Histochemical Demonstration Of Total Phosphorylase Activity For Diagnosis Of Carcinoma Cells In Human Stomach And Intestines

Modified techniques for the histochemical demonstration of phosphorylase of value in the diagnosis of cancer cells of human gastro-intestinal origin are described. Preservation of the enzyme activity is especially considered; this is facilitated by the use of thoroughly dried cryostat sections. Routine use of these techniques in the clinical laboratory is suggested. Using these procedures, carcinoma ceils of human stomach and intestines stained in 102 of 117 cases; normal control sections of human gastric epithelial cells are regularly unstained.     

Prospects for the use of nuclear transfer in human transplantation

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.     

MicroRNA Expression Profiles of Human iPS Cells,Retinal Pigment Epithelium Derived From iPS,and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.     

Characteristics of human embryonic neuronal cells procured by non-enzyme method

Isolation and culturing of human neuronal progenitor cells is of significant value for both fundamental research and therapeutic purposes. In this work, human embryonic neuronal cells were characterized as a heterogeneous population of progenitor cells with various differentiation potentials. During in vitro culturing the cells are capable of re-inoculating, proliferating, differentiating and migrating. While differentiating, these cells form neurons and glial cells. The present research demonstrates that depending upon the culturing conditions the embryonic neuronal cells may either form floating aggregates (incubation with embryonic serum), attach (incubation without serum), proliferate, or form neurospheres. Besides, peculiarities of aggregate differentiation during their incubation under various media are described.     

Current status of stem cell research: An editorial

The purpose of this editorial is to highlight recent developments in molecular biology tools and techniques in stem cell research and their applications to human diseases. Recent advancements in stem cell research and regenerative medicine are offering immense hope to cure human diseases and injuries, such as cancer, diabetes, Alzheimer's disease, Parkinson's disease, and traumatic brain injuries. In the last three decades, especially in the last decade, major breakthroughs have been seen in the conversion of adult stem cells into induced pluripotent stem cells, which in turn has led the way to developing stem cell therapies for human diseases. This article summarizes contributions of research into stem cell therapies.     

Trace metal imaging with high spatial resolution: applications in biomedicine

New generations of analytical techniques for imaging of metals are pushing hitherto boundaries of spatial resolution and quantitative analysis in biology. Because of this, the application of these imaging techniques described herein to the study of the organization and dynamics of metal cations and metal-containing biomolecules in biological cell and tissue is becoming an important issue in biomedical research. In the current review, three common metal imaging techniques in biomedical research are introduced, including synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). These are exemplified by a demonstration of the dopamine-Fe complexes, by assessment of boron distribution in a boron neutron capture therapy cell model, by mapping Cu and Zn in human brain cancer and a rat brain tumor model, and by the analysis of metal topography within neuromelanin. These studies have provided solid evidence that demonstrates that the sensitivity, spatial resolution, specificity, and quantification ability of metal imaging techniques is suitable and highly desirable for biomedical research. Moreover, these novel studies on the nanometre scale (e.g., of individual single cells or cell organelles) will lead to a better understanding of metal processes in cells and tissues.     

Interobserver and intraobserver differences in the diagnosis of urothelial cells. Comparison with classification by computer

The diagnostic performance of six human observers with various degrees of experience was tested against the diagnosis developed by consensus and by an automated, computer-generated cell-classification system. The study was based on randomly selected photographs of 200 urothelial cells, classified in groups of 50 as negative, atypical, suspicious and malignant, from smears of urine sediment. The performance of the human observers was tested in three separate sessions at suitable time intervals and evaluated by two analytical statistical techniques: the weighted kappa proposed by Cohen, to evaluate the degree of disagreement, and the log-linear analysis of association. The results indicated considerable interobserver and intraobserver differences, which appeared to be more closely related to the individual's perception of the visual targets than to the degree of experience or the target itself. The performance of the programmed computer was well within the midrange of the human diagnostic performance. This study indicated that under the experimental circumstances described in this paper, the diagnostic perception of cell images is highly subjective. The study implies that similar differences may exist elsewhere in diagnostic microscopy and suggests that the development of objective approaches to the morphologic classification of human disease may prove to be of clinical value.     

Numerical computation of distortions in magnetic fields and induced currents in physiological solutions produced by microscope objectives

Identifying distortions produced by commonly employed microscope objectives and their components in uniform DC and 60 Hz AC magnetic fields is important in imaging studies involving exposure of cells to spatially uniform or nonuniform magnetic fields. In this study, DC and 60 Hz AC magnetic flux densities were numerically computed in the presence of finite element models of various components of commonly utilized microscope objectives, as well as a model of a complete objective. Also computed were the distortions in the current density induced by an applied time-varying magnetic field in a physiological buffer contained within a Petri dish. We show that the magnetic flux density could be increased up to 65% in the presence of the nickel-chrome plating of an objective housing and that the presence of ferromagnetic components like a screw or spring could produce peaks that are 7% higher than the undistorted value of magnetic flux density. In addition, a slight tilt of 1% in the objective with respect to the magnetic field could cause a 93% deviation in magnetic flux density from the unperturbed value. These results correlate well with previously published experimental measurements that showed the presence of significant and sometimes asymmetric distortions in both DC and 60 Hz magnetic fields. Moreover, this study further reports that induced current density changed up to 37% compared to values in the absence of the objective. The existence of distortions in applied magnetic fields and induced currents could affect the interpretation of results of cell function studies if it is assumed that the cells are exposed to uniform magnetic flux densities in the presence of a microscope objective. Such assumptions of uniform magnetic flux density could also account for the lack of reproducibility in several studies that examined changes in intracellular calcium by imaging techniques.     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

Some theoretical and practical considerations for multivariate statistical cell classification useful in autologous stem cell transplantation and tumor cell purging.

BACKGROUND: As flow cytometric data becomes more complex, it becomes increasingly difficult to classify cells using conventional flow cytometry data techniques based on visual classification of the data by user-drawn regions. This paper shows some simple applications of multivariate statistical classification to classify flow cytometric data. METHODS: Discriminant Function Analysis (DFA) and Logistic Regression (LR) analysis techniques were evaluated with respect to their potential utility in the problem of detecting human breast cancer cells within normal bone marrow cells. Data sets having defined properties were employed to evaluate the potential utility of these statistical classification techniques whose performance was measured by ROC analysis. RESULTS: Two extreme but reasonable situations are presented: (1) data where the separation of cells was obvious by visual inspection and (2) data where major overlaps in the values of the individual FCM parameters made intuitive classification improbable. Both DFA and LR analysis were able to classify the cells of each type with acceptable accuracy and yield.CONCLUSIONS: The excellent empirical performance of both DFA and LR techniques, suggests that they offer promising approaches for classifying multiparameter FCM data using objective rules that may represent an improvement over commonly employed ad hoc approaches.     

Bacteriophage resistance inLactococcus

Lactic acid bacteria are industrial microorganisms used in many food fermentations.Lactococcus species are susceptible to bacteriophage infections that may result in slowed or failed fermentations. A substantial amount of research has focused on characterizing natural mechanisms by which bacterial cells defend themselves against phage. Numerous natural phage defense mechanisms have been identified and studied, and recent efforts have improved phage resistance by using molecular techniques. The study of how phages overcome these resistance mechanisms is also an important objective. New strategies to minimize the presence, virulence, and evolution of phage are being developed and are likely to be applied industrially.     

Human embryonic stem cell research: why the discarded-created-distinction cannot be based on the potentiality argument

Discussions about the use and derivation of pluripotent human embryonic stem cells are a stumbling block in developing public policy on stem cell research. On the one hand there is a broad consensus on the benefits of these cells for science and biomedicine; on the other hand there is the controversial issue of killing human embryos. I will focus on the compromise position that accepts research on spare embryos, but not on research embryos ('discarded-created-distinction', from now on d-c-d). I will point out that this viewpoint is hard to maintain. The main focus is that the 'revealed beliefs' of its defenders are inconsistent with their 'professed beliefs', more specifically with their main argument, i.e. the potentiality argument. I will point out that (1) the defenders of d-c-d actually grant a relative moral status to the human embryo, (2) this moral status is dependent on internal and external criteria of potentiality, (3) potentiality seen as a variable value that also depends on external criteria cannot justify d-c-d, and (4) an approach to human embryonic stem cell-research that would also allow the use of research embryos is more compatible with the feelings, attitudes and values of those who currently defend d-c-d and, therefore, could lead to a broader consensus and to actions that alleviate individual human suffering.     

Modeling chromosomes in mouse to explore the function of genes, genomic disorders, and chromosomal organization

One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures.     

用于疫苗生产的人二倍体细胞研究进展*

人二倍体细胞(human diploid cells, HDCs)作为制备疫苗的重要培养细胞基质备受人们的关注。由于人二倍体细胞与人类基因组相同、无外源因子、对多种病毒易感性、无潜在致瘤性、所制备的人二倍体疫苗(human diploid cell vaccine, HDCV)具有良好的免疫原性和安全性,适合于疫苗的工业化生产。当前,人群中使用的灭活疫苗、减毒疫苗或亚单位疫苗等均依赖于原代细胞、传代细胞和人二倍体细胞,其中用于疫苗制备的人二倍体细胞主要有WI-38、MRC-5、2BS和KMB-17等细胞系。然而,人二倍体细胞为有限性细胞,细胞的来源和培养技术等存在某些缺陷,进而影响其应用。综述了用于疫苗生产的人二倍体细胞及其疫苗制备技术的研究进展,并分析了存在的问题及改进策略。     

Exploring novel objective functions for simulating muscle coactivation in the neck

Musculoskeletal modeling allows for analysis of individual muscles in various situations. However, current techniques to realistically simulate muscle response when significant amounts of intentional coactivation is required are inadequate. This would include stiffening the neck or spine through muscle coactivation in preparation for perturbations or impacts. Muscle coactivation has been modeled previously in the neck and spine using optimization techniques that seek to maximize the joint stiffness by maximizing total muscle activation or muscle force. These approaches have not sought to replicate human response, but rather to explore the possible effects of active muscle. Coactivation remains a challenging feature to include in musculoskeletal models, and may be improved by extracting optimization objective functions from experimental data. However, the components of such an objective function must be known before fitting to experimental data. This study explores the effect of components in several objective functions, in order to recommend components to be used for fitting to experimental data. Four novel approaches to modeling coactivation through optimization techniques are presented, two of which produce greater levels of stiffness than previous techniques. Simulations were performed using OpenSim and MATLAB cooperatively. Results show that maximizing the moment generated by a particular muscle appears analogous to maximizing joint stiffness. The approach of optimizing for maximum moment generated by individual muscles may be a good candidate for developing objective functions that accurately simulate muscle coactivation in complex joints. This new approach will be the focus of future studies with human subjects.     

Induction of telomerase activity increase reprogramming efficiency of human dermal fibroblasts

Reprogramming of human cells is a perspective direction of development of Cell Biology with the distant prospect of using these methods for cellular-replacing therapy in clinical practice. One of the problems rising in this regard is low reprogramming efficiency and application of undesirable oncogenes in initial techniques. In this research, we had offered the alternative modified method of reprogramming human skin fibroblasts. It has been shown that the telomerase induction increases an exit of reprogrammed cells and allows excluding oncogene c-Myc from a used set of genes.     

Pluripotent human stem cells for the treatment of retinal disease

Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients.     

Integrating Concern for Animals into Personal Values

Recent scholarship in human–animal relationships demonstrates that new theoretical insights emerge when these relationships are seriously considered. We suggest that the most prominent measures of values in the environmental decision-making research literature, the Schwartz approach, may not adequately capture key aspects of how individuals value animals. Our research used confirmatory factor analysis and ordinary least squares regression techniques on data collected from an Amazon Mechanical Turk online survey. It expands on the values commonly used in the environmental decision-making literature to consider whether there is a distinct value dimension focused on animals. Our suggestion that a “concern for animals” value orientation constitutes a motivation that is separate from the other values commonly used in research on environmental decision-making finds support in our data. Traditional scaling methods indicate that the items we created have high reliability when used to form an unweighted additive scale. The results of confirmatory factor analysis are consistent with the argument that this “concern for animals” value orientation is distinct from biospheric altruism, humanistic altruism, self-interest, traditionalism, openness to change, and hedonism. Finally, our regressions using values and demographic, social, and political characteristics as controls show that an animal-focused value orientation and biospheric altruism have different relationships with identification with the animal rights movement than with identification with the environmental movement.