Hematological and plasma biochemical responses of crucian carp (Carassius auratus) to intraperitoneal injection of extracted microcystins with the possible mechanisms of anemia

Alterations in hematological indices such as decreases in blood cell counts (RBC), hematocrit (Ht) and hemoglobin (Hb) concentrations are key symptoms of anemia. However, few experiments were conducted to examine changes in hematological indices of fish exposed to microcystins that are believed to be fatal to circulatory systems of vertebrates. An acute toxicological experiment was designed to study hematological changes of crucian carp injected intraperitoneally (i.p.) with extracted microcystins at two doses, 50 and 200 mu g MC-LReqkg(-1) body weight. After being i.p. injected with microcystins, the fish exhibited behavioral abnormity. There were significant decreases in RBC in the high-dose group, and in Ht and Hb concentrations in both dose groups, while erythrocte sedimentation rate (ESR) significantly increased, indicating the appearance of normocytic anemia. There were no prominent changes in the three red cell indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH,), and mean corpuscular hemoglobin concentration (MCHC). Increases in blood urea nitrogen (BUN) and creatinine (CR) in both dose groups suggest the occurrence of kidney impairment. Alteration in blood indices was reversible at the low dose group. Conclusively, anemia induced by kidney impairment was a key factor to cause abnormity of swimming behaviors and high mortality of crucian carp. (c) 2007 Elsevier Ltd. All rights reserved.

Hemoglobin conjugated micelles based on triblock biodegradable polymers as artificial oxygen carriers

An artificial oxygen carrier is constructed by conjugating hemoglobin molecules to biodegradable micelles. Firstly a series of triblock copolymers (PEG-PMPC-PLA) in which the middle block contains pendant propargyl groups were synthesized and characterized. After the amphiphilic copolymer was self-assembled into core-shell micelles in aqueous solution, azidized hemoglobin molecules protected by carbon monoxide (CO) were conjugated to the micelles via click reaction between the propargyl and azido groups. The conjugation causes an increase of the micelle's mean diameter. Maximum conjugation ratio is 250 wt% in the hemoglobin-conjugated micelles (HCMs). Oxygen-binding ability of the HCMs was demonstrated by converting the CO-binding state of the HCMs into O-2-binding state.

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

Immunomodulation and antitumor activity of kappa-carrageenan oligosaccharides

The modulation of carrageenan oligosaccharides from Kappaphycus striatum on the immune system in S 180-bearing mice was investigated. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides (50, 100 and 200 mu g/g) for 14 days. The effects of carrageenan oligosaccharides on transplantable tumors and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells (QHS),. lymphocyte proliferation, the activity of natural killer cells (NK), production of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) were studied. Carrageenan oligosaccharides could significantly inhibit the growth of transplantable sarcoma S180 and increase macrophage phagocytosis, the form of antibody secreted by spleen cells, spleen lymphocyte proliferation, NK cells activity, serumal IL-2 and TNF-alpha level in S 180-bearing mice. Considering all these results, it is suggested that carrageenan oligosaccharides exert their antitumor effect by promoting the immune system. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Blood-gas properties of plateau zokor (Myospalax baileyi)

Plateau zokor (Myospalax baileyi) is one of the blind subterranean mole rats that spend their life solely underground in scaled burrows. It is one of the special species of the Qinghai-Tibet plateau. In their burrows, oxygen is low and carbon dioxide is high and their contents fluctuate with the change of seasons, soil types, rain and depth of burrows. However, plateau zokors show successful adaptation to that extreme environment. In this study, their adapting mechanisms to the hypoxic hypercapnic environment were analyzed through the comparison of their blood-gas properties with that of pikas (Ochotona curzniae) and Sprague-Dawley rats. The results indicated that plateau zokors had higher red blood corpuscle counts (8.11 +/- 0.59 (10(12)/L)) and hemoglobin concentrations (147 +/- 9.85 g/L), but hematocrit (45.9 +/- 3.29%) and mean corpuscular volume (56.67 +/- 2.57 fL) were lower than the other rodents. Their arterial blood and venous blood pH were 7.46 +/- 0.07 and 7.27 +/- 0.07. Oxygen pressure in arterial blood of plateau zokors was about 1.5 times higher than that of pikas and rats, and it was 0.36 and 0.26 times in their venous blood. Partial pressure for carbon dioxide in arterial and venous blood of plateau zokors was 1.5-fold and 2.0-fold higher, respectively, than in rats and pikas. Oxygen saturation of plateau zokors was 5.7 and 9.3 times lower in venous blood than that of pikas and rats, respectively. As result, the difference of oxygen saturation in arterial blood to venous blood was 2- and 4.5-fold higher in plateau zokors as that of pikas and rats, respectively. In conclusion, plateau zokors had a high tolerance to pH changes in tissues, together with strong capabilities to obtain oxygen from their hypoxic-hypercapnic environment. (c) 2006 Published by Elsevier Inc.

Integrated biomedical device for blood preparation

The separation of red blood cells from plasma flowing in microchannels is possible by bio-physical effects such as an axial migration effect and Zweifach-Fung bifurcation law. In the present study, subchannels are placed alongside a main channel to collect cells and plasma separately. The addition of a constriction in the main microchannel creates a local high shear force region, forcing the cells to migrate and concentrate towards the centre of the channel. The resulting lab-on-a-chip was manufactured using biocompatible materials. Purity efficiency was measured for mussel and human blood suspensions as different parameters including flow rate and geometries of parent and daughter channels were varied.

Effect of fluid dynamics and device mechanism on biofluid behaviour in microchannel systems: modelling biofluids in a microchannel biochip separator

Biofluid behaviour in microchannel systems is investigated in this paper through the modelling of a microfluidic biochip developed for the separation of blood plasma. Based on particular assumptions, the effects of some mechanical features of the microchannels on behaviour of the biofluid are explored. These include microchannel, constriction, bending channel, bifurcation as well as channel length ratio between the main and side channels. The key characteristics and effects of the microfluidic dynamics are discussed in terms of separation efficiency of the red blood cells with respect to the rest of the medium. The effects include the Fahraeus and Fahraeus-Lindqvist effects, the Zweifach-Fung bifurcation law, the cell-free layer phenomenon. The characteristics of the microfluid dynamics include the properties of the laminar flow as well as particle lateral or spinning trajectories. In this paper the fluid is modelled as a single-phase flow assuming either Newtonian or Non-Newtonian behaviours to investigate the effect of the viscosity on flow and separation efficiency. It is found that, for a flow rate controlled Newtonian flow system, viscosity and outlet pressure have little effect on velocity distribution. When the fluid is assumed to be Non-Newtonian more fluid is separated than observed in the Newtonian case, leading to reduction of the flow rate ratio between the main and side channels as well as the system pressure as a whole.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Granulocyte-macrophage precursor cell and colony-stimulating factor responses of mice infected with Salmonella typhimurium

The response of granulocyte-macrophage progenitor cells (in vitro colony-forming cells) and of colony-stimulating (CS) factor in serum were studied in mice infected intraperitoneally with 10(3) viable Salmonella typhimurium. Increases in the number of colony-forming cells in marrow and spleen and increases in the serum level of CS factor occurred during the infection. There was no evidence to suggest that progressive infection was associated with failure of macrophage production. Medium rich in CS factor increased the bactericidal activity of macrophages in vitro and it was suggested that CS factor could be involved in macrophage activation.

Evidence underlying the clinical management of diabetic macular oedema

Diabetic retinopathy (DR) is the leading cause of visual loss in the developed world in those of working age, and its prevalence is predicted to double by 2025. The management of diabetic retinopathy has traditionally relied on screening, on laser treatment delivered by ophthalmologists, and on optimising blood glucose and blood pressure. Recent evidence suggests that the role of systemic factors is more complex than originally thought, and that drugs such as ACE inhibitors, fibrates and glitazones may all influence the course of diabetic macular oedema. Antagonism of vascular endothelial growth factor offers a new therapeutic avenue that may transform the management of diabetic macular oedema. Several other therapeutic options are under investigation and development, including aminoguanidine, sorbinol, ruboxistaurin and autologous stem cell transfusion. © Royal College of Physicians, 2013.

Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor

The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Characterization of bioactive compounds of dealcoholized wine: valuation of distillation process

O vinho tinto é uma importante fonte de compostos fenólicos com atividade antioxidante e que estão relacionados com a prevenção de doenças cardiovasculares e cancro. Estes compostos são um sub-produto do processo de destilação vínica utilizado para produzir aguardente necessária para a produção de Vinho do Porto. Esta tese tem como objetivo valorizar os compostos fenólicos resultantes das destilarias de vinho, através do estudo da sua composição, das interações com o material polimérico do vinho, da sua estabilidade durante o armazenamento e avaliação dos seus potenciais efeitos biológicos in vitro. Isto irá permitir definir aplicações para estes compostos como ingredientes alimentares com propriedades funcionais. Dois vinhos tintos (RW1 e RW2) foram utilizados como fonte de compostos fenólicos. A fim de estudar estes compostos, cada vinho foi evaporado à pressão atmosférica, permitindo obter o respetivo vinho desalcolizado (DW1 e DW2). Os polissacarídeos e compostos fenólicos presentes nos vinhos desalcolizados foram fracionados por extração em fase sólida utilizando cartuchos C18 sep-pak. A fração hidrofóbica, rica em compostos fenólicos, foi separada em frações ricas em ácidos fenólicos, em procianidinas e em antocianinas, as quais foram usadas para avaliar a sua contribuição para a atividade antioxidante total e caracterização fenólica detalhada dos DW. Foram obtidas quantidades comparáveis de compostos fenólicos totais (1.3 g/L para RW1 e DW1, e 3.1 para RW2 e DW2), de taninos (1.2 g/L para RW1 e DW1 e 1.6 para RW2 e DW2) e de antocianinas (0.24 g/L para RW1 e DW1 e 0.41 para RW2 e DW2) para os vinhos e para os respetivos vinhos desalcolizados. A determinação da atividade antioxidante de RW e DW pelos métodos do DPPH e ABTS também originou valores semelhantes, permitindo inferir que o processo de destilação realizado não promoveu uma perda relevante de compostos fenólicos. A atividade antioxidante total de vinho deveu-se essencialmente à fração rica em antocianinas. Os dois DW foram dialisados para se obter o material polimérico dos vinhos (WPM1 e WPM2). O WPM1 e WPM2 apresentavam 1.1 e 1.3 g/L de material sólido, respetivamente. O WPM (WPM1 e WPM2) era composto por polissacarídeos (31 e 36%), proteínas (10 e 12%) e também por compostos fenólicos (32 e 43%). A análise de açúcares mostrou que as manoproteínas e as arabinogalactanas eram os principais polissacarídeos presentes. A extração do WPM com metanol deu origem a um material insolúvel em metanol (PMi) e a uma fração solúvel em metanol, que continuava a conter hidratos de carbono e compostos fenólicos, mostrando uma forte interação entre estes compostos. Para determinar a energia de ativação (Ea) da libertação dos compostos fenólicos de fracções de material polimérico do vinho, foram realizadas diálises do DW, WPM e PMi, utilizando-se quatro concentrações diferentes, a cinco temperaturas (5-40 °C). O valor da Ea foi 25 para o WPM e 61 kJ/mol para o PMi, mostrando que os compostos fenólicos do vinho podem estar associados de forma diferente à matriz polimérica e que uma fração pode estar, ainda, fortemente associada a esta matriz. A fim de avaliar a possível existência de interações seletivas com os compostos fenólicos, o WPM foi fracionado, permitindo a obtenção de uma fração rica em manoproteínas (MP), através de uma cromatografia de afinidade com concanavalina A e 3 frações ricas em arabinogalactanas (AG0, AG1 e AG2) obtidas por cromatografia de troca aniónica. Foi avaliada a difusão de nove antocianinas monoméricas através de uma membrana de diálise, em presença do WPM, e das frações ricas em MP e em AG. A diálise dos compostos fenólicos livres do vinho foi realizada como ensaio em branco. Todas as frações poliméricas mostraram capacidade para reter as antocianinas, embora em diferente extensão. Foi observada uma capacidade de retenção maior para as antocianinas acilglucosiladas do que para as antocianinas glucosiladas. A fração rica em AG teve uma maior contribuição para a capacidade de retenção das antocianinas pelo material polimérico vinho do que a fração rica em MP, principalmente quando as antocianinas estavam acetiladas. Com o objetivo de estudar formas para preservar, a longo prazo, as propriedades antioxidantes dos compostos fenólicos, o extrato de compostos fenólicos (PCE), em pó, foi armazenado em diferentes condições de luz e atmosfera, à temperatura ambiente durante 1 ano. Observou-se que o PCE armazenado no escuro, dentro de um exsicador sob atmosfera de azoto, preservou 95% da atividade antioxidante inicial. Também foram avaliadas as melhores condições para preservar as antocianinas quando em solução, armazenadas a duas temperaturas (5 e 30 ºC) durante 3 meses. A adição de 0.5 g/L de uma fração rica em polissacarídeos a um vinho armazenado a 30 ºC promoveu a proteção das antocianinas, especialmente das antocianinas cumaroiladas. Os potenciais efeitos biológicos dos compostos fenólicos foram avaliados em diferentes sistemas celulares in vitro utilizando as seguintes frações: WPM, WPS (polissacarídeos do vinho), WPC (compostos fenólicos do vinho), PA-E (fração rica em ácidos fenólicos), PR-E (fração rica em procianidinas) e APP-E (fração rica em antocianinas e procianidinas poliméricas). Foi observada uma maior viabilidade celular quando as células do carcinoma do cólon HT-29 foram expostas a dois agentes oxidantes (radiação UV e H2O2) em presença das frações PR-E e APP-E. Além disso, os extratos WPS, WPC, PR-E e APP-E mostraram propriedades anti-inflamatórias, avaliadas pela inibição da produção de NO por células de macrófagos RAW264.7, sendo o extrato APP-E (0.19 mg/mL) o que exibiu a maior capacidade anti-inflamatória. A fim de elucidar as propriedades antioxidantes dos extratos do vinho em células humanas, os glóbulos vermelhos (RBC) foram selecionados como um modelo metabolicamente simples. Os extratos WPM, WPS, WPC, PR-E, e APP-E mostraram efeito anti-hemolítico para a hemólise dos RBC provocada pelo peróxido de hidrogénio (H2O2) e pelo di-hidrocloreto de 2,2'-azo-bis(2-diaminopropano) (AAPH). Os resultados obtidos permitem concluir que o processo de desalcoolização dos vinhos à pressão atmosférica, preservou as principais características antioxidantes dos compostos fenólicos. Estes compostos podem contribuir para a defesa das células contra agentes oxidantes, nomeadamente por terem um potencial de atividades anti-inflamatória e anti-hemolítica, promovendo a viabilidade celular. A interação dos compostos fenólicos do vinho com o material polimérico permite inferir uma dosagem contínua e gradual das antocianinas vinho tinto após a sua ingestão, contribuindo para um período mais longo da sua exposição e, como consequência, dos seus potenciais benefícios para a saúde.

Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015