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PubMed1995

The ultrastructure of the foregut and its influence on bancroftian microfilariae ingestion in three Egyptian mosquito species.

Laboratory observations on the uptake of bancroftian microfilariae (mf.) by three Egyptian mosquito species revealed that microfilariae ingested by some tested species were badly damaged during ingestion. The foregut structures in Culex pipiens, Aedes caspius and Anopheles multicolor were investigated by light and scanning microscope techniques. In Ae. caspius which have well developed pharyngeal armatures, and An multicolor which have well developed cibarial armatures, high proportion of microfilariae were scored and damaged. Vice virsa, Cx. pipiens, considered as the main vector of bancroftian filariaris have neither developed pharyngeal nor cibarial pumps, the percentage of damaged mf was very small. The relationship between the injury of mf. in mosquito midgut and the presence of the foregut armatures was discussed.

Aedes↗
PubMed1998

Response of mosquitoes (Diptera: Culicidae) to carbon dioxide and octenol in Egypt.

Carbon dioxide and 1-octen-3-ol (octenol were evaluated individually, and in combination, as adult mosquito attractants at two sites in Qara, Matruh Governorate, Egypt. Four mosquito species were collected during the study: Aedes caspius, Anopheles multicolor, An. sergenti and Culex pipiens. Anopheles multicolor and Cx. pipiens were collected in insufficient numbers to allow statistical analysis. Significantly greater numbers of Ae. caspius and An sergenti were collected in CO2 and CO2 + octenol traps than octenol alone or the control at both locations (P < 0.05). Traps baited with octenol + CO2 resulted in greater capture of these two species at both locations; but results were not significant when compared with CO2 alone. There were no significant differences in number of mosquitoes collected between the octenol alone and control traps at both locations. At the release rate tested (0.5 mg/h at 21 degrees C), octenol was not an effective attractant for the mosquito species collected during the course of this study.

Animals↗
PubMed2025

Cytogenetics and genomics analysis of cold-hardy perennial wheatgrass: insights into agronomic performance, chromosome composition, and gene expression.

Intermedium wheatgrass (Thinopyrum intermedium), a perennial species with extensive root systems and high tolerance to cold, drought, and salinity, is a valuable genetic resource for the development of perennial crops. Over a decade-long selection process, two cold-hardy perennial wheatgrass lines were developed by crossing wheat-Thinopyrum partial amphiploids with Th. intermedium. These lines inherited key traits from Th. intermedium, including plant stature, spike morphology, and postharvest regrowth. Transcriptome-based single-nucleotide polymorphism tracing and sequential multicolor genomic in situ hybridization analyses revealed variations in the chromosome compositions of the perennial wheatgrass lines. The introgression of wheat chromosomes enhanced grain weight and size, while preserving the cold-hardy, perennial characteristics of the wheatgrass lines compared to Th. intermedium. Genome-wide gene expression was generally suppressed in the wheatgrass lines relative to Th. intermedium, particularly in conserved genes. This suppression was especially pronounced in genes involved in cell division and DNA repair pathways. In contrast, genes associated with cold tolerance and the water stress response were upregulated. We identified eight cold-tolerance genes in the Th. intermedium chromosomes and validated three of them, Thint.J05G452200, Thint.J05G452300, and Thint.V05G408900, using qRT-PCR. These genes encode proteins associated with cold tolerance and are potential candidates for further functional validation. Additionally, three chromosomes from homoeologous group 6 were introgressed, carrying six genes potentially associated with superior grain traits. Among them, TraesCS6D02G287800, which encodes a specific protein, exhibited high expression levels in both wheatgrass lines, suggesting its critical role in enhancing grain traits. Our results indicate that the suppression of grass gene expression, likely due to the introgression of wheat chromosomes and the upregulation of pathways related to cold tolerance and overwintering ability, contributes to the adaptive features of the wheatgrass lines. This study provides a genomic foundation for understanding gene expression regulation in distant hybrid progeny and offers valuable insights for designing new breeding strategies for perennial wheat or wheatgrass.

Chromosomes, Plant↗
PubMed1996

Bullous pemphigoid and epidermolysis bullosa acquisita. Differentiation by fluorescence overlay antigen mapping.

BACKGROUND AND DESIGN: From previous studies, we concluded that the fluorescence overlay antigen mapping (FOAM) technique could be of value to the differential diagnosis of the acquired subepidermal bullous skin disorders, bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). In these diseases, ultrastructural identification of the site of skin-bound IgG deposits at the epidermal basement membrane zone (EBMZ) may be essential to the correct diagnosis. Since ultrastructural studies are more expensive, time-consuming, and less widely available than immunofluorescence, we addressed the question of whether the FOAM technique can reliably identify the site of IgG deposits at the EBMZ, and distinguish BP from EBA. For this purpose, the technique was applied to perilesional skin from seven patients with BP and six with EBA, using computer-aided imaging of red-stained type VII collagen and green-stained IgG, according to previous findings. RESULTS: Digitized multicolor FOAM images of perilesional skin from patients with BP showed nonoverlap band patterns of green-stained lamina lucida IgG deposits (ultrastructurally proven) and red-stained type VII collagen. By contrast, FOAM images of EBA skin typically showed overlap patterns of green-stained sublamina densa IgG deposits and red-stained type VII collagen. These findings were observed also in skin tissue stored in Michel's transport medium or stored frozen for 15 years. CONCLUSIONS: The computer-aided FOAM technique may have great potential in distinguishing between IgG deposits above (BP) and just below (EBA) the lamina densa of the EBMZ in skin tissue. The technique is not as simple as saline-split skin methodology but offers more flexibility, and it certainly is quicker and less expensive than electron microscopy. Furthermore, the use of digitized fluorescence images offers improved possibilities for evaluating the various "linear" patterns of immune reactant deposition at the EBMZ in subepidermal bullous autoimmune skin diseases.

Adult↗
PubMed1999

De novo complete trisomy 5p: clinical report and FISH studies.

We describe a de novo trisomy 5p in a 1-year-old severely retarded boy. The complete short arm of chromosome 5 segregated as an additional marker chromosome in all metaphases. The marker was identified as 5p by conventional cytogenetic techniques (GTG, GBG, CBG) and molecular cytogenetic techniques (whole chromosome-painting probe, probes for the cri-du-chat region and the centromere, and additionally high-resolution multicolor banding using a chromosome 5-specific DNA probe cocktail). The clinical findings were similar to the established trisomy 5p phenotype including macrocephaly, facial abnormalities, tracheobronchial defects with subsequent respiratory infections, hypotonia, and psychomotor retardation. To the best of our knowledge this is the first description of an isolated complete 5p trisomy without involvement of the aberrant chromosome in any structural chromosomal rearrangements.

Centromere↗
PubMed1996

Flow cytometric immunophenotyping of non-Hodgkin's lymphomas and related disorders.

More than two decades have past since the recognition of non-Hodgkin's lymphomas (NHLs) as neoplasms of the immune system. During that time, a vast literature and knowledge base regarding the immunophenotypic and functional characteristics of neoplastic lymphocytes has been developed. Despite the accumulated wealth of knowledge, there remains no consensus as to the exact role of immunotypic and genotypic ancillary procedures in the evaluation of a lymph node biopsy. We review selected literature in this regard and provide an overview of the role of multicolor flow cytometry in establishing the diagnosis of specific NHL and related disorders.

B-Lymphocytes↗
PubMed1996

Quantification of CD1a, HLA-DR, and HLA class I expression on viable human Langerhans cells and keratinocytes.

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 +/- 19 x 10(3) molecules/cell) compared to viable Kc (785 +/- 110 x 10(3) molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 +/- 82 x 10(3) molecules/cell and 1600 +/- 133 x 10(3) molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases.

Adult↗
PubMed1998

Novel approach for simultaneous evaluation of cell phenotype, apoptosis, and cell cycle using multiparameter flow cytometry.

Apoptosis is a vital process for organism development and, when disrupted, can lead to abnormalities including cancer and autoimmune diseases. We demonstrate a novel multicolor flow cytometry approach for quantifying apoptosis and cell cycle information of phenotypically distinct populations, using less than 2 x 10(5) cells per sample. We used incorporation of Cy5-dUTP into DNA strand breaks by the terminal dUTP nucleotide end labeling (TUNEL) method to determine apoptosis, while cell cycle information was assessed with an ultraviolet DNA binding dye, DAPI. To simultaneously determine surface phenotype, we used paraformaldehyde fixation and a gentle permeabilization protocol combined with FITC- and PE-labeled surface antibodies. Using these fluorochromes, and three-laser instrumentation, we quantified apoptosis and cell cycle phase in lymphocyte subpopulations from heterogeneous human and murine cell sources, subjected to various culture conditions. Further, we used this method to detect divergent rates of apoptosis in a human, heterogeneous lymphocyte tumor population, demonstrating a potential application for clinical and/or research settings. Thus, we describe a six-parameter, four-color flow cytometry approach for evaluating apoptosis and cell cycle with dual surface labels. This method may also be useful as a generalized scheme to assess simultaneously two intracellular targets in a mixed cell population.

Animals↗
PubMed1998

A flow cytometer designed for fluorescence calibration.

In the development of suitable standards and calibration materials for fluorescence measurement, it becomes necessary to make accurate fluorescence measurements of these materials on flow cytometers. The results of such measurements may be affected by numerous sources of error, prominent among which are deviations of logarithmic amplifiers (log amps) from ideal response. To minimize the deleterious effects of log amps and multicolor fluorescence compensation circuitry on measurements, we built a flow cytometer with electronics incorporating high-precision peak detectors usable over a range from below 2 mV to 10 V, and we developed data acquisition software that transfers held peak values to a commercial 16-bit data acquisition system mounted in a personal computer running Windows 95. Fluorescence compensation is done in software, and transformation of the compensated data from a 16-bit linear to an 8-bit, 4-decade logarithmic scale is accomplished using a look-up table. Although dynamic range may be restricted by noise in the data acquisition system, high sensitivity can be achieved by photomultiplier tube gain adjustment, and it is likely that the use of a lower noise data acquisition system and/or digital processing of pulse information will enable operation over the full 4-decade dynamic range. Even at its current performance level, our instrument provides substantially better linearity over most of the scale than can be obtained using conventional electronics incorporating log amps; we believe this characteristic is critical for use in standards development.

Analog-Digital Conversion↗
PubMed1999

Signal to noise analysis of multiple color fluorescence imaging microscopy.

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.

Female↗
PubMed1996

ERBB2 and chromosome 17 centromere studies of ovarian cancer by fluorescence in situ hybridization.

More than 26,000 new cases of ovarian cancer are identified each year in the United States, with almost 75% of these malignancies in advanced stages at the time of diagnosis. Early-stage disease has a cure rate of up to 90%, but the long-term survival rate of patients with advanced disease is 5-20%. At this time, there are no biomarkers that are effective indicators of early ovarian cancer. Recently, immunohistochemical and Southern blot studies have suggested that overexpression/amplification of the oncogene ERBB2 (HER2/neu) is associated with aggressive ovarian malignancies; however, some studies have not supported this conclusion. Because tumor cells are known to be highly heterogeneous, we used fluorescence in situ hybridization (FISH) to study individual ovarian cancer cells for HER2/neu amplification and chromosome 17 centromere copy number. Simultaneous multicolor cohybridization of HER2/neu and chromosome 17 centromere alpha-satellite probes were carried out on 43 ovarian cancer samples. Ten of the forty-three samples showed moderate to high amplification of HER2/neu, with varying numbers of chromosome 17 centromeres present. In some cells the amplified HER2/neu was dispersed throughout the nucleus, whereas in other cells the amplified oncogenes were clustered together. Within a sample there was heterogeneity in oncogene and centromere copy number. In this small study, we were unable to identify a specific clinical correlation. However, FISH is a powerful method for the study of oncogene amplification in tumor samples.

Adult↗
PubMed1996

Micronuclei induced in round spermatids of mice after stem-cell treatment with chloral hydrate: evaluations with centromeric DNA probes and kinetochore antibodies.

The chromosomal effects of chloral hydrate (CH) on germ cells of male mice were investigated using two methods to detect and characterize spermatid micronuclei (SMN); (a) anti-kinetochore immunofluorescence (SMN-CREST) and (b) multicolor fluorescence in situ hybridization with DNA probes for centromeric DNA and repetitive sequences on chromosome X (SMN-FISH). B6C3F1 mice received single intraperitoneal (i.p.) injections of 82.7, 165.4, or 413.5 mg/kg and round spermatids were sampled at three time intervals representing cells treated in late meiosis, early meiosis, or as spermatogonial stem cells. No increases in the frequencies of SMN were detected for cells treated during meiosis using either SMN-CREST or SMN-FISH methods. After spermatogonial stem-cell treatment, however, elevated frequencies of SMN were detected by both methods. With SMN-FISH, dose trends were observed both in the frequencies of spermatids containing micronuclei and in the frequency of spermatids carrying centromeric label. These findings corroborate the recent report by Allen and colleagues [Allen JW et al.(1994): Mutat. Res. 323:81-88] that CH treatment of spermatogenic stem cells induced SMN. Furthermore, our findings suggest that chromosomal malsegregation or loss may occur in spermatids long after CH treatment of stem cells. Further studies are needed to understand the mechanism of action of the CH effect on stem cells and to determine whether similar effects are induced in human males treated with CH.

Anesthetics, Intravenous↗
PubMed1997

Induction of chromosomal aberrations in mouse zygotes by acrylamide treatment of male germ cells and their correlation with dominant lethality and heritable translocations.

The objectives of this research were: 1) to investigate the time course of the cytogenetic defects induced by acrylamide (AA) treatment (5 x 50 mg/kg) of male germ cells in first-cleavage zygote metaphases using PAINT/DAPI analysis, and 2) to characterize the correlation between chromosomal aberrations at first cleavage, dominant lethality, and heritable translocations. PAINT/DAPI analysis employs multicolor fluorescence in situ hybridization painting plus DAPI staining to detect both stable and unstable chromosomal aberrations at first-cleavage metaphase of the zygote. High levels of chromosomally defective zygotes were detected after mating at all postmeiotic stages (20-190-fold, P < 0.001). Early spermatozoa (6.5 d post-treatment) were the most sensitive, with 76% of the zygotes carrying cytogenetic defects. A significant 10-fold increase was also detected 27.5 d post-treatment, indicating that AA had a cytogenetic effect on meiotic stages. PAINT/DAPI analysis revealed that: 1) AA-induced chromosomal breaks occurred at random, and 2) the frequencies of symmetrical and asymmetrical exchanges were similar at all mating days, except 9.5 d after AA treatment, where significantly (P < 0.02) more asymmetrical aberrations were found. Furthermore, the proportions of zygotes carrying unstable and stable chromosomal aberrations followed a similar post-treatment time course as the proportions of dominant lethality among embryos and heritable translocations among offspring. These findings indicate that PAINT/DAPI analysis of zygotic metaphases is a promising method for detecting male germ cell mutagens capable of inducing chromosomal aberrations and for evaluating the associated risks for embryonic loss and balanced translocations at birth.

Acrylamide↗
PubMed2001

Isodicentric 7p, idic(7)(q11.2), in acute myeloid leukemia associated with older age and favorable response to induction chemotherapy: a new clinical entity?

Three adult de novo acute myeloid leukemias (AML M1, M2, and M4) with an isochromosome 7p are presented. No additional abnormalities were detected by G-band and multicolor, using combined binary ratio labeling, fluorescence in situ hybridization (FISH) analyses, indicating that the i(7p) was the sole, i.e., the primary, chromosomal aberration. Although the patients were elderly--68, 72, and 78 years old--they all responded very well to chemotherapy, achieving complete remission lasting more than a year. Further FISH analyses, using painting, centromeric, as well as 7q11.2-specific YAC probes, revealed that the i(7p) contained two centromeres and that the breakpoints were located in 7q11.2. Thus, the abnormality should formally be designated idic(7)(q11.2). The detailed mapping disclosed a breakpoint heterogeneity, with the breaks in 7q11.2 varying among the cases, being at least 1,310 kb apart. Furthermore, the breakpoints also differed within one of the cases, being located on both the proximal and the distal side of the most centromeric probe used. Based on our three patients, as well as on a previously reported 82-year-old patient with AML M2 and idic(7)(q11) as the only chromosomal change, we suggest that this abnormality, as the sole anomaly, is associated with AML in elderly patients who display a good response to induction chemotherapy and, hence, have a favorable prognosis. Furthermore, the heterogeneous breakpoints in 7q11.2 suggest that the important functional outcome of the idic(7)(q11.2) is the genomic imbalance incurred, i.e., gain of 7p and loss of 7q material, rather than a rearrangement of a specific gene.

Aged↗
PubMed2000

Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia.

Multicolor spectral karyotyping allows simultaneous visualization of all human chromosomes and screening for chromosomal rearrangements without a priori knowledge of any abnormalities involved. Based on this potentially increased sensitivity, we investigated, in a preliminary manner, whether spectral karyotyping could detect cytogenetic aberrations in karyotypically normal leukemia. The test population was comprised of 28 cryopreserved, cytogenetically normal acute myeloid leukemia (AML) samples from patients registered to a randomized trial for previously untreated AML (SWOG 9031). Two normal and 12 samples with known cytogenetic aberrations were used to validate and establish the diagnostic accuracy of the spectral karyotyping assay and instrumentation in a clinical setting. Enumeration and region-specific DNA fluorescence in situ hybridization (FISH) probes verified discrepant results. In the validation data set, spectral karyotyping refined complex karyotypic rearrangements in six cases and defined the chromosomal origin of a "jumping" homogeneously staining region; however, the technology was less sensitive in the detection of subtelomeric rearrangements and double minute chromosomes. In the test population, spectral karyotyping identified previously undetected cytogenetic aberrations in two cases (7%) of karyotypically normal AML: a cryptic 11q23 translocation in 20/20 cells and a minor monosomy 7 clone in 3/21 cells (FISH, 10.5%). Both of these abnormalities are considered to confer a poor prognosis when based on classical cytogenetic prognostic criteria. As an adjunct to classical cytogenetics and standard FISH analyses, the additive resolution of spectral karyotyping, in particular, with chromosome paints spiked with subtelomeric and/or locus-specific probes, may allow significant gains to be made in diagnostic accuracy and recognition of genotype/phenotype prognostic relationships, and in defining underlying biologic mechanisms in cancer. Genes Chromosomes Cancer 28:318-328, 2000.

Acute Disease↗
PubMed2001

A whole new way of looking at things: the use of Dark Reader technology to detect fluorophors.

The Dark Reader optical system (Clare Chemical Research, Denver, CO, USA) uses relatively low intensity broad-band visible blue light in combination with broad-band optical filters to detect fluorescence with a level of sensitivity that often surpasses that of UV transilluminators and can rival that of laser-based scanners. Applications of DR (Clare Chemical Research) devices include the detection of DNA and SYBR-stained protein samples following, and also during, electrophoresis. Unlike laser-based imaging systems, the fluorescence is directly visible to the user as well as being fully compatible with charge-coupled device (CCD) and Polaroid camera-based detection and imaging. Additionally, the DR optical system functions well in multicolor fluorophor environments. Because the Dark Reader does not emit any UV light, the extent of DNA damage incurred when visualizing DNA samples is drastically reduced compared to the damage produced by a UV device and this can have a significant benefit on downstream cloning protocols. Furthermore, dye photobleaching is minimal, extending the length of time that a fluorescent sample is visible. The inherent flexibility of the DR optical system allows many different configurations of the Dark Reader to be constructed such as transilluminators, hand lamps and integrated transilluminator-electrophoresis units.

DNA↗
PubMed2001

Fluorescent imaging and analysis with Typhoon 8600.

Current technologies in laser-based imaging systems for life science applications offer highly sensitive and quantitative detection of multiple labels. Typhoon 8600 variable mode imager is capable of fluorescence, storage phosphor, and chemiluminescence detection of gels and blots. The system delivers sensitive fluorescent detection of nucleic acids and proteins in both agarose and polyacrylamide gel formats. Detection and discrimination of multicolor fluorescent signals in fragment analysis, electromobility shift assays, Western blotting and other applications enhance the speed and accuracy of analysis.

DNA↗
PubMed2001

Hundredfold productivity of genome analysis by introduction of microtemperature-gradient gel electrophoresis.

Genome profiling, which employs temperature-gradient gel electrophoresis (TGGE) for DNA analysis, has recently been developed in identifying species by genotype. However, the performance of this technology like the general applications of TGGE was, though highly informative, limited in its ability due to methodological reasons. This study demonstrates that minimization of the gel for TGGE, to around one-tenth of its conventional size (approximately 2 cm), can be successfully introduced, resulting in a hundredfold higher performance (total evaluation of time, cost, and degree of parallel operations) than that of the conventional. Reproducibility was evaluated from the measures of the pattern similarity scores (PaSS) between band patterns (genome profiles) obtained with the conventional TGGE, and that with micro-TGGE (microTGGE) developed here, after extracting a set of featuring points from genome profiles. Size minimization, which leads to the reduction of the amount of samples required (cost-saving), is another great advantage, enhancing the employment of multicolor fluorescence technology. Since the further development of microbe-related fields such as epidemiology and microbial ecology inevitably require knowledge based on the identification of a great number of species and strains, microbe-related fields will receive the most optimal benefits from the technological improvements attained here.

Bacillus↗