Determinants of outcome in critically ill patients

Assessment of the outcome of critical illness is complex. Severity scoring systems and organ dysfunction scores are traditional tools in mortality and morbidity prediction in intensive care. Their ability to explain risk of death is impressive for large cohorts of patients, but insufficient for an individual patient. Although events before intensive care unit (ICU) admission are prognostically important, the prediction models utilize data collected at and just after ICU admission. In addition, several biomarkers have been evaluated to predict mortality, but none has proven entirely useful in clinical practice. Therefore, new prognostic markers of critical illness are vital when evaluating the intensive care outcome. The aim of this dissertation was to investigate new measures and biological markers of critical illness and to evaluate their predictive value and association with mortality and disease severity. The impact of delay in emergency department (ED) on intensive care outcome, measured as hospital mortality and health-related quality of life (HRQoL) at 6 months, was assessed in 1537 consecutive patients admitted to medical ICU. Two new biological markers were investigated in two separate patient populations: in 231 ICU patients and 255 patients with severe sepsis or septic shock. Cell-free plasma DNA is a surrogate marker of apoptosis. Its association with disease severity and mortality rate was evaluated in ICU patients. Next, the predictive value of plasma DNA regarding mortality and its association with the degree of organ dysfunction and disease severity was evaluated in severe sepsis or septic shock. Heme oxygenase-1 (HO-1) is a potential regulator of apoptosis. Finally, HO-1 plasma concentrations and HO-1 gene polymorphisms and their association with outcome were evaluated in ICU patients. The length of ED stay was not associated with outcome of intensive care. The hospital mortality rate was significantly lower in patients admitted to the medical ICU from the ED than from the non-ED, and the HRQoL in the critically ill at 6 months was significantly lower than in the age- and sex-matched general population. In the ICU patient population, the maximum plasma DNA concentration measured during the first 96 hours in intensive care correlated significantly with disease severity and degree of organ failure and was independently associated with hospital mortality. In patients with severe sepsis or septic shock, the cell-free plasma DNA concentrations were significantly higher in ICU and hospital nonsurvivors than in survivors and showed a moderate discriminative power regarding ICU mortality. Plasma DNA was an independent predictor for ICU mortality, but not for hospital mortality. The degree of organ dysfunction correlated independently with plasma DNA concentration in severe sepsis and plasma HO-1 concentration in ICU patients. The HO-1 -413T/GT(L)/+99C haplotype was associated with HO-1 plasma levels and frequency of multiple organ dysfunction. Plasma DNA and HO-1 concentrations may support the assessment of outcome or organ failure development in critically ill patients, although their value is limited and requires further evaluation.

Role of γ1 Laminin and Its KDI Peptide in the Central Nervous System

Since the 1980 s, laminin-1 has been linked to regeneration of the central nervous system (CNS) and promotion of neuronal migration and axon guidance during CNS development. In this thesis, we clarify the role of γ1 laminin and its KDI tripeptide in development of human embryonic spinal cord, in regeneration of adult rat spinal cord injury (SCI), in kainic acid-induced neuronal death, and in the spinal cord tissue of amyotrophic lateral sclerosis (ALS). We demonstrated that γ1 laminin together with α1, β1, and β3 laminins localize at the floor plate region in human embryonic spinal cord. This localization of γ1 laminin is in spatial and temporal correlation with development of the spinal cord and indicates that γ1 laminin may participate in commissural axon guidance during the embryonic development of the human CNS. With in vitro studies using the Matrigel culture system, we demonstrated that the KDI tripeptide of γ1 laminin provides a chemotrophic guidance cue for neurites of the human embryonic dorsal spinal cord, verifying the functional ability of γ1 laminin to guide commissural axons. Results from our experimental SCI model demonstrate that the KDI tripeptide enhanced functional recovery and promoted neurite outgrowth across the mechanically injured area in the adult rat spinal cord. Furthermore, our findings indicate that the KDI tripeptide as a non-competitive inhibitor of the ionotropic glutamate receptors can provide when administered in adequate concentrations an effective method to protect neurons against glutamate-induced excitotoxic cell death. Human postmortem samples were used to study motor neuron disease, ALS (IV), and the study revealed that in human ALS spinal cord, γ1 laminin was selectively over-expressed by reactive astrocytes, and that this over-expression may correlate with disease severity. The multiple ways by which γ1 laminin and its KDI tripeptide provide neurotrophic protection and enhance neuronal viability suggest that the over-expression of γ1 laminin may be a glial attempt to provide protection for neurons against ALS pathology. The KDI tripeptide is effective therapeutically thus far in animal models only. However, because KDI containing γ1 laminin exists naturally in the human CNS, KDI therapies are unlikely to be toxic or allergenic. Results from our animal models are encouraging, with no toxic side-effects detected even at high concentrations, but the ultimate confirmation can be achieved only after clinical trials. More research is still needed until the KDI tripeptide is refined into a clinically applicable method to treat various neurological disorders.

Heme oxygenase-1 in cardiovascular diseases

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.

Myotonic dystrophy type 2 (DM2) : Diagnostic methods and molecular pathology

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are the most common forms of muscular dystrophy affecting adults. They are autosomal dominant diseases caused by microsatellite tri- or tetranucleotide repeat expansion mutations in transcribed but not translated gene regions. The mutant RNA accumulates in nuclei disturbing the expression of several genes. The more recently identified DM2 disease is less well known, yet more than 300 patients have been confirmed in Finland thus far, and the true number is believed to be much higher. DM1 and DM2 share some features in general clinical presentation and molecular pathology, yet they show distinctive differences, including disease severity and differential muscle and fiber type involvement. However, the molecular differences underlying DM1 and DM2 muscle pathology are not well understood. Although the primary tissue affected is muscle, both DMs show a multisystemic phenotype due to wide expression of the mutation-carrying genes. DM2 is particularly intriguing, as it shows an incredibly wide spectrum of clinical manifestations. For this reason, it constitutes a real diagnostic challenge. The core symptoms in DM2 include proximal muscle weakness, muscle pain, myotonia, cataracts, cardiac conduction defects and endocrinological disturbations; however, none of these is mandatory for the disease. Myalgic pains may be the most disabling symptom for decades, sometimes leading to incapacity for work. In addition, DM2 may cause major socio-economical consequences for the patient, if not diagnosed, due to misunderstanding and false stigmatization. In this thesis work, we have (I) improved DM2 differential diagnostics based on muscle biopsy, and (II) described abnormalities in mRNA and protein expression in DM1 and DM2 patient skeletal muscles, showing partial differences between the two diseases, which may contribute to muscle pathology in these diseases. This is the first description of histopathological differences between DM1 and DM2, which can be used in differential diagnostics. Two novel high-resolution applications of in situ -hybridization have been described, which can be used for direct visualization of the DM2 mutation in muscle biopsy sections, or mutation size determination on extended DNA-fibers. By measuring protein and mRNA expression in the samples, differential changes in expression patterns affecting contractile proteins, other structural proteins and calcium handling proteins in DM2 compared to DM1 were found. The dysregulation at mRNA level was caused by altered transciption and abnormal splicing. The findings reported here indicate that the extent of aberrant splicing is higher in DM2 compared to DM1. In addition, the described abnormalities to some extent correlate to the differences in fiber type involvement in the two disorders.

Improving outcome of childhood bacterial meningitis by simplified treatment : Experience from Angola

Background Acute bacterial meningitis (BM) continues to be an important cause of childhood mortality and morbidity, especially in developing countries. Prognostic scales and the identification of risk factors for adverse outcome both aid in assessing disease severity. New antimicrobial agents or adjunctive treatments - except for oral glycerol - have essentially failed to improve BM prognosis. A retrospective observational analysis found paracetamol beneficial in adult bacteraemic patients, and some experts recommend slow β-lactam infusion. We examined these treatments in a prospective, double-blind, placebo-controlled clinical trial. Patients and methods A retrospective analysis included 555 children treated for BM in 2004 in the infectious disease ward of the Paediatric Hospital of Luanda, Angola. Our prospective study randomised 723 children into four groups, to receive a combination of cefotaxime infusion or boluses every 6 hours for the first 24 hours and oral paracetamol or placebo for 48 hours. The primary endpoints were 1) death or severe neurological sequelae (SeNeSe), and 2) deafness. Results In the retrospective study, the mortality of children with blood transfusion was 23% (30 of 128) vs. without blood transfusion 39% (109 of 282; p=0.004). In the prospective study, 272 (38%) of the children died. Of those 451 surviving, 68 (15%) showed SeNeSe, and 12% (45 of 374) were deaf. Whereas no difference between treatment groups was observable in primary endpoints, the early mortality in the infusion-paracetamol group was lower, with the difference (Fisher s exact test) from the other groups at 24, 48, and 72 hours being significant (p=0.041, 0.0005, and 0.005, respectively). Prognostic factors for adverse outcomes were impaired consciousness, dyspnoea, seizures, delayed presentation, and absence of electricity at home (Simple Luanda Scale, SLS); the Bayesian Luanda Scale (BLS) also included abnormally low or high blood glucose. Conclusions New studies concerning the possible beneficial effect of blood transfusion, and concerning longer treatment with cefotaxime infusion and oral paracetamol, and a study to validate our simple prognostic scales are warranted.

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

Avaliação do potencial anti-inflamatório agudo e crônico e imunossupressor do extrato etanólico de Pterodon polygalaeflorus através de estudos in vivo e in vitro

O gênero Pterodon pertence à família das Papilonaceas e inclui cinco espécies nativas do Brasil: P. pubescens Benth., P. emarginatus Vog., P. apparicioi Pedersoli e P. abruptus Benth., sendo a espécie objeto deste estudo a P. polygalaeflorus Benth.. Seus frutos são livremente comercializados em mercados da flora medicinal e utilizados pela medicina popular devido a propriedades anti-reumática, analgésica, antiinflamatória, dentre outros efeitos associados a esses frutos. O principal uso popular está relacionado ao efeito antiartrítico que parece se encontrar na fração oleosa do fruto. O objetivo deste trabalho foi avaliar o extrato etanólico de Pterodon polygalaeflorus (EEPpg) quanto ao seu potencial antiinflamatório crônico através do modelo de artrite induzida por colágeno (CIA) e seu efeito sobre os linfócitos in vitro, bem como sobre a expansão de células MAC-1+ induzida por adjuvante completo de Freund (AFC). A caracterização química do EEPpg foi realizada por cromatografia em camada delgada (TLC), cromatografia líquida de alta performance (HPLC) e cromatografia gasosa acoplada a espectrômetro de massa (GC-MS), através dos quais uma gama de compostos, incluindo terpenóides de polaridades variadas e flavonóides, foram observados. No modelo de CIA, o EEPpg reduziu significativamente parâmetros associados ao desenvolvimento e progressão da doença e à severidade da doença , inibindo em até 99% o seu desenvolvimento e levando a ausência de sinais clínicos evidentes após tratamento com as menores doses do extrato (0,01 mg/kg e 0,001 mg/kg). O tratamento com EEPpg também reduziu características histopatológicas típicas de articulações de animais com CIA, que também são observadas na artrite reumatóide. O EEPpg reduziu significativamente o peso dos linfonodos dos camundongos, bem como o número absoluto de segmentados, monócitos e linfócitos no sangue. In vitro, O EEPpg mostrou uma atividade anti-proliferativa dos esplenócitos estimulados com concanavalina A (Con A) ou lipopolissacarídeo (LPS) analisada através do ensaio de redução do sal de tetrazólio MTT, corroborada pelo seu efeito sobre o ciclo celular de linfócitos estimulados com Con A, onde o EEPpg nas concentrações de 5, 10 e 20 μg/mL reduziu significativamente, de maneira concentração-dependente, o número de células nas fases S+G2/M e aumentou na fase G0/G1 do ciclo celular. O efeito anti-proliferativo do EEPpg parece também estar associado ao aumento da apoptose dos linfócitos após estimulação com Con A, com aumento estatisticamente significativo no percentual de células mortas por apoptose nas maiores concentrações . O EEPpg inibiu a expansão de células Mac-1+ induzida por AFC no baço, porém não no peritônio. Esse resultado sugere um efeito inibidor do EEPpg sobre a migração celular para as articulações artríticas. Esses resultados contribuem para a validação do uso popular de P. polygalaeflorus contra doenças relacionadas a processos inflamatórios e imunes, sobretudo na artrite reumatóide, antes nunca demonstrado.

Efeito do uso de salbutamol nas propriedades mecânicas do sistema respiratório de indivíduos saudáveis, tabagistas e portadores de doença pulmonar obstrutiva crônica

A doença pulmonar obstrução crônica (DPOC) é caracterizada pela limitação de fluxo parcialmente reversível, classificada por níveis de obstrução pós-broncodilatador. Há várias evidências de que o FEV1 sozinho não é capaz de mostrar a broncodilatação de pacientes com DPOC, mesmo naqueles que apresentam melhora clínica. A técnica de oscilações forçadas (TOF) tem mostrado alta sensibilidade na detecção precoce de alterações mecânicas na DPOC, contudo o efeito broncodilatador na impedância respiratória de pacientes com DPOC ainda não está esclarecido. Objetiva avaliar a utilidade da TOF nos diferentes estágios de obstrução das vias aéreas; (2) avaliar a resposta da impedância respiratória ao salbutamol em indivíduos saudáveis ao exame espirométrico e pacientes com DPOC em diferentes graus de gravidade. Foram avaliados 25 indivíduos saudáveis sem história de tabagismo, 24 tabagistas e 151 pacientes com DPOC classificados em graus I, II, III e IV. Todos os sujeitos foram avaliados pela TOF seguida da espirometria, antes e após o uso do salbutamol spray. As curvas de resistência e reatância demonstraram alteração em todos os estágios de obstrução das vias aéreas após o uso do salbutamol. O grupo de risco apresentou alterações mecânicas semelhantes ao grupo leve (p=ns). Os parâmetros R0, Rm, Csr,din e Z4Hz apresentam desempenho diagnóstico adequado (AUC > 0,85) em todos os estágios de gravidade da doença. Todos os parâmetros de TOF e espirometria apresentaram diminuição após uso do salbutamol. Os indivíduos saudáveis apresentaram uma pequena diminuição comparada aos subgrupos de DPOC. A variação em termos absolutos da ΔZ4Hz e das derivadas da resistência, ΔR0, ΔRm, ΔS, apresentaram variação significativa (p<0,0001, p<0,003; p<0,04; p<0,0002, respectivamente) com o aumento da obstrução brônquica. Nas derivadas da reatância o ΔXm aumentou com a gravidade da doença (p<0,0002). Por outro lado, a ΔCrs,dyn não demonstrou diferença significativa com a gravidade da DPOC. Em termos percentuais os parâmetros da TOF apresentaram variação expressiva em ΔRm% (p<0,02), ΔS% (p<0,02) e ΔXm% (p<0,004) com o aumento da obstrução nas vias aéreas. Por outro lado, ΔR0%, ΔCrs,dyn% e ΔZ4Hz% não variaram entre os estágios da DPOC. A associação entre a broncodilatação nas vias aéreas e a impedância pulmonar foi fraca entre ΔXm vs ΔFVC (r=0,32, p<0,0001) e ΔZ4Hz% vs ΔFEV1% vs ΔFVC% (r=0.28, p<0,0005; r=0,29, p<0,0003, respectivamente). A TOF é útil na avaliação das alterações mecânicas nos diferentes níveis de obstrução das vias aéreas na DPOC. Demonstramos o benefício da medicação broncodilatadora, quantificando a melhora da ventilação através da TOF. A impedância respiratória diminui em todos os estágios da DPOC, o estágio leve melhorou tanto quanto o estágio muito grave. Isto sugere que a medida da impedância pulmonar não é dependente do volume como ocorre na espirometria e que a broncodilatação ocorre em todas as fases da progressão da DPOC.

青杨(Populus cathayana Rehd)雌雄植株对锈病和干旱胁迫的生理响应及差异蛋白质组比较

雌雄异株植物对环境的不同响应一直是一个有趣而新颖的研究领域，由于雌雄个体不同的繁殖成本及不同的生存策略，使得雌雄植株在生长、存活、生殖格局、空间分布、资源配置等方面已经表现出明显的不同，在生理和分子水平上也表现出明显的性别间差异。干旱是制约农林业发展的环境因子之一，叶锈病是对杨树危害最严重的病害之一，由于长期进化的结果，不同性别的植物必然对生物和非生物胁迫有着不同的响应。本文以雌雄异株的青杨为模式植物，研究雌雄间在生理、生化、亚细胞结构和蛋白质水平上对生物和非生物胁迫的差异响应。主要研究结果如下： （1） 青杨雌雄植株对锈病胁迫的生理生化差异响应 在正常的对照组中，雄株叶片比雌株叶片有着较高的活性氧自由基产生速率、较高的SOD、POD、PPO 和较低的CAT 活性；在锈病感染的早期阶段， SOD、POD、CAT 活性、活性氧自由基产生速率、H2O2 含量、膜脂过氧化程度和细胞膜的电渗率在雌雄株中都增加，而PPO 仅在雄株中增加明显，APX 仅在雌株中增加明显，并且雌株比雄株有着更严重的锈病感染程度、细胞膜的伤害程度和光合系统II 的破坏程度，雌株有更多的净光合速率、气孔导度和叶绿素a 含量的降低，在同工酶变化上，雌雄间对锈病也显示出不同的表达模式。结果显示，雄株比雌株对锈病有着更好的抗性和更有效的ROS 清除系统。 （2） 青杨雌雄植株对干旱胁迫的生理生化及亚细胞结构的差异响应 与较好水分条件相比，干旱下雄株比雌株有着更高的A-Ci 响应参数，如Rubisco 最大羧化速率、光呼吸速率、暗呼吸速率和最大电子传递速率等。干旱显著地增加了膜脂过氧化程度和游离脯氨酸含量，并且雄株比雌株表现出较低的膜脂过氧化程度，较高的总蛋白和游离脯氨酸含量。无论是中度干旱还是极度干旱，除了CAT 外，雄株比雌株表现为较强的抗氧化酶活性，在同工酶谱带上，雌雄间表现出不同的变化模式，并且有些条带是干旱影响应的，而有些条带是性别特异性的，这些性别特异性条带能够作为鉴定性别快速而准确的标记。干旱显著地影响了线粒体、叶绿体和细胞壁的结构，尤其在中度干旱胁迫下，雄株线粒体和叶绿体比雌株呈现出较好的完整性，并且雄株细胞壁要比雌株更厚。因此， 雄株比雌株表现出更强的干旱忍耐性和更高效的抗氧化酶系统。 （3） 青杨雌雄植株对干旱胁迫的蛋白质组差异响应 用双相电泳检测到雌雄间近1000 个蛋白点，通过对比发现对照组雌雄间有54 个差异蛋白点，干旱下雌雄间有108 个差异点，其中102 个被质谱成功鉴定。对照组雌雄间的差异蛋白主要集中在与光合作用相关蛋白、抗氧化酶、胁迫防御蛋白和一些调节基因表达的蛋白；干旱胁迫下雌雄间差异蛋白明显增多，主要有参与信号转导、调节基因表达、蛋白质加工、转录产物的转录翻译后修饰的调节性蛋白蛋白和参与氧化还原平衡、抗胁迫、细胞壁合成、光合作用、能量代谢、氨基酸代谢和脂肪酸代谢等的功能性蛋白。干旱下这些蛋白的表达量在雌雄中有的表现出相同的表达模式，如干旱下雌雄株中Rubisco 激活酶、小热激蛋白等表达都增加，而有的表现出相反的表达模式，如Rubisco 大亚基的降解片段、羰酸酯酶等在雄株中表达量上调而在雌株中却是下调。因此，雌雄间在蛋白质水平上对干旱胁迫响应的差异是显著的，也是复杂的。 It is an interesting and novel topic that dioecious plants possess different responses to environmental stress. As for the different productive cost and different survive strategy, different sexual plants have shown obviously morphological, physiological and molecular differences. Drought is one of the most worldwidely important environmental stress factors that limit plant growth and ecosystem productivity. Rust disease is one of the economically important diseases in many trees. As a result of the long evolutionary process, male and female plants should show different responses to abiotic and biotic stress. In this paper, using a dioeious tree of Populus cathayana Rehd as a model, we study the sexual differences to drought and rust disease stress in physiological, biochemical, sub-cellular and proteomics levels. The main results are follows: (1) The sexual differences in physiology and biochemistry of poplar to rust disease In controls, males showed higher production of superoxide radicals, higher activities of SOD, POD, PPO and lower CAT activity. Under rust disease, the activities of antioxidant, the content of ROS and the degree of cellular member destroyed were increased in both sexes, except for PPO in diseased males and APX in diseased females. However, females showed more seriously disease severity and cellular member and PS II destroyed degrees. Net photosynthesis rate, transpiration rate and chlorophyll a content were decreased more in diseased females than in males. There were also some different changes inantioxidant isozymes under rust disease. The results suggested that male poplar possessed a more effectively antioxidant system and were more resistant to rut disease than females. (2) The sexual differences in physiology and biochemistry of poplar to drought stress Under drought stress, there were higher rates of RuBP-saturated CO2 assimilation, dark respiration, photorespiratory release of oxygen, the max electron transportrate in CO2-saturated and carboxylation efficiency in males than in females. And males showed lower TBARS and higher proline content. Except for CAT, the activities of other antioxidants were higher in males than in females. Meanwhile, there were obviously differences in isozyme changes between teo sexes. Drought stress obviously destroyed the integralities of chloroplasts and mitochondria and the sexual differences in sub-cellular level were obviously under the moderate water stress. Male cell walls were more sensitive to drought stress than did female. The results suggested males were more resistant to drought stress. (3) The sexual differences in proteomics of poplar to drought stress By 2-D and MS analysis, we identified 102 different protein spots between males and females. Under control conditions, the different proteins were mainly in photosynthesis related proteins, antioxidants, stress response proteins and some gene expression related proteins. Under drought stress, the different proteins were focused on (i) regulated proteins such as signaling conduction, kinase, HSP, gene expressional regulation and protein modification, (ii) functional proteins such as photosynthesis, energy metabolism, antioxidant, redox, stress response, lipid metabolism and amino acid metabolism. Some protein showed the same expressional pattern, while some showed contrary expressional pattern. Thus, the results suggested that sexual differences in proteomics were significant and complex.

Reação de genótipos de girassol à mancha de alternaria (alternariaster helianthi) em condições de campo, nas safras 2013/2014 e 2014/2015.

RESUMO: A reação de 23 genótipos de girassol à mancha de Alternaria foi avaliada em dois experimentos de campo, conduzidos em Londrina, PR, nas safras 2013/2014 e 2014/2015. Os experimentos foram semeados em outubro de 2013 e outubro de 2014. A severidade da doença, que ocorreu por infecção natural das plantas pelo fungo, foi avaliada na fase de desenvolvimento R3, utilizando uma escala diagramática da doença. Após a colheita, também foram avaliados a produtividade, o peso de mil aquênios e o teor de óleo. Em cada experimento, verificou-se diferença estatística significativa entre os híbridos avaliados em condições de campo, tanto para a severidade da doença, quanto para os componentes de produção. Nenhum genótipo de girassol apresentou resistência completa à mancha de Alternaria. ABSTRACT: The reaction of 23 sunflower genotypes to Alternaria leaf spot disease was evaluated in two field experiments carried out in Londrina, state of Paraná, Brazil, during 2013/2014 and 2014/2015 growing seasons. The experiments were sown in October 2013 and October 2014. Alternaria disease severity, under natural conditions in the field, was evaluated at the R3 growth stage with reference to a diagrammatic scale developed for this disease. After harvesting, yield, 1000-seed weight and oil content were also evaluated. For each experiment, statistical significance was observed among the evaluated genotypes for disease severity and yield components. None of the sunflower genotypes showed complete resistance to Alternaria leaf spot.

Severidade de mancha de alternaria em genótipos de girassol no Cerrado do Distrito Federal.

RESUMO: Este trabalho teve como objetivo comparar a severidade da mancha de Alternaria de genótipos de girassol em ambientes do Cerrado do Distrito Federal. Três experimentos foram avaliados, dois na Embrapa Cerrados (Planaltina, DF) e um localizado na Embrapa Produtos e Mercado (Recanto das Emas, DF). Foram feitas análises ao longo do ciclo da cultura com um intervalo de 15 dias, sendo a primeira aos 35 dias após emergência. Cada planta foi analisada em três partes: terço inferior, terço médio e terço superior. Ao final de cada experimento, a área abaixo da curva de progresso da doença (AACPD) foi calculada para cada genótipo. De acordo com os resultados observados, o ambiente de sequeiro na Embrapa Cerrados, que deteve a maior média da severidade da doença, entre os ambientes avaliados. Dentre os genótipos avaliados, MG 360 mostrou menor severidade da doença no sequeiro e AGUARA 06 no irrigado da Embrapa Cerrados. O genótipo BSG 42 na Embrapa Produtos e Mercado, se destacou pela menor severidade do fungo. Houve influência significativa do ambiente nos resultados de severidade da doença, que está estreitamente relacionada com os fatores climáticos como temperatura e umidade relativa do ar. A compreensão dessas condições favoráveis ao desenvolvimento do fungo é essencial quando se avalia a resistência em genótipos de girassol. ABSTRACT: This study had the purpose of comparing the severity of Alternaria leaf spot on genotypes of sunflower in the Brazilian Cerrado. Three field trials were established, two at Embrapa Cerrados (Planaltina, DF) and one at Embrapa Produtos e Mercados (Recanto das Emas, DF). Evaluations were made during the crop cycle every 15 days, the first one happening 35 days after crop emergency. Each plant was analyzed in three parts: lower, middle and upper thirds. At the end of each trial the average area under the disease progress curve (AUDPC), for each third, was calculated for all genotypes. According to the observed results, the dryland environment at Embrapa Cerrados had the highest mean disease severity, among the studied environments. Between the evaluated genotypes, MG360 showed less severity in dryland and AGUARA 06 in the irrigated environment at Embrapa Cerrados. At Embrapa Produtos e Mercado the genotype that presented the best performance was BRS G42. Environmental conditions deeply affected the trials, mainly temperature and relative humidity. Their influence on Alternaria leaf spot epidemics in the Cerrado region must be studied in more detail, to avoid misinterpreting data when evaluating sunflower genotypes for resistance to this important fungal disease.

Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

EXPRESSION OF THE INFLAMMATORY REGULATOR A20 CORRELATES WITH LUNG FUNCTION IN PATIENTS WITH CYSTIC FIBROSIS

Abstract: Background: A20 and TAX1BP1 interact to negatively regulate NF-
-driven inflammation. A20 expression is altered in F508del/F508del
patients. Here we explore the effect of CFTR and CFTR genotype on A20 and
TAX1BP1expression. The relationship with lung function is also assessed.
Methods: Primary Nasal Epithelial cells (NECs) from CF patients
(F508del/F508del, n=8, R117H/F508del, n=6) and Controls (age-matched,
n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene
expression was determined by qPCR.
Results: Silencing of CFTR reduced basal A20 expression. Following LPS
stimulation A20 and TAX1BP1 expression was induced in control NECs and
reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del
had lower expression than R117H/F508del. A20, but not TAX1BP1 expression,
was proportional to FEV1 in all CF patients (r=0.968, p<0.001).
Conclusions: A20 expression is reduced in CF and is proportional to FEV1.
Pending confirmation in a larger study, A20 may prove a novel predictor
of CF inflammation/disease severity.

Structure-Phenotype Correlations of Human CYP21A2 Mutations in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by adrenal cortex. The predominant causes of the disorder are mutations in the CYP21A2 gene that encodes a Cytochrome P450 21-hydroxylase enzyme, which is central to steroidogenesis. The severity of the disease depends upon the extent of impaired enzymatic activity and can be classified under severe Classical form or the mild Non-Classical form, Molecular characterisation of CYP21A2 mutations can be used to predict clinical phenotype and disease severity based upon changes it brings in 21-hydroxylase enzyme structure. A humanized model of CYP21A2 has been used to map and investigate the structural role of all known disease-causing mutations. A structural explanation of clinical manifestation allows us to put forward criteria that might allow the prediction of clinical severity of the disease.

Pneumonia and lower respiratory infections in nursing home residents:Predictors of hospitalization and mortality

OBJECTIVES: To compare predictors of hospitalization and death in nursing home residents with pneumonia and other lower respiratory infections (LRIs). DESIGN: A nested cohort study. SETTING: Nine nursing homes in southern Ontario. PARTICIPANTS: Three hundred fifty-three nursing home residents with LRIs (enrolled in the control arm of a clinical trial). MEASUREMENTS: Comorbidities, vaccination status, age, health-related quality of life, functional status, and vital statistics were evaluated as potential predictors of hospitalization and mortality at 30 days. RESULTS: Moderate to high disease severity score on a practical severity scale was a strong independent predictor of hospitalization (odds ratio (OR)=7.12, P