THE FUNCTION OF HEAT-SHOCK PROTEINS IN STRESS TOLERANCE

We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid-resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance. Our experiments show that clone 2 cells were not protected from the killing effect of heat by pretreatment with sodium arsenite, whereas Faza 967 cells were. These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells. Stable heat-resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat-inducibility of hsp68. These results suggest that several hsps are needed to protect the critical biological processes at high temperature. The heat-resistant hepatoma cells also became resistant to several anticancer drugs. The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P-glycoprotein. Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P-gp may participate in protection against environmental stress such as heat.     

The histone-like C-terminal extension in ribosomal protein S6 in Aedes and Anopheles mosquitoes is encoded within the distal portion of exon 3

In eukaryotic cells, ribosomal protein S6 (RPS6) is the major phosphorylated protein on the small ribosomal subunit. In the mosquitoes Aedes aegypti and Aedes albopictus, the cDNA encoding RPS6 contains 300 additional nucleotides, relative to the Drosophila homolog. The additional sequence encodes a 100-amino acid, lysine-rich C-terminal extension of the RPS6 protein with 42-49% identity to histone H1 proteins from the chicken and other multicellular organisms. Using mass spectrometry we now show that the C-terminal extension predicted by the cDNA is present on RPS6 protein isolated from ribosomal subunits purified from Ae. albopictus cells. To expand our analysis beyond the genus Aedes, we cloned the rpS6 cDNA from an Anopheles stephensi mosquito cell line. The cDNA also encoded a lysine-rich C-terminal extension. However, in An. stephensi rpS6 the extension was approximately 70 amino acids longer than that in Ae. albopictus, and at the nucleotide level, it most closely resembled histone H1 proteins from the unicellular eukaryotes Leishmania and Chlamydomonas, and the bacterium Bordetella pertussis. To examine how the histone-like C-terminal extension is encoded in the genome, we used PCR-based approaches to obtain the genomic DNA sequence encoding Ae. aegypti and Ae. albopictus rpS6. The sequence encoding the histone-like C-terminal extension was contiguous with upstream coding sequence within a single open reading frame in Exon 3, indicating that the lysine-rich extension in mosquito RPS6 is not the result of an aberrant splicing event. An in silico investigation of the Anopheles gambiae genome based on the cDNA sequence from An. stephensi allowed us to map the An. gambiae gene to chromosome 2R, to deduce its exon-intron organization, and to confirm that Exon 3 encodes a C-terminal histone-like extension. Because the C-terminal extension is absent from Drosophila melanogaster, we examined a partial cDNA clone from a Psychodid fly, which shares a relatively recent common ancestor with the mosquitoes. The absence of the C-terminal extension in the Psychodid rpS6 cDNA suggests that the unusual RPS6 structure is restricted to a relatively small group of flies in the Nematocera.     

Adaptation of sympatric Achromatium spp. to different redox conditions as a mechanism for coexistence of functionally similar sulphur bacteria

Changes in the abundance of sympatric Achromatium spp. in response to the artificial manipulation of redox conditions in sediment microcosms was determined by fluorescence in situ hybridization (FISH). Adaptation to different redox conditions was shown to be one mechanism that supported the coexistence of functionally similar Achromatium spp. In sediment microcosms, in which the overlying water was oxygenated, Achromatium community size and composition remained unchanged over time. However, imposition of anoxic conditions induced changes in community structure. Anoxia caused a reduction in the relative abundance of Achromatium sp. RY8 (72 +/- 4% to 49 +/- 2%) and an increase in Achromatium sp. RY5 (19 +/- 5% to 32 +/- 3%) and a newly identified Achromatium sp., RYKS (14 +/- 4% to 27 +/- 2%). In anoxic microcosms supplemented with a single addition of nitrate at different initial concentrations the relative decline in Achromatium sp. RY8 was dependent on the initial nitrate concentration. In these experiments nitrate was rapidly removed. In contrast, when high levels of nitrate were maintained by periodic replacement of the overlying water with nitrate supplemented anoxic water, the composition of the Achromatium community remained stable over time. This suggested that all of the coexisting Achromatium spp. are obligate or facultative anaerobes, but, Achromatium sp. RY8 was more sensitive to sediment redox conditions than the other Achromatium species. Given the heterogeneous nature of sedimentary environments, redox-related niche differentiation may promote coexistence of sympatric Achromatium spp.     

Individual differences in the effects of mobile phone exposure on human sleep: rethinking the problem

Mobile phone exposure‐related effects on the human electroencephalogram (EEG) have been shown during both waking and sleep states, albeit with slight differences in the frequency affected. This discrepancy, combined with studies that failed to find effects, has led many to conclude that no consistent effects exist. We hypothesised that these differences might partly be due to individual variability in response, and that mobile phone emissions may in fact have large but differential effects on human brain activity. Twenty volunteers from our previous study underwent an adaptation night followed by two experimental nights in which they were randomly exposed to two conditions (Active and Sham), followed by a full‐night sleep episode. The EEG spectral power was increased in the sleep spindle frequency range in the first 30 min of non‐rapid eye movement (non‐REM) sleep following Active exposure. This increase was more prominent in the participants that showed an increase in the original study. These results confirm previous findings of mobile phone‐like emissions affecting the EEG during non‐REM sleep. Importantly, this low‐level effect was also shown to be sensitive to individual variability. Furthermore, this indicates that previous negative results are not strong evidence for a lack of an effect and, given the far‐reaching implications of mobile phone research, we may need to rethink the interpretation of results and the manner in which research is conducted in this field. Bioelectromagnetics 33:86–93, 2012. © 2011 Wiley Periodicals, Inc.     

MassSieve: Panning MS/MS peptide data for proteins

We present MassSieve, a Java‐based platform for visualization and parsimony analysis of single and comparative LC‐MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC‐MS/MS‐based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments.     

Real-time NMR kinetic studies provide global and residue-specific information on the non-cooperative unfolding of the beta-trefoil protein,interleukin-1beta

The interleukin-1beta (IL-1beta) structural motif is a beta-trefoil super fold created by six two-stranded beta-hairpins. Turns are thus particularly important in creating the topology and the arrangement of beta-strands in this structural motif. In contrast to the signals observed in optical studies, real-time NMR kinetic investigations of the denaturant-induced unfolding of interleukin-1beta provide direct, global, and residue-specific information on the structural nature of the unfolding reaction. Heterogeneity in the individual amino acid residue kinetics reveals a rugged unfolding landscape. The relative kinetic stability of native-like turns supports low temperature molecular dynamics predictions of turn-controlled unfolding.     

Projecting terrestrial biodiversity intactness with GLOBIO 4

Scenario‐based biodiversity modelling is a powerful approach to evaluate how possible future socio‐economic developments may affect biodiversity. Here, we evaluated the changes in terrestrial biodiversity intactness, expressed by the mean species abundance (MSA) metric, resulting from three of the shared socio‐economic pathways (SSPs) combined with different levels of climate change (according to representative concentration pathways [RCPs]): a future oriented towards sustainability (SSP1xRCP2.6), a future determined by a politically divided world (SSP3xRCP6.0) and a future with continued global dependency on fossil fuels (SSP5xRCP8.5). To this end, we first updated the GLOBIO model, which now runs at a spatial resolution of 10 arc‐seconds (~300 m), contains new modules for downscaling land use and for quantifying impacts of hunting in the tropics, and updated modules to quantify impacts of climate change, land use, habitat fragmentation and nitrogen pollution. We then used the updated model to project terrestrial biodiversity intactness from 2015 to 2050 as a function of land use and climate changes corresponding with the selected scenarios. We estimated a global area‐weighted mean MSA of 0.56 for 2015. Biodiversity intactness declined in all three scenarios, yet the decline was smaller in the sustainability scenario (?0.02) than the regional rivalry and fossil‐fuelled development scenarios (?0.06 and ?0.05 respectively). We further found considerable variation in projected biodiversity change among different world regions, with large future losses particularly for sub‐Saharan Africa. In some scenario‐region combinations, we projected future biodiversity recovery due to reduced demands for agricultural land, yet this recovery was counteracted by increased impacts of other pressures (notably climate change and road disturbance). Effective measures to halt or reverse the decline of terrestrial biodiversity should not only reduce land demand (e.g. by increasing agricultural productivity and dietary changes) but also focus on reducing or mitigating the impacts of other pressures.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Brief cognitive behavioral therapy in primary care: a hybrid type 2 patient-randomized effectivenessimplementation design

ABSTRACT: BACKGROUND: Despite the availability of evidence-based psychotherapies for depression and anxiety, they are underused in non-mental health specialty settings such as primary care. Hybrid effectiveness-implementation designs have the potential to evaluate clinical and implementation outcomes of evidence-based psychotherapies to improve their translation into routine clinical care practices. METHODS: This protocol article discusses the study methodology and implementation strategies employed in an ongoing, hybrid, type 2 randomized controlled trial with two primary aims: (1) to determine whether a brief, manualized cognitive behavioral therapy administered by Veterans Affairs Primary Care Mental Health Integration program clinicians is effective in treating depression and anxiety in a sample of medically ill (chronic cardiopulmonary diseases) primary care patients and (2) to examine the acceptability, feasibility, and preliminary outcomes of a focused implementation strategy on improving adoption and fidelity of brief cognitive behavioral therapy at two Primary Care-Mental Health Integration clinics. The study uses a hybrid type 2 effectiveness/implementation design to simultaneously test clinical effectiveness and to collect pilot data on a multifaceted implementation strategy that includes an online training program, audit and feedback of session content, and internal and external facilitation. Additionally, the study engages the participation of an advisory council consisting of stakeholders from Primary Care-Mental Health Integration, as well as regional and national mental health leaders within the Veterans Administration. It targets recruitment of 320 participants randomized to brief cognitive behavioral therapy (n = 200) or usual care (n = 120). Both effectiveness and implementation outcomes are being assessed using mixed methods, including quantitative evaluation (e.g., intent-to-treat analyses across multiple time points) and qualitative methods (e.g., focus interviews and surveys from patients and providers). Patient-effectiveness outcomes include measures of depression, anxiety, and physical health functioning using blinded independent evaluators. Implementation outcomes include patient engagement and adherence and clinician brief cognitive behavioral therapy adoption and fidelity. CONCLUSIONS: Hybrid designs are needed to advance clinical effectiveness and implementation knowledge to improve healthcare practices. The current article describes the rationale and challenges associated with the use of a hybrid design for the study of brief cognitive behavioral therapy in primary care. Although trade-offs exist between scientific control and external validity, hybrid designs are part of an emerging approach that has the potential to rapidly advance both science and practice. Trial registration NCT01149772 at http://www.clinicaltrials.gov/ct2/show/NCT01149772.     

Survival and Reproduction of Myxobolus cerebralis-Resistant Rainbow Trout Introduced to the Colorado River and Increased Resistance of Age-0 Progeny

Myxobolus cerebralis caused severe declines in rainbow trout populations across Colorado following its introduction in the 1980s. One promising approach for the recovery of Colorado’s rainbow trout populations has been the production of rainbow trout that are genetically resistant to the parasite. We introduced one of these resistant crosses, known as the GR×CRR (cross between the German Rainbow [GR] and Colorado River Rainbow [CRR] trout strains), to the upper Colorado River. The abundance, survival, and growth of the stocked GR×CRR population was examined to determine if GR×CRRs had contributed offspring to the age-0 population, and determine whether these offspring displayed increased resistance and survival characteristics compared to their wild CRR counterparts. Apparent survival of the introduced GR×CRR over the entire study period was estimated to be 0.007 (±0.001). Despite low survival of the GR×CRRs, age-0 progeny of the GR×CRR were encountered in years 2008 through 2011. Genetic assignments revealed a shift in the genetic composition of the rainbow trout fry population over time, with CRR fish comprising the entirety of the fry population in 2007, and GR-cross fish comprising nearly 80% of the fry population in 2011. A decrease in average infection severity (myxospores fish⁻¹) was observed concurrent with the shift in the genetic composition of the rainbow trout fry population, decreasing from an average of 47,708 (±8,950) myxospores fish⁻¹ in 2009 to 2,672 (±4,379) myxospores fish⁻¹ in 2011. Results from this experiment suggest that the GR×CRR can survive and reproduce in rivers with a high prevalence of M. cerebralis. In addition, reduced myxospore burdens in age-0 fish indicated that stocking this cross may ultimately lead to an overall reduction in infection prevalence and severity in the salmonid populations of the upper Colorado River.