A new method for estimating age-at-death from the first rib

A new method for estimating adult age-at-death from the first rib was developed as a modification of the Kunos et al. (Am J Phys Anthropol 110 (1999) 303-323) method. Data were collected on three aspects of the first rib (costal face, rib head, and tubercle facet) for 470 known-age males of Balkan ancestry collected as evidence during investigations conducted by the International Criminal Tribunal for the former Yugoslavia (ICTY). Ages-at-death range from 12 to 90 years (mean of 47.7 years). Several variables were extracted from the original study utilizing all three skeletal aspects of the first rib. This list was modified to 11 variables as preliminary tests on seriations of the samples were undertaken. A cumulative probit model with age measured on a log scale was used to calculate the mean and standard deviation of the ages-of-transition for each component. Multivariate analysis of the three components was also performed. The lowest correlation (r = 0.079, controlling for age) was between the geometric shape of the costal face and the surface texture of the tubercle facet. Assuming a correlation of zero, these two traits were used to calculate the highest posterior density regions for estimating individual ages-at-death. Age-at-death estimates generated from 50 and 95% posterior density regions indicate that this method captures age-related change reaching the ninth decade. The Bayesian statistical approach used here produced a valuable and promising new method for estimating age-at-death. Additional research is necessary to determine if these highest posterior density regions produce results highly correlated with age in other samples and its applicability to females.     

A new evaluation method for quantifying PI3K activity by HTRF assay

PIK3CA, coding a catalytic subunit of PI3K p110α, is frequently mutated in cancer. In previous studies, p110α with hotspot mutations such as E545K and H1047R were shown to be gain-of-function mutations. However, quantitative evaluation of these mutants was not well established. Recently, a new method for measuring PI3K activity using homogeneous time-resolved fluorescence (HTRF) has been developed. Using this method, we constructed a quantitative evaluation system for PI3K activity. Serial dilutions of standard PIP3 were subjected to the PI3K-HTRF assay in order to establish a regression line for calibration. The recombinant FLAG-tagged p110α proteins were engineered together with a regulatory subunit p85α in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled us to quantitatively evaluate the activities of hotspot mutants of p110α. We believe this method will also be applicable to the evaluation of p110α having uncharacterized mutations found in cancer.     

Solid-liquid interface method (SLIM): A new crystallization method for proteins

Despite impressive advances in theories, methods and technologies, crystallization still remains a serious bottleneck in structural determination of macromolecules. Here we present a novel solid-liquid interface method (SLIM) for protein crystallization, based on the pre-adding and drying of a crystallization reagent, and thereafter the dispensing of a protein solution to the dried media to initiate crystallization from the solid-liquid interface. Not only quick and easy to perform, the method also allows for a less concentrated protein solution for setting up crystallization trials.     

A universal method for automated gene mapping

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.     

A universal method for automated gene mapping

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.     

Universal fluorescent labeling (UFL) method for automated microsatellite analysis.

We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.     

Rapid automated multichannel microspectrofluorometry : A new method for studies on the cell-to-cell transfer of molecules

A new microchemical method is described to study the transfer of molecules between neighboring cells: rapid multichannel microfluorometry. The cell-to-cell transfer rates of metabolites or fluorescent tracers (microinjected by a microelectrophoretic or piezoelectric technique) are followed at pace with the in situ velocities of such processes via multisite topographic monitoring of fluorescence in injected cell and neighbors (continuously before, during and after microinjection). Furthermore, the sensitivity of the method allows the kinetic study of cell-to-cell transfer using metabolites (e.g., glucose-6-P) which are not fluorescent themselves, but which elicit associated changes in the redox state of endogenous fluorochromes, i.e. NAD(P) NAD(P)H transients.Since the cell-to-cell transfer of chemicals is known to proceed in various cells and tissues via intercellular junctions, the above technique was applied to a known coupled system (e.g. Chironomus salivary gland) and an uncoupled system (e.g. L cells). The cell-to-cell transfer of fluorescein was observable kinetically in Chironomus salivary glands, liver, Chang liver and Chang conjunctiva cultures. Equilibration of the dye between injected cell and neighbor was completed within a few hundred msec to a few sec, corresponding to an intercellular half total flux per unit concentration difference (g_f) of ˜2–10 × 10⁴ μm³/min. Such transfer was practically basent or a rare occurrence in L cells. In NCTC 8739 and L cells, the cell-to-cell transfer of glucose-6-P seemed to occur more frequently than that of fluorescein, suggesting the possibility that glucose-6-P (or a catabolite) may move through non-junctional regions of the cell membrane. Thus multichannel microfluorometry makes possible the study of tracer or metabolite transfer, without the need for multiple implantation of electrodes and with more kinetic detail than obtainable by other tracer techniques.     

水域生态系统中生物多样性经济价值评估的一个新方法

生物多样性的经济价值,是目前国内外有关生态系统服务价值研究中难点之一。生物多样性经济价值分为使用价值和非使用价值两类,前者包括直接和间接使用价值,后者包括选择价值、遗产价值和存在价值[1]。一般而言,对生物多样性的使用价值采用直接市场评价法较多,对非使用价值采用支付意愿法较多。但是这种基于支付意愿(Willingness to pay,WTP)调查的条件价值法(Contingent value method,CVM)评价生物多样性经济价值面临许多的困难,甚至非议[2—4]。许多学者认为,由于在生物多样性价值概念、计量方法、计量参数等方面的不规范性,导致生物多…     

An automated method for the evaluation of ram libido in real mating conditions

We investigated if sexual behaviour of rams can be assessed with an electronic Alpha-Detector (AD) which automatically records mounts of mating rams. To evaluate the rams’ libido (i.e. all sexual activities), we used six intact and six vasectomised rams in pen tests in three different seasons (late spring, autumn and early spring). The pen tests consisted of 30-min visual observations of each ram placed in a group of six Merino ewes (three ewes in oestrus and three ewes not in oestrus). In the pen tests, sexual behaviour was recorded and divided into two categories: pre-copulatory and copulatory. For validation purposes, during the pen tests the 12 rams were equipped with the AD and the number of times the 18 oestrous ewes were mounted were counted over a period of 3 days. Of the 1191 mounts visually identified in the six 30-min sessions, 1026 were recorded automatically by the AD (i.e. 94%). The paddock test is an automated method consisting of the same rams wearing an AD and placed in a flock of ~250 Merino ewes on two occasions (late spring (spring 1) and early spring of the following year (spring 2)), their copulatory activities were automatically recorded over a 5-day period. The results of the pen tests in the three seasons revealed no difference between the two types of rams (breeding v. detecting rams). Based on live observations high correlations (r=+0.81, P<0.003 for breeding and r=+0.76, P<0.02 for detecting rams) were found between pre-copulatory and copulatory behaviours. The libido of the two types of rams measured in pen tests showed high repeatability across the three seasons (83 and 75%, P<0.05 for copulatory and pre-copulatory behaviours, respectively). When measured automatically in paddock tests over two consecutive springs, even higher repeatability was observed in both breeding (94%; P<0.01) and detecting rams (97%; P<0.004) in the number of mounts. In addition, high correlations (+0.89<r<+0.94) between copulatory behaviours, as measured by live observations, and those measured by the AD were obtained. The automatic measurement of ram libido in paddock tests appears to be more reliable than pen tests and far less time consuming. We therefore recommend this automated method to estimate the libido of rams. In addition, this method can be used at any season of the year provided that ewes in oestrus are present in the flock.     

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

A new evaluation method for +Gz tolerance with loratadine by using a near-infrared spectroscopy

Background

Loratadine (Claritin^®), an over the counter antihistamine in U.S. and UK, is acceptable for use without adverse side effects by aircrew with mild or moderate allergic or other situations requiring an antihistamine. Although +Gz (head to foot direction) tolerance testing for aircrew with loratadine has not been documented in the published literature, it is commonly accepted that loratadine dose not effect +Gz tolerance. The purpose of this study was to offer and validate a new evaluation method for +Gz tolerance testing with loratadine by using a near-infrared spectroscopy (NIRS).

Methods

A double-blind, placebo-controlled, randomized, crossover protocol was used to administer 10 mg of loratadine or placebo in nine healthy subjects. The subjects didn't wear anti-G suit. The +Gz exposure profiles consisted of, in series, a gradual onset ran (0.1 G·sec^-1) to the subject's visual end-point (peripheral light loss) or loss of consciousness (GLOC), and rapid onset run (1.0 G·sec^-1) to the subject's same end-point. In this study, G-level tolerance was defined as the +Gz level at visual end-point and/or at GLOC. As a subject's G-duration tolerance, we measured the total time (seconds) during rapid onset run. Otherwise, to confirm the effect of loratadine on +Gz tolerance, we measured the cerebral NIRS variables (hemoglobin concentration changes and tissue oxygenation index) as a new quantitative method for +Gz tolerance during a centrifuge experiments.

Results

No significant differences were observed in +Gz tolerance (+Gz level, duration time and NIRS variables) between subjects taking loratadine and placebo.

Conclusion

Our results demonstrate that loratadine has no detectable effect on +Gz tolerance by using a new method with cerebral NIRS variables and the traditional method with +Gz level and duration time. This study represents the first use of a quantitative parameter such as cerebral NIRS variables to assess the effects of a drug on acceleration tolerance.

     

A method for the rapid automated assessment of olfactory function

We have developed a strategy for the rapid high-throughput screening of odor responsivity in genetically altered mice (in fact, any experimentally altered animal). Specifically, the report presents the development and validation of a fully automated procedure based on the evaluation of an animal's stimulus-induced reflexive breathing response (i.e. sniffing behavior) to both air and odorant stimuli. The method requires no training of the animal to be screened and the outcome of the evaluation yields an operationally defined measure. Briefly, using whole-body plethysmography, the procedure determines the numerical values for a set of 14 respiratory measures in response to the presentation of air and a well-above-threshold concentration of the odorant propanol. These measures of stimulus-induced sniffing are incorporated into a model that defines a single univariate measure of response behavior, or 'Sniffing Index', for each screened animal. The approach significantly discriminated between the reflexive sniffing response of a control group of mice and that of an experimentally defined manipulated group for which, a priori, we expected to observe a robust altered breathing response to odorant stimulation (i.e. non-odor-aversion-conditioned versus odor-aversion-conditioned C57BL/6J mice). Further, the procedure was able to significantly discriminate between a mutant phenotype with documented alterations in physiologic and behavioral function (namely, the OMP-null mutant), and their background strain. In addition, applying epidemiologic screening principles to the observed data, we established an operational procedure for the evaluation of unknown animals.     

A new method for evaluation of cavitation near mechanical heart valves

Evaluation of cavitation in vivo is often based on recordings of high-pass filtered random high-frequency pressure fluctuations. We hypothesized that cavitation signal components are more appropriately assessed by a new method for extraction of random signal components of the pressure signals. We investigated three different valve types and found a high correlation between the two methods (r2: 0.8806-0.9887). The new method showed that the cavitation signal could be extracted without a priori knowledge needed for setting the high-pass filter cut off frequency, nor did it introduce bandwidth limitation of the cavitation signal.     

A convenient automated method for the determination of proteolytic enzymes

An automated method for the assay of proteolytic enzymes is described. The insoluble dye-protein complex, Remazolbrilliant Blue-hide powder, is used as the substrate and is readily digested by trypsin, elastase, subtilisin, thermolysin, chymotrypsin, and to a lesser extent pronase, pepsin, and bacterial collagenase. The proteolytic activity of crude microbial culture preparations is expressed in terms of an equivalent concentration of crystalline trypsin, which itself can be readily determined within the concentration range 0.25–5.0 μg/ml.     

A non-radioactive automated method for DNA sequence determination

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.