In Vivo RNAi Screening Identifies Regulators of Actin Dynamics as Key Determinants of Lymphoma Progression

April 1, 2010Mouse models have markedly improved our understanding of cancer development and tumor biology. However, these models have shown limited efficacy as tractable systems for unbiased genetic experimentation. Here, we report the adaptation of loss-of-function screening to mouse models of cancer. Specifically, we have been able to introduce a library of shRNAs into individual mice using transplantable Eμ-myc lymphoma cells. This approach has allowed us to screen nearly 1,000 genetic alterations in the context of a single tumor-bearing mouse. These experiments have identified a central role for regulators of actin dynamics and cell motility in lymphoma cell homeostasis in vivo. Validation experiments confirmed that these proteins represent bona fide lymphoma drug targets. Additionally, suppression of two of these targets, Rac2 and twinfilin, potentiated the action of the front-line chemotherapeutic vincristine, suggesting a critical relationship between cell motility and tumor relapse in hematopoietic malignancies.National Institutes of Health (U.S.) (RO1 CA128803-01)Massachusetts Institute of Technology. Dept. of Biology (Training Grant)Massachusetts Institute of Technology. Undergraduate Research Opportunities ProgramNational Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1-U54-CA112967

Immediate response of myocardium to pressure overload includes transient regulation of genes associated with mitochondrial bioenergetics and calcium availability

Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca+2 ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability

Developing Pulmonary Vasculopathy in Systemic Sclerosis, Detected with Non-Invasive Cardiopulmonary Exercise Testing

BACKGROUND: Patients with systemic sclerosis (SSc) may develop exercise intolerance due to musculoskeletal involvement, restrictive lung disease, left ventricular dysfunction, or pulmonary vasculopathy (PV). The latter is particularly important since it may lead to lethal pulmonary arterial hypertension (PAH). We hypothesized that abnormalities during cardiopulmonary exercise testing (CPET) in patients with SSc can identify PV leading to overt PAH. METHODS: Thirty SSc patients from the Harbor-UCLA Rheumatology clinic, not clinically suspected of having significant pulmonary vascular disease, were referred for this prospective study. Resting pulmonary function and exercise gas exchange were assessed, including peakVO2, anaerobic threshold (AT), heart rate-VO2 relationship (O2-pulse), exercise breathing reserve and parameters of ventilation-perfusion mismatching, as evidenced by elevated ventilatory equivalent for CO2 (VE/VCO2) and reduced end-tidal pCO2 (PETCO2) at the AT. RESULTS: Gas exchange patterns were abnormal in 16 pts with specific cardiopulmonary disease physiology: Eleven patients had findings consistent with PV, while five had findings consistent with left-ventricular dysfunction (LVD). Although both groups had low peak VO2 and AT, a higher VE/VCO2 at AT and decreasing PETCO2 during early exercise distinguished PV from LVD. CONCLUSIONS: Previously undiagnosed exercise impairments due to LVD or PV were common in our SSc patients. Cardiopulmonary exercise testing may help to differentiate and detect these disorders early in patients with SSc

The C242T polymorphism of the p22-phox gene (CYBA) is associated with higher left ventricular mass in Brazilian hypertensive patients

<p>Abstract</p> <p>Background</p> <p>Reactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA) on left ventricular structure in Brazilian hypertensive subjects.</p> <p>Methods</p> <p>We cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441) and Vitória (n = 120)] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample.</p> <p>Results</p> <p>Genotype frequencies in both samples were consistent with the Hardy- Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height^2.7than those carrying the CC genotype in Campinas (76.8 ± 1.6 vs 70.9 ± 1.4 g/m^2.7; p = 0.009), and in Vitória (45.6 ± 1.9 vs 39.9 ± 1.4 g/m^2.7; p = 0.023) samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03).</p> <p>Conclusions</p> <p>The T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height^2.7and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.</p

Vocal Accuracy and Neural Plasticity Following Micromelody-Discrimination Training

Recent behavioral studies report correlational evidence to suggest that non-musicians with good pitch discrimination sing more accurately than those with poorer auditory skills. However, other studies have reported a dissociation between perceptual and vocal production skills. In order to elucidate the relationship between auditory discrimination skills and vocal accuracy, we administered an auditory-discrimination training paradigm to a group of non-musicians to determine whether training-enhanced auditory discrimination would specifically result in improved vocal accuracy.We utilized micromelodies (i.e., melodies with seven different interval scales, each smaller than a semitone) as the main stimuli for auditory discrimination training and testing, and we used single-note and melodic singing tasks to assess vocal accuracy in two groups of non-musicians (experimental and control). To determine if any training-induced improvements in vocal accuracy would be accompanied by related modulations in cortical activity during singing, the experimental group of non-musicians also performed the singing tasks while undergoing functional magnetic resonance imaging (fMRI). Following training, the experimental group exhibited significant enhancements in micromelody discrimination compared to controls. However, we did not observe a correlated improvement in vocal accuracy during single-note or melodic singing, nor did we detect any training-induced changes in activity within brain regions associated with singing.Given the observations from our auditory training regimen, we therefore conclude that perceptual discrimination training alone is not sufficient to improve vocal accuracy in non-musicians, supporting the suggested dissociation between auditory perception and vocal production

Decline in Diarrhea Mortality and Admissions after Routine Childhood Rotavirus Immunization in Brazil: A Time-Series Analysis

A time series analysis by Manish Patel and colleagues shows that the introduction of rotavirus vaccination in Brazil is associated with reduced diarrhea-related deaths and hospital admissions in children under 5 years of age

Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production

Despite recent therapeutic advancements, multiple myeloma (MM) remains incurable and new therapies are needed, especially for the treatment of elderly and relapsed/refractory patients. We have screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity and identified niclosamide, an anti-helminthic. Niclosamide, at clinically achievable non-toxic concentrations, killed MM cell lines and primary MM cells as efficiently as or better than standard chemotherapy and existing anti-myeloma drugs individually or in combinations, with little impact on normal donor cells. Cell death was associated with markers of both apoptosis and autophagy. Importantly, niclosamide rapidly reduced free light chain (FLC) production by MM cell lines and primary MM. FLCs are a major cause of renal impairment in MM patients and light chain amyloid and FLC reduction is associated with reversal of tissue damage. Our data indicate that niclosamides anti-MM activity was mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide. Niclosamide also modulated the nuclear factor-κB and STAT3 pathways in MM cells. In conclusion, our data indicate that MM cells can be selectively targeted using niclosamide while also reducing FLC secretion. Importantly, niclosamide is widely used at these concentrations with minimal toxicity

The C:N:P:S stoichiometry of soil organic matter

The formation and turnover of soil organic matter (SOM) includes the biogeochemical processing of the macronutrient elements nitrogen (N), phosphorus (P) and sulphur (S), which alters their stoichiometric relationships to carbon (C) and to each other. We sought patterns among soil organic C, N, P and S in data for c. 2000 globally distributed soil samples, covering all soil horizons. For non-peat soils, strong negative correlations (p < 0.001) were found between N:C, P:C and S:C ratios and % organic carbon (OC), showing that SOM of soils with low OC concentrations (high in mineral matter) is rich in N, P and S. The results can be described approximately with a simple mixing model in which nutrient-poor SOM (NPSOM) has N:C, P:C and S:C ratios of 0.039, 0.0011 and 0.0054, while nutrient-rich SOM (NRSOM) has corresponding ratios of 0.12, 0.016 and 0.016, so that P is especially enriched in NRSOM compared to NPSOM. The trends hold across a range of ecosystems, for topsoils, including O horizons, and subsoils, and across different soil classes. The major exception is that tropical soils tend to have low P:C ratios especially at low N:C. We suggest that NRSOM comprises compounds selected by their strong adsorption to mineral matter. The stoichiometric patterns established here offer a new quantitative framework for SOM classification and characterisation, and provide important constraints to dynamic soil and ecosystem models of carbon turnover and nutrient dynamics

High-Throughput NMR Assessment of the Tertiary Structure of Food Allergens

In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.1D (1)H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D (1)H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins