video stimuli.
The presence of recurrent high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among melanoma-prone families suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. To this end, we have developed a multiplex bead-based assay for high-throughput CDKN2A/CDK4 genotyping in the context of familial melanoma. Genomic DNA from 1,603 subjects (1,005 in training set and 598 in validation set) were amplified by multiplex PCR using five CDKN2A/CDK4 primer sets followed by multiplex allele-specific primer extension for 39 distinct germline variants. The products were then sorted and analyzed using the Luminex xMAP system. Genotypes were compared with previously determined sequence data. In the Toronto training cohort, all 145 samples with known variants were detected by the bead assay (100% concordance). Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77%). Overall, the bead assay correctly genotyped 1,540/1,603 (96.07%) of all individuals in the study and 1,540/1,545 (99.68%) of individuals whose variants were represented in the probe set. Out of a total of 62,517 allelic calls, 62,512 (99.99%) were correctly assigned. The multiplex bead-based assay is an accurate method for genotyping CDKN2A/CDK4 variants and is potentially useful in genotyping low-to-moderate melanoma risk single-nucleotide polymorphisms.
Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.
An improvement in overall survival among patients with metastatic melanoma has been an elusive goal. In this phase 3 study, ipilimumab--which blocks cytotoxic T-lymphocyte-associated antigen 4 to potentiate an antitumor T-cell response--administered with or without a glycoprotein 100 (gp100) peptide vaccine was compared with gp100 alone in patients with previously treated metastatic melanoma.
For carcinoma specimens with non-grossly identifiable lesions such as microcalcifications, difficulties may be encountered in locating these abnormalities and sampling the margins that are at risk. This is magnified in the case of skin-sparing procedures where the margin is a much greater surface area and is the operation of choice in patients with diffuse microcalcifications and/or multifocal in situ disease. The objective of this study was to determine the efficacy of specimen radiographs of mastectomy in identifying occult carcinoma associated with microcalcifications and assessing the resection margins. We reviewed the histology and corresponding specimen radiographs of 16 patients with diffuse and widespread microcalcifications and who underwent skin-sparing mastectomy. After the specimens were serially sectioned, specimen radiographs of each section of the specimens were obtained with digital mammography equipment. Findings in the specimen radiographs were used to direct the histologic sampling of the specimens. On gross examination, two (12.5%) mastectomy specimens had identifiable discreet masses; the lesions were 4 and 7 mm, respectively. Histologic examination revealed the presence of carcinoma in 13 (81%) cases; seven with both infiltrating and in situ carcinoma and six with in situ carcinoma alone. The remaining three patients demonstrated only changes of biopsy site without any residual carcinoma. Microcalcifications were associated with in situ carcinoma in all malignant cases. Among the 12 cases with microcalcifications present at or within 2 mm of the margins, in situ carcinoma was present at the margins in three cases and located within 2 mm of the margins in two cases. All margins were negative in all cases with microcalcifications that were at least 1 cm away from the resection margins. The average number of tissue blocks examined was 16.6 +/- 5.4. The average number of tissue blocks sampled among randomly selected mastectomy specimens was 15.2 +/- 5.4. There was no statistically significant difference in the number of blocks between mastectomy cases with specimen radiographs taken and those without (t-test). Our study suggests that specimen radiographs may assist in identifying occult breast carcinoma associated with microcalcifications and assessing the resection margins without increased sampling.
One of the clinical hallmarks of hereditary cancer susceptibility disorders is a younger-than-usual age at diagnosis. Familial aggregation of testicular germ cell tumor (TGCT) has been reported, but data on whether familial TGCT cases are diagnosed at an earlier age are inconclusive. Here we compared the age at diagnosis of familial TGCT cases with that of population cases in several countries. Familial TGCT is defined as affected individuals from families with >or=2 cases of TGCT. Age at diagnosis of familial cases from the United States, Canada, United Kingdom, Australia and New Zealand, Norway, and Hungary was compared to cases identified in population-based cancer registries from the respective country, using the generalized estimation equation method. Age at diagnosis was statistically significantly younger for familial TGCT cases from North America (P = 0.024), the United Kingdom (P < 0.0001), and Australia and New Zealand (P = 0.0033) compared with population cases. When stratified by histology, the difference in age at diagnosis distribution between familial and population cases was observed for seminoma cases from North America (P = 0.002) and the United Kingdom (P < 0.0001) and non-seminoma cases from the United Kingdom (P = 0.029) and Australia and New Zealand (P = 0.0023). In summary, we found that the age at diagnosis for familial TGCT cases is, on the average, 2-3 years younger than that for the population cases in North America, United Kingdom, and Australia and New Zealand. The younger age at diagnosis might be suggestive of a genetic basis for familial TGCT.
This phase III, randomized, double-blind, placebo-controlled study was conducted to evaluate the efficacy and safety of sorafenib with carboplatin and paclitaxel (CP) in patients with advanced melanoma who had progressed on a dacarbazine- or temozolomide-containing regimen.
Familial aggregation of testicular germ cell tumor (TGCT) has been reported, but it is unclear if familial TGCT represents a unique entity with distinct clinicopathologic characteristics. Here we describe a collection of familial TGCT cases from an international consortium, in an effort to elucidate any clinical characteristics that are specific to this population.
Anoxia or ischemia causes hyperexcitability and cell death in mammalian neurons. Conversely, in painted turtle brain anoxia increases ?-amino butyric acid (GABA)ergic suppression of spontaneous electrical activity, and cell death is prevented. To examine ischemia tolerance in turtle neurons, we treated cortical sheets with an in vitro mimic of the penumbral region of stroke-afflicted mammalian brain (ischemic solution, IS). We found that during IS perfusion, neuronal membrane potential (V(m)) and the GABA(A) receptor reversal potential depolarized to a similar steady state (-92 ± 2 to -28 ± 3 mV, and -75 ± 1 to -35 ± 3 mV, respectively), and whole-cell conductance (G(w)) increased >3-fold (from 4 ± 0.2 to 15 ± 1 nS). These neurons were electrically quiet and changes reversed after reperfusion. GABA receptor antagonism prevented the IS-mediated increase in G(w) and neurons exhibited enhanced electrical excitability and rapid and irreversible rundown of V(m) during reperfusion. These results suggest that inhibitory GABAergic mechanisms also suppress electrical activity in ischemic cortex. Indeed, after 4 hours of IS treatment neurons did not exhibit any apparent damage; while at 24 hours, only early indicators of apoptosis were present. We conclude that anoxia-tolerant turtle neurons are tolerant of exposure to a mammalian ischemic penumbral mimic solution.
Microphthalmia transcription factor (MITF)-associated (MiT) tumors are a family of rare malignancies, including alveolar soft part sarcoma (ASPS), clear cell sarcoma (CCS), and translocation-associated renal cell carcinoma (tRCC) that have dysregulated expression of oncogenic MITF family proteins. The MET receptor tyrosine kinase gene is transcriptionally activated by MITF family proteins, making MET a potential therapeutic target for MiT tumors. This study assessed the activity of tivantinib (ARQ 197), a selective MET inhibitor, in patients with MiT-associated tumors.
This paper proposes a method for detecting objects carried by pedestrians, such as backpacks and suitcases, from video sequences. In common with earlier work ,  on the same problem, the method produces a representation of motion and shape (known as a temporal template) that has some immunity to noise in foreground segmentations and phase of the walking cycle. Our key novelty is for carried objects to be revealed by comparing the temporal templates against view-specific exemplars generated offline for unencumbered pedestrians. A likelihood map of protrusions, obtained from this match, is combined in a Markov random field for spatial continuity, from which we obtain a segmentation of carried objects using the MAP solution. We also compare the previously used method of periodicity analysis to distinguish carried objects from other protrusions with using prior probabilities for carried-object locations relative to the silhouette. We have reimplemented the earlier state-of-the-art method  and demonstrate a substantial improvement in performance for the new method on the PETS2006 data set. The carried-object detector is also tested on another outdoor data set. Although developed for a specific problem, the method could be applied to the detection of irregularities in appearance for other categories of object that move in a periodic fashion.
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