Effect of botulinum neurotoxins from Mendoza of clostridium botulinum strains on cytoskeletal proteins of mammary tumor cells

The botulinum neurotoxin serotype A (BoNT A) produced by Clostridium botulinum, which causes botulism, is used for the treatment of multiple neurological diseases and its therapeutic action against cancer is currently being evaluated. In previous studies, we have shown that BoNT A from autochthonous soil strains (Su) have different properties than the reference A Hall strain. Among these, its molecular structure, its enzymatic activity against brain SNARE proteins and its greater specific toxic activity (AE) stand out. In cells from human mammary carcinoma (MCF-7) treated with BoNTs for 45 min, we found a marked effect on the expression of cytoskeletal proteins. Therefore, in this work, we delve into the study of the action of autochthonous BoNTs A and prototype A Hall on the distribution of actin and tubulin in these cells. Native forms of autochthonous BoNT (Su strains 1935 and 1891, Tupungato) and prototype A Hall were purified by saline precipitation. Their AE values (LD50 / mg protein) were established and their electrophoretic characteristics were evaluated under non-denaturing conditions. 250 LD50 of the BoNTs were incubated to MCF-7 cell cultures for 10 or 25 min. Later, the cells were fixed and processed for indirect immunofluorescence with the use of specific antibodies that recognize tubulin or actin. The samples were visualized by fluorescence microscopy. At the two times evaluated, the three types of BoNTs produced a marked redistribution of the actin cytoskeleton, patch form, on areas coinciding with the plasma membrane. Tubulin was redistributed to multiple areas with high signal density at 10 min of incubation only in the presence of BoNT 1891. At 25 min of incubation, the cells treated with BoNTs 1891 and 1935 showed this effect, while in those incubated with A Hall, the distribution of these proteins was not modified. The notable alterations in the distribution of components of the tumor cell cytoskeleton by BoNT from native strains of Mendoza soils open new perspectives for therapy against solid tumors.Fil: Chapana, Agostina Lucía. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guarniolo, D.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Carvelli, Flavia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sosa, E.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Fernández, R. A.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaFil: Sosa, M. A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Caballero, P. A.. Universidad Nacional de Cuyo. Facultad de Cs.médicas. Departamento de Patología. Area de Microbiología; ArgentinaIV Reunión Conjunta de Sociedades de Biología de la República ArgentinaArgentinaSociedad de Biología de CuyoSociedad Argentina de BiologíaSociedad de Biología de RosarioSociedad Chilena de Reproducción y DesarrolloAsociación de Biología de TucumánSociedad de Biología de Córdob

tetraxial textiles assessment of mesoscale mechanical modelling by experimental measurements

Tetraxial technical textiles were recently manufactured by a new loom developed to weave yarns in four directions. The textile has warp, weft, and two diagonal yarns oriented at symmetrical angles (typically ±45°) with respect to the warp direction. The peculiar yarns distribution could enhance the mechanical response of the textile in multiple directions aiming to almost isotropic in-plane behaviour. For the prediction of the mechanical performance of such tetraxial textiles, a reliable and accurate predictive model is of relevant importance. The present investigation aims to adopt the finite element numerical approach at the mesoscopic scale to predict the mechanical response for any in-plane loading of tetraxial textiles. An accurate modelling of the constitutive behaviour of the fibrous yarns was adopted considering a hyperelastic model. The modelling of the tetraxial unit cell allowed to have the mechanical behaviour for uniaxial and biaxial tensile and for shear loading conditions. The assessment of the accuracy of the numerical model was performed considering a huge experimental campaign dedicated to several hybrid tetraxial textiles. The comparison highlights the accuracy of the numerical model to predict the nonlinear behavior of the fabric for any loading condition and to provide the proper mechanical model for further optimization of tetraxial textiles supposed for different industrial applications

Predictive modeling of the current density and radiative recombination in blue polymer-based light-emitting diodes

The results of a combined experimental and modeling study of charge transport, recombination and light emission in blue organic light-emitting diodes (OLEDs) based on a polyfluorene derivative are presented. It is shown that the measured temperature-dependent current-voltage curves and the voltage-dependent current efficiency are accurately described using an OLED device model that is based on the separately determined unipolar electron and hole mobility functions. The recombination rate is calculated using the Langevin formula, including recombination of holes with free as well as trapped electrons. The light emission is obtained from the exciton formation profile using independently determined values of the exciton radiative decay probability, the average dipole orientation, and assuming a fraction of singlet excitons ¿S¿¿ = ¿(22±3)%, close to the quantum-statistical value. No additional free parameter is used. This shows that predictive one-dimensional device modeling of OLEDs is feasible

Tetraspanin (TSP-17) Protects Dopaminergic Neurons against 6-OHDA-Induced Neurodegeneration in <i>C. elegans</i>

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling

The Lantern Vol. 50, No. 2, Spring 1984

• The Storm • Je ne sais pas • The Ghetious Blastious • An Empty Cradle • The Playing Hands • Battle Hymn • A Limerick • Parting Thoughts • The River • Miss You • De la Tristeza • Two So Special • Time of the Unicorn • The Absence • Thru The Breeze • Is the World Really a Round Ball? • Brother • To Michael • Gravity • Refuge • Der Witwer • Plastic Flowers Never Die • Book on the Shelfhttps://digitalcommons.ursinus.edu/lantern/1124/thumbnail.jp

Hypoinsulinemia Regulates Amphetamine-Induced Reverse Transport of Dopamine

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level–dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders

Dysregulation of the Norepinephrine Transporter Sustains Cortical Hypodopaminergia and Schizophrenia-Like Behaviors in Neuronal Rictor Null Mice

A novel animal model highlights the link between Akt dysfunction, reduced cortical dopamine function, norepinephrine transporters, and schizophrenia-like behaviors

A randomised, double-blind, placebo-controlled trial of repeated nebulisation of non-viral cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy in patients with cystic fibrosis

Background: Cystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene leading to abnormal airway surface ion transport, chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco®, Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediated CFTR gene therapy formulation through preclinical and clinical development. Objective: To determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF. Design: This was a randomised, double-blind, placebo-controlled Phase IIb trial of the CFTR gene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers. Settings: Data were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database. Participants: Patients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV1) between 50% and 90% predicted and any combination of CFTR mutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised). Interventions: Subjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year. Main outcome measures: The primary end point was the relative change in percentage predicted FEV1 over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference. Results: There was a significant (p = 0.046) treatment effect (TE) of 3.7% [95% confidence interval (CI) 0.1% to 7.3%] in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) (p = 0.031) and CT gas trapping (p = 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age or CFTR mutation class. Subjects with a more severe baseline FEV1 had a FEV1 TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly (p = 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups. Conclusions: Monthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo. Limitations: Although encouraging, the improvement in FEV1 was modest and was not accompanied by detectable improvement in patients’ quality of life. Future work: Future work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration. Trial registration: ClinicalTrials.gov NCT01621867. Funding: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership