Montane refugia predict population genetic structure in the Large‐blotched Ensatina salamander

Understanding the biotic consequences of Pleistocene range shifts and fragmentation remains a fundamental goal in historical biogeography and evolutionary biology. Here, we combine species distribution models (SDM) from the present and two late Quaternary time periods with multilocus genetic data (mitochondrial DNA and microsatellites) to evaluate the effect of climate‐induced habitat shifts on population genetic structure in the Large‐blotched Ensatina (Ensatina eschscholtzii klauberi), a plethodontid salamander endemic to middle and high‐elevation conifer forest in the Transverse and Peninsular Ranges of southern California and northern Baja California. A composite SDM representing the range through time predicts two disjunct refugia, one in southern California encompassing the core of the species range and the other in the Sierra San Pedro Mártir of northern Baja California at the southern limit of the species range. Based on our spatial model, we would expect a pattern of high connectivity among populations within the northern refugium and, conversely, a pattern of isolation due to long‐term persistence of the Sierra San Pedro Mártir population. Our genetic results are consistent with these predictions based on the hypothetical refugia in that (i) historical measures of population connectivity among stable areas are correlated with gene flow estimates; and (ii) there is strong geographical structure between separate refugia. These results provide evidence for the role of recent climatic change in shaping patterns of population persistence and connectivity within the Transverse and Peninsular Ranges, an evolutionary hotspot.     

Effects of raising muscle glycogen synthesis rate on skeletal muscle ATP turnover rate in type 2 diabetes

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. We hypothesized that any impairment in insulin-stimulated muscle ATP production could merely reflect the lower rates of muscle glucose uptake and glycogen synthesis, rather than cause it. If this is correct, muscle ATP turnover rates in type 2 diabetes could be increased if glycogen synthesis rates were normalized by the mass-action effect of hyperglycemia. Isoglycemic- and hyperglycemic-hyperinsulinemic clamps were performed on type 2 diabetic subjects and matched controls, with muscle ATP turnover and glycogen synthesis rates measured using (31)P- and (13)C-magnetic resonance spectroscopy, respectively. In diabetic subjects, hyperglycemia increased muscle glycogen synthesis rates to the level observed in controls at isoglycemia [from 19 ± 9 to 41 ± 12 μmol·l(-1)·min(-1) (P = 0.012) vs. 40 ± 7 μmol·l(-1)·min(-1) in controls]. This was accompanied by a modest increase in muscle ATP turnover rates (7.1 ± 0.5 vs. 8.6 ± 0.7 μmol·l(-1)·min(-1), P = 0.04). In controls, hyperglycemia brought about a 2.5-fold increase in glycogen synthesis rates (100 ± 24 vs. 40 ± 7 μmol·l(-1)·min(-1), P = 0.028) and a 23% increase in ATP turnover rates (8.1 ± 0.9 vs. 10.0 ± 0.9 μmol·l(-1)·min(-1), P = 0.025) from basal state. Muscle ATP turnover rates correlated positively with glycogen synthesis rates (r(s) = 0.46, P = 0.005). Changing the rate of muscle glucose metabolism in type 2 diabetic subjects alters demand for ATP synthesis at rest. In type 2 diabetes, skeletal muscle ATP turnover rates reflect the rate of glucose uptake and glycogen synthesis, rather than any primary mitochondrial defect.     

Mechanical stretch induces epithelial-mesenchymal transition in alveolar epithelia via hyaluronan activation of innate immunity

Epithelial injury is a central event in the pathogenesis of many inflammatory and fibrotic lung diseases like acute respiratory distress syndrome, pulmonary fibrosis, and iatrogenic lung injury. Mechanical stress is an often underappreciated contributor to lung epithelial injury. Following injury, differentiated epithelia can assume a myofibroblast phenotype in a process termed epithelial to mesenchymal transition (EMT), which contributes to aberrant wound healing and fibrosis. We demonstrate that cyclic mechanical stretch induces EMT in alveolar type II epithelial cells, associated with increased expression of low molecular mass hyaluronan (sHA). We show that sHA is sufficient for induction of EMT in statically cultured alveolar type II epithelial cells and necessary for EMT during cell stretch. Furthermore, stretch-induced EMT requires the innate immune adaptor molecule MyD88. We examined the Wnt/β-catenin pathway, which is known to mediate EMT. The Wnt target gene Wnt-inducible signaling protein 1 (wisp-1) is significantly up-regulated in stretched cells in hyaluronan- and MyD88-dependent fashion, and blockade of WISP-1 prevents EMT in stretched cells. In conclusion, we show for the first time that innate immunity transduces mechanical stress responses through the matrix component hyaluronan, and activation of the Wnt/β-catenin pathway.     

Ozone inhalation promotes CX3CR1-dependent maturation of resident lung macrophages that limit oxidative stress and inflammation

Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that subpopulations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3 h) or filtered air. At 24 h after exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone-exposed mice, which were distinct from resident alveolar macrophages and identified by enhanced Gr-1(+) expression [Gr-1 macrophages (Gr-1 Macs)]. Further analysis showed that Gr-1(+) Macs exhibited high expression of MARCO, CX3CR1, and NAD(P)H:quinone oxioreductase 1. Gr-1(+) Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness, exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1β, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, are dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.     

Preliminary study of genetic variation in Hawaiian isolates of Beauveria bassiana [Hypocreales, Cordycipitaceae

There have been no previous surveys documenting genetic diversity in Beauveria bassiana (Balsamo) Vuillemin in Hawaii. We used PCR primers and DNA sequencing to genetically characterize 14 isolates of B. bassiana collected from insects in east Hawaii island (the largest Hawaiian island, known as the ‘Big Island’) and compared these with the ‘GHA’ strain found in the commercial product BotaniGard®. Twelve of the 14 Hawaiian isolates were unique and the GHA strain was not among those isolated from the wild. Our data provides evidence that genetic diversity of B. bassiana in Hawaii is high over small spatial scales.     

Ambient ozone primes pulmonary innate immunity in mice

Exposure to ozone in air pollution in urban environments is associated with increases in pulmonary-related hospitalizations and mortality. Because ozone also alters clearance of pulmonary bacterial pathogens, we hypothesized that inhalation of ozone modifies innate immunity in the lung. To address our hypothesis, we exposed C57BL/6J mice to either free air or ozone, and then subsequently challenged with an aerosol of Escherichia coli LPS. Pre-exposure to ozone resulted in enhanced airway hyperreactivity, higher concentrations of both total protein and proinflammatory cytokines in lung lavage fluid, enhanced LPS-mediated signaling in lung tissue, and higher concentrations of serum IL-6 following inhalation of LPS. However, pre-exposure to ozone dramatically reduced inflammatory cell accumulation to the lower airways in response to inhaled LPS. The reduced concentration of cells in the lower airways was associated with enhanced apoptosis of both lung macrophages and systemic circulating monocytes. Moreover, both flow cytometry and confocal microscopy indicate that inhaled ozone causes altered distribution of TLR4 on alveolar macrophages and enhanced functional response to endotoxin by macrophages. These observations indicate that ozone exposure increases both the pulmonary and the systemic biologic response to inhaled LPS by priming the innate immune system.     

Genetic basis of murine antibacterial defense to streptococcal lung infection

To evaluate the effect of genetic background on antibacterial defense to streptococcal infection, eight genetically diverse strains of mice (A/J, DBA/2J, CAST/Ei, FVB/NJ, BALB/cJ, C57BL/6J, 129/SvImJ, and C3H/HeJ) and tlr2-deficient mice (C57BL/6^tlr2−/−) were infected with three doses of Streptococcus zooepidemicus (500, 5,000, or 50,000 colony-forming units) by alveolar challenge. There was a range of susceptibility between the strains at each dose and time point (6, 24, and 96 h). At the lowest dose, the 129/SvImJ and C3H/HeJ strains had significantly higher bacterial counts at all time points after infection, when compared to A/J, DBA/2J, CAST/Ei, FVB/NJ, which were resistant to infection at the low dose of innoculum. At the medium dose, 129/SvImJ and C3H/HeJ had higher bacterial counts, while A/J, DBA/2J, and BALB/cJ showed reduced streptococcal growth. After the highest dose of Streptococcus, there were minimal differences between strains, suggesting the protective impact of modifier genes can be overcome. TLR2-deficient animals contained increased bacterial load with reduced cytokines after 96 h when compared to C57BL/6J controls suggesting a role of innate immunity in late antibacterial defense. Overall, we identify vulnerable (129/SvlmJ and C3H/HeJ) and resistant (A/J, FVB, and DBA) mouse strains to streptococcal lung infection, which demonstrate divergent genetic expression profiles. These results demonstrate that innate differences in pulmonary host defense to S. zooepidemicus are dependent on host genetic factors.     

Spatiotemporal Proteomic Analyses during Pancreas Cancer Progression Identifies Serine/Threonine Stress Kinase 4 (STK4) as a Novel Candidate Biomarker for Early Stage Disease

Pancreas cancer, or pancreatic ductal adenocarcinoma, is the deadliest of solid tumors, with a five-year survival rate of <5%. Detection of resectable disease improves survival rates, but access to tissue and other biospecimens that could be used to develop early detection markers is confounded by the insidious nature of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages in which surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing ∼2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage metastatic disease, respectively. A panel of seven up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated, and their levels were assessed in tissues collected at preinvasive, early invasive, and moribund stages of disease. Six of the seven markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker, and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma. Immunohistochemistry of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. Immunohistochemistry of a panel of human pancreas cancers confirmed that STK4 levels were increased in tumor epithelia relative to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage, including early (resectable), and therefore clinically meaningful, stages.In 2013 the American Cancer Society estimated there were 45,220 new cases of cancer of the pancreas, the most common form of which is pancreatic ductal adenocarcinoma (PDA),¹ with an estimated 38,430 deaths (1). The incidence of PDA increased on average by 1.5% per year from 2004 to 2008, a trend that is expected to continue. Because of the frequently asymptomatic character of PDA at early stages, diagnosis generally occurs only after locally advanced or metastatic spread, and the majority of patients therefore present with unresectable disease. This fact, combined with the notable resistance of PDA to chemotherapy, usually yields a very poor prognosis, with less than 20% survival past 12 months after diagnosis (2).Surgical resection with curative intent improves five-year survival rates from less than 5% to ∼20% (3), yet resection is possible in only 10% to 15% of patients because of the low probability of identifying resectable disease in asymptomatic patients (4). Accurate diagnosis of PDA can be further confounded by difficulties in distinguishing it from chronic pancreatitis (CP) and the associated fibrosis; conversely, pancreatitis can also be initiated by a tumor causing ductal obstruction (5). A non-invasive blood-based test would be ideal for early detection and diagnosis, but no validated markers currently exist with sufficient sensitivity and specificity to improve clinical decision making. Early stage tissue markers could complement current imaging (MRI, computed tomography, endoscopic ultrasound) and CA19.9 plasma screening modalities (6) and improve the identification of resectable disease, as well as provide further insight into pancreas cancer etiology.Mouse models that clinically, histologically, and molecularly recapitulate human PDA have been instrumental in elucidating the molecular mechanisms underlying PDA etiology and progression (7–11). Studies in these systems have shown that pancreas-specific expression of mutant Kras can initiate precursor pancreatic intraepithelial neoplasms (PanINs) that progress to invasive adenocarcinoma (7) and that the course, rate of progression, and metastatic capability of PDA are affected by the additional loss and/or mutation of key tumor suppressor genes (e.g. Dpc4/Smad4, Cdnk2a/ink4a, and Trp53), as well as the relative timing of these events (8, 10, 11). Accurate mouse models can also complement static genomic and proteomic studies of human samples because they enable spatiotemporal monitoring of markers from disease initiation to metastasis. Although many static analyses of putative biomarkers have been performed on resected tissues (12, 13), fewer studies have taken advantage of genetically engineered mouse models of PDA that progress from well-characterized precursor lesions to invasive and metastatic disease to discover protein biomarkers and chronicle their distribution and expression during disease progression (14, 15).Proteomic technologies enable the molecular characterization of biological samples at the structural and functional level (16), and multiplex antibody microarray platforms allow the characterization of multiple analytes simultaneously. Antibody microarrays have the added benefits of requiring minimal sample volumes and the ability to identify their cognate analytes with pico- to femtomolar sensitivities (17, 18). Given their low cost, immunoassay-based diagnostics are thought to be the most applicable proteomic technology in a clinical setting (19, 20).The KPC mouse model of PDA employs conditional pancreas-specific, endogenous expression of Kras^G12D and Trp53^R172H and spontaneously develops precursor PanINs with 100% penetrance that progress to invasive and metastatic PDA (8). We thus found this an ideal model to use in conjunction with a novel, high-dimensional, pancreas-cancer-tailored antibody microarray to assay for proteomic changes during disease progression (18, 21, 22). The microarray consisted of ∼2500 antibodies and represents, to our knowledge, the largest antibody array platform produced. Our goal was to follow the evolving primary tumor proteome from PanIN to invasive PDA. Using this approach, we identified and validated a panel of tissue markers differentiating early stages of tumorigenesis from both the normal organ and chronic pancreatitis. We identified the serine/threonine kinase STK4 as a novel early marker of PDA progression and confirmed its expression in human PDA.     

Modeling the Interruption of the Transmission of Soil-Transmitted Helminths by Repeated Mass Chemotherapy of School-Age Children

Background

The control or elimination of neglected tropical diseases has recently become the focus of increased interest and funding from international agencies through the donation of drugs. Resources are becoming available for the treatment of soil-transmitted helminth (STH) infection through school-based deworming strategies. However, little research has been conducted to assess the impact of STH treatment that could be used to guide the design of efficient elimination programs.

Methodology

We construct and analyse an age-structured model of STH population dynamics under regular treatment. We investigate the potential for elimination with finite rounds of treatment, and how this depends on the value of the basic reproductive number R₀ and treatment frequency.

Principal findings

Analysis of the model indicates that its behaviour is determined by key parameter groupings describing the basic reproduction number and the fraction of it attributable to the treated group, the timescale of material in the environment and the frequency and efficacy of treatment. Mechanisms of sexual reproduction and persistence of infectious material in the environment are found to be much more important in the context of elimination than in the undisturbed baseline scenario. For a given rate of drug use, sexual reproduction dictates that less frequent, higher coverage treatment is more effective. For a given treatment coverage level, the lifespan of infectious material in the environment places a limit on the effectiveness of increased treatment frequency.

Conclusions

Our work suggests that for models to capture the dynamics of parasite burdens in populations under regular treatment as elimination is approached, they need to include the effects of sexual reproduction among parasites and the dynamics infectious material in the reservoir. The interaction of these two mechanisms has a strong effect on optimum treatment strategies, both in terms of how frequently to treat and for how long.     

MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis

Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion. We show that this effect is largely attributed to the elongated and heavily glycosylated extracellular domain of MUC1 that protrudes high above the cell membrane and hence prevents activation of the cell surface anoikis-initiating molecules such as integrins and death receptors by providing them a mechanically ‘homing'' microenvironment. As overexpression of MUC1 is a common feature of epithelial cancers and as resistance to anoikis is a hallmark of both oncogenic epithelial–mesenchymal transition and metastasis, MUC1-mediated cell resistance to anoikis may represent one of the fundamental regulatory mechanisms in tumourigenesis and metastasis.Anoikis, the apoptotic process that occurs in cells that have lost adhesion to the extracellular matrix (ECM),^1,2 is a fundamental process for maintaining tissue homeostasis. It removes displaced epithelial/endothelial cells and thus prevents them from seeding to inappropriate sites. Resistance to anoikis contributes prominently to tumourigenesis and, in particular, to metastasis by allowing survival of cancer cells that have invaded into the blood or lymphatic circulation and thus facilitating their metastatic spread to remote sites.³Initiation of anoikis starts from the cell surface through activation of the cell surface anoikis-initiating molecules, for example, integrins, cadherins and death receptors, in response to loss of cell adhesion. Loss of the integrin-mediated cell basement matrix contact,⁴ loss of the E-cadherin-mediated cell–cell contact^5,6 or ligation of the cell surface death receptors with their ligands^4,7 all induce conformational changes or oligomerization of these cell surface anoikis-initiating molecules. This triggers a series of events leading to activation of either the caspase-8-mediated extrinsic apoptotic signalling pathway or the mitochondrion-mediated intrinsic apoptotic signalling pathway.MUC1 is a large transmembrane mucin protein that is expressed exclusively on the apical side of normal epithelial and some other cell types. MUC1 consists of a large extracellular domain, a transmembrane region and a short cytoplasmic tail. The MUC1 extracellular domain contains a variable number of tandem repeats that are heavily glycosylated (up to 50% of the MUC1 molecular weight) with complex O-linked mucin-type glycans⁸ and flanked by a unique N-terminal domain and an SEA domain. In the SEA domain, autocleavage takes place resulting in a heterodimer but both moieties remain firmly attached. The cytoplasmic tail of MUC1 contains 72 amino acids and harbours several phosphorylation sites and is able to interact with various growth factor receptors and intracellular signalling proteins.^{9, 10, 11}MUC1 is overexpressed up to at least 10-fold in epithelial cancers¹² and overexpression of MUC1 is closely associated with high metastatic potential and poor prognosis in many cancer types.¹³ In epithelial cancer cells, MUC1 loses its apical membrane polarization and becomes expressed over the entire cell surface.^14,15 In epithelial cancer cells, MUC1 also shows reduced expression of complex O-glycans and increased expression of short oncofetal oligosaccharides such as GalNAc-α (Tn antigen), sialylated GalNAc-α (sialyl-Tn antigen) and Galβ1,3GalNAc-α (Thomsen–Friedenreich, TF antigen).¹⁶ Immunological targeting of cancer-associated MUC1 has been under intensive investigation as a strategy for cancer treatment.^17,18 Our recent studies have shown that interaction of TF antigen on cancer-associated MUC1 with the galactoside-binding galectins promotes metastasis by enhancing tumour cell heterotypic adhesion to the vascular endothelium and also by increasing tumour cell homotypic aggregation for the potential formation of tumour emboli.^19–21In this report, we describe a new role of MUC1 in anoikis. We show that overexpression of MUC1 in epithelial cells prevents initiation of anoikis in response to loss of cell adhesion, an effect that is found to be attributed substantially to the MUC1 extracellular domain.