Modulation of the Maladaptive Stress Response to Manage Diseases of Protein Folding

Diseases of protein folding arise because of the inability of an altered peptide sequence to properly engage protein homeostasis components that direct protein folding and function. To identify global principles of misfolding disease pathology we examined the impact of the local folding environment in alpha-1-antitrypsin deficiency (AATD), Niemann-Pick type C1 disease (NPC1), Alzheimer''s disease (AD), and cystic fibrosis (CF). Using distinct models, including patient-derived cell lines and primary epithelium, mouse brain tissue, and Caenorhabditis elegans, we found that chronic expression of misfolded proteins not only triggers the sustained activation of the heat shock response (HSR) pathway, but that this sustained activation is maladaptive. In diseased cells, maladaptation alters protein structure–function relationships, impacts protein folding in the cytosol, and further exacerbates the disease state. We show that down-regulation of this maladaptive stress response (MSR), through silencing of HSF1, the master regulator of the HSR, restores cellular protein folding and improves the disease phenotype. We propose that restoration of a more physiological proteostatic environment will strongly impact the management and progression of loss-of-function and gain-of-toxic-function phenotypes common in human disease.     

Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans

Caenorhabditis elegans shows chemoattraction to 0.1-200 mM NaCl, avoidance of higher NaCl concentrations, and avoidance of otherwise attractive NaCl concentrations after prolonged exposure to NaCl (gustatory plasticity). Previous studies have shown that the ASE and ASH sensory neurons primarily mediate attraction and avoidance of NaCl, respectively. Here we show that balances between at least four sensory cell types, ASE, ASI, ASH, ADF and perhaps ADL, modulate the response to NaCl. Our results suggest that two NaCl-attraction signalling pathways exist, one of which uses Ca(2+)/cGMP signalling. In addition, we provide evidence that attraction to NaCl is antagonised by G-protein signalling in the ASH neurons, which is desensitised by the G-protein-coupled receptor kinase GRK-2. Finally, the response to NaCl is modulated by G-protein signalling in the ASI and ADF neurons, a second G-protein pathway in ASH and cGMP signalling in neurons exposed to the body fluid.     

A two-mutation model of radiation-induced acute myeloid leukemia using historical mouse data

From studies of the atomic bomb survivors, it is well known that ionizing radiation causes several forms of leukemia. However, since the specific mechanism behind this process remains largely unknown, it is difficult to extrapolate carcinogenic effects at acute high-dose exposures to risk estimates for the chronic low-dose exposures that are important for radiation protection purposes. Recently, it has become clear that the induction of acute myeloid leukemia (AML) in CBA/H mice takes place through two key steps, both involving the Sfpi1 gene. A similar mechanism may play a role in human radiation-induced AML. In the present paper, a two-mutation carcinogenesis model is applied to model AML in several data sets of X-ray- and neutron-exposed CBA/H mice. The models obtained provide good fits to the data. A comparison between the predictions for neutron-induced and X-ray-induced AML yields an RBE for neutrons of approximately 3. The model used is considered to be a first step toward a model for human radiation-induced AML, which could be used to estimate risks of exposure to low doses.     

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.     

Proteomic changes in the pressure overloaded right ventricle after 6 weeks in young rats: correlations with the degree of hypertrophy

Right ventricular (RV) hypertrophy is an important problem in congenital heart disease. We determined the alterations in phenotype that occur in the initial phase of RV hypertrophy and their possible correlations with the degree of hypertrophy. Therefore, we performed a differential proteomic profiling study on RV hypertrophy using an animal model of pulmonary artery banding (PAB) in parallel with hemodynamic characterization. The RV homogenates were subfractionated in myofilament and cytoplasmic proteins, which subsequently were separated by two-dimensional gel electrophoresis (2-DE), excised, and analyzed by mass spectrometry (MS). The cytoplasmic fraction showed expression changes in metabolic proteins, indicative of a shift from fatty acid to glucose as a substrate for energy supply. Up-regulation of three HSP-27s (1.9-, 1.7-, and 3.5-fold) indicated an altered stress response in RV hypertrophy. Detailed analysis by immunoblotting and MS showed that two of these HSP-27s were at least phosphorylated on Ser15. The myofilament fraction showed up-regulation of desmin and alpha-B-crystallin (1.4-and 1.3-fold, respectively). This alteration in desmin was confirmed by 1-DE immunoblots. Certain differentially expressed proteins, such as HSP-27, showed a significant correlation with the RV weight to the body weight ratio in the PAB rats, suggesting an association with the degree of hypertrophy.     

A gene frequency model for QTL mapping using Bayesian inference

Background

Information for mapping of quantitative trait loci (QTL) comes from two sources: linkage disequilibrium (non-random association of allele states) and cosegregation (non-random association of allele origin). Information from LD can be captured by modeling conditional means and variances at the QTL given marker information. Similarly, information from cosegregation can be captured by modeling conditional covariances. Here, we consider a Bayesian model based on gene frequency (BGF) where both conditional means and variances are modeled as a function of the conditional gene frequencies at the QTL. The parameters in this model include these gene frequencies, additive effect of the QTL, its location, and the residual variance. Bayesian methodology was used to estimate these parameters. The priors used were: logit-normal for gene frequencies, normal for the additive effect, uniform for location, and inverse chi-square for the residual variance. Computer simulation was used to compare the power to detect and accuracy to map QTL by this method with those from least squares analysis using a regression model (LSR).

Results

To simplify the analysis, data from unrelated individuals in a purebred population were simulated, where only LD information contributes to map the QTL. LD was simulated in a chromosomal segment of 1 cM with one QTL by random mating in a population of size 500 for 1000 generations and in a population of size 100 for 50 generations. The comparison was studied under a range of conditions, which included SNP density of 0.1, 0.05 or 0.02 cM, sample size of 500 or 1000, and phenotypic variance explained by QTL of 2 or 5%. Both 1 and 2-SNP models were considered. Power to detect the QTL for the BGF, ranged from 0.4 to 0.99, and close or equal to the power of the regression using least squares (LSR). Precision to map QTL position of BGF, quantified by the mean absolute error, ranged from 0.11 to 0.21 cM for BGF, and was better than the precision of LSR, which ranged from 0.12 to 0.25 cM.

Conclusions

In conclusion given a high SNP density, the gene frequency model can be used to map QTL with considerable accuracy even within a 1 cM region.     

Integrating comparative expression profiling data and association of SNPs with Salmonella shedding for improved food safety and porcine disease resistance

Salmonella in swine is a major food safety problem, as the majority of US swine herds are Salmonella-positive. Salmonella can be shed from colonized swine and contaminate (i) neighbouring pigs; (ii) slaughter plants and pork products; (iii) edible crops when swine manure is used as a fertilizer; and (iv) water supplies if manure used as crop fertilizer runs off into streams and waterways. A potentially powerful method of addressing pre-harvest food safety at the farm level is through genetic improvement of disease resistance in animals. In this research, we describe a successful strategy for discovering genetic variation at candidate genes associated with disease resistance in pigs. This involves integrating our recent global gene expression analysis of the porcine response to Salmonella with information from the literature about important candidate genes. We identified single-nucleotide polymorphisms (SNPs) in these functional candidate genes and genotyped three independent pig populations that had data on Salmonella faecal shedding or internal burden (total n = 377) at these loci. Of 31 SNPs genotyped, 21 SNPs segregated in at least two populations with a minor allele frequency of 15% or greater. Statistical analysis revealed thirteen SNPs associated with Salmonella faecal shedding or tissue colonization, with an estimated proportion of false positives (PFP) ≤0.2. The genes with associated SNPs included GNG3, NCF2, TAP1, VCL, AMT, CCR1, CD163, CCT7, EMP1 and ACP2. These associations provide new information about the mechanisms of porcine host response to Salmonella and may be useful in improving genetic resistance to this bacterium.     

Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison

Fifty-two strains from eight species of Fusarium were analyzed by rapid rRNA sequencing. Two highly variable stretches (138 and 214 nucleotides) of the 5' end of the 28S-like rRNA molecule were sequenced. Such stretches permit evaluation of the divergence between closely related species and even between varieties within a species. The phylogenetic tree computed from the number of nucleotide differences shows seven Fusarium species to be more closely related to one another than the eighth species, F. nivale, is to them. On the basis of these data, we discuss both the phylogenetic value of taxonomical criteria and the impact of our findings on the demarcation of the genus Fusarium. We conclude that this method is suitable for establishing a precise phylogeny between closely related species within a genus.