Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Significance levels in complex inheritance.

A LOD score >=3 is necessary but not sufficient to make a linkage test reliable, and this applies to complex inheritance as well as to major loci. Factors that affect this threshold are considered here. A LOD score as small as 2 is suggestive but is unreliable except as confirmation of either a significant linkage or a strong candidate locus. A threshold as great as 4 is unnecessarily conservative if multipoint tests are used sensibly. Marker density is not a major factor, and biases in the evaluation of LOD scores-especially inadequate allowance for estimation of nuisance parameters in multiple models-are paramount. Allelic association increases resolution for oligogenes within a candidate region and remains the only practical method to locate polygenes. The method sketched here combines multipoint linkage and allelic association to test efficiently for a regional candidate locus.     

A Unified Approach to Simultaneous Rank Test Procedures in the Unbalanced One‐way Layout

A general nonparametric approach to asymptotic multiple test procedures is proposed which is based on relative effects and which includes continuous as well as discontinuous distributions. The results can be applied to all relevant multiple testing problems in the one‐way layout and include the well known Steel tests as special cases. Moreover, a general estimator for the asymptotic covariance matrix is considered that is consistent even under alternative. This estimator is used to derive simultaneous confidence intervals for the relative effects as well as a test procedure for the multiple nonparametric Behrens‐Fisher problem.     

A generalized estimating equations approach to quantitative trait locus detection of non-normal traits

To date, most statistical developments in QTL detection methodology have been directed at continuous traits with an underlying normal distribution. This paper presents a method for QTL analysis of non-normal traits using a generalized linear mixed model approach. Development of this method has been motivated by a backcross experiment involving two inbred lines of mice that was conducted in order to locate a QTL for litter size. A Poisson regression form is used to model litter size, with allowances made for under- as well as over-dispersion, as suggested by the experimental data. In addition to fixed parity effects, random animal effects have also been included in the model. However, the method is not fully parametric as the model is specified only in terms of means, variances and covariances, and not as a full probability model. Consequently, a generalized estimating equations (GEE) approach is used to fit the model. For statistical inferences, permutation tests and bootstrap procedures are used. This method is illustrated with simulated as well as experimental mouse data. Overall, the method is found to be quite reliable, and with modification, can be used for QTL detection for a range of other non-normally distributed traits.     

Xenotransplant-associated infections

Xenotransplantation is considered increasingly as a solution to the current shortage of human organs for allotransplantation. In addition, it is being investigated as a treatment for a number of other diseases such as Parkinson's disease, diabetes mellitus, and acquired immunodeficiency disease. The potential risk of novel zoonotic infections is a concern associated with these procedures. Accordingly, the role of animal microbial agents must be critically examined. This review examines the concerns and proposed mechanisms for xenogeneic infections and details what is known and what still needs to be learned as the field of xenotransplantation progresses. Emphasis is placed on microbial agents of baboons and swine because they are currently the most common species considered as donor sources for xenotransplantation.     

Demographic incidence rates

Demographers commonly interpret incidence rates (for first marriages, say, or for births of a given order), cumulated over age for a closed cohort, as prevalences of corresponding demographic statuses. Since one is hard put to find an explicit justification for this interpretation in the literature, this note offers a mathematical proof that it is correct if and only if there is nondifferential mortality, as well as an extension to incidences for events which can happen more than once to an individual. Mortality incidence rates are discussed as well, and some of the evidence that cumulative incidence rates may get absurd values when computed from period data is reviewed.     

Role of autophagy in bone and muscle biology

Autophagy in eukaryotic cells is a constitutive process and functions as a homeostatic mechanism; it is up-regulated in response to specific stress stimuli such as starvation, hypoxia and as oxidative stress. In addition to playing a crucial role in adaptive responses to different stimuli, autophagy is also required for intracellular quality control. This second aspect is important to prevent the activation of pathological processes. Autophagy also plays a central role in cellular development and differentiation because it is involved in the regulation of energetic balance. This final aspect is critical for maintaining proper bone and muscle function as well as to prevent any pathological changes. Therefore, identifying new molecular targets involved in autophagy is critical to assure a good quality of life.     

Development of a high-throughput IMS-IMS-MS approach for analyzing mixtures of biomolecules

A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be +/-7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 h. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed.     

Biodiversity hotspots: hot for what?

In complex areas of international policy, such as biodiversity conservation, there is a risk that well-promoted strategies will be received by decision makers as a cure-all. The U.S.-based Conservation International is promoting biodiversity hotspots as a 'silver bullet' strategy for conserving most species for least cost. We assess the degree to which this goal is compatible with four social values that characterize the conservation movement. We find that biodiversity hotspots provide only a partial response because conservation does not treat all species as equal. We argue that explicit recognition of such values is fundamental to a structured debate contributing to the development of a common strategy for biodiversity conservation.     

Relationship of body composition to somatotype.

The purpose of this study is to determine the relationship in college-aged women between somatotype using both Sheldon's ('69) and Heath and Carter's ('67) procedures, and body composition, as measured by whole-body 40K counting and body density. Sheldon's endomorphy is closely associated with height and weight; Heath and Carter's first component is significantly related to weight and body fatness. Lean body mass (LBM) as a weight or as a percent is not closely related to Sheldon's mesomorphy or Heath and Carter's second component. However, when LBM and height are used as independent variables to estimate somatotype, both variables are significantly related to Heath and Carter's second component, accounting for 61% of the variance. Thus Heath and Carter's second component is significantly associated with LBM for a given body height. Most of the variation in Sheldon's ectomorphy and Heath and Carter's third component can be accounted for by weight and height. Sheldon's somatotype for all three components is not as closely related to body composition as Heath-Carter's. Body composition, as measured by either 40K counting or body density, is found to be important in accounting for variation in Heath and Carter's first and second components.     

The question of virginity testing in Turkey

Pre-marital sex for a woman is regarded as wrong in my country. As a result, it is socially forbidden for a woman to engage in this act. In order to present a woman as a virgin on her marriage day, she is subjected to pressure, and put under control both by her family and societal norms. However, a man is free and never made to suffer any of the above. A woman found to be a virgin on her first night of marriage is seen as a normal person while one suspected to have lost her virginity is made to undergo a series of medical examinations to bring clarity to her situation.     

Beyond directed evolution: Darwinian selection as a tool for synthetic biology

Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed.     

Plant-based insect repellents: a review of their efficacy, development and testing

Plant-based repellents have been used for generations in traditional practice as a personal protection measure against host-seeking mosquitoes. Knowledge on traditional repellent plants obtained through ethnobotanical studies is a valuable resource for the development of new natural products. Recently, commercial repellent products containing plant-based ingredients have gained increasing popularity among consumers, as these are commonly perceived as "safe" in comparison to long-established synthetic repellents although this is sometimes a misconception. To date insufficient studies have followed standard WHO Pesticide Evaluation Scheme guidelines for repellent testing. There is a need for further standardized studies in order to better evaluate repellent compounds and develop new products that offer high repellency as well as good consumer safety. This paper presents a summary of recent information on testing, efficacy and safety of plant-based repellents as well as promising new developments in the field.     

Vision Training Methods for Sports Concussion Mitigation and Management

There is emerging evidence supporting the use vision training, including light board training tools, as a concussion baseline and neuro-diagnostic tool and potentially as a supportive component to concussion prevention strategies. This paper is focused on providing detailed methods for select vision training tools and reporting normative data for comparison when vision training is a part of a sports management program. The overall program includes standard vision training methods including tachistoscope, Brock’s string, and strobe glasses, as well as specialized light board training algorithms. Stereopsis is measured as a means to monitor vision training affects. In addition, quantitative results for vision training methods as well as baseline and post-testing *A and Reaction Test measures with progressive scores are reported. Collegiate athletes consistently improve after six weeks of training in their stereopsis, *A and Reaction Test scores. When vision training is initiated as a team wide exercise, the incidence of concussion decreases in players who participate in training compared to players who do not receive the vision training. Vision training produces functional and performance changes that, when monitored, can be used to assess the success of the vision training and can be initiated as part of a sports medical intervention for concussion prevention.     

Plant oils as feedstock alternatives to petroleum – A short survey of potential oil crop platforms

Our society is highly depending on petroleum for its activities. About 90% is used as an energy source for transportation and for generation of heat and electricity and the remaining as feedstocks in the chemical industry. However, petroleum is a finite source as well as causing several environmental problems such as rising carbon dioxide levels in the atmosphere. Petroleum therefore needs to be replaced by alternative and sustainable sources. Plant oils and oleochemicals derived from them represent such alternative sources, which can deliver a substantial part of what is needed to replace the petroleum used as feedstocks.     

Defining life from death: Problems with the somatic integration definition of life

To determine when the life of a human organism begins, Mark T. Brown has developed the somatic integration definition of life. Derived from diagnostic criteria for human death, Brown’s account requires the presence of a life-regulation internal control system for an entity to be considered a living organism. According to Brown, the earliest point at which a developing human could satisfy this requirement is at the beginning of the fetal stage, and so the embryo is not regarded as a living human organism. This, Brown claims, has significant bioethical implications for both abortion and embryo experimentation. Here, we dispute the cogency of Brown’s derivation. Diagnostic criteria for death are used to determine when an organism irreversibly ceases functioning as an integrated whole, and may vary significantly depending on how developed the organism is. Brown’s definition is derived from a specific definition of death applicable to postnatal human beings, which is insufficient for generating a general definition for human organismal life. We have also examined the bioethical implications of Brown’s view, and have concluded that they are not as significant as he believes. Whether the embryo is classified as a human organism is of peripheral interest—a far more morally relevant question is whether the embryo is a biological individual with an identity that is capable of persisting during development.     

The epithelial integrin alphavbeta6 is a receptor for foot-and-mouth disease virus

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin alphavbeta3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, alphavbeta6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin beta6 subunit and expression of alphavbeta6 at the cell surface. Integrin alphavbeta6 is the major site for virus attachment on the beta6-transfected cells, and binding to alphavbeta6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the beta6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes alphavbeta6. These studies establish a role for alphavbeta6 as a cellular receptor for FMDV.     

Molecular aspects of sucrose transport and its metabolism to starch during seed development in wheat: A comprehensive review

Wheat is one of the most important crops globally, and its grain is mainly used for human food, accounting for 20% of the total dietary calories. It is also used as animal feed and as a raw material for a variety of non-food and non-feed industrial products such as a feedstock for the production of bioethanol. Starch is the major constituent of a wheat grain, as a result, it is considered as a critical determinant of wheat yield and quality. The amount and composition of starch deposited in wheat grains is controlled primarily by sucrose transport from source tissues to the grain and its conversion to starch. Therefore, elucidation of the molecular mechanisms regulating these physiological processes provides important opportunities to improve wheat starch yield and quality through biotechnological approaches. This review comprehensively discusses the current understanding of the molecular aspects of sucrose transport and sucrose-to-starch metabolism in wheat grains. It also highlights the advances and prospects of starch biotechnology in wheat.     

A template method for decomposing flow cytometry histograms of human chromosomes.

A new method for decomposing flow cytometry histograms of isolated human metaphase chromosomes is described and tested. The method is based on fitting a template, composed of the means of all chromosomes of a normal karyotype to the flow histogram. The utility of the method is demonstrated by application to flow measurements of chromosomes from a normal person and comparing the results with those obtained by conventional cytophotometry. The power of the method for detecting gross chromosomal abnormalities, such as trisomy 21, as well as more subtle variations such as a single translocation, is determined for simulated data.