The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the <Emphasis Type="Italic">cftr</Emphasis> knockout and heterozygous mouse

Background  

Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype.     

Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

Background

We developed a new version of the open source software package Peptrix that can yet compare large numbers of Orbitrap? LC-MS data. The peptide profiling results for Peptrix on MS1 spectra were compared with those obtained from a small selection of open source and commercial software packages: msInspect, Sieve? and Progenesis?. The properties compared in these packages were speed, total number of detected masses, redundancy of masses, reproducibility in numbers and CV of intensity, overlap of masses, and differences in peptide peak intensities. Reproducibility measurements were taken for the different MS1 software applications by measuring in triplicate a complex peptide mixture of immunoglobulin on the Orbitrap? mass spectrometer. Values of peptide masses detected from the high intensity peaks of the MS1 spectra by peptide profiling were verified with values of the MS2 fragmented and sequenced masses that resulted in protein identifications with a significant score.

Findings

Peptrix finds about the same number of peptide features as the other packages, but peptide masses are in some cases approximately 5 to 10 times less redundant present in the peptide profile matrix. The Peptrix profile matrix displays the largest overlap when comparing the number of masses in a pair between two software applications. The overlap of peptide masses between software packages of low intensity peaks in the spectra is remarkably low with about 50% of the detected masses in the individual packages. Peptrix does not differ from the other packages in detecting 96% of the masses that relate to highly abundant sequenced proteins. MS1 peak intensities vary between the applications in a non linear way as they are not processed using the same method.

Conclusions

Peptrix is capable of peptide profiling using Orbitrap? files and finding differential expressed peptides in body fluid and tissue samples. The number of peptide masses detected in Orbitrap? files can be increased by using more MS1 peptide profiling applications, including Peptrix, since it appears from the comparison of Peptrix with the other applications that all software packages have likely a high false negative rate of low intensity peptide peaks (missing peptides).     

Benefit with aromatase inhibitors in the adjuvant setting for postmenopausal women with breast cancer

The third-generation aromatase inhibitors, letrozole, anastrozole, and exemestane, have been shown to be effective both as alternatives to tamoxifen in first-line treatment of hormone-sensitive advanced breast cancer in postmenopausal women and following failure of first-line tamoxifen for endocrine therapy. These 3 agents are now being investigated as adjuvant therapy of early breast cancer, as alternative or complementary treatments to the standard, tamoxifen. Three treatment strategies are under investigation: replacement of tamoxifen as adjuvant therapy for 5 years (early adjuvant therapy), sequencing of tamoxifen before or after an aromatase inhibitor during the first 5 years (early sequential adjuvant therapy), or following 5 years of tamoxifen (extended adjuvant therapy). In the first adjuvant trial (Arimidex, Tamoxifen Alone or in Combination [ATAC]), anastrozole was significantly superior to tamoxifen in reducing risk of disease recurrence, and recently, the Breast International Group (BIG) trial BIG 1-98 demonstrated the significant superiority of letrozole over tamoxifen in improving disease-free survival. A large trial (International Collaborative Cancer Group [ICCG] trial 96) investigated sequencing of 2 to 3 years of exemestane after 2 to 3 years of tamoxifen and found that switching to exemestane was significantly superior in disease-free survival compared with continuing on tamoxifen. The Arimidex or Nolvadex (ARNO) and the small ITA (Italian Tamoxifen Arimidex) trials similarly sequenced anastrozole after tamoxifen and also found that sequencing reduced the hazard of recurrence compared with remaining on tamoxifen. Trial MA.17 evaluated extended adjuvant therapy with letrozole vs placebo following 5 years of tamoxifen. Disease-free survival was significantly improved with letrozole vs placebo, irrespective of whether patients had lymph node-positive or node-negative tumors. All 3 aromatase inhibitors were generally well tolerated. Results of these trials indicate that aromatase inhibitors provide important benefits relative to tamoxifen in each of these adjuvant treatment settings, but the optimal approach still needs to be defined. Other trials continue to investigate some of these adjuvant treatment strategies.     

Inflammatory responses to induced infectious endometritis in mares resistant or susceptible to persistent endometritis

Background

Canine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines.

Results

Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser⁴⁷³, mTOR complex 1 (mTORC1) at Ser²⁴⁴⁸, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser⁶⁵ was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines.

Conclusions

Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both in vivo and in vitro.     

Automated genome mining for natural products

Background  

Discovery of new medicinal agents from natural sources has largely been an adventitious process based on screening of plant and microbial extracts combined with bioassay-guided identification and natural product structure elucidation. Increasingly rapid and more cost-effective genome sequencing technologies coupled with advanced computational power have converged to transform this trend toward a more rational and predictive pursuit.     

Increased activation of blood neutrophils after cigarette smoking in young individuals susceptible to COPD

Background

Cigarette smoking is the most important risk factor for Chronic Obstructive Pulmonary Disease (COPD). Only a subgroup of smokers develops COPD and it is unclear why these individuals are more susceptible to the detrimental effects of cigarette smoking. The risk to develop COPD is known to be higher in individuals with familial aggregation of COPD. This study aimed to investigate if acute systemic and local immune responses to cigarette smoke differentiate between individuals susceptible or non-susceptible to develop COPD, both at young (18-40 years) and old (40-75 years) age.

Methods

All participants smoked three cigarettes in one hour. Changes in inflammatory markers in peripheral blood (at 0 and 3 hours) and in bronchial biopsies (at 0 and 24 hours) were investigated. Acute effects of smoking were analyzed within and between susceptible and non-susceptible individuals, and by multiple regression analysis.

Results

Young susceptible individuals showed significantly higher increases in the expression of FcγRII (CD32) in its active forms (A17 and A27) on neutrophils after smoking (p = 0.016 and 0.028 respectively), independently of age, smoking status and expression of the respective markers at baseline. Smoking had no significant effect on mediators in blood or inflammatory cell counts in bronchial biopsies. In the old group, acute effects of smoking were comparable between healthy controls and COPD patients.

Conclusions

We show for the first time that COPD susceptibility at young age associates with an increased systemic innate immune response to cigarette smoking. This suggests a role of systemic inflammation in the early induction phase of COPD.

Trial registration

Clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00807469","term_id":"NCT00807469"}}NCT00807469

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0121-2) contains supplementary material, which is available to authorized users.     

Peroxiredoxin I and II in Human Eyes: Cellular Distribution and Association with Pterygium and DNA Damage

Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2’-deoxyguanosine antibody with the nuclear dye 4’,6’-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Rainfall-driven sex-ratio genes in African buffalo suggested by correlations between Y-chromosomal haplotype frequencies and foetal sex ratio

Background  

The Y-chromosomal diversity in the African buffalo (Syncerus caffer) population of Kruger National Park (KNP) is characterized by rainfall-driven haplotype frequency shifts between year cohorts. Stable Y-chromosomal polymorphism is difficult to reconcile with haplotype frequency variations without assuming frequency-dependent selection or specific interactions in the population dynamics of X- and Y-chromosomal genes, since otherwise the fittest haplotype would inevitably sweep to fixation. Stable Y-chromosomal polymorphism due one of these factors only seems possible when there are Y-chromosomal distorters of an equal sex ratio, which act by negatively affecting X-gametes, or Y-chromosomal suppressors of a female-biased sex ratio. These sex-ratio (SR) genes modify (suppress) gamete transmission in their own favour at a fitness cost, allowing for stable polymorphism.     

Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer

The purpose of this study was to investigate the association between tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and prognosis in patients with primary breast cancer and to analyze whether measurement of TIMP-1 in tumor extracts added prognostic information to that obtained from measurements of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). An established sandwich enzyme-linked immunosorbent assay was thoroughly validated for the measurement of total TIMP-1 in tumor tissue extracts and used to determine levels of total TIMP-1 in 341 detergent-extracted tumor tissue samples from patients with primary breast cancer. The median age of the patients was 56 years (range, 29-75 years), and 164 were lymph node-negative, and 177 were lymph node-positive. The median follow-up time of the patients was 8.5 years (range, 7.3-11.3 years), and during follow-up 153 patients experienced recurrence of disease, and 136 patients died. In univariate survival analysis, we found a significant association between tumor tissue TIMP-1 level and both shorter recurrence-free survival (p = 0.0004) and shorter overall survival (p = 0.03). In multivariate survival analysis, higher tumor tissue TIMP-1 levels significantly and independently predicted shorter recurrence-free survival (p < 0.05, hazard ratios >1, comparing quartiles II-IV with I). In addition, we found that measurement of TIMP-1 levels added prognostic information to that obtained from measurement of PAI-1. In conclusion, high levels of TIMP-1 in tumor tissue extracts are significantly associated with a poor prognosis in patients with primary breast cancer. Furthermore TIMP-1 adds prognostic information to that obtained from PAI-1. However, further validation in independent data sets is needed.