Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.     

An in vitro investigation of the acetabular labral seal in hip joint mechanics

Labrum pathology may contribute to early joint degeneration through the alteration of load transfer between, and the stresses within, the cartilage layers of the hip. We hypothesize that the labrum seals the hip joint, creating a hydrostatic fluid pressure in the intra-articular space, and limiting the rate of cartilage layer consolidation. The overall cartilage creep consolidation of six human hip joints was measured during the application of a constant load of 0.75 times bodyweight, or a cyclic sinusoidal load of 0.75+/-0.25 times bodyweight, before and after total labrum resection. The fluid pressure within the acetabular was measured. Following labrum resection, the initial consolidation rate was 22% greater (p=0.02) and the final consolidation displacement was 21% greater (p=0.02). There was no significant difference in the final consolidation rate. Loading type (constant vs. cyclic) had no significant effect on the measured consolidation behaviour. Fluid pressurisation was observed in three of the six hips. The average pressures measured were: for constant loading, 541+/-61kPa in the intact joint and 216+/-165kPa following labrum resection, for cyclic loading, 550+/-56kPa in the intact joint and 195+/-145kPa following labrum resection. The trends observed in this experiment support the predictions of previous finite element analyses. Hydrostatic fluid pressurisation within the intra-articular space is greater with the labrum than without, which may enhance joint lubrication. Cartilage consolidation is quicker without the labrum than with, as the labrum adds an extra resistance to the flow path for interstitial fluid expression. However, both sealing mechanisms are dependent on the fit of the labrum against the femoral head.     

A new approach for the joint analysis of multiple ChIP-seq libraries with application to histone modification

Most approaches for analyzing ChIP-Seq data are focused on inferring exact protein binding sites from a single library. However, frequently multiple ChIP-Seq libraries derived from differing cell lines or tissue types from the same individual may be available. In such a situation, a separate analysis for each tissue or cell line may be inefficient. Here, we describe a novel method to analyze such data that intelligently uses the joint information from multiple related ChIP-Seq libraries. We present our method as a two-stage procedure. First, separate single cell line analysis is performed for each cell line. Here, we use a novel mixture regression approach to infer the subset of genes that are most likely to be involved in protein binding in each cell line. In the second step, we combine the separate single cell line analyses using an Empirical Bayes algorithm that implicitly incorporates inter-cell line correlation. We demonstrate the usefulness of our method using both simulated data, as well as real H3K4me3 and H3K27me3 histone methylation libraries.     

A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation,Motility and Invasion

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl⁻ mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.     

Serum selenium and single-nucleotide polymorphisms in genes for selenoproteins: relationship to markers of oxidative stress in men from Auckland, New Zealand

There is controversy as to the recommended daily intake of selenium (Se), and whether current New Zealand diets are adequate in this nutrient. Various functional single-nucleotide polymorphisms (SNPs) polymorphisms may affect the efficacy of Se utilisation. These include the glutathione peroxidases GPx1 rs1050450, GPx4 rs713041, as well as selenoproteins SEPP1 rs3877899, SEL15 rs5845, SELS rs28665122 and SELS rs4965373. This cross-sectional study measured serum Se levels of 503 healthy Caucasian men in Auckland, New Zealand, between ages 20–81. The Se distribution was compared with activities of the antioxidant enzymes glutathione peroxidase and thioredoxin reductase, and DNA damage as measured by the single cell gel electrophoresis assay, both without and with a peroxide-induced oxidative challenge. Serum Se was measured using inductively coupled plasma-dynamic reaction cell-mass spectrometry, while selenoprotein SNPs were estimated using TaqMan^® SNP genotyping assays. While antioxidant enzyme activities and DNA damage recorded after a peroxide challenge increased with increasing serum selenium, the inherent DNA damage levels in leukocytes showed no statistically significant relationship with serum selenium. However, these relationships and dietary Se requirements at the individual level were modified by several different SNPs in genes for selenoproteins. The GPx1 rs1050450 C allele was significantly associated with GPx activity. Significant correlations between serum Se level and GPX activity were seen with all genotypes except for homozygous minor allele carriers, while the GPx1 rs1050450 CT genotype showed the highest correlation. Several genotypes showed significant correlations between serum Se and TR activity with SEPP1 rs3877899 GG genotype showing the highest correlation. A significant decreasing trend in DNA damage with increasing serum Se was seen among GPx1 rs1050450 CC and GPx4 rs713041 TT genotype carriers up to a serum Se level of 116 and 149 ng/ml, respectively. In the absence of this genetic information, we would recommend a serum Se concentration in the region of 100–150 ng/ml as providing a useful compromise.     

Oligo- and Polymetastatic Progression in Lung Metastasis(es) Patients Is Associated with Specific MicroRNAs

Rationale

Strategies to stage and treat cancer rely on a presumption of either localized or widespread metastatic disease. An intermediate state of metastasis termed oligometastasis(es) characterized by limited progression has been proposed. Oligometastases are amenable to treatment by surgical resection or radiotherapy.

Methods

We analyzed microRNA expression patterns from lung metastasis samples of patients with ≤5 initial metastases resected with curative intent.

Results

Patients were stratified into subgroups based on their rate of metastatic progression. We prioritized microRNAs between patients with the highest and lowest rates of recurrence. We designated these as high rate of progression (HRP) and low rate of progression (LRP); the latter group included patients with no recurrences. The prioritized microRNAs distinguished HRP from LRP and were associated with rate of metastatic progression and survival in an independent validation dataset.

Conclusion

Oligo- and poly- metastasis are distinct entities at the clinical and molecular level.     

Docosahexaenoic Acid Supplementation Does Not Improve Western Diet-Induced Cardiomyopathy in Rats

Obesity increases risk for cardiomyopathy in the absence of hypertension, diabetes or ischemia. The fatty acid milieu, modulated by diet, may modify myocardial structure and function, lending partial explanation for the array of cardiomyopathic phenotypy. We sought to identify gross, cellular and ultrastructural myocardial changes associated with Western diet intake, and subsequent modification with docosahexaenoic acid (DHA) supplementation. Wistar and Sprague-Dawley (SD) rats received 1 of 3 diets: control (CON); Western (WES); Western + DHA (WES+DHA). After 12 weeks of treatment, echocardiography was performed and myocardial adiponectin, fatty acids, collagen, area occupied by lipid and myocytes, and ultrastructure were determined. Strain effects included higher serum adiponectin in Wistar rats, and differences in myocardial fatty acid composition. Diet effects were evident in that both WES and WES+DHA feeding were associated with similarly increased left ventricular (LV) diastolic cranial wall thickness (LVW_cr/d) and decreased diastolic internal diameter (LVID_d), compared to CON. Unexpectedly, WES+DHA feeding was associated additionally with increased thickness of the LV cranial wall during systole (LVW_cr/s) and the caudal wall during diastole (LVW_ca/d) compared to CON; this was observed concomitantly with increased serum and myocardial adiponectin. Diastolic dysfunction was present in WES+DHA rats compared to both WES and CON. Myocyte cross sectional area (CSA) was greater in WES compared to CON rats. In both fat-fed groups, transmission electron microscopy (TEM) revealed myofibril degeneration, disorganized mitochondrial cristae, lipid inclusions and vacuolation. In the absence of hypertension and whole body insulin resistance, WES+DHA intake was associated with more global LV thickening and with diastolic dysfunction, compared to WES feeding alone. Myocyte hypertrophy, possibly related to subcellular injury, is an early change that may contribute to gross hypertrophy. Strain differences in adipokines and myocardial fatty acid accretion may underlie heterogeneous data from rodent studies.     

Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an "inflammatory" monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.     

Contemporary and historical evolutionary processes interact to shape patterns of within-lake phenotypic divergences in polyphenic pumpkinseed sunfish, Lepomis gibbosus

Historical and contemporary evolutionary processes can both contribute to patterns of phenotypic variation among populations of a species. Recent studies are revealing how interactions between historical and contemporary processes better explain observed patterns of phenotypic divergence than either process alone. Here, we investigate the roles of evolutionary history and adaptation to current environmental conditions in structuring phenotypic variation among polyphenic populations of sunfish inhabiting 12 postglacial lakes in eastern North America. The pumpkinseed sunfish polyphenism includes sympatric ecomorphs specialized for littoral or pelagic lake habitats. First, we use population genetic methods to test the evolutionary independence of within-lake phenotypic divergences of ecomorphs and to describe patterns of genetic structure among lake populations that clustered into three geographical groupings. We then used multivariate analysis of covariance (MANCOVA) to partition body shape variation (quantified with geometric morphometrics) among the effects of evolutionary history (reflecting phenotypic variation among genetic clusters), the shared phenotypic response of all populations to alternate habitats within lakes (reflecting adaptation to contemporary conditions), and unique phenotypic responses to habitats within lakes nested within genetic clusters. All effects had a significant influence on body form, but the effects of history and the interaction between history and contemporary habitat were larger than contemporary processes in structuring phenotypic variation. This highlights how divergence can be better understood against a known backdrop of evolutionary history.