Rac signaling in breast cancer: a tale of GEFs and GAPs

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.     

An <Emphasis Type="Italic">L</Emphasis><Subscript>1</Subscript>-regularized logistic model for detecting short-term neuronal interactions

Interactions among neurons are a key component of neural signal processing. Rich neural data sets potentially containing evidence of interactions can now be collected readily in the laboratory, but existing analysis methods are often not sufficiently sensitive and specific to reveal these interactions. Generalized linear models offer a platform for analyzing multi-electrode recordings of neuronal spike train data. Here we suggest an L ₁-regularized logistic regression model (L ₁ L method) to detect short-term (order of 3 ms) neuronal interactions. We estimate the parameters in this model using a coordinate descent algorithm, and determine the optimal tuning parameter using a Bayesian Information Criterion. Simulation studies show that in general the L ₁ L method has better sensitivities and specificities than those of the traditional shuffle-corrected cross-correlogram (covariogram) method. The L ₁ L method is able to detect excitatory interactions with both high sensitivity and specificity with reasonably large recordings, even when the magnitude of the interactions is small; similar results hold for inhibition given sufficiently high baseline firing rates. Our study also suggests that the false positives can be further removed by thresholding, because their magnitudes are typically smaller than true interactions. Simulations also show that the L ₁ L method is somewhat robust to partially observed networks. We apply the method to multi-electrode recordings collected in the monkey dorsal premotor cortex (PMd) while the animal prepares to make reaching arm movements. The results show that some neurons interact differently depending on task conditions. The stronger interactions detected with our L ₁ L method were also visible using the covariogram method.     

Conditional deletion of calcium-modulating cyclophilin ligand causes deafness in mice

Calcium-modulating cyclophilin ligand (Caml) is a ubiquitously expressed cytoplasmic protein that is involved in multiple signaling and developmental pathways. An observation in our laboratory of a protein-protein interaction between Caml and the cytoplasmic region of Cadherin23 led us to speculate that Caml might be important in the inner ear and play a role in the development and/or function of hair cells. To address this question, we generated a mouse line in which Caml expression was eliminated in Atoh1-expressing cells of the inner ear upon administration of tamoxifen. Tamoxifen was administered immediately after birth to neonates to assess the effect of loss of Caml in the inner ear during postnatal development. Hearing in treated animals was tested by auditory brain stem response (ABR) analysis and cochlear pathology was evaluated by light microscopy. Lack of Caml expression in the inner ear leads to severe loss of cochlear hair cells and complete deafness. Elucidating the role of Caml in the inner ear will aid our understanding of the molecular pathways important for auditory development and function.     

Diagnosed hypertension in Canada: incidence, prevalence and associated mortality

Background:

Hypertension is a leading risk factor for cardiovascular diseases. Our objectives were to examine the prevalence and incidence of diagnosed hypertension in Canada and compare mortality among people with and without diagnosed hypertension.

Methods:

We obtained data from linked health administrative databases from each province and territory for adults aged 20 years and older. We used a validated case definition to identify people with hypertension diagnosed between 1998/99 and 2007/08. We excluded pregnant women from the analysis.

Results:

This retrospective population-based study included more than 26 million people. In 2007/08, about 6 million adults (23.0%) were living with diagnosed hypertension and about 418 000 had a new diagnosis. The age-standardized prevalence increased significantly from 12.5% in 1998/99 to 19.6% in 2007/08, and the incidence decreased from 2.7 to 2.4 per 100. Among people aged 60 years and older, the prevalence was higher among women than among men, as was the incidence among people aged 75 years and older. The prevalence and incidence were highest in the Atlantic region. For all age groups, all-cause mortality was higher among adults with diagnosed hypertension than among those without diagnosed hypertension.

Interpretation:

The overall prevalence of diagnosed hypertension in Canada from 1998 to 2008 was high and increasing, whereas the incidence declined during the same period. These findings highlight the need to continue monitoring the effectiveness of efforts for managing hypertension and to enhance public health programs aimed at preventing hypertension.Globally, raised blood pressure is the leading risk factor for death, accounting for about 13% of all deaths,^1,2 and it is the strongest risk factor for lost years of healthy life.¹ Left untreated, hypertension can increase the risk of stroke, coronary artery disease, dementia, heart and kidney failure, and other chronic diseases.^3–6 Managing hypertension through lifestyle modification or the use of antihypertensive medications, or both, can help mitigate these outcomes.⁷ Over the past decades in Canada, mortality associated with cardiovascular diseases has decreased,⁸ partly because of increased awareness and diagnosis of hypertension and better control of blood pressure.^9,10 However, the prevalence of hypertension remains high, and currently there are no mechanisms to track new cases at the national level.To date, information about hypertension in Canada has been mainly obtained by health surveys conducted at the provincial or national levels. Such surveys typically provide prevalence (not incidence) data and include limited data about trends over time.^11–15 National health surveys in Canada are resource intensive, do not include information about people who live in remote areas or institutions, and may underestimate hypertension prevalence because of recall bias and non-response.¹⁶ The use of administrative data that is population-based and routinely collected, such as physician claims and hospital discharge data, allows for a more comprehensive picture of this condition. Other important advantages of using administrative data include the readiness of the data to be analyzed, cost-efficiency, wide geographic coverage and the relatively complete capture of patient contact with the health care system (i.e., less prone to selection bias).Several recent studies in Canada and the United States have established valid methods for using administrative data to identify cases of hypertension.^16–23 In a study conducted in Ontario involving women and men aged 20 years and older, Tu and colleagues found that the prevalence and incidence of diagnosed hypertension were 24.5% in 2005 and 3.2% in 2004, respectively.²⁴ We used the same validated case definition to examine the prevalence and incidence of diagnosed hypertension in Canada from 1998/99 to 2007/08 by age and by province and territory. We also compared all-cause mortality by age and sex among those with and without diagnosed hypertension.     

Baseline Q waves as a prognostic modulator in patients with ST-segment elevation: insights from the PLATO trial

Background:

Baseline Q waves may provide additional value compared with time from the onset of symptoms in predicting outcomes for patients with ST-segment elevation. We evaluated whether baseline Q waves superseded time from symptom onset as a prognostic marker of one-year mortality in patients with ST-segment elevation acute coronary syndrome. Our study was derived from data from patients undergoing primary percutaneous coronary intervention within 24 hours in the PLATelet inhibition and patient Outcomes trial

Methods:

Q waves on the baseline electrocardiogram were evaluated by a blinded core laboratory. We assessed the associations between baseline Q waves and time from symptom onset to percutaneous coronary intervention with peak biomarkers, ST-segment resolution on the discharge electrocardiogram, and one-year all-cause and vascular mortality.

Results:

Of 4341 patients with ST-segment elevation, 46% had baseline Q waves. Compared to those without Q waves, those with baseline Q waves were older, more frequently male, had higher heart rates, more advanced Killip class and had a longer time between the onset of symptoms and percutaneous coronary intervention. They also had higher one-year all-cause mortality than patients without baseline Q waves (baseline Q waves: 4.9%; no baseline Q waves: 2.8%; hazard ratio [HR] 1.78, 95% confidence interval [CI] 1.29–2.45, p < 0.001). Complete ST-segment resolution was greatest and all-cause mortality lowest among those with symptom onset three hours or less before percutaneous coronary intervention and no baseline Q waves. After multivariable adjustment, baseline Q waves, but not time from symptom onset, were associated with a significant increase in all-cause mortality (adjusted HR 1.42, 95% CI 1.10–2.01, p = 0.046) and vascular mortality (adjusted HR 1.58, 95% CI 1.09–2.28, p = 0.02).

Interpretation:

The presence of baseline Q waves provides useful additional prognostic insight into the clinical outcome of patients with ST-segment elevation. Clinical Trials.gov registration no. {"type":"clinical-trial","attrs":{"text":"NCT00391872","term_id":"NCT00391872"}}NCT00391872The clinical outcome of patients with ST-segment elevation myocardial infarction (STEMI) is directly related to the extent of myocardial necrosis.¹ Because the extent of necrosis is strongly influenced by the duration of symptoms, time is a key clinical proxy for the stage of evolution of STEMI.² The length of time from the onset of symptoms is important in strategies for triage and management and for gauging prognosis. Although time from the occurrence of epicardial artery occlusion in a laboratory experimental model can be measured precisely, time from the onset of symptoms is often difficult to accurately estimate because of subjectivity and reliance on recall. Thus, establishing a more reliable method for determining the stage of myocardial infarction (MI) evolution in patients with STEMI would be useful for evaluating the potential for myocardial salvage and guiding clinical management.There is evidence that the assessment of Q waves on the baseline electrocardiogram (ECG) in the region of ST-segment elevation may be a useful predictor of left ventricular dysfunction and outcomes in patients with STEMI given streptokinase within four to six hours of the onset of symptoms.^3,4 Because prior studies of the predictive value of baseline Q waves focused on patients receiving fibrinolytic therapy, we extended this question to a large population of patients with STEMI who were at high risk of adverse clinical outcomes (e.g., death, ardiogenic shock and heart failure) and undergoing mechanical reperfusion with percutaneous coronary intervention in the Assessment of Pexelizumab in Acute Myocardial Infarction (APEX-AMI)⁵ trial within six hours of symptom onset. A key finding of this study was that Q waves were a key prognostic factor of 90-day mortality and the composite measure of death, cardiogenic shock and heart failure; in addition, Q waves were better than time from symptom onset in predicting these 90-day outcomes.⁶ Whether these findings are applicable to a more general STEMI population studied prospectively is unclear.Given the increasing uptake of therapy for STEMI with primary percutaneous coronary intervention and the continuing challenges in achieving timely reperfusion, we sought to validate these findings in a more contemporary cohort. The PLATelet inhibition and patient Outcomes (PLATO) study not only provided this opportunity in a large population, but it also extended our evaluation to patients with less stringent ST-segment elevation entry criteria (1 mm in two contiguous leads) randomized over a wider entry window (24 h from symptom onset) and followed for a longer period (1 yr).⁷ In the current study, we aimed to prospectively evaluate whether Q waves in the region of qualifying ST-segment elevation on the baseline ECG provided additional value compared with time from symptom onset as a predictor of all-cause mortality in patients with ST-segment elevation undergoing primary percutaneous coronary intervention in the PLATO trial.⁷ We also assessed associations with vascular death, a prespecified component of the primary outcome in the PLATO trial.     

Cryptic lineages and Pleistocene population expansion in a Brazilian Cerrado frog

Diversification of South American species endemic to open habitats has been attributed to both Tertiary events and Pleistocene climatic fluctuations. Nonetheless, phylogeographical studies of taxa in these regions are few, precluding generalizations about the timing and processes leading to differentiation and speciation. We inferred population structure of Hypsiboas albopunctatus, a frog widely distributed in the Brazilian Cerrado. Three geographically distinct lineages were recovered in our phylogeny. The Chapada dos Guimarães (CG) clade was the first to diverge from other populations and contains multiple haplotypes from a single population in western Cerrado, probably representing a cryptic species. The southeast clade (SE) includes populations along the southeastern limit of the range within the historical distribution of the Brazilian Atlantic forest. Finally, the Central Cerrado (CC) group includes haplotypes from the interior of Brazil that are paraphyletic relative to the SE clade. Analyses of historical demography indicate significant population expansion in the CC and SE populations, likely associated with colonization of newly formed open habitats. The divergence of populations in the CG clade occurred in the late Miocene, concordant with the uplift of the central Brazilian plateau. Divergence of the SE clade from the CC occurred during the mid‐Pleistocene. Thus, both Tertiary geological events and Pleistocene climatic fluctuations promoted divergences among lineages. Our study reveals a complex history of diversification in the Cerrado, a morphoclimatic domain highly threatened because of anthropogenic habitat alteration. We identified surprisingly deep divergences in a widely distributed frog, indicating that the Cerrado is not a barrier‐free habitat and that its diversity is likely underestimated.     

Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and partitioning to cellular triacylglycerol in McArdle-RH7777 cells

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.     

Cytoplasmic targeting motifs control localization of toll-like receptor 9

Toll-like receptors (TLRs) are essential for host defense. Although several TLRs reside on the cell surface, nucleic acid recognition of TLRs occurs intracellularly. For example, the receptor for CpG containing bacterial and viral DNA, TLR9, is retained in the endoplasmic reticulum. Recent evidence suggests that the localization of TLR9 is critical for appropriate ligand recognition. Here we have defined which structural features of the TLR9 molecule control its intracellular localization. Both the cytoplasmic and ectodomains of TLR9 contain sufficient information, whereas the transmembrane domain plays no role in intracellular localization. We identify a 14-amino acid stretch that directs TLR9 intracellularly and confers intracellular localization to the normally cell surface-expressed TLR4. Truncation or mutation of the cytoplasmic tail of TLR9 reveals a vesicle localization motif that targets early endosomes. We propose a model whereby modification of the cytoplasmic tail of TLR9 results in trafficking to early endosomes where it encounters CpG DNA.