重组纤维连接蛋白EDA-EDB融合蛋白的分子克隆

目的构建重组pET28a-EDA-EDB质粒,制备重组纤维连接蛋白EDA-EDB融合蛋白。方法采用基因重组技术,将EDA、EDB基因片段连接,再将该重组基因插入pET28a表达载体,表达重组融合EDA-EDB蛋白后,利用6×His/Ni-NTA纯化系统进行纯化,免疫印迹法检测。结果成功连接EDA、EDB基因片段,重组pET28a-EDA-EDB质粒;在大肠杆菌BL21(DE3)中高度表达重组蛋白,获得较纯的重组蛋白,免疫印迹法证实为FN的组分。结论EDA-EDB重组蛋白能采用pET系统在大肠杆菌中表达,并可通过6×His/Ni-NTA纯化系统进行纯化而获得免疫原。     

柱切换HPLC法分析尿中吗啡和O^6—单乙酰吗啡的研究

目的建立用柱切换HPLC技术分析尿中吗啡和06-单乙酰吗啡的方法.方法尿样用硼砂缓冲液(pH9.2)稀释后进入预处理柱(YWG-C18,33mm×5.0mm,10μm),用H2O洗去杂质,再用CH3OHH2O(6040)将被分析组分洗脱进入分析柱(Lichrospher(R)100CN,125mm×4.0mm,5μm),分析流动相为CH3OH磷酸盐缓冲液(pH6.86)=2278.紫外检测器波长为286nm.结果尿中吗啡和06--单乙酰吗啡的线性范围分别为50～1 600n/ml和100～1 600n/ml.吗啡和O6--单乙酰吗啡的精密度均小于4%.吗啡和O6-单乙酰吗啡的检测限均为40n/ml.结论用CSHPLC测定尿中吗啡和O6-单乙酰吗啡,方法准确、灵敏、快速、简便.     

Evidence-based prevention strategies of suicide behavior in the world: implications for Slovenia

In this review we consider how Slovenia could consider tackling its high rate of suicide (overall 29 per 100,000, 46 in males, 13 in females). First, we consider the evidence for risk factors that may contribute to Slovenia's high rate of suicide. Second, we describe the interventions to try to reduce the impact of these factors and the evidence for such interventions. We categorize interventions in terms of their operation at either the population level or that of high-risk groups. However, it should be borne in mind that settings often assumed to provide access to population groups, such as general practice and schools, do not reach some people who are likely to be at high risk; for example those who have dropped out of school or who have been excluded from a GP's list. We focus particularly on those for high-risk groups, as a number of East-European countries with high suicide rates such as Slovenia, Hungary, and the Baltic republics are currently considering a shift toward more community-based mental health services. The provision of community mental health services in Slovenia would provide an opportunity to study their impact on the suicide rate. However, we conclude that their development should be accompanied by other initiatives operating at population levels. This multilevel approach acknowledges the complexity of the etiology of suicide, the impossibility of reaching all those at risk through services and the lack of strong evidence for any one intervention.     

Deaths associated with liposuction: case reports and review of the literature

Tumescent liposuction is a common cosmetic procedure that is performed as an outpatient service in physician's offices and is largely believed to be safe. The protuberant areas of the body containing the undesirable fat deposits are injected with normal saline containing lidocaine and epinephrine for pain control and hemostasis, and the waterlogged cells are suctioned out via cannula through a small incision. We recently encountered three cases in which deaths were attributed to this procedure. Two showed fat embolization in the lung and one died from fluid overload. The osmium tetroxide post-fixed lung sections showed fat emboli in the interstitial capillaries and arterioles. We reviewed the recent literature and found that pulmonary thromboemboli, fat embolization, fluid overload, and lidocaine and epinephrine intoxication are found at autopsy in many cases. Forensic pathologists responsible for determining the cause and manner of death should become familiar with the postmortem findings and risks of liposuction therapy and communicate them to their clinical colleagues and communities.     

Detection of ketosis in vitreous at autopsy after embalming

Ketosis occurs in ketoacidosis or malnourishment. When either is suspected in relation to a death, it may be important to analyze for ketosis at autopsy. We encountered a case where starvation was suspected in a deceased nursing home resident, where the body had been embalmed prior to autopsy. Gas chromatography (GC) was unable to separate acetone from formaldehyde, a component of embalming fluid. The Acetest is a simple test that can detect acetone and acetoacetate in body fluids. We validated the Acetest with GC on vitreous. The Acetest and GC were consistent except at very low levels of acetone or acetoacetate. The sensitivity of the Acetest for acetoacetate in vitreous was 10 mg/dL, consistent with early starvation. Significant interference from embalming fluid did not occur. The Acetest was negative in the described case. The Acetest is a simple and useful test for the detection of ketosis in embalmed autopsies.     

A kinetic model describing the pharmacokinetics of ethyl glucuronide in humans

The glucuronide conjugation is a minor pathway of ethanol metabolism. The determination of ethyl glucuronide (EG) in serum and urine has gained importance in forensic and other legal decisions. To prospectively calculate the serum concentration of this non-oxidative ethanol metabolite, the computer program developed includes a parameter fitting routine. Multiple ethanol doses can be handled.The mathematical modeling was based on the following assumptions and simplifications, respectively. A single enzyme system is responsible for ethanol conjugation at one distinct site; the distribution of EG into the systemic circulation is delayed; the elimination of EG follows first-order kinetics.The concentration of EG was calculated using three kinetic parameters: a rate constant for the first-order formation of EG from serum ethanol, a transfer constant for an obstructed transfer of EG from the formation site (FS) into the central compartment (CC) and an exponential elimination constant.The program was applied to the data collected from 21 drinking experiments. The fitting algorithm optimized the three kinetic parameters, until the sum of concentration error squares of the data points was minimized. The means+/-standard deviation of the rate constant for the first-order formation of EG from serum ethanol was 0.0011+/-0.0006 h(-1), the transfer constant for an obstructed transfer of EG from the FS into the CC was 0.43+/-0.1996 h(-1) and the exponential elimination constant was 3.0+/-1.45 h(-1).Using the range of these parameters, it is now possible to calculate minimum and maximum serum concentrations of EG based on ethanol doses and drinking times. The comparison of calculated and measured concentrations can prove the plausibility of an alleged ethanol consumption. This can be crucial when the serum ethanol concentration (SEC) itself is not meaningful.     

Bile analysis of drugs in postmortem cases

Bile is, in certain cases, collected together with blood from different sites (heart, brain, femoral), urine and other organs or matrices. This study reports comparative results obtained from the analysis of blood and bile for different drugs found: acetaminophen, amphetamine and related compounds, several antidepressants, several benzodiazepines, cocaine and its metabolites, dextropropoxyphene and its metabolite, hydroxyzine, methadone and metabolite, morphine and codeine, levomepromazine, thioridazine, propranolol, tramadol and its metabolite. Several findings are presented: (1) There were no significant differences in the levels of the compounds among the samples of blood obtained from different sites. (2) Levels in bile are generally several fold higher than those in blood. The mean bile to blood ratios vary from about 1 (for acetaminophen, amphetamine) to about 2000 (for desmethylclobazam). (3) In certain cases (16 over 44), although the drug or its metabolite was not detected in blood from different sites, it was detected in bile. As other authors had advocated, it is very useful to ask the pathologist to take the gall bladder with its contents together with the other samples, in order that the sample of bile can be used in the comprehensive toxicological analysis and therefore be complementary to the other fluids or matrices. An additional advantage for using bile is that the concentrations of drugs or their metabolites are generally several fold higher than their blood concentrations.     

Quantitation using GC-TOF-MS: example of bromazepam

Time-of-flight mass spectrometry (TOF-MS) offers new perspectives for forensic toxicology. Qualitative and quantitative analyses of a mixture of three selected benzodiazepines (diazepam, nordazepam and bromazepam) were used to compare gas chromatography (GC-TOF-MS, quadrupole GC-MS, GC-ECD) and liquid chromatography (HPLC-DAD) data. Method validation parameters like LOD, LOQ, S/N-ratios reflect the capabilities of GC-TOF-MS. Five-point calibrations for bromazepam in human peripheral blood (50, 100, 160, 200, 300 ng/ml) using medazepam as internal standard (1000 ng/ml) were performed. The calibrations using GC-TOF-MS (using the fragments of m/z 236 and 288), GC-ECD (dual system) and HPLC-DAD (at 235 nm) all showed correlation coefficients close or superior to 0.99. Quadrupole GC-MS data was not used in the comparison of extracted samples due to the low sensitivity in the full scan mode. Two analyses of real cases concerning bromazepam are presented. In the first case, the presence or absence of bromazepam could not be established with both HPLC-DAD and GC-ECD due to background signals. The extracted ion chromatograms and spectrum traces after the analysis with the GC-TOF-MS could clearly excluded the presence of bromazepam. The second case illustrates the quantitation of bromazepam, where both HPLC-DAD and GC-ECD were unable to give satisfactory results, again due to interfering background signals. The analyses performed on the GC-TOF-MS-system demonstrated high sensitivity and also high selectivity due to the high quality of mass spectra obtained. The advantages of GC-TOF-MS make it a promising analytical technique for forensic toxicology.