Uso de plasma rico em plaquetas em queimaduras Modelo experimental em ratos

As queimaduras são eventos comuns e recorrentes no dia a dia dos atendimentos médicos. Há uma constante busca para entender a sua fisiopatologia, no intuito de minimizar seus resultados devastadores. Plasma rico em plaquetas (PRP) é um concentrado de plaquetas com capacidade de liberação local de múltiplos fatores de crescimento (FC) que aceleram a cicatrização. Este estudo consiste em dois experimentos: o primeiro visa validar um modelo experimental para a criação de queimaduras de tamanho e profundidade padronizados e, o segundo, avalia o uso de PRP em queimaduras. Para o desenvolvimento de queimaduras na validação do modelo experimental, foi idealizado um equipamento que permitisse o controle preciso da temperatura, além da utilização em conjunto de uma técnica inovadora de fixação que garante pressão constante no momento das queimaduras. Para o primeiro experimento, foram utilizados 12 ratos, por grupo, submetidos a queimaduras de 60 oC, 70 oC ou 80 oC por dez segundos, com um equipamento que desenvolvemos. Metade dos animais de cada grupo foi morta no terceiro dia e suas feridas foram analisadas por histologia, e, na outra metade, a ferida foi mensurada e acompanhada até o seu fechamento. No uso de PRP em queimaduras, foram avaliadas queimaduras de segundo grau (SG), segundo grau com diabetes mellitus induzido (SGD) e queimaduras de terceiro grau (TG). Noventa animais foram distribuídos em três grupos (SG, SGD e TG), onde, em cada um, dez animais foram tratados, dez serviram de controle e dez foram utilizados para o preparo do PRP. As áreas das feridas foram acompanhadas até o vigésimo primeiro dia, quando os animais foram mortos e biópsias de pele foram realizadas. Os resultados da validação do modelo mostram que as queimaduras produzidas com 60 oC foram de SG superficial (28% da derme envolvida); com 70 oC foram de SG profundo (72% da derme envolvida); e com 80 oC foram de TG (100% da derme envolvida). Em relação ao uso de PRP em queimaduras, observou-se que nos grupos tratados SG e SGD houve aceleração do fechamento da ferida e redução no número de células CD31, CD163, CD68, MPO e TGF-β positivas, e aumento do número de células MMP2 positivas. A neoepiderme foi mais fina nos controles dos grupos SG e SGD, e o tecido de granulação foi reduzido nos controles SGD e TG. O modelo utilizado é seguro e confiável para produzir queimaduras regulares e uniformes, de diâmetros variados, pela capacidade do controle fino da temperatura e pelo posicionamento do animal, e reprodutíveis. PRP parece acelerar a cicatrização de queimaduras de SG e SGD, mas não de TG.

水稻中一个编码富含脯氨酸蛋白基因家族的表达和功能研究

OsPRP1是水稻中特有的，编码一类富含脯氨酸蛋白（proline-rich protein, PRP）的基因家族，该家族有4个成员。在GenBank中，仅找到了一个结构相对接近的玉米PRP蛋白，但是对它的研究仅限于序列上的某些描述，而且在序列上，仍然与这4个OsPRP1蛋白有明显的差别。OsPRP1蛋白明显有别于前人（1999）所描述的4类PRP蛋白。它们是植物中一类新的小分子量的PRP蛋白，仅有一个保守结构域而属于DUF1210蛋白超家族的成员。 四个OsPRP1基因在基因组中紧密串联排列，而且编码区高度保守，基因结构也高度一致，证明它们来源于同一个祖先基因，是通过基因复制的方式产生的。PAML分析也证明它们在进化时间上的相互距离并不远。而在进化过程中由于执行生理功能上的某种重要性，在编码区表现出很的保守性，因而很难在RNA水平和蛋白水平上研究四个OsPRP1基因的表达差异。但是它们在表达调节区（如启动子区）显示出明显的差异。在克隆启动子的基础上，用GUS报告基因的策略，没有检测到它们在拟南芥中的表达活性，说明它们具有一定的种属特异性。而在水稻中，这四个基因的表达显示出了明显的时序和空间差异，既表现出在组织器官及其发育阶段上的特异性和变化，又在一定程度上表现出交叉，出现了功能上的分化和重叠。根据启动子区上的顺式元件，检测了这些基因对6种植物生长调节物质和3种非生物胁迫因子的应答反应，证明它们的表达调节也表现出显著的差异和分化。因此，四个基因的进化出现了功能退化、亚功能化和产生新功能等多种命运，比理论模型预期的要复杂得多，可能在执行生理功能和对环境反应上具有各自的生物学意义。 植物细胞壁是多种碳水化合物和蛋白质相互交织在一起的亲水性网络。在保护和支撑原生质、细胞通信、细胞分化、细胞对生物的和非生物胁迫的抗性等方面发挥着重要作用。目前已知的大多数植物PRP蛋白被描述成细胞壁结构性蛋白。OsPRP1基因家族编码的蛋白质都有一个N-端信号肽，暗示它们可能是一类分泌蛋白。我们用OsPRP1.1::GFP融合蛋白进行了亚细胞定位，证明了OsPRP1蛋白的确也是细胞壁相关蛋白。用原核表达体系表达了GST-融合蛋白，制备抗体，通过免疫印迹证明这些蛋白不溶于温和的提取缓冲溶液中，但可以被高盐和强碱溶液溶解，且分子量增加了三倍。证明在体内可能出现修饰、与细胞壁其它组分相交联的现象。 在这四个基因中，只有OsPRP1.2能够在水稻根中特异表达。根的原位杂交实验证明，OsPRP1在分化成熟程度低的细胞中大量表达，而在分化成熟程度高的细胞中几乎不表达。GUS染色的结果同样发现，基因在维管柱特别是中柱鞘附近的薄壁细胞的表达比别的细胞要强得多，而且在根的生长方向上表现出与发育相关的表达特征。说明OsPRP1基因可能参与了这些细胞的分化、发育过程。而基因表达的组织器官特异性的差异和对不同刺激因素的不同反应意味着它们可能参与了多种生理过程。为此构建了一个RNAi的表达载体，转化水稻，Southern杂交实验得到9个单位点插入的T2代独立株系，其中有6个株系与野生型对照相比，主根的早期伸长受到显著抑制，主根的一级侧根地数量减少，根尖分生区的细胞在轴向上的伸长受到了抑制。结合表达定位和原位杂交的结果，我们对它们在植物生长发育中的功能进行了深入探讨。

典型草原优势植物养分回收特性对养分和水分梯度变化的响应

养分回收是植物衰老时，养分从衰老组织向活的组织体转移的一种过程。该过程延长了养分在植物体内的滞留时间，提高了植物保持养分的能力，因此是植物适应养分贫瘠生境的策略之一。全球气候变化，包括降水格局改变和氮（N）沉降增加，改变了生态系统正常的生物地球化学循环，因此可能会对植物的养分回收特性产生影响。研究不同N、磷（P）、水梯度上，植物物种养分回收特性的响应格局，对于预测N沉降增加和降水格局改变对物种养分利用策略的潜在影响，具有一定的理论与实践意义。本研究以中国科学院植物研究所多伦恢复生态学实验站长期施N肥实验（0，1，2，4，8，16，32，64 g N m-2 yr-1等8个水平）为研究平台研究了克氏针茅（Stipa krylovii）群落中优势植物养分回收随N素添加梯度的变化，同时结合三个盆栽控制实验（施N肥实验：0，0.5，1，2，4，8，16，32，64，128 g N m-2等10个水平;施P肥实验：梯度同施N肥实验;控水实验：3600，4000，4500，5143，6000，7200，9000，12000，18000，36000 mL pot-1等10个水平），主要探讨了羊草（Leymus chinensis）养分回收效率（从衰老组织中回收转移的养分百分数，RE）和养分回收度（以枯叶中养分浓度衡量，RP）以及其它叶片养分特性(绿叶养分浓度和比叶面积SLA)对环境因子改变的响应格局。同时，我们还调查了枯叶C:N比和C:P比等参数，研究环境因子改变对凋落物分解的影响。 1）连续4年施N肥显著降低了五个物种叶片N素回收度（NRP），对P素回收度（PRP）的影响在各物种间差异较大，但低N提高了多数物种的PRP;物种间，冷蒿（Artemisia frigida）RP（枯叶N和P浓度分别为14.3±2.0 mg g-1和0.68±0.09 mg g-1）最低，砂韭（Allium bidentatum）（N：5.2±0.2 mg g-1，P：0.12±0.01 mg g-1）最高。沿施N梯度，N素回收效率（NRE）的变化趋势在物种间差异较小但在方法间（叶干重水平，叶面积水平和单株水平）差异较大，而P素回收效率（PRE）的变化在物种间和方法间差别都较大。叶干重水平和叶面积水平上，NRE在四个物种中表现出显著降低的趋势，PRE只在糙隐子草（Cleistogenes squarrosa）和星毛委陵菜（Potentilla acaulis）中显著降低，其它三个物种变化不显著。单株水平上，所有物种NRE（除了克氏针茅）和PRE均与施N量梯度无显著性关系。物种间，砂韭的RE最高（＞80.0%），冷蒿和星毛委陵菜最低（＜60.0%）。方法间，叶片水平上的RE均高于单株水平上。沿施N肥梯度，两个禾本科物种SLA无显著变化规律，而其它三个物种SLA表现出先增加后变化不大的趋势。物种间，最高和最低的SLA分别表现在冷蒿和克氏针茅。沿施N肥梯度，五个物种C:N比呈先显著降低后缓慢降低的趋势。物种间，最大和最小的C:N比分别出现在砂韭和冷蒿。 2）盆栽施N肥实验中，一定范围内，施N肥显著提高了羊草地上地下生物量、SLA和绿叶N浓度，显著降低了C:N比、NRP、NRE和PRE，但对绿叶P浓度、叶片PRP和C:P比无明显影响。平均枯叶N浓度和枯叶P浓度分别为16.2 mg g-1和1.01 mg g-1，平均NRE和PRE分别为46.1﹪和58.1﹪。10月时，地下生物量和养分积累均高于地上部分。 3）盆栽施P肥实验中，一定范围内，施P肥显著地提高了羊草地上地下生物量、SLA、绿叶N浓度和绿叶P浓度，显著降低了C:N比、C:P比、NRP、PRP和PRE，但对NRE无显著影响。平均枯叶N浓度和枯叶P浓度分别为9.9 mg g-1和7.43 mg g-1，平均NRE和PRE分别为58.2﹪，平均PRE为56.1﹪。10月时地下部分生物量和N库积累均高于地上部分，而P库在两个部分间差别不大。 4）盆栽控水实验中，一定范围内，供水量增加显著增加了羊草地上地下生物量、SLA、NRP、PRP、PRE、C:N比和C:P比，显著降低了绿叶N浓度，但对绿叶P浓度和NRE无显著性影响。平均枯叶N浓度和枯叶P浓度分别为10.4 mg g-1和0.32 mg g-1，平均NRE和PRE分别为54.4﹪和76.8﹪。10月时，地下部分生物量和养分积累均高于地上部分。 以上结果表明，N、P和水分因子的改变影响了植物生物量和养分分配、叶片养分特性、养分回收能力以及枯叶分解质量等，且不同梯度影响程度也不同。因此，未来全球变化包括N沉降增加和降水格局改变可能影响植物养分利用策略和凋落物分解特性，进而可能对植被-土壤系统养分循环产生影响。

分光光度法测定单一稀土元素的研究

“分光光度法测定单一稀土元素的研究”论文包括三部分内容：一、用导数分光光度法测定单一稀土元素。研究了稀土离子－钛铁试剂水溶液体系的三阶及四阶导数吸收光谱。在0.05-0.3N的NaOH溶液中，Pr、Nd、Sm、Eu、Dy、Ho、Er、Tm与钛铁试剂形成具有f-f跃迁吸收光谱的络合物。在350-810nm波长区间它们有各自的特征吸收峰。其高阶导数光谱不仅灵敏度增大了许多倍，且选择性也有较大的提高。如对钕的测定是特效的，而其它稀土之间虽然存在着程度不同的干扰，但用校正系数法均可方便地扣除这些干扰。本体系的常规吸收光谱的灵敏度是高氯酸体系的1.5-19.5倍，三阶导数（半峰）灵敏度是高氯酸盐的常规吸收光谱法的6-166倍，四阶导数则为其的22-401倍。如以峰－峰法测量四阶导数光谱，铒的导数摩尔吸光系数已达3.62 * 10~3。温度对导数光谱略有影响，但可以通过简单方法校正。本文拟定了用稀土－钛铁试剂的高阶导数光度法测定混合稀土中Pr、Nd、Sm、Dy、Ho、Er、Tm的方法，和文献报导的导数光度法相比，本法具有灵敏度高、分析的元素多，不用有机溶剂等优点，适合于各种混合稀土氧化物中Pr、Nd、Sm、Dy、Ho、Er、Tm的准确快速测定。二、钸－钛铁试剂显色反应机理的探讨及混合稀土中钸和铕的测定。钛铁试剂与钸、铕有形成在可见光谱区域具有光谱吸收的络合物。铕的络合物在420nm处的摩尔吸光系数为75。本文着重对于钸和钛试剂的显色反应机理进行了探讨。通过各种实验证实只有四阶钸和钛铁试剂形成红色络合物（其最大吸收峰位于500nm，摩尔吸光系数为4128）。而三阶钸与钛铁试剂仅能形成无色络合物。但在碱性介质中， 空气或浓度中的氧可将三阶钸的络合物迅速氧化为四价钸的红色络合物。因此，不需要预先将三价钸氧化后再进行显色，可直接在形成三价钸的络合物后，再通过空气氧化来显色。从而提出了分光光度法准确快速测定混合稀土中百分之十到万分之几的钸的简便方法。此外，拟定了在微酸性溶液中分光光度法测定混合稀土中铕的分析方法，除大量的钸和镱外，其它稀土对铕的测定无干扰。三、计算光度分析法测定单一稀土元素的探讨。本工作采用三个显色体系（二甲酚橙、偶氮氯膦III、均三氯偶氮胂），达到了用计算光度分析法同时测定多个单一稀土元素的目的。比较了全主元高斯消长法、求病态方程组解的方法、抛物线拟合法，黄金分割法，BFS变R度法，布罗伊佥变R度法，梯度牛顿法、PRP其轭梯度法、FR共轭梯度法、包维尔算法、可变多面体方法，线性规划改进的单纯形法，可变误差多面体方法，网格法，复合形法等十七个计算方法的结果，着重探扩充了测定点数、波长位置、目标函数、约束条件、组分数目、组分含量范围及实验误差、一个体系和多个体系对结果的影响。获得了一些有指导意义的经验规律。其中以抛物线拟合、黄金分割、可变误差多面体等方法较为优越。合成样品五组分和八组分混合稀土氧化物分析获得较为满意的结果。

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

CDNA cloning and mRNA expression of the lipopolysaccharide- and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri

Lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP) play a crucial role in the innate immune response of invertebrates as a pattern recognition protein (PRP). The scallop LGBP gene was obtained from Chlamys farreri challenged by Vibrio anguillarum by randomly sequencing cDNA clones from a whole body cDNA library, and by fully sequencing a clone with homology to known LGBP genes. The scallop LGBP consisted of 1876 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 440 amino acids with the estimated molecular mass of 47.16 kDa and a predicted isoelectric point of 5.095. The deduced amino acid sequence showed a high similarity to that of invertebrate recognition proteins from blue shrimp, black tiger shrimp, mosquito, freshwater crayfish, earthworms, and sea urchins, with conserved features including a potential polysaccharide-binding motif, a glucanase motif, and N-glycosylation sites. The temporal expression of LGBP genes in healthy and V. anguillarum-challenged C farreri scallop, measured by real-time semiquantitative reverse transcription polymerase chain reaction (PCR), showed that expression was up-regulated initially, followed by recovery as the stimulation cleared. Results indicated that scallop LGBP was a constitutive and inducible acute-phase protein that could play a critical role in scallop-pathogen interaction. (C) 2004 Elsevier B.V. All rights reserved.

Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing microinvaders as a pattern recognition protein (PRP). The cDNA of Zhikong scallop Chlamys farreri C-type lectin (designated CFLec-1) was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of CFLec-1 was 1785 bp, consisting of a 5'-terminal untranslated region (UTR) of 66 bp and an unusually long 3' UTR of 1040 bp with seven polyadenylation signal sequences AATAAA and a poly(A) tail. The CFLec-1 cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15 amino acid residues and a mature protein of 206 amino acids. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deduced amino acid sequence. The expression pattern of CFLec-1 transcripts in healthy and bacterial challenged scallops was studied by semi-quantitative RT-PCR. mRNA transcripts of CFLec-1 could be mainly detected in the tissues of haemocytes, gill, gonad and mantle of unchallenged scallops, whereas the expression of CFLec-1 transcripts was increased in all the tested tissues after heat-killed Vibrio anguillarum challenge. The temporal expression of CFLec-1 mRNA in haemolymph challenged by Micrococcus luteus and V anguillarum was both up-regulated and reached the maximum level at 8 and 16 It post stimulation, respectively, and then dropped back to the original level. In order to investigate its immune functions, CFLec- I was recombined and expressed in Escherichia coli BL21(DE3)-pLysS as a fusion protein with thioredoxin. The recombinant CFLec-1 agglutinated bacteria E. coli JM109 in vitro, and the agglutination was Ca2+ dependent which could be inhibited by EDTA. But it did not agglutinate M. luteus, Candida lipolytica and animal erythrocytes including rabbit, rat, mouse, chicken, human group A, human group B, human group O. Meanwhile, the recombinant CFLec-1 could inhibit the growth of both E. coli JM 109 and M. luteus, but no inhibition activity against V anguillarum. These result indicated that CFLec-1 was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Treatment of effluents contaminated with prions. Chemical reactor design

The control and elimination of prionic infective agents that may be present in the effluents, turns out to be a complicated mechanism inside a High Containment bological Facility. There are two ways to carry out this neutralization: Installation of thermal systems to ensure achieve a minimum temperature of 134 ° C sterilization plateau for a residence time of 18 minutes, and the use of chemical reactors based on the addition of sodium hypochlorite so the mixture maintained 2% of free chlorine during the reaction period. This study presents the design phases, elements and benefits, of a chemical reactor that allows the treatment of prion effluents in order to serve as a model to biocontainment facilities with areas of animal experimentation, who want to develop their work with prions.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrPC) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPC has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8–24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP0/0 T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF- and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T-cell activation and effector function.

The human prion protein residue 129 polymorphism lies within a cluster of epitopes for T cell recognition.

T cell immune responses to central nervous system-derived and other self-antigens are commonly described in both healthy and autoimmune individuals. However, in the case of the human prion protein (PrP), it has been argued that immunologic tolerance is uncommonly robust. Although development of an effective vaccine for prion disease requires breaking of tolerance to PrP, the extent of immune tolerance to PrP and the identity of immunodominant regions of the protein have not previously been determined in humans. We analyzed PrP T cell epitopes both by using a predictive algorithm and by measuring functional immune responses from healthy donors. Interestingly, clusters of epitopes were focused around the area of the polymorphic residue 129, previously identified as an indicator of susceptibility to prion disease, and in the C-terminal region. Moreover, responses were seen to PrP peptide 121-134 containing methionine at position 129, whereas PrP 121-134 [129V] was not immunogenic. The residue 129 polymorphism was also associated with distinct patterns of cytokine response: PrP 128-141 [129M] inducing IL-4 and IL-6 production, which was not seen in response to PrP 128-141 [129V]. Our data suggest that the immunogenic regions of human PrP lie between residue 107 and the C-terminus and that, like with many other central nervous system antigens, healthy individuals carry responses to PrP within the T cell repertoire and yet do not experience deleterious autoimmune reactions.

High Percentage Inorganic Arsenic Content of Mining Impacted and Nonimpacted Chinese Rice

Two approaches were undertaken to characterize the arsenic (As) content of Chinese rice. First, a national market basket survey (n = 240) was conducted in provincial capitals, sourcing grain from China's premier rice production areas. Second, to reflect rural diets, paddy rice (n = 195) directly from farmers fields were collected from three regions in Hunan, a key rice producing province located in southern China. Two of the sites were within mining and smeltery districts, and the third was devoid of large-scale metal processing industries. Arsenic levels were determined in all the samples while a subset (n = 33) were characterized for As species, using a new simple and rapid extraction method suitable for use with Hamilton PRP-X100 anion exchange columns and HPLC-ICP-MS. The vast majority (85%) of the market rice grains possessed total As levels <150 ng g(-1). The rice collected from mine-impacted regions, however, were found to be highly enriched in As, reaching concentrations of up to 624 ng g(-1). Inorganic As (As(i)) was the predominant species detected in all of the speciated grain, with As(i) levels in some samples exceeding 300 ng g(-1). The As(i) concentration in polished and unpolished Chinese rice was successfully predicted from total As levels. The mean baseline concentrations for As(i) in Chinese market rice based on this survey were estimated to be 96 ng g(-1) while levels in mine-impacted areas were higher with ca. 50% of the rice in one region predicted to fail the national standard.

Arsenic-glutathione complexes - Their stability in solution and during separation by different HPLC modes

Complexes of arsenic compounds and glutathione are believed to play an essential part in the metabolism and transport of inorganic arsenic and its methylated species. Up to now, the evidence of their presence is mostly indirect. We studied the stability and Chromatographic behaviour of glutathione complexes with trivalent arsenic: i.e. As^III(GS)₃, MA ^III(GS)₂ and DMA^III(GS) under different conditions. Standard ion chromatography using PRP X-100 and carbonate or formic acid buffer disintegrated the complexes, while all three complexes are stable and separable by reversed phase chromatography (0.1% formic acid/acetonitrile gradient). As^III(GS)₃ and MA^III(GS)₂ were more stable than DMA^III(GS), which even under optimal conditions tended to degrade on the column at 25 °C. Chromatography at 6 °C can retain the integrity of the samples. These results shed more light on the interpretation of a vast number of previously published arsenic speciation studies, which have used Chromatographic separation techniques with the assumption that the integrity of the arsenic species is guaranteed. © The Royal Society of Chemistry 2004.

Pan-retinal photocoagulation and other forms of laser treatment and drug therapies for non-proliferative diabetic retinopathy: systematic review and economic evaluation

BACKGROUND: Diabetic retinopathy is an important cause of visual loss. Laser photocoagulation preserves vision in diabetic retinopathy but is currently used at the stage of proliferative diabetic retinopathy (PDR).

OBJECTIVES: The primary aim was to assess the clinical effectiveness and cost-effectiveness of pan-retinal photocoagulation (PRP) given at the non-proliferative stage of diabetic retinopathy (NPDR) compared with waiting until the high-risk PDR (HR-PDR) stage was reached. There have been recent advances in laser photocoagulation techniques, and in the use of laser treatments combined with anti-vascular endothelial growth factor (VEGF) drugs or injected steroids. Our secondary questions were: (1) If PRP were to be used in NPDR, which form of laser treatment should be used? and (2) Is adjuvant therapy with intravitreal drugs clinically effective and cost-effective in PRP?

ELIGIBILITY CRITERIA: Randomised controlled trials (RCTs) for efficacy but other designs also used.

REVIEW METHODS: Systematic review and economic modelling.

RESULTS: The Early Treatment Diabetic Retinopathy Study (ETDRS), published in 1991, was the only trial designed to determine the best time to initiate PRP. It randomised one eye of 3711 patients with mild-to-severe NPDR or early PDR to early photocoagulation, and the other to deferral of PRP until HR-PDR developed. The risk of severe visual loss after 5 years for eyes assigned to PRP for NPDR or early PDR compared with deferral of PRP was reduced by 23% (relative risk 0.77, 99% confidence interval 0.56 to 1.06). However, the ETDRS did not provide results separately for NPDR and early PDR. In economic modelling, the base case found that early PRP could be more effective and less costly than deferred PRP. Sensitivity analyses gave similar results, with early PRP continuing to dominate or having low incremental cost-effectiveness ratio. However, there are substantial uncertainties. For our secondary aims we found 12 trials of lasers in DR, with 982 patients in total, ranging from 40 to 150. Most were in PDR but five included some patients with severe NPDR. Three compared multi-spot pattern lasers against argon laser. RCTs comparing laser applied in a lighter manner (less-intensive burns) with conventional methods (more intense burns) reported little difference in efficacy but fewer adverse effects. One RCT suggested that selective laser treatment targeting only ischaemic areas was effective. Observational studies showed that the most important adverse effect of PRP was macular oedema (MO), which can cause visual impairment, usually temporary. Ten trials of laser and anti-VEGF or steroid drug combinations were consistent in reporting a reduction in risk of PRP-induced MO.

LIMITATION: The current evidence is insufficient to recommend PRP for severe NPDR.

CONCLUSIONS: There is, as yet, no convincing evidence that modern laser systems are more effective than the argon laser used in ETDRS, but they appear to have fewer adverse effects. We recommend a trial of PRP for severe NPDR and early PDR compared with deferring PRP till the HR-PDR stage. The trial would use modern laser technologies, and investigate the value adjuvant prophylactic anti-VEGF or steroid drugs.

STUDY REGISTRATION: This study is registered as PROSPERO CRD42013005408.

FUNDING: The National Institute for Health Research Health Technology Assessment programme.

SWOT analysis of a pediatric rehabilitation programme : a participatory evaluation fostering quality improvement

Purpose. To present the results of a strengths, weaknesses, opportunities and threats (SWOT) analysis used as part of a process aimed at reorganising services provided within a pediatric rehabilitation programme (PRP) in Quebec, Canada and to report the perceptions of the planning committee members regarding the usefulness of the SWOT in this process. Method. Thirty-six service providers working in the PRP completed a SWOT questionnaire and reported what they felt worked and what did not work in the existing model of care. Their responses were used by a planning committee over a 12- month period to assist in the development of a new service delivery model. Committee members shared their thoughts about the usefulness of the SWOT. Results. Current programme strengths included favourable organisational climate and interdisciplinary work whereas weaknesses included lack of psychosocial support to families and long waiting times for children. Opportunities included working with community partners, whereas fear of losing professional autonomy with the new service model was a threat. The SWOT results helped the planning committee redefine the programme goals and make decisions to improve service coordination. SWOT analysis was deemed as a very useful tool to help guide service reorganisation. Conclusions. SWOT analysis appears to be an interesting evaluation tool to promote awareness among service providers regarding the current functioning of a rehabilitation programme. It fosters their active participation in the reorganisation of a new service delivery model for pediatric rehabilitation.

Norton de Matos, o improvável republicano: Um olhar sobre Portugal e o império entre Afonso Costa e Salazar

Discutir o papel político de José Norton de Matos (1867-1955) na galáxia republicana portuguesa entre 1910 e 1955 é o objectivo principal desta tese. A alteração do prisma de análise mais habitual quando se fala de Norton, do colonial para o político, foi acompanhada pela opção de privilegiar os seus escritos contemporâneos, em detrimento das muitas análises retrospectivas posteriormente publicadas, e de cotejar a sua voz com múltiplas vozes de Portugal e do império, entre muitas outras com que se cruzou. Tentámos compreender como é que um liberal como Norton, monárquico de tradição embora não de filiação, usa as suas credenciais coloniais para entrar na política republicana à boleia da polémica do cacau escravo e da Sociedade Portuguesa Anti-Esclavagista. Com a aproximação a Bernardino Machado, aos Jovens Turcos, à maçonaria e à facção do PRP dominada por Afonso Costa, insere-se na órbita democrática e afonsista e ganha o cargo de governador-geral de Angola. Aos africanos, cuja definição legislativa como uma categoria distinta da de cidadão (o indígena) antecipa em 1913, leva as luzes da educação laica e a liberdade de trabalho mas paradoxalmente convive com as escolas das missões e o trabalho forçado. A fibra de político que revela na gestão das tensões entre as elites coloniais e metropolitanas irá ser apurada como ministro da Guerra. Discutimos, em especial, o seu papel político num ambicioso projecto: transformar dezenas de milhares de portugueses, pobres, analfabetos e vindos de meios rurais com parcas simpatias republicanas, em cidadãos e soldados da República. A improvisação, que possibilitou que o Corpo Expedicionário Português fosse combater em França ao lado dos britânicos, teve o rasgo de asa e os problemas inerentes à megalomania que o caracterizava. No Verão de 1917, o prestígio com que regressa à capital após difíceis negociações com o governo de Lloyd George torna-o um sério concorrente de Afonso Costa embora com ele partilhe também o ónus da crescente impopularidade da guerra. Reencontrar-se-ão, após o exílio sidonista, na Conferência da Paz em Paris mas, ao contrário de Costa, que não regressará à política activa, Norton tornar-se-á o altocomissário mais marcante e polémico da nova República do pós-guerra. Após a queda do regime e dois novos exílios, será Norton de Matos a fazer os compromissos necessários para permanecer no país e, aí, combater a Ditadura Militar e o Estado Novo de Salazar. Quando, em 1931, consegue unir a oposição em torno da Aliança Republicano-Socialista, começa a impor-se como símbolo da I República. É com esse estatuto que integrará as frentes unidas antifascistas dos anos 40 que culminarão na sua candidatura à Presidência da República em 1948/1949. O modo como consegue fazer a ponte entre os vários ciclos e gerações da oposição e, simultaneamente, resistir às pressões para romper a unidade com os comunistas na fase mais crispada da Guerra Fria será uma última manifestação do seu talento político, cuja análise poderá ajudar a enriquecer o debate sobre essa realidade plural e multipolar que foi a galáxia republicana na primeira metade do século XX.