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Characterization and phenotypic analysis of differentiating CD34+ human bone marrow cells in liquid culture.

Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.

Antigens, CD↗

Differential coexpression analysis using microarray data and its application to human cancer.

MOTIVATION: Microarrays have been used to identify differential expression of individual genes or cluster genes that are coexpressed over various conditions. However, alteration in coexpression relationships has not been studied. Here we introduce a model for finding differential coexpression from microarrays and test its biological validity with respect to cancer. RESULTS: We collected 10 published gene expression datasets from cancers of 13 different tissues and constructed 2 distinct coexpression networks: a tumor network and normal network. Comparison of the two networks showed that cancer affected many coexpression relationships. Functional changes such as alteration in energy metabolism, promotion of cell growth and enhanced immune activity were accompanied with coexpression changes. Coregulation of collagen genes that may control invasion and metastatic spread of tumor cells was also found. Cluster analysis in the tumor network identified groups of highly interconnected genes related to ribosomal protein synthesis, the cell cycle and antigen presentation. Metallothionein expression was also found to be clustered, which may play a role in apoptosis control in tumor cells. Our results show that this model would serve as a novel method for analyzing microarrays beyond the specific implications for cancer.

Algorithms↗

Fourier-domain low-coherence interferometry for differential mode delay analysis of an optical fiber.

We propose a novel mode analysis and differential mode delay measurement method for an optical fiber using Fourier-domain low-coherence interferometry. A spectral interferometer based on a Mach-Zehnder interferometer setup was used with a broadband source and an optical spectrum analyzer to detect relative temporal delays between the guided modes of a few-mode optical fiber by analyzing spectral interference signals. We have shown that experimental results of the proposed method agree well with those results obtained by using a conventional time-domain measurement method. We have demonstrated that this new mode analysis technique has high sensitivity (<60 dB) and very good resolution (<1 ps/m).

Journal Article↗

Automated pattern ranking in differential display data analysis.

Gene expression analysis by differential display (DD) is limited by the labor-intensive visual evaluation of the electrophoretic data traces. We describe a flexible method for computer-assisted ranking of expression patterns in data from DD experiments. The method is based on a pairwise alignment and comparison of the quantitative trace data with respect to specific expression patterns defined by the investigator. The observed patterns are ranked according to a score value that identifies the most potential findings to be confirmed visually instead of the vast amount of original results. This two-step approach, enabled by the efficient computer algorithm for gene expression pattern comparison, will increase the percentage of true-positive findings chosen for the tedious downstream processing, while minimizing the cost and labor involved in large scale DD data analysis.

Algorithms↗

Atlas-level single-cell integration and clustering-free differential expression analysis with GEDI 2.0.

MOTIVATION: GEDI is a generative framework for multi-sample, multi-condition single-cell analysis that performs batch correction, latent representation learning, and clustering-free differential expression within a unified model. However, the original implementation suffered from prohibitive memory use and runtime, preventing its application to modern atlas-scale datasets. RESULTS: We present GEDI 2.0, a complete high-performance reimplementation featuring a standalone C++ computational core with pre-allocated workspaces, strict sparse-matrix preservation, optimized BLAS routines, and multi-threaded block-coordinate descent. Across extensive benchmarks spanning up to 500 000 cells and 10 000 features, GEDI 2.0 achieves 40%-63.6% mean reduction in peak memory, 2.98&#xd7; mean single-threaded speedups, and up to 11.5&#xd7; acceleration with parallel execution, while maintaining full numerical equivalence to the original method. These improvements enable GEDI 2.0 to analyze million-cell datasets, a scale not achievable with the legacy implementation. GEDI 2.0 provides R and Python interfaces and seamless interoperability with common single-cell workflows. AVAILABILITY AND IMPLEMENTATION: Source code, documentation, reproducible codebase, and tutorials are available at https://github.com/csglab/gedi2.

Single-Cell Analysis↗

Development of a simple and rapid method based on polymerase chain reaction-based restriction fragment length polymorphism analysis to differentiate Helicobacter, Campylobacter, and Arcobacter species.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of amplified DNA fragment of the 16S and 23S rRNA genes was performed on 35 Helicobacter, 24 Campylobacter, and 15 Arcobacter strains. PCR amplification generated a 1004-bp fragment of 16S rDNA and a 2.6-Kbp fragment of 23S rDNA from each strain. The amplicons were digested with DdeI and HpaII, respectively. For both assays, distinctive profiles were obtained for each genus. 23S rDNA PCR-RFLP analysis with HpaII enzyme identified Campylobacter and Helicobacter strains at the species level. Analysis of 16S rRNA gene with DdeI enzyme was not useful for the specific identification of Campylobacter and Arcobacter, although it discriminated among Helicobacter species. The PCR-RFLP technique allowed for the discrimination among these three related genus with only one restriction enzyme; therefore it can be a simple, rapid, and useful method for routine identification.

Arcobacter↗

Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes.

Codon usage data has been compiled for 110 yeast genes. Cluster analysis on relative synonymous codon usage revealed two distinct groups of genes. One group corresponds to highly expressed genes, and has much more extreme synonymous codon preference. The pattern of codon usage observed is consistent with that expected if a need to match abundant tRNAs, and intermediacy of tRNA-mRNA interaction energies are important selective constraints. Thus codon usage in the highly expressed group shows a higher correlation with tRNA abundance, a greater degree of third base pyrimidine bias, and a lesser tendency to the A+T richness which is characteristic of the yeast genome. The cluster analysis can be used to predict the likely level of gene expression of any gene, and identifies the pattern of codon usage likely to yield optimal gene expression in yeast.

Base Composition↗

Differential cell analysis of cytocentrifuged bronchoalveolar fluid samples affected by the area counted.

OBJECTIVE: To investigate variations in the differential cell counts between the quadrants of cytocentrifuged bronchoalveolar lavage (BAL) fluid preparations and to evaluate the diagnostic impact of these differences in interstitial lung diseases (ILD). STUDY DESIGN: BAL fluid samples obtained from 30 patients suspected of having ILD or pneumonia were cytocentrifuged and additionally stained with May-Grünwald-Giemsa stain. Two observers differentiated 200 cells in each quadrant as well as in a circular pattern around the center of the cytocentrifuge spot. RESULTS: Lymphocytes and alveolar macrophages were not randomly distributed on the cytocentrifuge spot. Ten samples of patients with histologically confirmed ILD were selected to test the diagnostic impact using a validated computer program. The predicted diagnosis did not correspond to the histologic diagnosis for one quadrant from 1 of these 10 samples (sarcoidosis instead of idiopathic pulmonary fibrosis), whereas the differential cell counts performed around the center of the cytocentrifuge spot provided the correct diagnosis in all cases. CONCLUSION: BAL fluid differential cell counts varied between the quadrants of the cytocentrifuge spot. The center of the cytocentrifuge spot appeared to be the most reliable area. Therefore, cell counting is recommended in a circular pattern around the center of the cytocentrifuge spot.

Bronchoalveolar Lavage Fluid↗

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody.

We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.

Adult↗

Dielectric analysis and Differential Scanning Calorimetry of water-in-oil microemulsions.

The system investigated is a multicomponent thermodynamically stable liquid consisting of water spherical droplets, (approximately 10 nm diameter), stabilized by a surfactant shell and dispersed in a continuous oil phase (microemulsions). A comparative analysis is carried out among the results obtained with three different experimental approaches, namely, dielectric analysis, Thermally Stimulated Depolarization, and Differential Scanning Calorimetry. The different measurement procedures are briefly described. The attention was focused on the system water concentration. Two microemulsions, differing only in the oil used as continuous phase, were studied. For any concentration a comparison between the results obtained with the different experimental methods was undertaken. The evolution with water addition of the water/oil interphase region was analysed in some detail.

Journal Article↗

Analysis of differentially expressed transcripts of fungal elicitor- and wound-treated wild rice (Oryza grandiglumis).

Suppression subtractive hybridization was used to construct a subtracted library (ogfw) from plants of a wild rice species, Oryza grandiglumis, subjected to a fungal elicitor and physical wounding. To screen the differentially expressed transcripts in the library, we applied a reverse Northern blot analysis to a cDNA microarray containing 1,152 random clones. Based on the average expression ratio, we selected 156 clones showing an elevated expression level. The elevated expression levels and overall expression profiles over time were verified by Northern blot analysis. A comparative functional categorization of the subtracted expressed sequence tags (ESTs) of the ogfw library against ESTs isolated from blast-infected O. sativa showed that the functional categories of cell rescue, defense and virulence, transcription, and cellular transport and transport mechanism of the ogfw library were threefold higher in the former than in the latter. These subtracted ESTs can be presumed to be related to the defense/resistance system and will be used to investigate the defense mechanisms of wild rice and to provide new insights into the genome of wild rice, which in turn will assist molecular breeding strategies of cultivated rice.

Amino Acid Sequence↗

Differentiation, ageing, and terminal differentiation: a semantic analysis.

The largely unsolved problems in the theoretical analysis of differentiation and ageing involve a substantial component of linguistic (semantic) difficulties. Some of these are simple traps of ambiguity, resulting from metaphorical or analogical employment of established terms--for example, "terminal differentiation" (loss of division potential in vitro) as a borrowing from "differentiation" as used by developmental biologists, or "commitment" by analogy with "determination". Some difficulties represent a failure to adopt (at least provisionally) an operational (empirical) view--for example, failure to ask what is the nature of the evidence for the view that a fertilized ovum is totipotential, or to scrutinize the evidence for the view that cells "terminally differentiated" in vitro in a conventional medium are in fact moribund under all conditions, or to examine more closely the view that the differentiated state and the cycling state are mutually exclusive. With respect to the problem of ageing, we review some of the critical experiments on "terminal differentiation" or "clonal senescence". We then proceed to consider some of the models that have been proposed, including a molecular model proposed by the author which appears to overcome some of the objections to other models. Some of the models exemplify the results of what are ultimately semantic vices. The problems with which these remarks began should indeed yield to the immense and novel resources of molecular biology. But the development of complete analyses demands not only good luck and delicate technique but also critical semantic clarity and severity. Given the best tools, we shall solve major theoretical problems only if we understand quite fully what problem it is that we are trying to solve--and the history of science illustrates that this is not as elementary a matter as it sounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗

Ontogenetic derivation and cell differentiation in photosynthetic tissues of C3 and C4 Cyperaceae.

Four variants of Kranz anatomy occur in the Cyperaceae. Three of these anatomical types (fimbristyloid, chlorocyperoid, and eleocharoid) are unique among taxa with C(4) photosynthesis in that the photosynthetic carbon reduction tissue (PCR, functional equivalent of bundle sheath) is located within the vascular strand and is separated from the primary carbon assimilation tissue (PCA, positional equivalent of mesophyll) by the mestome sheath layer. In the fourth anatomical type, rhynchosporoid, PCR tissue is located in the position of the mestome sheath. In this study, we compared two aspects of development of PCR and PCA tissues in representatives of the C(3) and C(4) types: (1) ontogenetic derivation and (2) cellular differentiation. Analysis of the planes of cell division associated with procambial strand formation indicated that PCR tissue is always derived from the procambium, while PCA tissue is derived from the ground meristem. These cell lineages remain distinct after the initial organization of vascular strands. Analysis of cell differentiation using accumulation of cell-type-specific photosynthetic enzymes as markers of differentiation indicated that, with one exception, a low level of non-cell-specific enzyme accumulation preceded abundant and cell-specific accumulation of photosynthetic enzymes at the distal end of the leaf elongation zone. Enzyme accumulation coincided spatially (and temporally) with structural aspects of cell differentiation. Previous cladistic analyses have indicated that these anatomical types represent separate evolutionary origins of the C(4) pathway, and the differences in developmental pathways observed here reflect these independent origins from C(3) ancestors.

Journal Article↗

cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora.

For studies of differential gene expression in prokaryotes, methods for synthesizing representative cDNA populations are required. Here, a technique is described for the synthesis of cDNA from the potato pathogens Erwinia carotovora subsp. atroseptica (Eca) and Erwinia carotovora subsp. carotovora (Ecc) using a combination of short oligonucleotide (11-mer) primers that were known to anneal to conserved sequences in the 3' regions of enterobacterial genes. Specific PCR amplifications with primers designed to anneal to 14 known genes from either Eca or Ecc revealed the presence of the corresponding transcripts in cDNA, suggesting that the cDNA represented a broad genomic coverage. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed genes in Eca, including one that shows significant similarity, at the protein level, to an avirulence gene from Xanthomonas campestris pv. raphani. Northern analysis was used to confirm that differentially amplified cDNA fragments were derived from differentially expressed genes. This is the first report of the use of cDNA-AFLP to study differential gene expression in prokaryotes.

Blotting, Northern↗

The use of immunohistologic analysis in differentiating cutaneous T-cell lymphoma from psoriasis and dermatitis.

BACKGROUND AND DESIGN: --Many investigators have applied immunohistologic analysis to skin biopsy specimens to distinguish cutaneous T-cell lymphoma (CTCL) from benign inflammatory diseases, such as psoriasis and dermatitis, which can clinically mimic early-stage CTCL. We studied the number and distribution of epidermal cells labeled with various monoclonal antibodies in normal skin and in psoriatic, dermatitic, and CTCL lesions by the immunoperoxidase technique. RESULTS: --Extensive staining of keratinocytes (KCs) with HLA-DR was seen in 27 of 41 patients with CTCL, but in only one of 14 patients with psoriasis and zero of 10 patients with dermatitis. CD2+ and CD3+ cells were present in the middle and upper epidermis of CTCL lesions in much greater numbers than in normal, dermatitic, and psoriatic skin. The percent epidermal area covered by CD1+ cells in psoriatic lesions (1.13%) and dermatitic lesions (1.65%) was significantly lower than that found in CTCL lesions (3.60%). CONCLUSION: --Epidermal immunohistologic patterns using anti-CD1, anti-CD2, anti-CD3, and anti-HLA-DR antibodies have the potential to distinguish CTCL from psoriasis and dermatitis in clinically ambiguous cases.

Adult↗

[Analysis of differential protein expression profile between retinoic acid resistant and sensitive acute promyelocytic leukemia cells].

BACKGROUND & OBJECTIVE: This study was to compare the protein expression profiles between retinoic acid (RA) resistant and sensitive acute promyelocytic leukemia (APL) cells by proteomic research method. METHODS: Total cellular proteins extracted from a RA sensitive cell line NB4 and a RA resistant cell line MR2 were separated by two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). High quality 2D-PAGE protein profiles were obtained and analyzed by PDQuest v7.1 analysis software to screen differentially expressed protein spots. Those sports were identified by mass spectrometry. RESULTS: 2-DE patterns of APL cell lines with high-resolution and reproducibility were obtained. The average spots for MR2 and NB4 cells were 890+/-45 and 912+/-56, respectively. 57 significantly differentially expressed protein spots were screened, among which 23 protein spots were identified to be up-regulated and 34 down-regulated in MR2 cells compared with NB4 cells. Ten proteins were identified by mass spectrometry, with a successful identification rate of 70%. The identified proteins could be classified into different categories: oncogenes,cell cycle regulator and signal transducer. CONCLUSION: The utilization of 2D-PAGE is effective in identifying the protein expression profiles between RA resistant and sensitive APL cells, therefore this study may provide a novel clue to elucidate the drug resistant mechanisms of all-trans retinoic acid.

Antineoplastic Agents↗