Dapper Antagonist of Catenin-1 Cooperates with Dishevelled-1 during Postsynaptic Development in Mouse Forebrain GABAergic Interneurons

Synaptogenesis has been extensively studied along with dendritic spine development in glutamatergic pyramidal neurons, however synapse development in cortical interneurons, which are largely aspiny, is comparatively less well understood. Dact1, one of 3 paralogous Dact (Dapper/Frodo) family members in mammals, is a scaffold protein implicated in both the Wnt/β-catenin and the Wnt/Planar Cell Polarity pathways. We show here that Dact1 is expressed in immature cortical interneurons. Although Dact1 is first expressed in interneuron precursors during proliferative and migratory stages, constitutive Dact1 mutant mice have no major defects in numbers or migration of these neurons. However, cultured cortical interneurons derived from these mice have reduced numbers of excitatory synapses on their dendrites. We selectively eliminated Dact1 from mouse cortical interneurons using a conditional knock-out strategy with a Dlx-I12b enhancer-Cre allele, and thereby demonstrate a cell-autonomous role for Dact1 during postsynaptic development. Confirming this cell-autonomous role, we show that synapse numbers in Dact1 deficient cortical interneurons are rescued by virally-mediated re-expression of Dact1 specifically targeted to these cells. Synapse numbers in these neurons are also rescued by similarly targeted expression of the Dact1 binding partner Dishevelled-1, and partially rescued by expression of Disrupted in Schizophrenia-1, a synaptic protein genetically implicated in susceptibility to several major mental illnesses. In sum, our results support a novel cell-autonomous postsynaptic role for Dact1, in cooperation with Dishevelled-1 and possibly Disrupted in Schizophrenia-1, in the formation of synapses on cortical interneuron dendrites.     

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca²⁺ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca²⁺ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca²⁺ level after activation; time to reach peak cytosolic free Ca²⁺ and the EC₅₀ of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca²⁺ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca²⁺ signaling.     

Estimation of Parameters Influencing Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon (Salmo salar)

Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml⁻¹. Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×10⁷ pfu fish⁻¹ hour⁻¹ one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k =  4.37 d⁻¹ were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites.     

Disability Affects the 6-Minute Walking Distance in Obese Subjects (BMI>40 kg/m2)

Introduction

In obese subjects, the relative reduction of the skeletal muscle strength, the reduced cardio-pulmonary capacity and tolerance to effort, the higher metabolic costs and, therefore, the increased inefficiency of gait together with the increased prevalence of co-morbid conditions might interfere with walking. Performance tests, such as the six-minute walking test (6MWT), can unveil the limitations in cardio-respiratory and motor functions underlying the obesity-related disability. Therefore the aims of the present study were: to explore the determinants of the 6-minute walking distance (6MWD) and to investigate the predictors of interruption of the walk test in obese subjects.

Methods

Obese patients [body mass index (BMI)>40 kg/m²] were recruited from January 2009 to December 2011. Anthropometry, body composition, specific questionnaire for Obesity-related Disabilities (TSD-OC test), fitness status and 6MWT data were evaluated. The correlation between the 6MWD and the potential independent variables (anthropometric parameters, body composition, muscle strength, flexibility and disability) were analysed. The variables which were singularly correlated with the response variable were included in a multivariated regression model. Finally, the correlation between nutritional and functional parameters and test interruption was investigated.

Results

354 subjects (87 males, mean age 48.5±14 years, 267 females, mean age 49.8±15 years) were enrolled in the study. Age, weight, height, BMI, fat mass and fat free mass indexes, handgrip strength and disability were significantly correlated with the 6MWD and considered in the multivariate analysis. The determination coefficient of the regression analysis ranged from 0.21 to 0.47 for the different models. Body weight, BMI, waist circumference, TSD-OC test score and flexibility were found to be predictors of the 6MWT interruption.

Discussion

The present study demonstrated the impact of disability in obese subjects, together with age, anthropometric data, body composition and strength, on the 6-minute walking distance.     

RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics

Rho family GTPases regulate diverse processes in human melanoma ranging from tumor formation to metastasis and chemoresistance. In this study, a combination of in vitro and in vivo approaches was utilized to determine whether RHOJ, a CDC42 homologue that regulates melanoma chemoresistance, also controls melanoma migration. Depletion or overexpression of RHOJ altered cellular morphology, implicating a role for RHOJ in modulating actin cytoskeletal dynamics. RHOJ depletion inhibited melanoma cell migration and invasion in vitro and melanoma tumor growth and lymphatic spread in mice. Molecular studies revealed that RHOJ alters actin cytoskeletal dynamics by inducing the phosphorylation of LIMK, cofilin, and p41‐ARC (ARP2/3 complex subunit) in a PAK1‐dependent manner in vitro and in tumor xenografts. Taken together, these observations identify RHOJ as a melanoma linchpin determinant that regulates both actin cytoskeletal dynamics and chemoresistance by activating PAK1.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

Control of (Multi)Drug Resistance and Tuberculosis Incidence over 23 Years in the Context of a Well-Supported Tuberculosis Programme in Rural Malawi

Background

The rise in tuberculosis (TB) incidence following generalized HIV epidemics can overwhelm TB control programmes in resource-limited settings, sometimes accompanied by rising rates of drug resistance. This has led to claims that DOTS-based TB control has failed in such settings. However, few studies have described the effect of a sustained and well-supported DOTS programme on TB incidence and drug resistance over a long period. We present long-term trends in incidence and drug resistance in rural Malawi.

Methods

Karonga District in northern Malawi has an adult HIV prevalence of ∼10%. A research group, the Karonga Prevention Study, collaborates with the National Tuberculosis Programme to support core TB control activities. Bacteriological, demographic and clinical (including HIV status) information from all patients starting TB treatment in the District have been recorded since 1988. During that period isolates from each culture-positive TB patient were exported for drug sensitivity testing. Antiretroviral therapy (ART) has been widely available since 2005.

Results

Incidence of new smear-positive adult TB peaked at 124/100,000/year in the mid-90s, but has since fallen to 87/100,000/year. Drug sensitivity information was available for 95% (3132/3307) of all culture-positive cases. Initial resistance to isoniazid was around 6% with no evidence of an increase. Fewer than 1% of episodes involved a multi-drug resistant strain.

Discussion

In this setting with a generalised HIV epidemic and medium TB burden, a well-supported DOTS programme enhanced by routine culture and drug sensitivity testing may well have reduced TB incidence and maintained drug resistance at low levels.     

Origin of a Core Bacterial Gene via Co-option and Detoxification of a Phage Lysin

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