Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

Sputum microbiome profiles identify severe asthma phenotypes of relative stability at 12 to 18 months

1. Download : Download high-res image (203KB)
2. Download : Download full-size image

     

The asymmetries in morphology, desoxyribonucleic acid, protein and ribonucleic acid of the two hemispheres induced by monolateral removal of the olfactory bulb and retrobulbar structures in neonatal rats

Monolateral removal of the olfactory bulb and the olfactory penduncular structures in the neonatal rat resulted in a profound morphological and biochemical asymmetry between the two hemispheres. The experimental hemisphere, ipsilateral to the lesion, protruded into the space normally occupied by the olfactory bulb and showed enlarged ventricles. The brain loci were displaced rostrally in this hemisphere.The desoxyribonucleic acid, ribonucleic acid and protein contents of the experimental hemisphere at 25 and 60 days of age were all significantly lower than that found in the control hemisphere contralateral to the lesion.To resolve between the two possible causes of this asymmetry (i.e. atrophy of the experimental or hypertrophy of the control hemisphere) hemispheres of the asymmetric brain were compared with homonymous hemispheres of unoperated normal rats. This comparison revealed that the asymmetry is basically due to an excess of desoxyribonucleic acid synthesis in the control hemisphere, which continues even after postnatal day 25, on the one hand, and a dearth of protein in the experimental hemisphere on the other. This finding implies two separate mechanisms for the processes that underlie the asymmetries observed for these two substances.Our results demonstrate two important characteristics of the developing brain. Firstly they indicate that removal of the olfactory bulb and the olfactory peduncle can produce considerable changes in the hemispheres, and secondly they unravel the strong latent potential of the brain for cell proliferation beyond the usual period of cell division in the brain. As in these experiments regulation of cell division is affected, this system might serve as a model for the study of aberrant cell division found in tumor formation and the process of carcinogenesis.     

RNA interference against enterovirus 71 infection

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.     

The pharmacology of fever

The ability to minimise, if not prevent, large variations in deep body temperature that would otherwise result from some environmental conditions is a homeostatic function of unquestioned benefit that is demonstrated only by the more highly evolved animals. Nevertheless, body temperature is raised above normal values in many pathological conditions. This increase in temperature or fever is an active and co-ordinated response, which indicates the involvement of the CNS. Central injection and lesion studies have shown that the brain, in particular the PO/AH, is the site of action of fever-inducing agents, termed pyrogens. Electrophysiological data show that pyrogens modify the activity of central thermosensitive neurones as if to increase heat gain and decrease heat loss. The common response of fever to pyrogens of diverse origins is attributable to fever being mediated by an endogenous pyrogen released by phagocytic cells in the host. The mechanism by which central neuronal function is disturbed by pyrogens present in the periphery is not known. Tracer studies have yet to demonstrate the passage of a pyrogen across the blood-brain barrier. The possible involvement of several putative neuro- transmitters and modulators in fever has been reviewed here, but most compounds have not been studied sufficiently to allow firm conclusions to be drawn. Much of the data is limited to the effects of the putative mediators on normal thermoregulation but, even when the effect is hyperthermia, such observations do not necessarily indicate a role for the endogenous material in fever. Dose-response curves for agonists and the effects of antagonists are often undetermined. This shortfall in data is due to some extent to the nature of fever; a central response in vivo over several hours. Although fever may enhance other host reactions to combat infection and inflammation, neither this benefit nor the undesirability of antipyretic therapy has been demonstrated unequivocally in either homeothermic laboratory animals or humans. Consequently, antipyretic drugs continue to be used clinically to alleviate the fever, malaise and/or pain commonly associated with disease. The drugs in common usage are the nonsteroidal antipyretic analgesics, many of which also have an anti-flammatory effect. The primary mode of action of these drugs as antipyretics appears at present to be the inhibition of cyclo-oxygenase and a consequent reduction of prostanoid material in pyrogen-sensitive areas of the brain. PGEs in the PO/AH have received most study to date, but other mediators in other parts of the CNS, where the density of pyrogen receptors may be sparse, cannot be discounted and await further investigation.     

Topoanatomical measurements of protein-bound radioactivity in the rat brain after intraventricular application of [14C]leucine

The pattern of distribution of protein-bound radioactivity was studied in rats 4 h after stereotaxic injection of [¹⁴C]leucine into the lateral ventricle. For quantitative estimation of the distribution of radioactivity, the semiconductographic method was used. The patterns revealed in this way were in general agreement with patterns obtained by the autoradiographic technique. The semiconductographic measurements indicated a high labelling, especially of some thalamic and hypothalamic nuclei. With the exception of the base of the telencephalon, the cortical structures were very poorly labelled. A steep gradient from the ventricular system to the brain tissue was observed. Very high labelling of blood vessels and leptomeningeal cells, especially in the vicinity of the optic chiasma, was observed.This quantitative analysis of the distribution pattern indicated a marked difference in uptake of the radiolabelled amino acid from the cerebrospinal fluid and calls for caution in the interpretation of experiments in which the relation between brain function and macromolecular synthesis is studied by the intraventricular injection of a radiolabelled precursor.A method is also described which makes it possible to determine the density distribution within a non-radioactive tissue slice in a simple way.     

Dysregulation of the Hypothalamus-Pituitary-Adrenal Axis in Male and Female, Genetically Obese (ob/ob) Mice

The autosomal, recessive obesity of ob/ob mice is associated with hypercorticosteronemia and amelioration of most symptoms of obesity following adrenalectomy. Increased adrenocorticotropic hormone (ACTH) secretion has been hypothesized on the basis of several reports of higher pituitary ACTH content in ob/ob mice compared to lean littermates. However, the only measurement of ACTH blood concentration found lower levels in ob/ob mice than in leans suggesting that hypercorticosteronemia might result solely from an enhanced adrenal response to ACTH and also suggesting that the ob/ob's elevated pituitary ACTH content might be due to decreased ACTH secretion rather than increased ACTH synthesis. In our study, basal serum ACTH levels were higher in ob/ob males and females compared to sex-matched lean littermates. Anterior pituitary ACTH synthesis was also elevated as indicated by increased content of ACTH and proopiomelanocortin mRNA in obese mice of both sexes; however hypothalamic corticotropin-releasing factor content was not different in lean and obese mice. Basal serum ACTH and corticosterone (CS) levels showed normal circadian rhythm in both phenotypes and sexes, but the circadian increase in CS level was much greater in obese mice than in leans despite equal serum ACTH increases in the two phenotypes. Ether stress at both peak and trough of the circadian rhythm also stimulated much larger serum CS increases in obese mice even though ACTH increases were again equal in the two phenotypes. Taken together, these results strongly indicate that ob/ob mice have increased synthesis and secretion of pituitary ACTH despite the presence of chronically elevated serum CS. This hyperactivity of the hypothalamo-pituitary-adrenal axis appears to be most pronounced in ob/ob females since pituitary ACTH content was equal in obese males and females despite much higher circulating CS levels in the females. Furthermore, the results also indicate an enhanced response to ACTH by the adrenal cortex of the obese mouse. Thus, ob/ob mice exhibit abnormal hypothalamo-pituitary-adrenal axis function with hyperactivity occurring at the level of pituitary ACTH synthesis/secretion as well as at the level of adrenocortical response to ACTH.     

体复康对运动性疲劳大鼠脑组织中阿黑皮素原基因表达动态变化的影响

目的 :从基因转录水平阐明运动性疲劳产生及药物调节的机理。方法 :采用生物素标记阿黑皮素原 (POMC)cRNA探针 (Bio cRNA)原位杂交法结合图像分析 ,观察中药复方体复康对运动性疲劳大鼠不同脑区POMCmRNA表达。结果 :对于慢性运动应激 ,体复康能增强POMC在海马CA1 区、下丘脑部位的基因表达 ,而能降低POMC在额叶皮层的基因表达。长时期 (共 7周 )大强度的跑台运动 (速度由 1 5m/min递增至 3 5m/min ,运动时间为每天 2 0～2 5min)的大鼠 ,末次运动后POMC基因表达在额叶皮层、海马CA1 下降 (前者至运动结束后 3h仍未恢复 ,后者至运动结束后 3 0min即恢复 ) ,在下丘脑则增强 ;体复康能增强末次运动后POMC在额叶皮层、海马CA1 区、下丘脑等脑区的基因表达 ,尤其是在运动结束后 3h最为显著。结论 :POMCmRNA表达在运动性疲劳大鼠不同脑区呈现不同的动态变化趋势 ,体复康对其POMCmRNA表达有重要动态调节作用。     

注射用核糖核酸Ⅱ专项评价方法的构建与安全合理性

目的：探讨注射用核糖核酸Ⅱ安全与合理性专项评价研究。方法：本研究按月份分层随机抽取某院2016年9月-2017年8月临床使用核糖核酸Ⅱ的病例270份，药品说明书定量评价、药物利用研究及药品不良反应/事件Logistic回归分析综合评价方法，初步评价核糖核酸Ⅱ的安全合理性。结果：依据药品说明书评价结果提示合理性较高，其中给药剂量（100%），溶媒选择（100%），溶媒剂量（72.59%），给药浓度（74.07%），给药频次（98.15%），给药时间（100%），6项指标12个月内波动幅度与均值保持一致（90.80%）；药物利用研究结果提示，存在超剂量、超浓度给药情况（aDDDs=5.68，aDDCs=7.37，dDUI=1.19，cDUI=1.53）。研究期内发现4例不良反应，均为一般不良反应，经Logistic回归分析提示与药物使用浓度有关。结论：此种评价方法可有效评价药物临床使用的安全合理性，应加强我院核糖核酸Ⅱ临床监测工作，尤其是超剂量、超浓度给药情况。     

Role of viral RNA and lipid in the adverse events associated with the 2010 Southern Hemisphere trivalent influenza vaccine

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL 2010 SH trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. In an accompanying study, we described the contribution to these adverse events of the 2010 SH influenza strains as expressed in the CSL 2010 SH TIV using in vitro cytokine/chemokine secretion from whole blood cells and induction of NF-κB activation in HEK293 reporter cells. The aim of the present study was to identify the root cause components that elicited the elevated cytokine/chemokine and NF-κB signature. Our studies demonstrated that the pyrogenic signal was associated with a heat-labile, viral-derived component(s) in the CSL 2010 SH TIV. Further, it was found that viral lipid-mediated delivery of short, fragmented viral RNA was the key trigger for the increased cytokine/chemokine secretion and NF-κB activation. It is likely that the FS reported in children <5 years were due to a combination of the new influenza strains included in the 2010 SH TIV and the CSL standard method of manufacture preserving strain-specific viral components of the new influenza strains (particularly B/Brisbane/60/2008 and to a lesser extent H1N1 A/California/07/2009). These combined to heighten immune activation of innate immune cells, which in a small proportion of children <5 years of age is associated with the occurrence of FS. The data also demonstrates that CSL TIVs formulated with increased levels of splitting agent (TDOC) for the B/Brisbane/60/2008 strain can attenuate the pro-inflammatory signals in vitro, identifying a potential path forward for generating a CSL TIV indicated for use in children <5 years.