An approach to identify functional homologues and suppressors of genes in fission yeast

We have developed a procedure using a bank of temperature-sensitive (ts) mutants of fission yeast to identify mutants which can be rescued by expression of a plasmid-borne gene of interest. The procedure has been used to identify new ts alleles of cdc2 and swi7/pol1, a ts mutant rescued by actin, and to identify a ts allele of cdc11 which can be rescued by combined mammalian Myc and Max expression. The procedure should also be useful as an alternative approach to identify genes in fission yeast which are functionally homologous to genes of interest from other organisms. Received: 28 February 1997     

Mixed-up nuts: identification of peanuts and tree nuts by children.

BACKGROUND: Peanuts and tree nuts frequently cause severe allergic reactions. Nut avoidance is the key treatment, and accurate identification of nuts is essential for successful avoidance. OBJECTIVES: To determine the age at which nut-allergic and nonallergic children can accurately identify various nuts and whether nut-allergic children can identify nuts they should avoid. METHODS: A "nut box" was constructed containing samples of 11 common nuts and pine nuts. Nut-allergic and nonallergic children were asked to identify the nuts, and their responses were compared and correlated by age. Nut-allergic children were asked to identify the nut(s) that they should not eat. RESULTS: One hundred children (37 allergic and 63 nonallergic) were enrolled. The mean number of nuts correctly identified was only 2.7 per child and increased with age, but there was large variation. Fifty-nine children identified 2 or fewer nuts. Peanuts in the shell were identified most often (89% of children), followed by peanuts out of the shell (52%). Other nuts were identified less commonly, ranging from 32% for pistachios to 0% for Brazil nuts. Nut-allergic children were not better able to correctly identify tree nuts and were less able in the case of peanuts. Of the nut-allergic children, 10 (27%) could not identify the peanut or tree nut to which they were allergic. CONCLUSIONS: In general, children, including those who are allergic to nuts, can identify few nuts. This lack of recognition could put them at increased risk for unintentional ingestion. As part of an overall educational plan, nut-allergic children should be taught not only to avoid but also to identify the nut to which they are allergic.     

Typing the HLA-B locus by a nested primer approach and oligonucleotide hybridization

A system for intermediate level identification of the HLA-B locus alleles was devised. This system can be extended to identify individual alleles in any sample. The first step used primers which amplify all HLA-B alleles. This amplicon was subjected to SSOP hybridization to allow intermediate level typing of samples. In the second step, group-specific primers were utilized to obtain specific amplification of groups consisting of a few alleles. The oligotypes within each group were identified by the use of SSOP. The separation of groups of alleles by amplification allowed the use of a limited number of probes to identify oligotypes present in a sample. Additional probes can be added as new alleles are identified, increasing the flexibility of the system. HLA typing software was developed to determine the resolution of the system and to identify HLA oligotypes. PCR-SSOP methods are in wide use and have been extensively validated. The procedures reported here will be relatively easy to implement for large-scale DNA-based typing of the HLA-B locus.     

Understanding the pathogenesis of psoriasis, psoriatic arthritis, and autoimmunity via a fusion of molecular genetics and immunology

The goal of my laboratory is to understand the molecular genetics basis of the inflammatory skin disease psoriasis and associated psoriatic arthritis. In performing these studies my colleagues and I have begun to identify common pathways leading to autoimmunity as well, because some of the defective pathways leading to autoimmunity are the same in different autoimmune diseases. Some of these pathways are involved in determining the activation status of inflammatory cells in the resting state. Other pathways are likely to determine target organ specificity and will be unique to a particular disease. Our approaches rely on genetic studies with cases and families to identify the causative variants, and then functional studies to identify the role of these variants in the predisposition to psoriasis and autoimmunity. The advantage of genetics approaches to understanding diseases such as those of the immune system is that one can identify novel genes and pathways that were not previously suspected as being involved in either the immune system or tolerance.     

Monoclonal hybridoma screening by analysis of immunoglobulin light chains

A technique is described for the characterization of immunoglobulin light chains of hybridomas in culture. Immunoglobulins biosynthetically labelled with 14C are obtained from culture supernatants. Following complete reduction and alkylation light chains are separated from heavy chains and most other labelled contaminants by urea-formate gel electrophoresis. They are subsequently analyzed by isoelectric focusing using a simple transfer procedure. The method can be used to analyze up to 30 samples at a time and has a potential for the distinction of 750 light chains. The technique is especially useful (1) to determine the monoclonality of antibodies at an early stage in production, (2) to identify and classify antibodies having different structures but similar specificities, (3) to identify any alterations which may occur in quantity or quality of antibodies in long term culture, (4) to identify different hybridomas which produce antibodies of identical light chain subgroup.     

Evaluation of regulatory potential and conservation scores for detecting cis-regulatory modules in aligned mammalian genome sequences

Techniques of comparative genomics are being used to identify candidate functional DNA sequences, and objective evaluations are needed to assess their effectiveness. Different analytical methods score distinctive features of whole-genome alignments among human, mouse, and rat to predict functional regions. We evaluated three of these methods for their ability to identify the positions of known regulatory regions in the well-studied HBB gene complex. Two methods, multispecies conserved sequences and phastCons, quantify levels of conservation to estimate a likelihood that aligned DNA sequences are under purifying selection. A third function, regulatory potential (RP), measures the similarity of patterns in the alignments to those in known regulatory regions. The methods can correctly identify 50%-60% of noncoding positions in the HBB gene complex as regulatory or nonregulatory, with RP performing better than do other methods. When evaluated by the ability to discriminate genomic intervals, RP reaches a sensitivity of 0.78 and a true discovery rate of approximately 0.6. The performance is better on other reference sets; both phastCons and RP scores can capture almost all regulatory elements in those sets along with approximately 7% of the human genome.     

The neuropathology of brain metastases

Metastatic brain disease frequently complicates extra central nervous system (CNS) neoplastic disease, with an increase in reported incidence over time. Brain parenchyma is the commonest anatomical site, with other lesions involving the spinal cord, dura and tissues surrounding the CNS. Metastases are usually characterised by a well-defined border with surrounding brain, although some can show an infiltrative edge. The use of appropriate immunohistochemical panels can help identify the origin of most tumours, and molecular testing should be performed according to the site of origin even if performed on a previous specimen due to potential changes in molecular characteristics. Reliable detection of leptomeningeal metastasis using CSF cytology relies on examination of an adequate volume of fluid; immunocytochemistry and flow cytometry can also be useful in the correct settings. Advances in the field include liquid biopsies, where circulating biomarkers are examined, and the use of methylation profiling to identify primary tumours.     

The neuropathology of brain metastases

Metastatic brain disease frequently complicates extra central nervous system (CNS) neoplastic disease, with an increase in reported incidence over time. Brain parenchyma is the commonest anatomical site, with other lesions involving the spinal cord, dura and tissues surrounding the CNS. Metastases are usually characterised by a well-defined border with surrounding brain, although some can show an infiltrative edge. The use of appropriate immunohistochemical panels can help identify the origin of most tumours, and molecular testing should be performed according to the site of origin even if performed on a previous specimen due to potential changes in molecular characteristics. Reliable detection of leptomeningeal metastasis using CSF cytology relies on examination of an adequate volume of fluid; immunocytochemistry and flow cytometry can also be useful in the correct settings. Advances in the field include liquid biopsies, where circulating biomarkers are examined, and the use of methylation profiling to identify primary tumours.     

Endogenous avidin-binding activity in human lymphoid tissue.

Biotin-avidin systems can be used as an alternative to indirect antibody sandwich methods in the detection of T lymphocyte subsets in cryostat sections of human lymphoid tissue. Appreciable endogenous avidin binding activity (EABA) has been found, however, in human lymph nodes and tonsils. Such EABA can be a source of false positive staining when biotin-avidin detection systems are used to identify cells in cryostat sections. The finding that avidin binding cells may also contain endogenous peroxidase activity and are morphologically similar to histiocytes suggests that such cells may be of histiocytic lineage. EABA is not seen in intrafollicular "tingible body" macrophages, however, and only rarely in medullary sinus histiocytes. Thus further studies are necessary to identify the lineage of avidin binding cells in lymphoid tissues. EABA can be effectively blocked by treatment of cryostat sections with 1% avidin followed by 0.01% biotin before specific staining with biotinylated antibodies and avidin-peroxidase or avidin-fluorochrome conjugates.     

Proteome analysis of the sera from Chinese Parkinson's disease patients

Clinical proteomics is a powerful tool that can be used to identify proteins that are differentially expressed in disease states, leading to greater understanding of the molecular and cellular events that contribute to disease. The aim of this study was to identify protein changes in the sera from Chinese Parkinson's disease (PD) patients, with the goal of finding biomarkers for PD diagnosis, and to elucidate the events occurring at the onset of PD. Using differential display to identify proteins with altered expression in PD patients, we obtained 15 protein spots corresponding to 13 different gene products that were likely to be involved in PD. Two-dimensional gel electrophoresis and mass spectrometry were used to identify differentially expressed proteins, 7 of which have never previously been associated with PD patients. They are likely to be involved in antioxidation, lipid metabolism, intracellular transport, cell proliferation and immunoregulation. The altered levels of these proteins may be related to the pathophysiological mechanisms of PD. As a result, some of these proteins could be considered as candidate biomarkers.     

Tools for building,analyzing and evaluating HLA haplotypes from families

The highly polymorphic classical human leukocyte antigen (HLA) genes display strong linkage disequilibrium (LD) that results in conserved multi-locus haplotypes. For unrelated individuals in defined populations, HLA haplotype frequencies can be estimated using the expectation-maximization (EM) method. Haplotypes can also be constructed using HLA allele segregation from nuclear families. It is straightforward to identify many HLA genotyping inconsistencies by visually reviewing HLA allele segregation in family members. It is also possible to identify potential crossover events when two or more children are available in a nuclear family. This process of visual inspection can be unwieldy, and we developed the “HaplObserve” program to standardize the process and automatically build haplotypes using family-based HLA allele segregation. HaplObserve facilitates systematically building haplotypes, and reporting potential crossover events. HLA Haplotype Validator (HLAHapV) is a program originally developed to impute chromosomal phase from genotype data using reference haplotype data. We updated and adapted HLAHapV to systematically compare observed and estimated haplotypes. We also used HLAHapV to identify haplotypes when uninformative HLA genotypes are present in families. Finally, we developed “pould”, an R package that calculates haplotype frequencies, and estimates standard measures of global (locus-level) LD from both observed and estimated haplotypes.     

Automated image analysis of digital colposcopy for the detection of cervical neoplasia

Digital colposcopy is a promising technology for the detection of cervical intraepithelial neoplasia. Automated analysis of colposcopic images could provide an inexpensive alternative to existing screening tools. Our goal is to develop a diagnostic tool that can automatically identify neoplastic tissue from digital images. A multispectral digital colposcope (MDC) is used to acquire reflectance images of the cervix with white light before and after acetic-acid application in 29 patients. A diagnostic image analysis tool is developed to identify neoplasia in the digital images. The digital image analysis is performed in two steps. First, similar optical patterns are clustered together. Second, classification algorithms are used to determine the probability that these regions contain neoplastic tissue. The classification results of each patient's images are assessed relative to the gold standard of histopathology. Acetic acid induces changes in the intensity of reflected light as well as the ratio of green to red reflected light. These changes are used to differentiate high-grade squamous intraepithelial (HGSIL) and cancerous lesions from normal or low-grade squamous intraepithelial (LGSIL) tissue. We report diagnostic performance with a sensitivity of 79% and a specificity of 88%. We show that diagnostically useful digital images of the cervix can be obtained using a simple and inexpensive device, and that automated image analysis algorithms show a potential to identify histologically neoplastic tissue areas.     

Anomalies and Specific Functions in the Clinical Identification of Defense Mechanisms

Standard teaching about defense mechanisms generally focuses on definitions, which do not readily aid the clinician in identifying defenses whenever individuals use them. This report demonstrates a process by which the clinician can identify when a defense is used, which ones are likely being used, and with what aim. Clinicians first notice that a defense may be operating whenever the other individual presents with anomalies in the expression of affect, behavior, speech, or its content. Some of these anomalies are described. Next, to identify the specific defense or general level of defensive functioning used, the clinician must identify the specific function of the defense in context using a process of guided clinical inference. This report examines 2 verbatim examples from recorded interviews of one case to demonstrate this process. The examples present a microcosm of clinical concerns that have a surprising relationship to the individual's course and prognosis.     

基于独立分量分析的脑电中眼电伪迹消除

利用独立分最分析的方法对脑电中眼电伪迹成分进行剔除。针对扩腮熵最大算法能够同时分离超高斯和亚高斯信号的特点，将脑电信号分解成独立分量，利用伪迹脑地形图的特征，将伪迹分最分离，得到不含伪迹的脑电信号。实验结果表明。该算法具有较强的稳健性和实用性。     

Genome-wide identification of replication origins in yeast by comparative genomics

We discovered that sequences essential for replication origin function are frequently conserved in sensu stricto Saccharomyces species. Here we use analysis of phylogenetic conservation to identify replication origin sequences throughout the Saccharomyces cerevisiae genome at base pair resolution. Origin activity was confirmed for each of 228 predicted sites--representing 86% of apparent origin regions. This is the first study to determine the genome-wide location of replication origins at a resolution sufficient to identify the sequence elements bound by replication proteins. Our results demonstrate that phylogenetic conservation can be used to identify the origin sequences responsible for replicating a eukaryotic genome.     

Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species

Mycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.     

Texture Analysis and Characteristic Identification About Plaque Tissues of IVUS

Intravascular ultrasound can provide clear real-time cross-sectional images, including lumen and plaque. In practice, to identify the plaques tissues in different pathological changes is very important. However, the grayscale differences of them are not so apparent. In this paper a new textural characteristic space vector was formed by the combination of Co-occurrence Matrix and fraction methods. The vector was projected to the new characteristic space after multiplied by a projective matrix which can best classify those plaques according to the Fisher linear discriminant. Then the classification was completed in the new vector space. Experimental results found that the veracity of this classification could reach up to 88%, which would be an accessorial tool for doctors to identify each plaque.     

运用蛋白质芯片技术鉴定干扰素的亚型

目的：采用自制的6株干扰素单克隆抗体，在本实验室建立的金基底蛋白质芯片平台上对IFN-α1b、IFN-α2a、IFN-α2b、IFN-β及IFN-γ进行鉴定。方法：金基底蛋白质芯片用已知亚型的IFN半成品包被，封闭后与自制的6株干扰素单抗反应，再与Cy3-羊抗鼠IgG反应，反应后GenePix4100A芯片扫描仪扫描读数，将结果与ELISA法、Western blot法比较。同样方法检测IFN成品。结果：金基底蛋白质芯片检测技术结果表明，该平台鉴定干扰素的亚型具有特异性，与ELISA法、Western blot法能得出一致的结论。结论：运用本实验室建立的蛋白质芯片平台可以对干扰素的亚型进行鉴定，具有高通量，操作简单，样品用量少，重复性强等优点。     

Proclets in healthcare

Healthcare processes can be characterized as weakly-connected interacting light-weight workflows coping with different levels of granularity. Classical workflow notations fall short in supporting these kind of processes. Although these notations are able to describe the life-cycle of individual cases and allow for hierarchical decomposition, they primarily support monolithic processes. However, they are less suitable for healthcare processes. The Proclets framework is one formalism that provides a solution to this problem. Based on a large case study, describing the diagnostic process of the gynecological oncology care process at the Academic Medical Center (AMC), we identify the limitations of “monolithic workflows”. Moreover, by using the same case study, we investigate whether healthcare processes can be described effectively using Proclets. In this way, we provide a comparison between the Proclet framework and existing workflow languages and identify research challenges.