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Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O 2 ) partial pressure (Po 2 ) gradients that drive O 2 uptake. Following hatch, larval fishes lack a developed gill and the skin is the dominant site of gas transfer, yet few studies have addressed the contribution of convective water flow to cutaneous O 2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O 2 transfer and thus aid in O 2 uptake. In zebrafish ( Danio rerio ), the frequency of pectoral fin movements increased in response to hypoxia at 4 days postfertilization (dpf), but the response was blunted by 15 dpf, when the gills become the dominant site of O 2 uptake, and was absent by 21 dpf. In rainbow trout ( Oncorhynchus mykiss ), Po 2 measured at the skin surface of ventilating larvae was lower when the pectoral fins had been surgically removed, directly demonstrating that fins contribute to convective flow that dissipates cutaneous Po 2 boundary layers. Lack of pectoral fins compromised whole animal O 2 consumption in trout during hypoxia, but this effect was absent in zebrafish. Overall, our findings support a respiratory role of the pectoral fins in rainbow trout, but their involvement in zebrafish remains equivocal.

We investigated the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), prosurvival kinase Akt, oxidative stress, and autophagy in the cytotoxicity of parkinsonian neurotoxin 1-methyl-4-phenyl piridinium (MPP+) towards SH-SY5Y human neuroblastoma cells. MPP+-mediated oxidative stress, mitochondrial depolarization, and apoptotic cell death were associated with rapid (within 2 h) activation of AMPK, its target Raptor, and prosurvival kinase Akt. Antioxidants N-acetylcysteine and butylated hydroxyanisole suppressed MPP+-induced cytotoxicity, AMPK, and Akt activation. A genetic or pharmacological inhibition of AMPK increased MPP+-triggered production of reactive oxygen species and cell death, and diminished Akt phosphorylation, while AMPK activation protected SH-SY5Y cells from MPP+. On the other hand, genetic or pharmacological inactivation of Akt stimulated MPP+-triggered oxidative stress and neurotoxicity, but did not affect AMPK activation. At later time-points (16-24 h), MPP+ inhibited the main autophagy repressor mammalian target of rapamycin, which coincided with the increase in the levels of autophagy marker microtubule-associated protein 1 light-chain 3B. MPP+ also increased the concentration of a selective autophagic target sequestosome-1/p62 and reduced the levels of lysosomal-associated membrane protein 1 and cytoplasmic acidification, suggesting that MPP+-induced autophagy was coupled with a decrease in autophagic flux. Nevertheless, further pharmacological inhibition of autophagy sensitized SH-SY5Y cells to MPP+-induced death. Antioxidants and AMPK knockdown reduced, whereas genetic inactivation of Akt potentiated neurotoxin-triggered autophagy. These results suggest that MPP+-induced oxidative stress stimulates AMPK, which protects SH-SY5Y cells through early activation of antioxidative Akt and late induction of cytoprotective autophagy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: We investigated whether a school-based physical activity intervention would lead to improvements in working memory, inhibition and cognitive flexibility in adolescents aged 13-15 years., Methods: The adolescents at the active school ( n  = 108) participated in an intervention that included increased physical activity for 20 min/day, focused on aerobic activity with low cognitive demands for an entire school year. The adolescents at the control school ( n  = 59) received no extra physical activity. At the beginning (baseline) and end (follow-up) of the school year, the participants performed tests of executive function (working memory, inhibition and cognitive flexibility) and performed tests of physical fitness and health., Results: There was no change in executive functioning at follow-up when comparing the schools. However, only 46% complied with the intervention. When non-compliers were excluded from the analyses, the results remained the same, except for a small but significant increase in working memory for the active school as compared to the control school., Conclusion: These results indicate that compliance with the intervention was low and that aerobic exercise with low cognitive load does not produce improvements in executive functioning., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2019.)

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O 2 levels. The HVR is a critical defense mechanism that serves to delay the negative consequences of hypoxia on aerobic respiration. However, the physiological significance of the HVR in larval fishes is unclear as they do not have a fully developed gill and rely primarily on cutaneous gas transfer. Using larval zebrafish (4, 7, 10 and 15 days post-fertilization; dpf), we examined HVR under three levels of hypoxia (25, 45 and 60 mmHg). The larvae exhibited widely different HVRs as a function of developmental age and level of the hypoxia. Yet, critical O 2 tensions ( P crit ) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O 2 sensors were used to measure a significant decrease in buccal cavity water O 2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O 2 from the buccal cavity. To assess the physiological significance of the HVR, ventilatory water flow was prevented in larvae at 4 and 7 dpf by embedding their heads in agar. An increase in P crit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O 2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O 2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish., (© 2019. Published by The Company of Biologists Ltd.)

Although the medical profession strives for safe prescribing, most medications are unique challenges even when prescribed by an experienced provider. In this article we discuss the pitfalls associated with drug interactions between commonly used antibiotics and anticoagulants, the complexities associated with the administration of novel reversible anticoagulants, the often-overlooked severe adverse drug reactions from commonly used classes of medications such as corticosteroids, the nuances of managing an acetaminophen overdose, and uncommon yet serious adverse events associated with the use of contraceptive hormone drugs., (© 2019, The American College of Clinical Pharmacology.)

Hypoxia-inducible factor (Hif) 1α, an extensively studied transcription factor, is involved in the regulation of many biological processes in hypoxia including the hypoxic ventilatory response. In zebrafish, there are two paralogs of Hif-1α (Hif-1A and Hif-1B), but little is known about the specific roles or potential sub-functionalization of the paralogs in response to hypoxia. Using knockout lines of Hif-1α paralogs, we examined their involvement in the hypoxic ventilatory response, measured as ventilation frequency ( f V ) in larval and adult zebrafish ( Danio rerio ). In wild-type zebrafish, f V increased across developmental time (4, 7, 10 and 15 days post--fertilization, dpf) in response to hypoxia (55 mmHg). In contrast, the Hif-1B knockout fish did not exhibit an increase in hypoxic f V at 4 dpf. Similar to wild-type, as larvae of all knockout lines developed, the magnitude of f V increased but to a lesser degree than in the wild-type larvae, until 15 dpf at which point there was no difference among the genotypes. In adult zebrafish, only in Hif-1B knockout fish was there an attenuation in f V during sustained exposure to 30 mmHg for 1 h but there was no effect when fish were exposed for a shorter duration to progressive hypoxia. The mechanism of action of Hif-1α, in part, may be through its downstream target, nitric oxide synthase, and its product, nitric oxide. Overall, the effect of each Hif-1α paralog on the hypoxic ventilatory response of zebrafish varies over development and is dependent on the type of hypoxic stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: The degree by which mechanisms underlying phenotypic convergence are similar among taxa depends on the number of evolutionary paths available for selection to act upon. Likelihood of convergence will be influenced by an interplay of factors such as genetic architecture, phylogenetic history and population demography. To determine if there is convergence or divergence in mechanisms underlying phenotypic similarity, we assessed whether gene transcription patterns differed among species with similar levels of hypoxia tolerance., Results: Three species of marine fish from the superfamily Cottoidea (smoothhead sculpin [Artedius lateralis], sailfin sculpin [Nautichthys oculofasciatus] and Pacific staghorn sculpin [Leptocottus armatus]), all of which have previously been shown to share the same level of hypoxia tolerance, were exposed to short-(8 h) and longer-term (72 h) hypoxia and mRNA transcripts were assessed using a custom microarray. We examined hypoxia-induced transcription patterns in metabolic and protein production pathways and found that a high proportion of genes associated with these biological processes showed significant differences among the species. Specifically, the data suggest that the smoothhead sculpin, unlike the sailfin sculpin and the Pacific staghorn sculpin, relied on amino acid degradation rather than glycolysis or fatty acid oxidation to generate ATP during hypoxia exposure. There was also variation across the species in the transcription of genes involved in protein production (e.g. mRNA processing and protein translation), such that it increased in the smoothhead sculpin, decreased in the sailfin sculpin and was variable in the Pacific staghorn sculpin., Conclusions: Changes in metabolic and protein production pathways are part of the key responses of fishes to exposures to environmental hypoxia. Yet, species with similar overall hypoxia tolerance exhibited different transcriptional responses in these pathways, indicating flexibility and complexity of interactions in the evolution of the mechanisms underlying the hypoxia tolerance phenotype. The variation in the hypoxia-induced transcription of genes across species with similar hypoxia tolerance suggests that similar whole-animal phenotypes can emerge from divergent evolutionary paths that may affect metabolically important functions.

Biocatalytic potential of Streptomyces strains isolated from the rhizosphere of plants and from mycorrhizosphere of fungi has been investigated. A total of 118 Streptomyces isolates were selected and functionally screened for 10 different biotechnologically important enzymatic activities: hydrolase (cellulase, cutinase, gelatinase, lipase, protease, polyhydroxyalkanoate (PHA) depolymerase), phenol oxidase and peroxidase (laccase, tyrosinase, and lignin peroxidase), and aminotransferase. Out of 118 tested Streptomyces spp., 90% showed at least one enzymatic activity. The most abundant were enzymes involved in the biomass degradation, as the production of cutinase, cellulase, and lignin peroxidase were detected in 31%, 40%, and 48% of the isolates, respectively. The improved specific activities of lipase (isolates BV315 and BV100) and tyrosinase (isolates BV87 and BV88) were shown in comparison with the industrially relevant activities of Pseudomonas strains. Plant rhizosphere soils were more prolific source of Streptomyces strains with biocatalytic potential in comparison with mycorrhizosphere soils. Overall, 284 enzyme activities among 118 Streptomyces isolates have been detected. This is the first comprehensive screening of Streptomyces isolates from rhizosphere and mycorrhizosphere soils for novel biocatalysts, showing that specific environmental habitats, such as rhizosphere soils, are "treasure troves" of Streptomyces with biocatalytic potential., (© 2018 International Union of Biochemistry and Molecular Biology, Inc.)

In aquatic environments, hypoxia is a multi-dimensional stressor that can vary in O 2 level (partial pressure of O 2 in water, P w O 2 ), rate of induction and duration. Natural hypoxic environments can therefore be very different from one another. For the many fish species that have evolved to cope with these different hypoxic environments, survival requires adjusting energy supply and demand pathways to maintain energy balance. The literature describes innumerable ways that fishes combine aerobic metabolism, anaerobic metabolism and metabolic rate depression (MRD) to accomplish this, but it is unknown whether the evolutionary paths leading to these different strategies are determined primarily by species' phylogenetic histories, genetic constraint or their native hypoxic environments. We explored this idea by devising a four-quadrant matrix that bins different aquatic hypoxic environments according to their duration and P w O 2  characteristics. We then systematically mined the literature for well-studied species native to environments within each quadrant, and, for each of 10 case studies, described the species' total hypoxic response (THR), defined as its hypoxia-induced combination of sustained aerobic metabolism, enhanced anaerobic metabolism and MRD, encompassing also the mechanisms underlying these metabolic modes. Our analysis revealed that fishes use a wide range of THRs, but that distantly related species from environments within the same matrix quadrant have converged on similar THRs. For example, environments of moderately hypoxic P w O 2  favoured predominantly aerobic THRs, whereas environments of severely hypoxic P w O 2  favoured MRD. Capacity for aerial emergence as well as predation pressure (aquatic and aerial) also contributed to these responses, in addition to other biotic and abiotic factors. Generally, it appears that the particular type of hypoxia experienced by a fish plays a major role in shaping its particular THR., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

In the present study, crude bacterial extract containing violacein is investigated for the preparation of antimicrobial polyamide fabrics. The optimal culture conditions of Janthinobacterium lividum (JL) for maximum biomass and violacein production were found to be 25°C, pH 7.0, while the addition of ampicillin of 0.2 mg mL -1 in the small scale increased violacein production 1.3-fold. In scale-up trials, the addition of 1% (v/v) glycerol in a fed-batch bioreactor, resulted in fivefold extracted crude violacein increase with final concentration of 1.828 g L -1 . Polyamide 6.6 fabrics were dyed following three different processes; through simultaneous fermentation and dyeing (SFD), by incubating the fabric in the sonicated bacterial culture after fermentation and by using cell-free extract containing violacein. Maximum color change (Δ E ) and color strength ( K / S ) obtained for SFD fabrics were 74.81 and 22.01, respectively, while no alteration of fastness and staining of dye at acid and alkaline perspiration or at water was indicated. The dyed fabrics presented significant antifungal activity against Candida albicans, C. parapsilosis , and C. krusei , as well as antibacterial properties against Escherichia coli, Staphylococcus aureus , and the S. aureus MRSA. We have shown that J. lividum cultures can be successfully used for violacein production and for simultaneous dying of fabrics resulting in dyed fabrics with antimicrobial properties without utilization of organic solvents.

About 20,100 research publications dated 2000-2017 were recovered searching the PubMed and Web of Science databases for Streptomyces, which are the richest known source of bioactive molecules. However, these bacteria with versatile metabolism are powerful suppliers of biocatalytic tools (enzymes) for advanced biotechnological applications such as green chemical transformations and biopharmaceutical and biofuel production. The recent technological advances, especially in DNA sequencing coupled with computational tools for protein functional and structural prediction, and the improved access to microbial diversity enabled the easier access to enzymes and the ability to engineer them to suit a wider range of biotechnological processes. The major driver behind a dramatic increase in the utilization of biocatalysis is sustainable development and the shift toward bioeconomy that will, in accordance to the UN policy agenda "Bioeconomy to 2030," become a global effort in the near future. Streptomyces spp. already play a significant role among industrial microorganisms. The intention of this minireview is to highlight the presence of Streptomyces in the toolbox of biocatalysis and to give an overview of the most important advances in novel biocatalyst discovery and applications. Judging by the steady increase in a number of recent references (228 for the 2000-2017 period), it is clear that biocatalysts from Streptomyces spp. hold promises in terms of valuable properties and applicative industrial potential.

During periods of severe hypoxia or anoxia, Carassius spp. are known for their ability to produce ethanol as their anaerobic end product, which diffuses into the environment thereby reducing the osmotic and acidotic load associated with "anaerobic" glycolysis. However, the relationship between alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities, key ethanol metabolizing enzymes, and hypoxia tolerance among Carassius spp. and their closely related non-ethanol-producing cyprinids remains unclear. To address this, we quantified the activity levels of key anaerobic enzymes in liver and muscle in species of cyprinids over 48 h of severe hypoxia exposure (0.7 kPa). As predicted, muscle ADH activity was highest in the two most hypoxia-tolerant species (Carassius spp.), with very low levels present in the other species examined. However, liver ADH activities showed an inverse relationship with hypoxia tolerance, with the most hypoxia-tolerant fish having the lowest ADH activity. There was no correlation between hypoxia tolerance and ALDH and LDH activities in muscle or liver. All species produced lactate, reaching their highest levels after 8 h, but returning to near-baseline levels by 48 h of sustained exposure to hypoxia, suggesting lactate oxidation or depressed ATP demand. Liver glycogen content was not affected by 48 h hypoxia exposure in the most hypoxia-tolerant species, whereas the least tolerant species consumed the majority of the liver glycogen stores, which is probably due to the greater relative hypoxia exposure experienced by these species. Our findings that liver ADH activities were inversely related to hypoxia tolerance suggests that in all but Carassius spp., the ethanol metabolizing pathways in cyprinids is largely similar to that observed in other vertebrates and plays a role in the detoxification of ethanol. Furthermore, conservation of glycogen stores may be the result of metabolic-depressing pathways in the more tolerant species, regardless of the ability to produce ethanol, or adaptations that improve oxygen uptake to reduce metabolic demands due to hypoxia.

Vertebrate hypoxia tolerance can emerge from modifications to the oxygen (O2) transport cascade, but whether there is adaptive variation to O2 binding at the terminus of this cascade, mitochondrial cytochrome c oxidase (COX), is not known. In order to address the hypothesis that hypoxia tolerance is associated with enhanced O2 binding by mitochondria we undertook a comparative analysis of COX O2 kinetics across species of intertidal sculpins (Cottidae, Actinopterygii) that vary in hypoxia tolerance. Our analysis revealed a significant relationship between hypoxia tolerance (critical O2 tension of O2 consumption rate; Pcrit), mitochondrial O2 binding affinity (O2 tension at which mitochondrial respiration was half maximal; P50), and COX O2-binding affinity (apparent Michaelis-Menten constant for O2 binding to COX; Km,app O2). The more hypoxia tolerant species had both a lower mitochondrial P50 and lower COX Km,app O2, facilitating the maintenance of mitochondrial function to a lower O2 tension than in hypoxia intolerant species. Additionally, hypoxia tolerant species had a lower overall COX Vmax but higher mitochondrial COX respiration rate when expressed relative to maximal electron transport system respiration rate. In silico analyses of the COX3 subunit postulated as the entry point for O2 into the COX protein catalytic core, points to variation in COX3 protein stability (estimated as free energy of unfolding) contributing to the variation in COX Km,app O2. We propose that interactions between COX3 and cardiolipin at four amino acid positions along the same alpha-helix forming the COX3 v-cleft represent likely determinants of interspecific differences in COX Km,app O2., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Biologicals are a rapidly expanding class of medications used in the treatment of many different conditions. This article reviews the common characteristics of this class and the requirements for safe and effective use in patients. Several vignettes are included to illustrate common challenges., (© 2017, The American College of Clinical Pharmacology.)

We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G 2 M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Differences in physiology related to young or old age and/or organ system impairment alter the absorption, distribution, metabolism, and excretion of many medications and consequently their effectiveness and toxicity. This module discusses common alterations in medication use and dosage that are required in the pediatric age group, in the elderly, and in patients with renal or hepatic disease., (© 2016, The American College of Clinical Pharmacology.)

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Transcriptionally mediated phenotypic plasticity as a mechanism of modifying traits in response to an environmental challenge remains an important area of study. We compared the transcriptional responses to low oxygen (hypoxia) of the hypoxia-tolerant intertidal fish, the tidepool sculpin (Oligocottus maculosus) with the closely related hypoxia-intolerant subtidal fish, the silverspotted sculpin (Blepsias cirrhosus) to determine whether these species use different mechanisms to cope with hypoxia. Individuals from each species were exposed to environmental O(2) tensions chosen to yield a similar level of tissue hypoxia, and gene transcription was assessed in the liver over time. There was an effect of time in hypoxia, where the greatest transcriptional change in the silverspotted sculpin occurred between 3 and 24 h in contrast to the tidepool sculpin where the largest transcriptional change occurred between 24 and 72 h of hypoxia. A number of genes showed similar hypoxia-induced transcription patterns in both species (e.g. genes associated with glycolysis and apoptosis) suggesting they are involved in a conserved hypoxia response. A large set of genes showed divergent transcriptional patterns in the two species, including fatty acid oxidation and oxidative phosphorylation, suggesting that these biological processes may contribute to explaining variation in hypoxia tolerance in these species. When both species were exposed to a single environmental O(2) tension, large transcriptional responses were seen in the hypoxia-intolerant silverspotted sculpin while almost no response was observed in the hypoxia-tolerant tidepool sculpin. Overall, divergent transcription patterns in response to both magnitude and duration of hypoxia provide insights into the processes that may determine an animal's capacity to tolerate frequent bouts of hypoxia in the wild., (© 2014 John Wiley & Sons Ltd.)

Cancer patients frequently exhibit a deficiency in Type-1 (but not Type-2 or regulatory) CD4(+) T cell responses against tumor-associated antigens (TAA), which may limit protection against disease progression or responsiveness to immunotherapy in these individuals. Since such deficiency was acutely evident in patients with active disease (AD), where chronic stimulation of anti-tumor CD4(+) T cells would be expected and activation-induced cell death may be prevalent, we employed MHC Class II-peptide tetramers to characterize the frequency and apoptotic status of TAA- vs. influenza (FluM1) virus-specific CD4(+) T cells in the peripheral blood of HLA-DR*0401(+) patients with melanoma or renal cell carcinoma. We observed that Flu-specific CD4(+) T cells ranged from 0.17 to 3.89%, while up to approximately 1% of CD4(+) T cells reacted against individual TAA epitopes derived from the EphA2 or MAGE-6 proteins. The frequencies of EphA2 and MAGE-6-specific CD4(+) T cells in patients were significantly correlated with AD and gender of the patient (i.e., females > males), while frequencies of Flu-specific CD4(+) T cells were distributed within a normal range in all patients. Notably, patient CD4(+) T cells reactive with MHC class II-TAA (but not MHC class II-Flu) tetramers were significantly enriched for a pro-apoptotic (Annexin-V(+)) phenotype, particularly amongst the Th1 (T-bet(+)) subset. These results suggest that the preferential sensitivity of TAA (but not viral)-specific CD4(+) Th1 cells to apoptosis in melanoma patients with AD will need to be overcome for optimal clinical benefit of immunotherapeutic approaches to be realized.

The Salmoniform whole-genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity-responsive paralogs of the Na(+) , K(+) ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue-specific expression patterns. We found that these paralogs arose during a small-scale duplication event prior to the Salmoniform, but after the teleost, whole-genome duplication. The 'freshwater paralog' (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the 'saltwater paralog' (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na(+) , K(+) ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity-responsive Na(+) , K(+) ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole-genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity-responsive Na(+) , K(+) ATPase α1 paralogs in fishes., (© 2014 John Wiley & Sons Ltd.)

Effects of muscle fatigue on force coordination and task performance of various manipulation tasks are explored. Grip force (GF; normal force component acting at the digits-object contact area) and load force (LF; tangential component that lifts and holds objects) were recorded prior to and after fatiguing the distal (DAM; i.e., GF producing) and proximal arm muscles (PAM; LF producing). Results reveal a deterioration of GF scaling (i.e., averaged GF-LF ratio), GF-LF coupling (their correlation), and task performance (ability to exert a prescribed LF pattern) associated with DAM, but not PAM fatigue. Deteriorated force coordination clearly increases the likelihood of dropping an object; however, the observed selective effects of DAM and PAM fatigue represent a novel finding deserving of further research.

Discordance between mitochondrial and nuclear DNA has been noted in many systems. Asymmetric introgression of mitochondria is a common cause of such discordances, although in most cases the drivers of introgression are unknown. In the yellow-rumped warbler, evidence suggests that mtDNA from the eastern, myrtle warbler, has introgressed across much of the range of the western form, the Audubon's warbler. Within the southwestern United States myrtle mtDNA comes into contact with another clade that occurs in the Mexican black-fronted warbler. Both northern forms exhibit seasonal migration, whereas black-fronted warblers are nonmigratory. We investigated the link between mitochondrial introgression, mitochondrial function, and migration using novel genetic, isotopic, biochemical, and phenotypic data obtained from populations in the transition zone. Isotopes suggest the zone is coincident with a shift in migration, with individuals in the south being resident and populations further north becoming increasingly more migratory. Mitochondrial respiration in flight muscles demonstrates that myrtle-type individuals have a significantly greater acceptor control ratio of mitochondria, suggesting it may be more metabolically efficient. To our knowledge this is the first time this type of intraspecific variation in mitochondrial respiration has been measured in wild birds and we discuss how such mitochondrial adaptations may have facilitated introgression., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

We assessed hypoxia tolerance in 11 species of fish from the superfamily Cottoidea (commonly called sculpins) that are known to differ in their critical O(2) tensions (P(crit)) and examined whether hypoxia tolerance correlated with larger substrate stores and higher maximal activity of enzymes associated with anaerobic adenosine triphosphate production (especially glycolysis). Among the sculpins studied, there was large variation in time to loss of equilibrium (LOE(50)) at 6.4 ± 0.1 torr, with values ranging between 25 and 538 min, and the variation in LOE(50) was correlated with P(crit). Our measures of time to LOE(50) and P(crit) were regressed against maximal enzyme activities of lactate dehydrogenase (LDH), pyruvate kinase (PK), creatine phosphokinase (CPK), and citrate synthase (CS) as well as the concentrations of glycogen, glucose, and creatine phosphate in the brain, liver, and white muscle. In the brain, there was a phylogenetically independent relationship between P(crit) and tissue LDH, PK, CPK, and CS activities expressed relative to tissue mass. Hypoxia-tolerant sculpins (those with low P(crit) values) had higher levels of brain LDH, PK, CPK, and CS than did hypoxia-sensitive sculpins. Similarly, LOE(50) regressed against brain LDH, PK, and CPK activities expressed relative to tissue mass, with the more hypoxia-tolerant species (i.e., those with higher LOE(50)) having higher enzyme activities. However, when the phylogenetic relationship among our sculpins was taken into account, only the relationship between hypoxia tolerance and LDH activity remained significant. When enzyme activities were expressed relative to total soluble protein in the tissue, the only relationships that remained were between brain LDH activity and P(crit) and LOE(50). In liver and white muscle, there were no relationships between the measures of hypoxia tolerance and enzyme activity or metabolite content. Overall, our analysis suggests that hypoxia-tolerant sculpins maintain higher maximal activities of some of the enzymes involved in anaerobic metabolism in the brain, and this may be an adaptation to hypoxia.

The hippocampus is crucial for episodic autobiographical memory retrieval. Functional neuroimaging evidence suggests that it is similarly engaged in recent and remote retrieval when memories are matched on vividness and personal importance. Far fewer studies have investigated the nature of hippocampal-neocortical coactivation in relation to memory remoteness. The purpose of this study was to examine hippocampal activity and functional connectivity as a function of memory age. Unlike most studies of autobiographical memory, we included autobiographical memories formed in the days and weeks before scanning, in addition to truly remote memories on the order of months and years. Like previous studies, we found that the hippocampus was active bilaterally regardless of memory age, with anterior activity increasing up to 1 yr and then decreasing, and with posterior activity being less sensitive to memory age. More importantly, hippocampal functional connectivity varied with memory age. Retrieving recent memories (≤1 yr) showed a late coactivation of the hippocampus and areas of the autobiographical memory network, whereas retrieving remote memories (10 yrs) showed an early negative coactivation of the hippocampus and left inferior frontal gyrus followed by a positive coactivation with anterior cingulate. This finding may reflect that the hippocampus is more strongly integrated with the autobiographical memory network for recent than for remote memories, and that more effort is required to recover remote memories., (Copyright © 2011 Wiley Periodicals, Inc.)

Purpose: The purpose of the paper was to assess physical recovery in post stroke patients., Work Method: Prospective cohort study was conducted. The study involved 30 post stroke patients from the city of Nis, aged 40-69. To assess the functional status of the Barthel index (BI) was used. Functional status and BI scores were determined by two times, on admission to the Clinic for rehabilitation and three months after discharge from the Clinic., Work Results: A total number of patients was 30 (20 men and 10 women). There were twice more men than women (66.7% vs 33.3%, p < 0.001). The average age of all patients was 58.93 +/- 6.86. The majority of patients, were from the age group 60 to 69 years. Left-side hemiparesis had 23(77.%) of all patients (16 men and 7 women). Right-side hemiparesis had 7 (23%) patients (4 men and 3 women). Ischemic as a cause of stroke was found in 92% of patients and hemorrhage in 8%. The values of BI score on admission ranged from 30 to 100, with a mean BI score in all patients on admission to the Clinic was the 57. Three months after discharge from rehabilitation in all patients there was an increase in BI score in relation to the value of BI on admission. The average value of BI score was 79. The determined difference is statistically significant (p < 0.01)., Discussion: All the patients achieved an increase in BI score indicating their improved status after the applied rehabilitation treatment., Conclusion: At the end of the study 57% of the patients could move independently. Patients with a left-sided lesions had better self care of patients with right-sided lesions. Mobility was better in patients with left-sided lesions at the start of rehabilitation. On completion of the rehabilitation, patients with right-sided lesions still needed external help.

AlphaVbeta3 (alphavbeta3) is an important molecule for tumor-induced angiogenesis and is upregulated in metastatic melanoma (MM). We proposed to study the mechanism of action of etaracizumab, a monoclonal antibody targeting alphavbeta3, in MM. Patients with MM and biopsiable tumor were treated with etaracizumab in 3 dose cohorts starting from 8 mg/kg. Tumor saturation by etaracizumab using LM609 immunohistochemical staining of tumor sections was the primary endpoint. Subsequent dose cohorts were defined based on the tumor saturation by etaracizumab. Secondary end points were analysis of clinical benefit and changes from baseline of several tumor and peripheral blood biomarkers. Eighteen patients were enrolled at 3 dose levels. Etaracizumab showed better melanoma cell saturation at the 8mg/kg and 1 mg/kg dose compared with the 4 mg/kg dose and better vascular endothelial cell saturation at 8 mg/kg compared with lower dose groups. Etaracizumab demonstrated an acceptable safety profile. The optimal biologic dose out of those selected for investigation was 8 mg/kg. Patients treated at the highest dose may have had better clinical benefit secondary to suppression of the activated immediate downstream effector of alphavbeta3 signaling, FAK, in melanoma cells, but this alone did not ultimately affect melanoma cell proliferation or apoptosis. No apparent antiangiogenic or immunomodulatory effects of etaracizumab were noted.

Behavioral responses to progressive hypoxia exposure were assessed in several species of fish from the family Cottidae (sculpins), which are distributed along the near-shore marine environment and differ in their hypoxia tolerance. The use of aquatic surface respiration (ASR) and aerial emergence as a response to progressive decreases in environmental O(2) differed between intertidal and subtidal sculpins. Intertidal sculpins consistently displayed ASR followed by emergence behaviors, while the subtidal species performed these behaviors at low frequency or not at all. There was a significant negative correlation between the O(2) thresholds for the onset of ASR and critical O(2) tensions (P(crit), a measure of hypoxia tolerance), such that the hypoxia-tolerant species performed ASR at higher O(2) tensions than the more hypoxia-sensitive species. There was no relationship between the O(2) thresholds for emergence and P(crit). When restricted from accessing the water surface during progressive decreases in O(2), all species of sculpin displayed the same sequence of behaviors including agitation, attempts to escape, quiescence, and unresponsiveness. The only behavior from this suite that correlated with P(crit) was the first sign of agitation, which occurred at a lower O(2) tension in the most hypoxia-tolerant species. With the application of phylogenetically independent contrasts, all the relationships between behavior and P(crit) were nonsignificant, which limits our capacity to separate selection-driven changes in behavior from the phylogenetic signal.

Patient M.L. [Levine, B., Black, S. E., Cabeza, R., Sinden, M., Mcintosh, A. R., Toth, J. P., et al. (1998). Episodic memory and the self in a case of isolated retrograde amnesia. Brain, 121, 1951-1973], lost memory for events occurring before his severe traumatic brain injury, yet his anterograde (post-injury) learning and memory appeared intact, a syndrome known as isolated or focal retrograde amnesia. Studies with M.L. demonstrated a dissociation between episodic and semantic memory. His retrograde amnesia was specific to episodic autobiographical memory. Convergent behavioral and functional imaging data suggested that his anterograde memory, while appearing normal, was accomplished with reduced autonoetic awareness (awareness of the self as a continuous entity across time that is a crucial element of episodic memory). While previous research on M.L. focused on anterograde memory of laboratory stimuli, in this study, M.L.'s autobiographical memory for post-injury events or anterograde autobiographical memory was examined using prospective collection of autobiographical events via audio diary with detailed behavioral and functional neuroanatomical analysis. Consistent with his reports of subjective disconnection from post-injury autobiographical events, M.L. assigned fewer "remember" ratings to his autobiographical events than comparison subjects. His generation of event-specific details using the Autobiographical Interview [Levine, B., Svoboda, E., Hay, J., Winocur, G., & Moscovitch, M. (2002). Aging and autobiographical memory: dissociating episodic from semantic retrieval. Psychology and Aging, 17, 677-689] was low, but not significantly so, suggesting that it is possible to generate episodic-like details even when re-experiencing of those details is compromised. While listening to the autobiographical audio diary segments, M.L. showed reduced activation relative to comparison subjects in midline frontal and posterior nodes previously identified as part of the autobiographical memory network. Reductions were also evident in M.L. in association with personal semantic stimuli (e.g., recordings describing personal habits and routines). These data suggest an association between M.L.'s impoverished recollection of autobiographical material and reduced activation in midline sectors of the autobiographical memory network that support the autonoetic, first-person element of episodic memory.

EphA2, a member of the receptor tyrosine kinase family, is commonly expressed by a broad range of cancer types, where its level of (over)expression correlates with poor clinical outcome. Because tumor cell expressed EphA2 is a nonmutated "self" protein, specific CD8(+) T cells are subject to self-tolerance mechanisms and typically exhibit only moderate-to-low functional avidity, rendering them marginally competent to recognize EphA2(+) tumor cells in vitro or in vivo. We have recently reported that the ability of specific CD8(+) T cells to recognize EphA2(+) tumor cells can be augmented after the cancer cells are pretreated with EphA2 agonists that promote proteasomal degradation and up-regulated expression of EphA2/class I complexes on the tumor cell membrane. In the current study, we show that treatment of EphA2(+) tumor cells with the irreversible heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), similarly enhances their recognition by EphA2-specific CD8(+) T-cell lines and clones in vitro via a mechanism that is dependent on proteasome and transporter-associated protein function as well as the retrotranslocation of EphA2 into the tumor cytoplasm. When 17-DMAG and agonist anti-EphA2 monoclonal antibodies are coapplied, T-cell recognition of tumor cells is further increased over that observed for either agent alone. These studies suggest that EphA2 represents a novel heat shock protein 90 client protein and that the treatment of cancer patients with 17-DMAG-based "pulse" therapy may improve the antitumor efficacy of CD8(+) T effector cells reactive against EphA2-derived epitopes.

The ability of an organism to acquire O(2) from its environment is key to survival and can play an important role in dictating a species' ecological distribution. This study is the first, to our knowledge, to show a tight, phylogenetically independent correlation between hypoxia tolerance, traits involved in dictating O(2) extraction capacity and the distribution of a group of closely related fish species, sculpins from the family Cottidae, along the nearshore marine environment. Sculpins with higher hypoxia tolerance, measured as low critical O(2) tensions (P(crit)), inhabit the O2 variable intertidal zones, while species with lower hypoxia tolerance inhabit the more O(2) stable subtidal zone or freshwater. Hypoxia tolerance is phylogenetically independently associated with an enhanced O(2) extraction capacity, with three principal components accounting for 75 per cent of the variation in P(crit): routine O(2) consumption rate; mass-specific gill surface area; and whole blood haemoglobin (Hb)- O(2)-binding affinity (P(50)). Variation in whole blood Hb-O(2)P(50) is strongly correlated with the intrinsic O(2)-binding properties of the purified Hb while the differences in the concentration of the allosteric Hb modulators, ATP and GTP, provide a Hb system with substantial plasticity for survival in a highly O(2) variable environment.

The EphA2 receptor tyrosine kinase is an attractive therapeutic target that is commonly overexpressed on solid tumors, with the degree of overexpression associated with disease progression, metastatic potential, and poor prognosis. Agonistic mAbs or ligand (ephrinA1)-Fc fusion protein are capable of inducing EphA2 internalization and degradation, thereby (at least transiently) eliminating the influence of this oncoprotein. We and others have also shown that EphA2 contains multiple peptide epitopes that can be recognized by effector CD4(+) and CD8(+) T cells isolated from tumor-bearing patients. Herein, we show that "agonist" reagents that trigger the proteasome-dependent degradation of tumor cell EphA2 result in the improved presentation of peptides derived from (both the extracellular and intracellular domains of) EphA2 in MHC class I complexes expressed on the tumor cell membrane for at least 48 h, as manifested by increased recognition by EphA2-specific CD8(+) T cells in vitro. We also observed that while delivery of ephrinA1-Fc fusion protein or agonist mAb into EphA2(+) tumor lesions promotes EphA2 degradation in situ, this single administration of agent does not dramatically alter tumor progression in a humanized SCID model. However, when combined with the adoptive transfer of normally nontherapeutic (human) anti-EphA2 CD8(+) CTL, this dual-agent regimen results in complete tumor eradication. These results suggest that strategies targeting the conditional proteasome-mediated destruction of tumor cell EphA2 may enable EphA2-specific CD8(+) T cells (of modest functional avidity) to realize improved therapeutic potential.

Severe hypoxia exposure and exhaustive exercise in goldfish both elicit a strong activation of substrate-level phosphorylation with the majority of the metabolic perturbations occurring in the white muscle. Approximately half of the muscle glycogen breakdown observed during severe hypoxia exposure was accounted for by ethanol production and loss to the environment, which limited the extent of muscle glycogen recovery when animals were returned to normoxic conditions. Ethanol production in goldfish is not solely a response to anoxia/hypoxia exposure however, as a transient increase in ethanol production was observed during the early stages of recovery from exhaustive exercise. These data suggest that ethanol production is a ubiquitous "anaerobic" end product, which accumulates whenever metabolic demands exceed mitochondrial oxidative potential. Exhaustive exercise and hypoxia exposure both caused a 7 to 8 micromol g(-1) wet mass increase in muscle [lactate] and the rates of recovery following these perturbations were similar. The rates of muscle PCr and pHi recovery after hypoxia exposure and exhaustive exercise were similar with levels returning to controls values within 0.5 h. Surprisingly, liver [glycogen] was not depleted during exposure to severe hypoxia, however, during recovery from both hypoxia and exercise dramatically different responses in liver [glycogen] were noted. During the early stages of recovery, liver [glycogen] transiently increased to high levels after exhaustive exercise, while during recovery from hypoxia there was a transient decrease in liver glycogen over the same time frame. Overall, this points to the liver playing a dramatically different role in facilitating recovery from exercise compared with hypoxia exposure.

Populations of the common killifish Fundulus heteroclitus are found along a latitudinal temperature gradient in habitats with high thermal variability. The objectives of this study were to assess the effects of temperature and population of origin on killifish swimming performance (assessed as critical swimming speed, U(crit)). Acclimated fish from northern and southern killifish populations demonstrated a wide zone (from 7 degrees to 33 degrees C) over which U(crit) showed little change with temperature, with performance declining significantly only at lower temperatures. Although we observed significant differences in swimming performance between a northern and a southern population of killifish in one experiment, with northern fish having an approximately 1.5-fold-greater U(crit) than southern fish across all acclimation temperatures, we were unable to replicate this finding in other populations or collection years, and performance was consistently high across all populations and at both low (7 degrees C) and high (23 degrees C) acclimation temperatures. The poor swimming performance of southern killifish from a single collection year was correlated with low muscle [glycogen] rather than with other indicators of fuel stores or body condition. Killifish acclimated to 18 degrees C and acutely challenged at temperatures of 5 degrees , 18 degrees , 25 degrees , or 34 degrees C showed modest thermal sensitivity of U(crit) between 18 degrees and 34 degrees C, with performance declining substantially at 5 degrees C. Thus, much of the zone of relative thermal insensitivity of swimming performance is intrinsic in this species rather than acquired as a result of acclimation. These data suggest that killifish are broadly tolerant of changing temperatures, whether acute or chronic, and demonstrate little evidence of local adaptation in endurance swimming performance in populations from different thermal habitats.

We present the first results of a study of the Gacka River spring area, an important Croatian drinking water resource. Stable isotope data (obtained at the Stable Isotope Laboratory SILab in Rijeka) are used in combination with hydrological and meteorological data. The delta18O values give information on the mean catchment altitudes of the three main springs of Gacka River (Majerovo vrelo, Tonkovica vrelo and Pecina). Hydrological, meteorological and stable isotope data indicate good mixing of infiltrated and ground waters. The precipitation d-excess shows a seasonally varying influence of continental and maritime air masses.

A patient from the University of Pittsburgh is presented who developed autoimmunity during IL-2 based combination therapy. IL-2 was originally described as a "T cell growth factor" capable of expanding previously activated T cells, enhancing the cytotoxicity of antigen-specific cytotoxic T cells and NK cells. High dose Interleukin 2 (HDIL2) is now FDA-approved for therapy of patients with metastatic melanoma and renal cell carcinoma, based on its ability to induce durable responses in 5-10% of patients. The antitumor effect of HDIL2 is incompletely understood, but it appears that this regimen alters the balance of immigrant T effector cells in relation to T suppressor cells. It promotes a less immunosuppressive tumor microenvironment, inducing tumor regression in a subset of patients that is yet to be defined. The antitumor activity of IL-2, as for other agents that promote durable antitumor activity against melanoma such as interferon alpha and anti-CTLA4 antibody, is frequently associated with development of autoimmunity as observed in this patient. We present studies investigating the mechanisms for the therapeutic benefit of HDIL2 in melanoma.

We acclimated two species of sculpin, the freshwater prickly sculpin (Cottus asper) and the closely related marine Pacific staghorn sculpin (Leptocottus armatus) to freshwater ( approximately 0 g/L), brackish water (15 g/L), and seawater (30 g/L) for at least 4 wk and examined the relationships between respiration, ion regulation, gill morphology, and freshwater tolerance. The prickly sculpin successfully acclimated to all three salinities and did not experience appreciable changes in plasma osmolality, [Cl-], or mortality. Gill Na+/K+-ATPase activity was lowest in prickly sculpins acclimated to freshwater, their native salinity, and increased during acclimation to seawater. Furthermore, prickly sculpins acclimated to freshwater had a 30% higher P(crit) than fish acclimated to brackish water or seawater; P(crit) is the environmental P(O2) below which an animal can no longer maintain a routine (.-)M(O2), and an increase in P(crit) represents a compromise of respiratory gas exchange. The higher P(crit) observed in prickly sculpins acclimated to freshwater is likely a consequence of their having small, relatively thick gills that increase in thickness (by approximately 1 microm) during freshwater exposure. In contrast, the marine Pacific staghorn sculpin successfully acclimated to brackish water and seawater, but high mortality (25%) was observed after 3 wk of exposure to freshwater. Pacific staghorn sculpins exposed to freshwater suffered significant, 15%-20%, reductions in plasma osmolality and [Cl-], and these losses in plasma ions resulted in a 1.4-fold increase in gill Na+/K+-ATPase activity. Pacific staghorn sculpins have large, thin gills that are not modified in response to salinity acclimation, and as a result, these animals show no respiratory compromise during freshwater acclimation, as evidenced by the lack of change in P(crit), but show significant ion regulatory disturbance. Overall, this study suggests that gill thickening and the resulting respiratory compromise are necessary for freshwater tolerance in sculpins.

Dendritic cells (DCs) are potent antigen presenting cells and represent attractive candidates for use in novel immunotherapies for patients with renal cell carcinoma (RCC), a disease that has proven refractory to conventional treatment modalities, such as chemotherapy and radiotherapy. Given the perceived need to augment antitumor type-1 immunity (TC1 and Th1) as a therapeutic end point, and the known functional plasticity of DC populations that may display heterogeneous capacity to promote T-cell responses, we sought to identify a preferred DC preparation with this capacity. We compared 2 different preparations of monocyte-derived DC using interferon-alpha (IFN-alpha) (IFN-DC and alphaDC1) with classic DCs "matured" (mDCs) using interleukin-1beta/interleukin-6/tumor necrosis factor-alpha/prostaglandin E2, for their ability to promote autologous TC1 antitumor responses from RCC patients in vitro. IFN-alpha-conditioned DC promoted significantly higher numbers of RCC-specific CD8+ T cells exhibiting a cytotoxic phenotype after in vitro stimulation (IVS) than cytokine cocktail-mDCs. Furthermore, IVS using IFN-DCs was able to diminish regulatory-type T cells among CD4+ T-cell responder populations versus IVS using conventional mDC-based vaccines. These data emphasize an important role for IFN-alpha in modulating the immunologic functions of DCs toward a polarized DC1-type capable of coordinately promoting TH1-type and TC1-type T-cell mediated immunity and supports the translational development of patient-derived IFN-alpha-conditioned DC for use in novel immunotherapies for patients with RCC, and in whom, endogenous tumor-specific TC1 effector cells may be dysfunctional, anergic, or prone to undergo apoptosis.

Animals living in the intertidal zone experience regular, predictable fluctuations in physical parameters including temperature, oxygen and salinity and rely on behavioural, physiological and biochemical mechanisms to cope with environmental variation. In the present study, behavioural strategies induced by aquatic hypoxia (e.g. emergence) were performed at similar oxygen tensions across laboratory, mesocosm and field environments; the number of individuals performing these behaviours at any one time was similar in mesocosms and the field. The use of aquatic surface respiration (ASR) was more plastic than emergence behaviour, occurring at a lower oxygen tension in juveniles than adults and being influenced by the addition of alarm substance. Oxygen uptake was lower in air than in water in adults but, in contrast, oxygen uptake was not influenced by the respiratory medium in juveniles. In the laboratory, 72 h of forced emergence did not affect whole body concentrations of lactate but when ASR and emergence were prevented within mesocosm environments there was a significant elevation of lactate. The present study highlights the benefits of transcending traditional laboratory/field boundaries allowing the responses of laboratory-held animals to environmental fluctuation to be integrated with how these animals perform in their natural environment.

Purpose: Although T-helper (Th) epitopes have been previously reported for many tumor antigens, including MAGE-A6, the relevant HLA-DR alleles that present these peptides are expressed by only a minority of patients. The identification of tumor antigenic epitopes presented promiscuously by many HLA-DR alleles would extend the clinical utility of these peptides in vaccines and for the immunomonitoring of cancer patients., Experimental Design: A neural network algorithm and in vitro sensitization assays were employed to screen candidate peptides for their immunogenicity., Results: The MAGE-A6(140-170), MAGE-A6(172-187), and MAGE-A6(280-302) epitopes were recognized by CD4+ T cells isolated from the majority of normal donors and melanoma patients evaluated. Peptide-specific CD4+ T cells also recognized autologous antigen-presenting cell pulsed with recombinant MAGE-A6 (rMAGE) protein, supporting the natural processing and MHC presentation of these epitopes. Given the strong primary in vitro sensitization of normal donor CD4+ T cells by the MAGEA6(172-187) epitope, suggestive of potential cross-reactivity against an environmental stimulus, we identified a highly homologous peptide within the Mycoplasma penetrans HF-2 permease (MPHF2) protein. MPHF2 peptide-primed CD4+ T cells cross-reacted against autologous APC pulsed with the MAGE-A6(172-187) peptide or rMAGE protein and recognized HLA-matched MAGE-A6+ melanoma cell lines. These responses seemed heteroclitic in nature because the functional avidity of MPHF2 peptide-primed CD4+ T cells for the MAGE-A6(172-187) peptide was approximately 1,000 times greater than that of CD4+ T cells primed with the corresponding MAGE-A6 peptide., Conclusions: We believe that these novel "promiscuous" MAGE-A6/MPHF2 Th epitopes may prove clinically useful in the treatment and/or monitoring of a high proportion of cancer patients.

The goal of this study was to investigate the effects of adenosine and its stable analogue 2-chloroadenosine (CADO) on the cytotoxic activity and cytokine production by human antimelanoma specific CD8+ and CD4+ T-helper type 1 (Th1) clones. The cytotoxic activity of CD8+ T cells was inhibited by adenosine and CADO. Using Lab MAP multiplex technology, we found that adenosine inhibits production of various cytokines and chemokines by CD8+ and CD4+ T cells. Studies with CGS21680, a specific agonist of adenosine A2A receptor (AdoRA2A), and ZM241385, an AdoRA2-selective antagonist, indicate that the inhibitory effects of adenosine are mediated via cyclic AMP (cAMP)-elevating AdoRA2A, leading to protein kinase A (PKA) activation. Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKAI and PKAII, we found that activation of PKAI, but not of PKAII, mimicked the inhibitory effects of adenosine on T-cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKA inhibitor peptide 14-22) failed to abrogate the inhibitory effects of CADO. In contrast, Rp-8-Br-cAMPS that antagonizes binding of cAMP to the regulatory I subunit and PKA activation was efficient in blocking the inhibitory effect of adenosine on the functional activity of T cells. Our findings on the ability of adenosine to inhibit the effector function of antimelanoma specific T cells suggest that intratumor-produced adenosine could impair the function of tumor-infiltrating T lymphocytes. Thus, blocking the inhibitory activity of tumor-produced adenosine might represent a new strategy for improvement of cancer immunotherapy.

Executive functions are highly sensitive to the effects of aging and other conditions affecting frontal lobe function. Yet there are few validated interventions specifically designed to address executive functions, and, to our knowledge, none validated in a healthy aging sample. As part of a large-scale cognitive rehabilitation randomized trial in 49 healthy older adults, a modified Goal Management Training program was included to address the real-life deficits caused by executive dysfunction. This program emphasized periodic suspension of ongoing activity to establish goal hierarchies and monitor behavioral output. Tabletop simulated real-life tasks (SRLTs) were developed to measure the processes targeted by this intervention. Participants were randomized to two groups, one of which received the intervention immediately and the other of which was wait-listed prior to rehabilitation. Results indicated improvements in SRLT performance and self-rated executive deficits coinciding with the training in both groups. These gains were maintained at long-term follow-up. Future research will assess the specificity of these effects in patient groups.

Quantitative neuroimaging is increasingly used to study the effects of traumatic brain injury (TBI) on brain structure and function. This paper reviews quantitative structural and functional neuroimaging studies of patients with TBI, with an emphasis on the effects of diffuse axonal injury (DAI), the primary neuropathology in TBI. Quantitative structural neuroimaging has evolved from simple planometric measurements through targeted region-of-interest analyses to whole-brain analysis of quantified tissue compartments. Recent studies converge to indicate widespread volume loss of both gray and white matter in patients with moderate-to-severe TBI. These changes can be documented even when patients with focal lesions are excluded. Broadly speaking, performance on standard neuropsychological tests of speeded information processing are related to these changes, but demonstration of specific brain-behavior relationships requires more refined experimental behavioral measures. The functional consequences of these structural changes can be imaged with activation functional neuroimaging. Although this line of research is at an early stage, results indicate that TBI causes a more widely dispersed activation in frontal and posterior cortices. Further progress in analysis of the consequences of TBI on neural structure and function will require control of variability in neuropathology and behavior.

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87-111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87-111 stimulates Th1-type and Th-2/Th0-type CD4(+) T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4(+) T cells and CD4(+) T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-gamma and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4(+) T cells recognizing peptide NY-ESO-1 87-111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87-111 in the large number of cancer patients with NY-ESO-1-expressing tumors.

The NY-ESO-1 and LAGE-1 genes are expressed by many human cancers, but not by normal tissues, with the exception of testis and placenta. The NY-ESO-1 and LAGE-1 genes give rise to multiple MHC class I and class II-presented epitopes derived from the open reading frames (ORF) 1 and 2. Here, we have investigated whether NY-ESO-1/LAGE-1 ORF2 encodes promiscuous MHC class II-restricted epitopes. Using a set of overlapping peptides from the ORF2 protein sequence and autologous dendritic cells (DCs) from normal donors and melanoma patients, we have identified three HLA-DRB1*0401-restricted peptide sequences from the LAGE-1 ORF2 that are capable of stimulating T-helper 1-type melanoma-reactive CD4+ T cells. From these bulk CD4+ T cells, we have generated CD4+ T-cell clones able to recognize not only peptide-pulsed DCs but also autologous DCs loaded with the LAGE-1 ORF2 protein. We have demonstrated that these peptides not only bind to multiple HLA-DR molecules apart from HLA-DRB1*0401 but also stimulate CD4+ T cells when presented in the context of these HLA-DR molecules. Furthermore, our binding data have delineated two additional sequences capable of broadly binding to multiple HLA-DR molecules. Altogether, these data support the immunogenicity of NY-ESO-1/LAGE-1 ORF2 gene products and clearly demonstrate their capability to stimulate T-helper 1 type CD4+ T cells. Because of the role of these cells in promoting long-lasting antitumor CTL responses, our data provide a rationale for cancer vaccine trials with peptides derived from the NY-ESO-1/LAGE-1 ORF2 for a large fraction of patients with NY-ESO-1/LAGE-1(+) tumors.

Studies in the field of radiation chemistry have shown that the greatest damage induced by ionizing radiation appears as a consequence of uncontrolled production of free radicals in the living organism, which have their origin in oxidative stress induced by ionizing radiation. Ionizing radiation also has an influence on the content of bio-essential elements in mammalian tissues. Analysis of the concentration of magnesium (Mg) seven days after whole body irradiation with a single dose of 4.2 Gy of gamma rays from a 60Co source, showed a slight reduction in Mg content in the following analyzed tissues: front brain (cerebral hemispheres with underlying structures except pituitary gland and hypothalamus) and adrenal glands from irradiated animals as compared to the non-irradiated controls, this difference however being not statistically significant. No significant differences in the content of Mg were detected in the pituitary glands between the two groups of animals. In contrast, a significantly higher concentration of Mg seven days after irradiation was measured in the hind brain-containing cerebellum with lateral lobes and flocculi (p < 0.005), hypothalamus (p < 0.0001) and testes (p < 0.05) of irradiated animals, when compared to the non-irradiated controls. This accumulation of Mg, in particular tissues of the Central Nervous System (CNS) and endocrine glands regulated by the pituitary gland and hypothalamus, may indicate the differential role Mg++ ions have in keeping the homeostasis of certain tissues, those of the brain, adrenal glands and testes after exposure to ionizing radiation.

The effects of supplementation with selenous yeast and ionizing radiation on selenium (Se) content and distribution were evaluated in rat tissues (liver, kidney, spleen, heart, muscle, blood, front brain, hind brain, hypothalamus, pituitary, adrenal glands, testes, and hair). This study had 16 Se-supplemented (0.5 micrograms Se/d) and 16 placebo adult male Wistar rats. One half of the animals (eight Se-supplemented and eight placebos) were irradiated with a single dose of 4.2 Gy from a Co-60 source and sacrificed 7 d after irradiation along with nonirradiated animals and analyzed for Se content determination. The data obtained showed that selenous yeast supplementation increased Se levels in rat tissues (highest increases in hypothalamus, 161%; hind brain, 126%; spleen, 110%; and adrenal gland, 105%). Ionizing radiation induced significant changes in Se content and distribution (decrease in liver, blood, hair, femoral muscle, spleen, and hypothalamus; increase in kidney, testes, adrenal glands, and brain of placebo group). Supplementation with selenous yeast reduces changes in Se content and distribution after irradiation. It seems that the animal tissue susceptibility to oxidative damage may be correlated to their ability to retain Se in tissues.