A method of one-step enzyme labelling of short oligonucleotide probes for filter hybridisation

Here we describe a method of labelling short oligonucleotide probes with enzyme without purification or chemical modifications. Biotinylated oligonucleotides as short as 10 nt are coupled with streptavidin-conjugated enzyme, hybridised and detected with enzyme-triggered chemiluminescence. The detection of hybridisation signal is linear for two orders of magnitude of target dilution. It is shown to be comparable in sensitivity with standard procedures and with radioactive detection. The method is quick, simple and has potential for automation of large-scale oligo-nucleotide hybridisation and multiplex sequencing.     

A new retroviral vector for detecting mutations and chromosomal instability in mammalian cells

A retroviral vector carrying both forward (neo) and backward (herpes simplex virus thymidine kinase or HSV-TK gene) selection markers was constructed as a substrate for mutational assay in mammalian cells. The cells infected with this virus are first selected with G418, mutagenized and then selected with the anti-herpes drug acyclovir (ACV). Since HSV-TK, but not the host TK, is capable of converting ACV to a toxic metabolite, cells retaining the intact HSV-TK gene fail to survive, while the cells carrying a mutated HSV-TK gene or which have lost the gene can form colonies in the presence of ACV, making it possible to detect the genetic defects in a positive manner. It is also possible to discriminate between small mutations and large deletions by checking the presence of the linked marker, neo. As a model experiment, we prepared an uncloned pool of rat fibroblast cells (CREF) infected with this virus and irradiated them with increasing doses of ultraviolet light. Dose-dependent increases in the number of ACV-resistant colonies were observed. Structural analysis of the HSV-TK gene in these clones revealed point mutations or small deletions in the majority of the cases. Since it requires no pre-existing genetic markers in the host cells, this system may be used for a wide variety of mammalian cells and provides a useful tool to assess both their susceptibility to various mutagens and their genomic instability.     

Use of 5-nitroindole-2'-deoxyribose-5'-triphosphate for labelling and detection of oligonucleotides

The 5'-triphosphate of 5-nitroindole-2'-deoxyriboside has been shown to be a good substrate for terminal deoxynucleotidyl transferase (TdT). An antibody has been prepared for the detection of 5-nitroindole and has been used for the detection of 5-nitroindole tailed DNA both in single-stranded form and after hybridisation to a template. This is therefore a new method for the detection of nucleic acid probes.     

A simple method for synthesis of B-cell clonospecific probes

A sensitive PCR-based method was developed to produce B-cell clonogenic probes without the need for sequencing and specific oligonucleotide synthesis. Specificity and sensitivity were assessed and found to be comparable to that achieved using established methods. Possible applications include the detection of MRD, bone marrow involvement with lymphoma, and the contamination of autologous bone marrow or peripheral blood progenitor cell harvests with malignant cells carrying IgH rearrangements.     

A new method of infant monitoring: for use at home and hospital

AIM: To test a New Zealand originated, designed and funded remote infant heart rate monitor in the home and hospital settings (temporarily named the King Monitor) for accuracy and reliability. METHODS: The units were pretested using ECG simulators and on infants already being monitored in the neonatal unit. Longer term trials on hospital infants and infants being simultaneously monitored at home were then conducted. RESULTS: Interference and electrode problems were corrected during the pretesting phase. The unit worked accurately when compared with the standard neonatal heart and respiratory rate monitor in hospital and appeared in some infants to give earlier warning of problems than the standard home apnoea monitor. CONCLUSION: This simple to use monitor worked reliably and accurately under a wide variety of settings and with varying sized infants. In addition, the lack of direct connection between the infant and the control unit allowed freedom of movement of normal infants around the cot or bassinet. The monitor will require to be adapted for portable use at home and during travel.     

A nonradioactive method for improved restriction analysis and fingerprinting of large P1 artificial chromosome clones

The P1 artificial chromosome (PAC) cloning system is very useful for physical mapping, however, the large insert sizes cause difficulty in routine restriction analysis. In order to facilitate restriction mapping and fingerprinting, we have developed a simple, nonradioactive method for end-labeling and detection of restriction fragments from PAC clones. This method is very easy to implement, gives good differentiation of restriction fragments, and uses comparatively small amounts of DNA. We have used this method for restriction analysis of PAC clones containing inserts from human as well as from lower vertebrates. The method should also be applicable to other large-insert plasmid systems.     

COREX用煤及检测块煤热解新方法

COREX工艺是目前惟一工业化的熔融还原炼铁新工艺,其优势之一为可以尽可能少用焦炭,能直接使用块煤。针对COREX工艺给出了其用煤要求,为了减少其焦炭使用量,发挥其直接用煤优势,必须找出焦炭与块煤使用的匹配关系,对块煤运输和装卸产生的粉煤总结出几种利用途径。通过观测用于COREX-3000的块煤在熔化气化炉中的裂解和脱气过程,提出了块煤热爆裂性能和脱气性能测试的实验室可视化试验方法。     

A simple method for the study of cytosolic content of oligonucleotides in cells

BACKGROUND: Nonuniform attenuation in the thorax can generate artifacts in single-photon emission computed tomographic myocardial perfusion studies that mimic coronary artery disease. In this article we present both phantom and simulation data, as well as clinical data, in support of an emission-based method that provides reliable correction for attenuation effects without the need for a transmission measurement. METHODS AND RESULTS: The attenuation map is derived from the measured distribution of 99mTc-labeled macroaggregated albumin in the lungs and a radioactive binder wrapped about the thorax. This information is acquired as part of a dual-isotope acquisition during the rest 201Tl study. Segmentation is used to define the interiors of lung and body compartments, which are assigned a single attenuation coefficient for each of the two tissue types. The appropriateness of this approach was investigated by examining the measured attenuation coefficients in a group of 80 individuals (40 male, 40 female) from positron emission tomographic transmission studies. The correction technique was evaluated with computer simulations, a physical phantom, and clinical data acquired from 20 patients. Analysis of the positron emission tomographic data found a small SD in the mean attenuation coefficients for the body (<5%) and lungs (<15%). The application of emission-based attenuation-correction technique produced a substantial reduction in the magnitude of the attenuation artifact in images obtained from both the phantom and the simulation studies. The emission-based attenuation-correction technique was easily applied to myocardial perfusion studies, where it had a significant effect, resulting in changes in interpretation for nine of 20 patients. CONCLUSIONS: The results of this study provide strong support for the concept that an attenuation map can be generated with fixed attenuation values in place of those that are directly measured. Thus the emission-based attenuation-correction technique can be considered an inexpensive alternative to transmission-based correction methods. Because the emission-based correction technique does not require any additional hardware, it has the major advantage of being applicable to all single-photon emission computed tomographic systems.     

A trial of the clinical use of a new method of electrosleep

A novel device and technique of interference electric sleep therapy have been developed and tried in 125 children and 324 adults with different diseases. The highest clinical response, especially in children, was achieved on Elson unit.     

A new method for labelling saxitoxin and its binding to non-myelinated fibres of the rabbit vagus, lobster walking leg, and garfish olfactory nerves

1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from tritiated water to the toxin. 2. The radiochemical purity of the labelled toxin has been directly determined, rather than being based on indirect biochemical means, as in previous experiments with Wilzbach-labelled STX and TTX. 3. The specific activity of the labelled toxin, 66 d..m.f-mole-1, corresponds with one tritium atom per molecule STX, an improvement of about 300-fold over other means of labelling TTX and STX. 4. The binding of this toxin to rabbit, lobster and garfish olfactory nerve fibres has been re-examined. 5. The density of sodium channels calculated on the basis of the binding of the toxin is about four to six-times the values previously reported.     

An AFLP-based procedure for the efficient mapping of mutations and DNA probes in barley

A strategy based upon AFLP markers for high-efficiency mapping of morphological mutations and DNA probes to linkage groups in barley is presented. First, 511 AFLP markers were placed on the linkage map derived from the cross Proctor x Nudinka. Second, loci controlling phenotypic traits were assigned to linkage groups by AFLP analysis, using F2 populations consisting of 30-50 mutant plants derived from crosses of the type "mutant x Proctor" and "mutant x Nudinka." To map DNA probes, 67 different wild-type barley lines were selected to generate F2 populations by crossing with Proctor and Nudinka. F2 plants that were polymorphic for a given RFLP fragment were classified into genotypic classes. Linkage of the RFLP polymorphism to 1 of the 511 AFLP loci was indicated by cosegregation. The use of the strategy is exemplified by the mapping of the mutation branched-5 to chromosome 2 and of the DNA probes Bkn2 and BM-7 to chromosomes 5 and 1, respectively. Map expansion and marker order in map regions with dense clustering of markers represented a particular problem. A discussion considering the effect of noncanonical recombinant products on these two parameters is provided.     

A new method for measuring menstrual blood loss and its use in screening women before endometrial ablation

OBJECTIVE: 1. To develop and validate a method for measuring menstrual blood loss in a routine setting, and 2. To assess the value of measuring menstrual blood loss before endometrial ablation. DESIGN: A prospective, observational study. SETTING: Four Yorkshire hospitals: The General Infirmary at Leeds, St. James's University Hospital, Leeds, St. Luke's Hospital, Bradford and The Friarage Hospital, Northallerton. PARTICIPANTS: Three hundred and seventy-two women who had been offered endometrial ablation for menorrhagia. MEASUREMENT: Sanitary material was washed with a nonionic detergent in a known volume of water. The haemoglobin in a sample of solution was measured by mixing with sodium carbonate for spectrophotometric analysis. INTERVENTIONS: The menstrual blood loss result was revealed to each women. Electrosurgical endometrial ablation was performed for those who decided to have surgery. MAIN OUTCOME MEASURES: Proportion of women with normal menstrual blood loss (< or = 80 mL) who avoided surgery. Comparison of endometrial ablation outcome in women with and without genuine menorrhagia. RESULTS: Thirty-six women (10%) with normal menstrual blood loss who declined surgery continued to avoid surgery after a mean of 27 months. Two hundred and ninety-two women were followed up for one year after endometrial ablation. Those with genuine menorrhagia (n = 122) were less likely to be dissatisfied (9% vs 18%) (OR 2.5, 95% CI 1.1-4.7) or to require hysterectomy (4% vs 7%) (OR 1.8, 95% CI 0.6-5.2) than women with normal menstrual blood loss (n = 170). CONCLUSIONS: The objective diagnosis of menorrhagia can be undertaken in a routine setting and may provide some women, who have a normal menstrual blood loss, sufficient reassurance to refrain from surgery. Women with genuine menorrhagia have a better outcome after endometrial ablation than those with normal menstrual blood loss.     

Evaluation of a new method for abdominal compression for use in oesophageal investigation

The use of conventional extra-abdominal compression with a constant cuff pressure was shown to give various pressures intra-abdominally in different subjects. A feedback system was therefore used, in which the extra-abdominal pressure is controlled by the intra-abdominal pressure. For an intra-abdominal pressure increase of 15 mm Hg the external cuff pressure in 20 subjects varied from 21 to 98 mm Hg. There was, however, an almost linear relationship between increased intra-abdominal pressure and cuff pressure. The extra-abdominal pressure required to obtain a certain intra-abdominal pressure also varied with the size of the cuff. To obtain relevant and comparable results in studies of hiatal incompetence and gastro-oesophageal reflux, the feedback system is recommended and the size of the intra-abdominal cuff should be stated.     

A new model for phenotypic suppression of frameshift mutations by mutant tRNAs

According to the prevailing model, frameshift-suppressing tRNAs with an extra nucleotide in the anticodon loop suppress +1 frameshift mutations by recognizing a four-base codon and promoting quadruplet translocation. We present three sets of experiments that suggest a general alternative to this model. First, base modification should actually block such a four-base interaction by two classical frameshift suppressors. Second, for one Salmonella suppressor tRNA, it is not mutant tRNA but a structurally normal near cognate that causes the +1 shift in-frame. Finally, frameshifting occurs in competition with normal decoding of the next in-frame codon, consistent with an event that occurs in the ribosomal P site after the translocation step. These results suggest an alternative model involving peptidyl-tRNA slippage at the classical CCC-N and GGG-N frameshift suppression sites.     

基于行为分布的DDoS攻击检测方法

分布式拒绝服务(distfibuted denial of service,DDoS)攻击能够在短时间内产生巨量的数据包耗尽目标主机或网络的资源,经过研究发现这些伪造的数据包在一个特定的时间内有着合法数据包所不具备的函数特点.因此,本文提出了行为分布的模型,一旦有可疑流流入服务器,则开始计算这些可疑流的行为分布差异,如果该差异小于一个设定的阈值,则判断有DDoS攻击发生;反之则为合法的数据访问.根据NS-3的模拟实验,证明该模型能够有效的从合法访问中区分出DDoS攻击流,对提前控制DDoS攻击的发生具有重要的意义.