Current knowledge on the strain typing of the pathogenic fungus Histoplasma capsulatum var. capsulatum: a review of the findings

The classification of microbial strains is currently based on different typing methods, which must meet certain criteria in order to be widely used. Phenotypic and genotypic methods are being employed in the epidemiology of several fungal diseases. However, some problems associated to the phenotypic methods have fostered genotyping procedures, from DNA polymorphic diversity to gene sequencing studies, all aiming to differentiate and to relate fungal isolates or strains. Through these studies, it is possible to identify outbreaks, to detect nosocomial infection transmission, and to determine the source of infection, as well as to recognize virulent isolates. This paper is aimed at analyzing the methods recently used to type Histoplasma capsulatum, causative agent of the systemic mycosis known as histoplasmosis, in order to recommend those that yield reproducible and accurate results.     

Purification from human plasma of a heparin-released lipase with activity against triglyceride and phospholipids.

A triglyceride lipase different from lipoprotein lipase, but measurable only after intravenous heparin injection, has been isolated from human plasma by sequential use of heparin-Sepharose and concanavalin A-Sepharose affinity chromatography. Using these procedures, phospholipase A1 activity was found to chromatograph identically with the triglyceride lipase. The constancy of the ratio of activities after isoelectric focusing (pI 4.1) and during thermal deactivation indicates that this enzyme has hydrolase activity against both triglycerides and phospholipids. This conclusion was supported further by the homogeneity of the protein as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

The biological significance of malondialdehyde determination in the assessment of tissue oxidative stress

Although lipid peroxidation of biological samples may be assessed by different chemical and physical methods, those based on the measurement of malondialdehyde formed from the breakdown of endoperoxides during the last stages of the oxidation of a polyunsaturated fatty acid, appear as the most widely used. Among the various methods to evaluate malondialdehyde, which include direct spectrophotometry or high pressure liquid chromatography, the reaction with thiobarbituric acid to form a colored adduct appears as a more rapid, inexpensive and sensitive technique. This method, however, is subject to some interferences which, if not considered, may lead to erroneous results. The present review emphasizes the significance of malondialdehyde measurement in biological samples, the chemical conditions of its reaction with thiobarbituric acid and the different procedures to isolate and determine the malondialdehyde-thiobarbituric acid adduct.     

A novel method for measurement of triglyceride lipase activity: suitable for microgram and nanogram quantities of tissue

The measurement of triglyceride lipase activity in microgram and nanogram quantities of tissue is reported. The method involves quantitation of glycerol released from a triglyceride substrate, which is shown to provide a value of approximately one-third of that obtained by quantitation of free fatty acid release. Influences on glycerol release, including pH optimum, NaCl inhibition, and activation by serum and heparin are characterized. Two separate assays are described for the measurement of glycerol that yield identical results with nanogram quantities of tissue. The advantage of one assay is its simplicity, while the advantage of the other is that it can be adjusted to measure very small tissue samples (nanogram) with the use of microanalytical procedures (i.e., enzymatic amplification of the NAD+ product of glycerol analysis). Sensitivity of the method is demonstrated by the analysis of triglyceride lipase activity in nanogram samples of single soleus muscle fibers. Measurement of picomole quantities of glycerol produced by lipase activity in single muscle fibers represents at least a 1,000-fold increase in sensitivity compared to currently available methods.     

A general strategy to determine the congruence between a hierarchical and a non-hierarchical classification

Background  

Classification procedures are widely used in phylogenetic inference, the analysis of expression profiles, the study of biological networks, etc. Many algorithms have been proposed to establish the similarity between two different classifications of the same elements. However, methods to determine significant coincidences between hierarchical and non-hierarchical partitions are still poorly developed, in spite of the fact that the search for such coincidences is implicit in many analyses of massive data.     

Fed-batch culture technology

The use of fed-batch procedures offers distinct advantages over other modes of operation of bioreactors, and is a widely researched technique. These advantages are discussed; some uses of fed-batch procedures and the associated methods of modelling and control are reviewed.     

Serum Triglyceride: Method of Estimation and Levels in Normal Humans

Previously described glycerol methods for estimation of serum triglyceride were modified. Conditions during saponification and colour development were altered so as to minimize the possibility of glycerol loss. Using this modification, serum triglyceride was determined in 100 healthy men and women, aged 15 to 79 years. There was a log normal distribution. In both sexes the level increased up to the sixth decade and then decreased. In each decade men had higher levels than women. The geometric mean (and 95% limits) for men was 129 mg. % (68-248); for women, 105 mg. % (54-206); and for the entire group, 117 mg. % (59-233). Comparison of results from several laboratories using different methods showed wide variation in serum triglyceride levels.     

Development and application of probes for labeling the actin cytoskeleton in living plant cells

The actin cytoskeleton is one of the most important components of eukaryotic cytoskeletons. It participates in numerous crucial procedures of cells and has been studied by using various methods. The development and application of appropriate probes for actin visualization is the first and foremost step for functional analysis of actin in vivo. Since the actin cytoskeleton is a highly dynamic and sensitive structure, methods previously used to visualize actin often harm cells and cannot reveal the native state of the actin cytoskeleton in living cells. The development of labeling technologies for living plant cells, especially the emergence and application of green fluorescent protein-tagged actin markers, has provided new insights into the structure and function of the actin cytoskeleton in vivo. There has been a number of probes for actin labeling in living plant cells though they each present different advantages and defects. In this review, we discuss and compare those widely used methods for actin visualization and analysis.     

基于PCR技术的miRNA定量检测方法

microRNA(miRNA)是一类广泛存在于真核生物中,不编码蛋白质的短序列RNA,它广泛参与真核生物的生长发育、新陈代谢和应激反应等生命活动。但绝大多数miRNA的生物学功能还不清楚,通过灵敏的定量检测方法,了解miRNA在不同组织部位的时空表达,是探究其功能的重要环节。现着重介绍了2类7种基于PCR技术的miRNA定量检测方法的基本原理和实验流程,并分析了这些定量检测技术间的异同和适用范围。     

Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.     

Escaping the Bonferroni iron claw in ecological studies

I analyze some criticisms made about the application of alpha-inflation correction procedures to repeated-test tables in ecological studies. Common pitfalls during application, the statistical properties of many ecological datasets, and the strong control of the tablewise error rate made by the widely used sequential Bonferroni procedures, seem to be responsible for some 'illogical' results when such corrections are applied. Sharpened Bonferroni-type procedures may alleviate the decrease in power associated to standard methods as the number of tests increases.
More powerful methods, based on controlling the false discovery rate (FDR), deserve a more frequent use in ecological studies, especially in those involving large repeated-test tables in which several or many individual null hypotheses have been rejected, and the most significant p-value is relatively large.
I conclude that some reasonable control of alpha inflation is required of authors as a safeguard against striking, but spurious findings, which may strongly affect the credibility of ecological research.     

Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei

This protocol describes a methodology for the genetic transformation of the rodent malaria parasite Plasmodium berghei and the subsequent selection of transformed parasites expressing green fluorescent protein (GFP) by flow-sorting. It provides methods for: transfection of the schizont stage with DNA constructs that contain gfp as the selectable marker; selection of fluorescent mutants by flow-sorting; and injection of flow-sorted, GFP-expressing parasites into mice and the subsequent collection of transformed parasites. The use of two different promoters for the expression of GFP is described; these two promoters require slightly different procedures for the selection of mutants. The protocol enables the collection of transformed parasites within 10-12 days after transfection. The genetic modification of P. berghei is widely used to investigate gene function in Plasmodium sp. The application of flow-sorting to the selection of transformed parasites increases the possibilities of parasite mutagenesis, by effectively expanding the range of selectable markers.     

Effect of an anti-lipoprotein lipase serum on plasma triglyceride removal.

Anti-lipoprotein lipase sera injected intravenously in roosters blocked quantitatively the catabolism of very low density lipoprotein (VLDL) triglyceride. Antibodies were produced in rabbits immunized with highly purified lipoprotein lipase (LPL, glycerol ester hydrolase, E C 3.1.1.3) prepared from chicken adipose tissue. Following anti-LPL serum injection there was a linear increase in plasma triglyceride concentration. The rate of entry of triglyceride in plasma was estimated from the rate of triglyceride accumulation in the plasma of animals injected with anti-LPL serum, or from the disappearance curve of biologically labelled VLDL. In instances where both measurements were conducted in the same animals there was very close agreement between the two procedures. Inhibition of VLDL triglyceride catabolism of anti-LPL serum provided a way to characterize newly secreted VLDL that exhibited a broad spectrum of particle sizes with a median of 625 A degrees. They contained 76.2 +/- 1.2% triglyceride and had a high ratio of free to ester cholesterol (2.46 +/- 0.45). In control VLDL samples there was 46.1% triglyceride, and the ratio of free to ester cholesterol was 1.19. The complete inhibition of triglyceride removal by an antiserum prepared against adipose tissue LPL demonstrates that the NaCl-inhibited, serum-activated lipase prepared by affinity chromatography on heparin-Sepharose and concanavalin A-Sepharose columns is the enzyme responsible in vivo for the catabolism of VLDL triglyceride. Further, the kinetics of triglyceride accumulation in the plasma provide evidence that the site of degradation of VLDL triglyceride is within the plasma compartment.     

New resampling method for evaluating stability of clusters

Background  

Hierarchical clustering is a widely applied tool in the analysis of microarray gene expression data. The assessment of cluster stability is a major challenge in clustering procedures. Statistical methods are required to distinguish between real and random clusters. Several methods for assessing cluster stability have been published, including resampling methods such as the bootstrap.     

Absorption of triglycerides in the absence of lipase

Medium chain triglycerides are considered to be readily absorbed intact in the absence of pancreatic lipase, unlike long chain triglycerides. Commercial medium chain triglyceride oils comprise various medium chain fatty acids from 6 to 12 carbons in length resulting in triglyceride molecules of different sizes and molecular weights. The effect of molecular weight and hence fatty acid chain length on the efficiency of intact medium chain triglyceride absorption is unknown. Therefore, this study measured, using a single-pass marker perfusion technique, intestinal jejunum absorption of five medium chain and one long chain triglycerides in anesthetized Sprague-Dawley rats. The molecular weights of the five medium chain triglycerides were 470.7, 498.8, 526.8, 554.9, 639.0, and the long chain triglyceride, 885.4. Residual luminal pancreatic lipase was removed prior to lipid perfusion. This study demonstrated that medium chain triglycerides were absorbed in the absence of lipase whereas long chain triglyceride was not. There was no significant variation in the absorption of the five different medium chain triglycerides perfused. The molecular weight of the medium chain triglyceride did not affect its intact absorption by the small intestine.     

Comparative study of lipid extraction from microalgae by organic solvent and supercritical CO2

Pavlova sp. was employed to evaluate the efficiency of different lipid extraction methods. The microalgal crude lipids content determined using the mixed solvent with ultrasonic method was 44.7 wt.%. The triglyceride content obtained by the mixed solvent method was 15.6 wt.%. The extraction yield was the FAME yield divided by the maximum FAME (15.9 wt.%). The extraction yield was improved by cell disruption prior to extraction, and the highest triglyceride extraction yield of 98.7% was observed using the supercritical fluid extraction (SFE) method with bead-beating. The results indicate that the SFE method is effective and provides higher selectivity for triglyceride extraction though the total lipid extracted was less than that using solvent extraction.     

小鼠胚胎干细胞建系技术研究进展

目前,对小鼠胚胎干细胞的研究较为深入,并已成为研究细胞分化及信号转导、新基因发现及功能鉴定、器官发生、人类疾病和药物开发等的有效手段。胚胎干细胞建系是一项基础性工作。虽然技术日趋成熟,有些品系小鼠的胚胎干细胞建系已是常规技术,但不同品系小鼠胚胎干细胞的建系效率仍有很大差异,建系途径和方法各有特点,一个品系胚胎干细胞的建系方法不一定都适用于其他品系。本文从小鼠胚胎干细胞建系的途径、分离操作技术、培养体系等方面进行综述,并就与之相关的有些问题提出思考和对策。     

P-values for group sequential testing

Group sequential testing procedures are widely employed in long-termclinical trials. We discuss several methods for computing theP-value following a group sequential trial based on differentoutcome-space orderings. We use a criterion defined in thispaper to compare these methods and point out interesting differences.     

Endogenous triglyceride turnover in liver and plasma of the dog

Radioactive glycerol and S(f) > 20 lipoproteins labeled with it were used to study turnover of plasma S(f) > 20 and hepatic triglyceride in anesthetized dogs. From specific activity-time curves of these lipids after an injection of labeled material, a tentative and incomplete model for the kinetics of endogenous hepatic and plasma triglyceride was defined and partially validated. Pool sizes and turnover rates of triglyceride in liver and S(f) > 20 lipoproteins of plasma were then calculated in seven dogs. Hepatic triglyceride was composed of two compartments: 60% metabolically inert and 40% metabolically active. Although communication between these hepatic compartments surely occurred during the time course of these studies, it was not sufficient to be detected by our present methods. The metabolically active compartment turned over as a single pool but with two destinations: a quite variable proportion (an average of 61%) was secreted into plasma as S(f) > 20 triglyceride, and an average of 39% was presumably hydrolyzed within the liver. The fractional turnover rate of plasma S(f) > 20 triglyceride was 2-3 times that of hepatic triglyceride. This finding, and the parallel decline of specific activities of plasma S(f) > 20 and liver triglyceride after injection of labeled glycerol, confirm the rate-determining role of hepatic triglyceride. In this respect the dog differs importantly from man. Though turnover rates of plasma S(f) > 20 triglyceride fell in the same range in men and dogs, the relationship of turnover rate to plasma concentration of this lipid differed greatly between them. The model for the dog does resemble that previously reported for man, however, in the lack of major recycling of intact plasma triglyceride between the liver and plasma. Lack of such recycling, however, does not exclude return of plasma triglyceride into a hepatic triglyceride sink. The amount of such unidirectional uptake, if any, could not be determined by these techniques.     

Solving the Problem of Comparing Whole Bacterial Genomes across Different Sequencing Platforms

Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.