Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Dysregulation of autophagy by HIV-1 Nef in human astrocytes

Viruses often exploit autophagy, a common cellular process of degradation of damaged proteins, organelles, and pathogens, to avoid destruction. HIV-1 dysregulates this process in several cell types by means of Nef protein. Nef is a small HIV-1 protein which is expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HIV-Associated Neurocognitive Disorders (HAND). In order to explore its effect in the CNS with respect to autophagy, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus vector (Ad-Nef). We observed that Nef expression triggered the accumulation of autophagy markers, ATG8/LC3 and p62 (SQSMT1). Similar results were obtained with Bafilomycin A1, an autophagy inhibitor which blocks the fusion of autophagosome to lysosome. Furthermore co-expression of tandem LC3 vector (mRFP-EGFP-LC3) and Ad-Nef in these cells produced mainly yellow puncta (mRFP+, EGFP+) strongly suggesting that autophagosome fusion to lysosome is blocked in PHFA cells in the presence of Nef. Together these data indicate that HIV-1 Nef mimics Bafilomycin A1 and blocks the last step of autophagy thereby helping HIV-1 virus to avoid autophagic degradation in human astrocytes.     

Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10(-6) to 10(-1) mol L(-1) urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.     

Potential biofuel additive from renewable sources--Kinetic study of formation of butyl acetate by heterogeneously catalyzed transesterification of ethyl acetate with butanol

Butyl acetate holds great potential as a sustainable biofuel additive. Heterogeneously catalyzed transesterification of biobutanol and bioethylacetate can produce butyl acetate. This route is eco-friendly and offers several advantages over the commonly used Fischer Esterification. The Amberlite IR 120- and Amberlyst 15-catalyzed transesterification is studied in a batch reactor over a range of catalyst loading (6–12 wt.%), alcohol to ester feed ratio (1:3 to 3:1), and temperature (303.15–333.15 K). A butanol mole fraction of 0.2 in the feed is found to be optimum. Amberlite IR 120 promotes faster kinetics under these conditions. The transesterifications studied are slightly exothermic. The moles of solvent sorbed per gram of catalyst decreases (ethanol > butanol > ethyl acetate > butyl acetate) with decrease in solubility parameter. The dual site models, the Langmuir Hinshelwood and Popken models, are the most successful in correlating the kinetics over Amberlite IR 120 and Amberlyst 15, respectively.     

Variation in volatiles of Astragalus gombiformis Pomel

The essential oils obtained by hydrodistillation (Clevenger apparatus) from aerial parts of Astragalus gombiformis were analysed by gas chromatography coupled with mass spectrometry (GC/MS). This study showed that the A. gombiformis essential oils are complex mixtures of important natural compounds, which varied qualitatively and quantitatively between cultivated and wild plants and between phenological stages of development. All analysed oils are characterized by the constant presence of phytol, 6,10,14-trimethyl-2-pentadecanone, 4-terpineol, and gamma-terpinene. This study is the first report on the chemical composition of essential oils from A. gombiformis and indicates that these oils should be more studied.     

Synthesis and evaluation of library of betulin derivatives against the botulinum neurotoxin A protease

Botulinum neurotoxins (BoNTs) are the most toxic proteins currently known. Current treatments for botulinum poisoning are all protein based with a limited window of opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a new therapeutic strategy for the treatment of botulism as it may provide an effective post-exposure remedy. As such, a small library of 40 betulin derivatives was synthesized and screened against the light chain of BoNT serotype A (LC/A); five positive hits (IC₅₀ <100 μM) were uncovered. Detailed evaluation of inhibition mechanism of three most active compounds revealed a competitive model, with sub-micromolar K_i value for the best inhibitor (7). Unfortunately, an in vitro cell-based assay did not show any protection of rat cerebellar neurons against BoNT/A intoxication by 7.     

Ubiquitously expressed hematological and neurological expressed 1 downregulates Akt-mediated GSK3β signaling, and its knockdown results in deregulated G2/M transition in prostate cells

As the molecular mechanism of β-catenin deregulation is not well understood, and stabilized β-catenin is known to translocate into the nucleus and activate genes for proliferation, a novel regulatory factor, hematological and neurological expressed 1 (HN1), for Akt-GSK3β-β-catenin axis is reported here. In our studies, HN1 gene structure was characterized. HN1 expression was found to be epidermal growth factor-responsive in PC-3 cells, and protein expression was also upregulated in PC-3 and LNCaP but not in DU145 cells. Additionally, HN1 was found to be downregulated by the specific AKT inhibitor wortmannin but not with PI3K or MAPK inhibitors, LY294002 and PD98059, respectively, in PC-3 and MCF-7 cells. Further, siRNA-mediated knockdown of HN1 resulted in considerable increase in Akt((S473)) and GSK3β((S9),(Y216)) phosphorylations; moreover, subsequent accumulation of β-catenin, increase in c-myc expression, and nuclear accumulation of cyclin D1 were observed in PC-3 cells. Knockdown of HN1 also resulted in prolongation of G(1) phase in cell cycle, increasing tetraploidy, presumably because of cells escaping from abnormal mitosis in PC-3 cells. Consistently, overexpression of HN1 reversed the cell-cycle-specific observations, resulted in accumulation of cells in G(2)/M, and reduced the proliferation rate, which were investigated using flow cytometry and methylthiazol tetrazolium assays. As activating mutations of β-catenin have been demonstrated in late-stage tumors, and β-catenin stabilization was correlated with poor prognosis in previous reports, epidermal growth factor-upregulated HN1 expression might have a role in deregulating the AKT-GSK3β((S9))-mediated signaling as a novel compensating mechanism.     

Formulation and Stability Testing of Itraconazole Crystalline Nanoparticles

Itraconazole (ITZ) crystalline nanoparticles were prepared using relatively simple, low-cost sonoprecipitation technique, in which both the solvent and antisolvent were organic in nature. The effect of stabilizer type (hydroxypropyl methylcellulose, hydroxypropyl cellulose, Inutec SP1®, and pluronic F127), drying method (oven and freeze drying) and matrix former used (Avicel PH101, and Aerosil®200) on the dissolution performance as a key characteristic of nanocrystals was evaluated. In 10 min, all of the prepared nanocrystals showed 3.77−8.59 times improvement in percent drug dissolved compared to pure ITZ. Concerning the effect of stabilizer type, the following rank order can be given: pluronic F127 ≥ hydroxypropyl cellulose ≥ hydroxypropyl methylcellulose (HPMC) > inutec SP1. Freeze-dried ITZ nanocrystals containing Avicel PH 101 showed better dissolution rate compared to other nanocrystals. The chemical structure of itraconazole nanocrystals was not changed as revealed by Fourier transform infrared. Stability study of selected nanocrystals (F5, F7, and F8) revealed physical and chemical stability of F7 and F8, while a decrease in dissolution rate of F5 was observed (although being chemically stable) when stored under high relative humidity conditions. Although inutec is less potent than pluronic F127 and HPMC regarding their effect on dissolution rate enhancement, it is equipotent to pluronic F127 in preserving the rapid drug dissolution.Key words: itraconazole, nanocrystals, nanoparticles, stability study     

VIP and endothelin receptor antagonist: An effective combination against experimental pulmonary arterial hypertension

Background

Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy?

Methods

Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival.

Results

Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days.

Conclusions

1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.     

Effect of hydroxytyrosol‐rich preparations on phenolic‐linked antioxidant activity of seeds

Hydroxytyrosol‐rich extract (HRE) and hydroxytyrosol‐rich olive mill wastewater (HROMW) were used as exogenous growth enhancers to stimulate tomato seedling vigor. The tomato seeds soaking in 10% w/v HROMW or HRE solutions were optimum in maximally enhancing seedling performance according to biochemical seed vigor parameters. Biochemical parameters as the average glucose‐6‐phosphate dehydrogenase (G6PDH) activity in HRE‐treated seeds (915.11 nmoles min^?1 mg^?1 protein) was higher than control (629.58 nmoles min^?1 mg^?1 protein) and correlated with the increased phenolic content (3530 μg g^?1 fw) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH)‐based antioxidant activity (70.60%), respectively. Some key enzymes, guaiacol peroxidase (GPX) (6100.65 nmoles min^?1 mg^?1 protein) and catalase (2.04 μmoles min^?1 mg^?1 protein), were also higher in response to treatments and correlated with enhanced phenolic content and antioxidant activity. This study supports the hypothesis that the exogenous phenolic application stimulates the pentose phosphate pathway through an over‐expression of endogenous phenolic synthesis and an increase in free‐radical scavenging antioxidant activity. Therefore, the current study indicates the enhancement of seed vigor by HRE especially and HROMW as reflected by the stimulation of biochemical responses.