Cationic gemini lipids containing polyoxyethylene spacers as improved transfecting agents of plasmid DNA in cancer cells

Lipoplex nano-aggregates have been analyzed through biophysical characterization (electrostatics, structure, size and morphology), and biological studies (transfection efficiency and cell viability) in five cancer cell lines. Lipoplexes were prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, constituted by a zwitterionic lipid (DOPE) and a gemini cationic lipid (GCL) synthesized in this work, bis(hexadecyl dimethyl ammonium) oxyethylene], referred to as (C16Am)(2)(C2O)(n), (where n is the oxyethylene spacer length, n = 1, 2 or 3, between the ammonium heads). Cryo-TEM micrographs show nano-aggregates with two multilamellar structures, a cluster-type (at low-to-medium GCL composition) and a fingerprint-type that coexists with the cluster-type at medium GCL composition and appears alone at high GCL composition. SAXS diffractograms show that these lipoplexes present three lamellar structures, two of them coexisting at low and high GCL composition. The optimized transfection efficiency (TE) of pDNA was higher for lipoplexes containing GCLs with a longer (n = 3) or shorter (n = 1) polyoxyethylene spacer, at high GCL composition (alpha - 0.7) with low charge ratio (rho(eff) 2). In the all cancer cell lines studied, the TE of the optimized formulations was much better than those of both lipofectamine 2000 and lipoplexes with GCLs of the bis(hexadecyl dimethyl ammonium) alkane series recently reported. Probably, (a) the coexistence of two lamellar structures at high GCL composition synergizes the TE of these lipid vectors, (b) the orientation of the polyoxyethylene region in (C16Am)(2)(C2O)(3)/DOPE may occur in such a way that the spacing between two cationic heads becomes smaller than that in (C16Am)(2)(C2O)(2)/DOPE which is poor in terms of TE, and (c) the synergistic interactions between serum proteins and (C16Am)(2)(C2O)(n)/DOPE-pDNA lipoplexes containing a polyoxyethylene spacer improve TE, especially at high GCL content. Lipoplexes studied here show very low levels of toxicity, which confirm them as improved vectors of pDNA in gene therapy.

A delocalizable cationic headgroup together with an oligo-oxyethylene spacer in gemini cationic lipids improves their biological activity as vectors of plasmid DNA

Lipoplex nano-aggregates constituted of plasmid DNA (pDNA) pEGFP-C3 and mixed cationic liposomes, consisting of several percentages of a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl imidazolium) oxyethylene series, referred to as (C(16)Im)(2)(C2O)(n), with oxyethylene spacers (n = 1, 2 or 3) between the imidazolium cationic groups and the DOPE zwitterionic helper lipid, have been characterized by various biophysical and biological approaches carried out at several GCL compositions (alpha), and either the mass or the effective charge ratio of the lipoplex. The electrochemical study by zeta-potential confirms that the three GCLs yield a 10% lower effective charge than the nominal one, while compacted pDNA yields only a 25% effective negative charge. The SAXS study reveals, irrespective of the spacer length (n) and effective charge ratio (rho(eff)), the presence of two lamellar structures, i.e., one (L-alpha,L-main) in the whole GCL composition and another (L-alpha,L-DOPE,L-rich) with higher periodicity values that coexists with the previous one at low GCL composition (alpha = 0.2). The cryo-TEM analysis shows two types of multilamellar structures consisting of cationic lipidic bilayers with pDNA sandwiched between them: a cluster-type (C-type) at low alpha = 0.2 and a fingerprint-type (FP-type) at alpha >= 0.5, both with similar interlamellar spacing (d) in agreement with the L-alpha,L-main structure determined by SAXS. Transfection efficacies (TEs) of each lipid mixture were determined in four different cell lines (HEK293T, HeLa, Caco-2 and A549) at several alpha and rho(eff) values in the absence and presence of serum (FBS). The optimized formulations (alpha = 0.2 and rho(eff) = 2.0) substantially transfect cells much better than a commercial transfection reagent, Lipofectamine 2000 and previously studied efficient lipoplexes containing other cationic head groups or spacers both in the absence and presence of serum. The activity of optimized formulations may be attributed to the combination of several factors, such as: (a) the fusogenic character of DOPE which results in higher fluidity of the lipoplexes at alpha = 0.2, (b) the coexistence of two lamellar structures at alpha = 0.2 that synergizes the TE of these lipid vectors, and mainly (c) the higher biocompatibility of the GCLs reported in this work due to the presence of two imidazolium cationic groups together with an oligo-oxyethylene spacer. The length of the spacer in the GCL seems to have less impact, although (C(16)Im)(2)(C2O)(n)/DOPEpDNA lipoplexes with n = 1 and 3 show higher gene transfection than n = 2. All the optimum formulations reported herein are all highly efficient with negligible levels of toxicity, and thus, may be considered as very promising gene vectors for in vivo applications.

气流化学激光理论研究的若干进展

以超声速HF/DF化学激光和超声速氧碘化学激光(COIL)为代表的气流化学激光(GCL),因其科学意义、军事和工业应用价值,近30多年来得到了突飞猛进的发展.由于超声速膨胀混合流在控制强放热反应动力学和热力学过程方面的特殊本领,使气体动力学在高功率GCL的发展中起着关键性的作用.高功率GCL性能的分析计算自然也沿用非平衡气体动力学的方法,假定气流(包括激光能级分子和原子)为连续介质,谱线为均匀加宽,并联立求解气体动力学方程组,增益动力学和基于光强迭加原则的辐射传输诸方程,称为速率方程(RE)模型.20世纪70年代后期又提出和发展了GCL性能计算的半气体动理学(SGK)模型,在SGK模型中仍假定气流为连续介质,但同时考虑了激光能级分子微观热运动的贡献,谱线加宽的非均匀加宽效应,并用双参数摄动法求解激光能级分子速度分布函数方程组(即广义Boltzmann方程组),因此SGK模型是一个同时考虑宏观和微观尺度运动的跨尺度模型.本文综述RE模型和SGK模型以及用它们预测GCL性能的若干研究进展,同时简评等增益模型和腔模(模图样)理论研究的一些进展.最后从气体动力学的角度提出一些值得进一步研究的课题.

Postnatal development of the retina in root vole Microtus oeconomus

Vision plays an important role in the living habits of animals, especially in feeding. We investigated the postnatal development of retina in root vole Microtus oeconornus. The result shows that the retina of the M. oeconornus is very primitive before postnatal day （PD） 3. The neuroblastic layer does not differentiate and makes up more than half of the retina layer. The outer plexiform layer （OPL） first comes into existence at PDS. At PD6, as the presence of the OPL becomes obvious, the outer nuclear layer （ONL） and inner nuclear layer （INL） are much clearer. At PD18, the retina is similar to an adult retina and each layer becomes distinct. The thickness and cell density of the ganglion cell layer （GCL） and ONL during different postnatal days were also examined. These results show that the thickness and density of ONL increase during ontogeny, while the thickness and density of GCL decrease. Compared with Rattus norvegicus, Apodemus agrarius , Cricetulus triton, Microtus mandarinus , Myospalax cansus , Spermophilus dauricus and Sciurotamias davidianus, the histological structure of the retina of M. oeconornus is between that of nocturnal and diurnal rodents.

Sustainability study of the application of geosynthetic clay liners in hostile and aggressive environments

This paper discusses the sustainable performance of geosynthetic clay liners (GCLs) which are popularly specified as “leachate retaining” or as “water proofing” membranes in the geo-environmental construction industry. Geosynthetic clay liners (GCLs) are composite matting comprising of bentonite clay with two covering geosynthetics. These are innovative labour saving construction material, developed over the last three decades. The paper outlines the variety of Geosynthetic Clay Liners (GCLs) can be classified essentially into two distinctly different forms viz; (a) air dry (< 8% m/c) with granular or powdered bentonite or (b) bentonite cake factory prehydrated to a moisture content (~40% m/c) beyond its shrinkage limit and vacuum extruded as a clay cake to enhance its sustainable performance. The dominant mineral in bentonite clay is the three-layered (2:1) clay mineral montmorillonite. High quality bentonites need to be used in the GCL manufacture. Sodium montmorillonite has the desired characteristic of high swelling capacity, high cation exchange capacity and the consequently very low hydraulic conductivity, providing the basis for the hydraulic sealing medium in GCLs. These encapsulate the active montmorillonite clay minerals which depend on the water and chemical balance between the sealing element and the surrounding geo environment. Quantitative mineralogical analyses and an assessment of the adsorbed cation regime, diffusion coefficients and clay leachate compatibility must necessarily be an integral part of the site appraisal to ensure acceptable long term sustainability and performance. Factors influencing the desired performance of bentonite in the GCLs placed in difficult construction and hostile chemical environments are discussed in this paper. Accordingly, the performance specifications for GCLs are identified and the appropriateness of enhancing the cation exchange capacity with polymer treatment and the need for factory prehydration of the untreated sodium bentonite is emphasised. The advantage of factory prehydrating the polymer treated bentonite to fluid content beyond its shrinkage limit and subsequently factory processing it to develop laminated clay is to develop a GCL that has enviable sealing characteristics with a greater resistance to geochemical attack and cracking. Since clay liners are buried in the ground as base liners, capping layer or as structural water proofing membrane, they can easily avoid strict quality and performance monitoring being “out of sight, out of mind!”. It is very necessary that barrier design for leachate containment must necessarily be in accordance with legislative requirement Assessment of long term hydraulic conductivities and clay-leachate compatibility assessment is deemed necessary. The derogatory factors affecting the sustainable performance of the bentonite in GCLs placed in difficult construction and hostile chemical environments are discussed. Sustainability concepts incorporated in waste management practice must aim to achieve 100% recycling and fully implement the handling of solid waste in developing countries with relatively lower labour costs.

Mutation screening of the glutamate cysteine ligase modifier (GCLM) gene in patients with schizophrenia.

BACKGROUND: Experimental evidences show that glutathione and its rate-limiting synthesizing enzyme, the glutamate-cysteine ligase (GCL), are involved in the pathogenesis of schizophrenia. Furthermore, genetic association has been previously reported between two single nucleotide polymorphisms lying in noncoding regions of glutamate cysteine ligase modifier (GCLM) gene, which specifies for the modifier subunit of GCL and schizophrenia. OBJECTIVE: We wanted to investigate the presence of GCLM true functional mutations, likely in linkage disequilibrium with the previously identified single nucleotide polymorphism alleles, in the same set of cases that allowed the detection of the original association signal. METHODS: We screened all the coding regions of GCLM and their intronic flanking vicinities in 353 patients with schizophrenia by direct DNA sequencing. RESULTS: Ten sequence variations were identified, five of which were not previously described. None of these DNA changes was within the GCLM coding sequence and in-silico analysis failed to indicate functional impairment induced by these variations. Furthermore, screening of normal controls and downstream statistical analyses revealed no significant relationship of any of these DNA variants with schizophrenia. CONCLUSION: It is unlikely that functional mutations in the GCLM gene could play a major role in genetic predisposition to schizophrenia and further studies will be required to assess its etiological function in the disease.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Cambios maculares y de capa de fibras nerviosas ganglionares posterior a la vitrectomía pars plana y sus factores pronósticos en la fundación oftalmológica nacional

OBJETIVOS: Determinar los factores pronóstico, cambios maculares morfológicos y de capa de fibras nerviosas ganglionares posterior a vitrectomía pars plana, en la Fundación Oftalmológica Nacional. MATERIALES Y MÉTODOS: Estudio longitudinal de antes y después (3y6 meses) de la vitrectomía pars plana (VPP) en pacientes con membrana epirretiniana, agujero macular, síndrome de tracción vítreo macular y opacidades vítreas no-inflamatorias. Se realizó seguimiento clínico y con tomografía de coherencia óptica. RESULTADOS: Grupo de 60 pacientes (mujeres 65.0%), edad promedio 65.45+9.49años y tiempo de enfermedad promedio 23+29.79meses. Las indicaciones de VPP (n=60ojos) fueron agujero macular (38.3%) y membrana epirretiniana (36.7%). Se encontró diferencia significativa entre grosor del complejo capa de células ganglionares (CCG)+capa plexiforme interna (CPI) inicial y 3 meses (p=0.039), correlación entre grosor del complejo CCG+CPI al tercer y sexto mes (r=0.704,p<0.001) y grosor del complejo CCG+CPI al tercer mes con grosor foveal central (CFT) al tercer y sexto mes (r=–0.594,p<0.001 y r=–0.595,p=0.001). Mayores de 65años tenían menor grosor de CFNG a 6meses (r=-0.528,p=0.007). El grosor de CFNG promedio y la presencia de la zona elipsoide inicial fueron factores pronósticos de buena agudeza visual al tercer mes de VPP (r2=0.414,p=0.018, y r2=0.414,p=0.010). CONCLUSIÓN: El grosor de CFNG y la presencia de la zona elipsoide inicial tienen alta capacidad predictiva de buena agudeza visual al tercer mes de VPP, y, correlación inversa entre grosor del complejo CCG+CPI con CFT al tercer y sexto mes de VPP.

The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor-mediated signalling

Key point summary • Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells. • Mutant mice with deficits in Ca2+ channel auxiliary α2δ-2 subunits are used as models of cerebellar ataxia. • Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing. • We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter-matched control mice. • These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ-2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias. Abstract Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’ du2J mouse model of ataxia and absence epilepsy represents a clean knock-out of the auxiliary Ca2+ channel subunit, α2δ-2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell (GC) layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone-Purkinje cell(IN-PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN-PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)-mediated modulation of spontaneous neuronal spike firing and CB1Rmediated presynaptic inhibition of synaptic transmission at IN-PC synapses in both +/du2J and du2J/du2J mutants; effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

TRPV1 receptors are involved in protein nitration and Muller cell reaction in the acutely axotomized rat retina

We report here the protein expression of TRPV1 receptor in axotomized rat retinas and its possible participation in mechanisms involved in retinal ganglion cell (RGC) death. Adult rats were subjected to unilateral, intraorbital axotomy of the optic nerve, and the retinal tissue was removed for further processing. TRPV1 total protein expression decreased progressively after optic nerve transection, reaching 66.2% of control values 21 days after axotomy. The number of cells labeled for TRPV1 in the remnant GCL decreased after 21 days post-lesion (to 63%). Fluoro-jade B staining demonstrated that the activation of TRPV1 in acutely-lesioned eyes elicited more intense neuronal degeneration in the GCL and in the inner nuclear layer than in sham-operated retinas. A single intraocular injection of capsazepine (100 mu M), a TRPV1 antagonist, 5 days after optic nerve lesion, decreased the number of GFAP-expressing Muller cells (72.5% of control values) and also decreased protein nitration in the retinal vitreal margin (75.7% of control values), but did not affect lipid peroxidation. Furthermore, retinal explants were treated with capsaicin (100 mu M), and remarkable protein nitration was then present, which was reduced by blockers of the constitutive and inducible nitric oxide synthases (7-NI and aminoguanidine, respectively). TRPV1 activation also increased GFAP expression, which was reverted by both TRPV1 antagonism with capsazepine and by 7-NI and aminoguanidine. Given that Muller cells do not express TRPV1, we suppose that the increased GFAP expression in these cells might be elicited by TRPV1 activation and by its indirect effect upon nitric oxide overproduction and peroxynitrite formation. We incubated Fluorogold pre-labeled retinal explants in the presence of capsazepine (1 mu M) during 48 h. The numbers of surviving RGCs stained with fluorogold and the numbers of apoptotic cells in the GCL detected with TUNEL were similar in lesioned and control retinas. We conclude that TRPV1 receptor expression decreased after optic nerve injury due to death of TRPV1-containing cells. Furthermore, these data indicate that TRPV1 might be involved in intrinsic protein nitration and Muller cell reaction observed after optic nerve injury. (C) 2010 Elsevier Ltd. All rights reserved.

Monopoly power and trade protection: cross- industry evidence

This paper investigates the impact of industry concentration on trade policy. Annual panel-databases of Brazilian industries for the years 1988 through 1994 were used. The regressions reported here are robust to openness indicator. concentration index, control variables and sample size, and suggest that the higher the concentration of a given industry the higher its leveI of trade protection. In the period of study the country experienced a major trade liberalization, but the results in the paper show that the reduction in protection was smaller in more concentrated sectors. Assuming that concentration is ;1 gCl()d proX\' for mOllopoh' po\\'er as it reduces the free-rider problem in coordinating a lobby the results in this paper indicates that interest groups with control over specific markets in fact are able to obtain policy advantages that reduce (international) competition.

Bundle Block Adjustment of CBERS-2B HRC Imagery Combining Control Points and Lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Uso da lidocaína isolada ou associada à quetamina ou ao butorfanol, em anestesia epidural em cães: avaliação cardiorrespiratória e analgésica

Pós-graduação em Anestesiologia - FMB

Efetividade de duas soluções desinfetantes e da irradiação por micro-ondas na desinfecção de próteses totais contaminadas com staphylococcus aureus resistente à meticilina (MRSA)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)