Studies of the role of MAP kinase-activated protein kinase-5 (MK5) in reactive and reparative fibrosis in the murine heart

MK5 est une sérine/thréonine kinase, identifiée à l'origine comme une protéine kinase régulée/activée par p38 (PRAK), activée par les p38 MAPK et MAPK atypiques ERK3 et ERK4. Bien que MK5 soit exprimée dans le cœur, sa fonction physiologique commence seulement à être étudiée. Nous avons étudié les effets de la surcharge de pression chronique induite par la constriction aortique transversale (TAC) et les effets de l'infarctus du myocarde (IM) induit par la ligature permanente de l'artère coronaire descendante antérieure gauche (LADG) chez des souris hétérozygotes (MK5+/-). Aussi, nous avons étudié in vitro le rôle de MK5 dans la fonction de fibroblaste cardiaque en utilisant des fibroblastes de génotype MK5+/+, MK5+/-, MK5-/- et des fibroblastes avec un knockdown de MK5 par siRNA (MK5-kd). À l'âge de douze semaines, les souris MK5+/- étaient plus petites que les souris MK5+/+. La fonction systolique était diminuée chez les souris MK5+/-. Deux semaines après TAC, les poids cardiaque/longueur du tibia (PC/LT) ont augmenté de manière similaire et significative dans les 2 groupes. L’augmentation de l'ARNm du collagène de type 1α1 (COL1A1) MK5+/+ était atténuée de manière significative chez les souris MK5+/-. Huit semaines après TAC, PC/LT était significativement moins chez les souris TAC-MK5+/-. La progression de l'hypertrophie était atténuée dans les cœurs MK5+/-. L’immunoréactivité de MK5 a été détectée dans les fibroblastes cardiaques mais pas dans les myocytes. Ces données suggèrent que MK5 dans les fibroblastes cardiaques joue un rôle pro-fibrotique et pro-hypertrophique important dans le remodelage cardiaque pathologique. Nous avons examiné l'effet de MK5+/- dans la fibrose réparatrice après un IM. Les taux de mortalité chez les 2 groupes avec LADG ne différaient pas 7 jours suivant l'IM mais ils étaient plus élevés chez les souris MK5+/- sur une période de 21 jours. La principale cause de décès était la rupture cardiaque. Les fonctions systolique et diastolique sont également altérées chez les 2 groupes avec LADG. La zone de cicatrice et le collagène dedans la cicatrice ont diminué dans les cœurs de MK5+/- 8 jours après l'IM. L’infiltration des cellules inflammatoires était similaire dans les deux groupes avec LADG. L’angiogenèse était significativement élevée dans la zone péri-infarctus de cœurs du MK5+/-. Ces données suggèrent que MK5 peut jouer un rôle dans la régulation de la fonction des fibroblastes cardiaques. C’est pourquoi, nous avons examiné la mobilité et la prolifération du fibroblastes qui étaient diminuées chez les fibroblasts MK5-/-. Le transcriptome pour les protéines impliquées dans le remodelage de la matrice extracellulaire différait selon le génotype des fibroblastes. La sécrétion de COL1A1 et fibronectine étaient significativement augmentée dans les fibroblastes MK5-/- et MK5-kd. La contraction du myofibroblaste a été diminuée dans les fibroblastes MK5-kd. Ces données suggèrent que MK5 est impliqué dans la régulation de multiples aspects de la fonction des fibroblasts cardiaques. En conclusion, nous avons montré un rôle de MK5 dans le remodelage cardiaque pathologique ainsi qu’un rôle de MK5 dans la fonction des fibroblastes cardiaques.MK5 is a serine/threonine kinase, originally identified as a p38 Regulated/Activated Protein Kinase (PRAK), activated by p38 MAPK and the atypical MAPKs ERK3 and ERK4. Although MK5 is expressed in the heart, its physiological function is just beginning to be studied. Herein, we studied the effects of chronic pressure overload induced by transverse aortic constriction (TAC) and the effects of myocardial infarction (MI) induced by permanent ligation of the left anterior descending coronary artery (LADL) in heterozygous mice for a functional knockout of MK5 (MK5+/-). We also studied the role of MK5 in cardiac fibroblast function and in extracellular remodeling in healthy heart in vitro using MK5 wild type (MK5+/+), haplodeficient (MK5+/-), deficient (MK5-/-), and siRNA-mediated knockdown (MK5-kd) fibroblasts. At twelve weeks of age, MK5+/- mice were smaller than age-matched wild-type littermates (MK5+/+). Left ventricular end-diastolic diameter and systolic function were reduced in MK5+/- mice. Two weeks post-TAC, heart weight/tibia length ratios (HW/TL) were similarly and significantly increased in both MK5+/+ and MK5+/- hearts. However, eight weeks post-TAC, HW/TL ratios were significantly lower in TAC-MK5+/- mice compared to TAC-MK5+/+ mice. Thus, the progression of hypertrophy in response to chronic pressure overload was attenuated in MK5+/- hearts. Furthermore, two weeks of pressure overload induced increase in collagen type 1 α 1 (COL1A1) mRNA in MK5+/+ mice and this increase was significantly attenuated in MK5+/- mice. As MK5 immunoreactivity was detected in cardiac fibroblasts but not myocytes, these findings suggest that MK5 within cardiac fibroblasts plays an important pro-fibrotic and pro-hypertrophic role in cardiac remodeling during chronic pressure overload. We then examined the effect of reduced MK5 expression on reparative fibrosis following MI. Mortality rates for MK5+/+ and MK5 +/- did not differ significantly over seven days post-MI. In contrast, mortality was higher in MK5+/- mice over twenty-one days. Systolic and diastolic functions were similarly impaired in both MK5+/+ and MK5+/- mice post-MI. Scar area and scar collagen content were reduced in MK5+/- hearts eight days post-MI. Inflammatory cell infiltration was similar in both ligated groups whereas angiogenesis was significantly greater in the peri-infarct zone in LADL-MK5+/- hearts. These results suggest that MK5 may play a role in regulating cardiac fibroblast function. Finally, we examined the effect of reduced MK5 expression on fibroblast function. Motility and proliferation were reduced in MK5-/- fibroblasts compared to MK5+/+ and MK5+/- fibroblasts. The transcriptome for proteins involved in extracellular matrix remodeling (ECM) differed depending on fibroblast genotype. Similarly, collagen 1-α1 and fibronectin secretion was increased in MK5-/- and MK5-kd fibroblasts compared with MK5+/+. In addition, knocking down MK5 decreased myofibroblast contraction. Taken together, these data suggest that MK5 is involved in regulating multiple aspects of cardiac fibroblast function. In conclusion, we have shown a role for MK5 in cardiac remodeling during chronic pressure overload and myocardial infarction. Moreover, we have demonstrated a role for MK5 in cardiac fibroblast function and extracellular matrix remodeling

The role of sign in students' modeling of scalar equations

We describe students revising the mathematical form of physics equations to match the physical situation they are describing, even though their revision violates physical laws. In an unfamiliar air resistance problem, a majority of students in a sophomore level mechanics class at some point wrote Newton's Second Law as F = -ma; they were using this form to ensure that the sign of the force pointed in a direction consistent with the chosen coordinate system while assuming that some variables have only positive value. We use one student's detailed explanation to suggest that students' issues with variables are context-dependent, and that much of their reasoning is useful for productive instruction.Comment: 5 pages, 1 figure, to be published in The Physics Teache

ENHANCEMENT OF DISSOLUTION CHARACTERISTICS OF CLOPIDOGREL BISULPHATE BY PRONIOSOMES

Objective: The present investigation aims to formulate and evaluate proniosomes of clopidogrel bisulphate for improving its dissolution characteristics. Methods: The slurry method was used for the preparation of proniosomes of clopidogrel using cholesterol, sorbitan monostearate (Span 60) and maltodextrin as a carrier. Clopidogrel proniosomes were evaluated for their entrapment efficiency and in vitro drug release. The best formula (F1) that achieved maximum drug release was further evaluated by measurement of the angle of repose, morphological examination, determination of vesicle size, determination of zeta potential, Fourier transform infrared spectroscopy and differential thermal analysis. The in vivo behavior of the selected proniosomal formula (F1) was studied by measuring the antiplatelet activity in adult male mice. Results: The entrapment efficiency of clopidogrel proniosomes was in the range of 83.04±1.99 to 90.14±0.30. % drug released from proniosomal formulations was in the range of 79.73±0.35 to 97.70±1.10 % within 4 h. Clopidogrel proniosomes significantly enhanced the in vitro release of clopidogrel compared with the plain drug that achieved 61.77±2.22 % drug release. F1 significantly (p ≤ 0.001) increased the bleeding time and bleeding volume and significantly (p ≤ 0.05) prolonged prothrombin time and decreased prothrombin activity and increased the international normalized ratio (INR) compared to plain clopidogrel. Conclusion: The present investigation introduced proniosomes as a promising carrier for clopidogrel that could enhance its dissolution and pharmacological effect

Two N-terminal regions of the Sendai virus L RNA polymerase protein participate in oligomerization

AbstractThe RNA dependent RNA polymerase of Sendai virus consists of a complex of the large (L) and phosphoprotein (P) subunits where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. We previously showed that the L protein forms an oligomer [Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235–245] and mapped the L oligomerization domain between amino acids 1 and 174 of the protein [Çevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 313, 525–536]. An internal deletion encompassing amino acids 20 to 178 of the L protein lost polymerase activity but still formed an L–L oligomer. The first 25 amino acids of paramyxovirus L proteins are highly conserved and site-directed mutagenesis within this region eliminated the biological activity of the L protein but did not have any effect on P–L or L–L interactions. Moreover deletion of amino acids 2–18 in L abolished biological activity, but again the L–L binding was normal demonstrating that the oligomerization domain of L protein resides in two N-terminal regions of the protein. Therefore, sequences between both aa 2–19 and aa 20–178 can independently mediate Sendai L oligomerization, however, both are required for the activity of the protein

Cancer drug therapy and stochastic modelling of “nano-motors”

Controlled inhibition of kinesin motor proteins is highly desired in the field of oncology. Among other interventions, the selective Eg5 competitive and allosteric inhibitors is the most successful targeted chemotherapeutic regime/options, inducing cancer cell apoptosis and tumor regression with improved safety profile. Though promising, this approach is under clinical trials, for the discovery of efficient and least harmful Eg5 inhibitors. The aim of present research is to bridge the computational modelling approach with drug design and therapy of cancer cells. Thus a computational model, interfaced with the clinical data of “Eg5 dynamics” and “inhibitors” via special functions is presented in this article. Comparisons are made for the drug efficacy and the threshold values are predicted through numerical simulations

A review on hyperthermia via nanoparticle-mediated therapy

Hyperthermia treatment, generated by magnetic nanoparticles (MNPs) is promising since it is tumour-focused, minimally invasive and uniform. The most unique feature of magnetic nanoparticles is their reaction to and manipulation by a magnetic force which is responsible for enabling their potential as heating mediators for cancer therapy. With magnetic nanoparticle hyperthermia, a tumour is preferentially loaded with systemically administered nanoparticles with high-absorption cross section for transduction of an extrinsic energy source to heat. To maximize the energy deposited in the tumour while limiting the exposure in healthy tissues, the heating is achieved by exposing the region of tissue containing magnetic nanoparticles to an alternating magnetic field. The magnetic nanoparticles dissipate heat from relaxation losses thereby heating localized tissue above normal physiological ranges. Besides thermal efficiency, the biocompatibility of magnetite nanoparticles assists in their deployment as efficient drug carriers for targeted therapeutic regimes. In the present article we provide a state-of-the-art review focused on progress in nanoparticle induced hyperthermia treatments which have several potential advantages over both global and local hyperthermia treatments achieved without nanoparticles. Green bio-nanotechnology has attracted substantial attention and has demonstrable abilities to improve cancer therapy. Furthermore we have listed the challenges associated with this treatment along with future opportunities in this field which it is envisaged will be of interest to biomedical engineers, bio-materials scientists, medical researchers and pharmacological research groups

\u3cem\u3eMonilinia vaccinii-corymbosi\u3c/em\u3e sensitivity to demethylation inhibitor fungicides and its effect on Monilinia blight control in wild blueberry fields

Monilinia vaccinii-corymbosi (Reade) Honey (M.vc), the causal agent of Monilinia blight of wild blueberry, is controlled primarily by fungicide applications. Demethylation-inhibiting fungicides (DMIs) have been used for over 30 years for Monilinia blight control due to flexibility of use (i.e., ability to use after an infection period) and disease control effectiveness and consistency. In the present study, the sensitivity of ten M.vc isolates to three DMIs- propiconazole, difenoconazole and prothioconazole-desthio were evaluated in vitro by a mycelial growth inhibition assay. In addition, four field trials were conducted during two crop seasons: 2012 and 2013, to examine the efficacy of these DMIs to control Monilinia blight. All the tested DMIs were effective in inhibiting mycelial growth of M.vc isolates, although the mean EC50 values differed significantly. In field experiments, three of four trials had significant treatment effect on disease incidence and severity of vegetative buds. Prothioconazole-desthio and propiconazole provided consistent control against Monilinia blight. Conversely, difenoconazole was effective in in vitro analysis, but did not demonstrate satisfactory Monilinia blight control in all field trials. In the 2012 trials, both prothioconazole-desthio and propiconazole reduced disease incidence of vegetative buds by 100% compared to the untreated control. Prothioconazole- desthio reduced disease development in 2013 with 94 and 99.8% less incidence, and 75 and 99.5% less severity. Similarly, propiconazole also reduced incidence of vegetative buds by 88% and 99.8%, and severity by 54% and 99.7%. No phytotoxic symptoms were observed in any of the field trials. The results of the study serve as a benchmark to monitor shifts in M.vc sensitivity to these fungicides in the future

Acaricidal efficiency of solar 50 % new emulsifiable concentrate formulation against the two-spotted spider mite (TSSM) Tetranychus urticae Koch (Acari: Tetranychidae) under laboratory and greenhouse conditions

Oils are some of the most efficient and secure alternatives to synthetic fungicides, acaricides and insecticides used as pesticides for decades. Around the world, mineral oils are a potential pesticide against many pests. To provide novel active ingredients and new pesticide formulations to the pesticide industry, the major goal of this research was to formulate one of the petroleum fractions and test its acaricidal efficiency against two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae). Solar's physical features were put to the test. Then, it was prepared as an emulsifiable concentrate following the guidelines provided by specialized pesticide organizations for this kind of formulation. The novel formulation was subsequently biologically tested against T. urticae adults in the lab, and it demonstrated good acaricidal activity with an LC50 of 4548 ppm. Under greenhouse conditions, it was also tested against T. urticae immature, adults (males and females) and number of deposited eggs. There was a direct correlation for all stages between concentration, the percentage of immature and adult mortality, and the percentage of egg-hatching inhibition. In the case of the immature, 100% mortality was shown after 7 days of treatment. However, in the case of adult males and females, 100% mortality was shown after 3 days of treatment. Additionally, after 14 days from treatment, it entirely stopped egg depositing. The new formulation might be applied to manage the TSSM.