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PubMed2000

Glutathione S-transferase theta 1 gene deletion and risk of acute myeloid leukemia.

Individuals with a homozygous deletion of the glutathione S-transferase theta 1 (GSTT1) gene lack GSTT1 enzymatic detoxification of environmental carcinogens by conjugation with glutathione. The GSTT1 gene deletion has been associated with carcinogen-induced chromosomal changes in lymphocytes, and some but not all epidemiological evidence has suggested that the GSTT1 gene deletion may increase susceptibility to myelodysplasia. We conducted a case-control study to test whether individuals with an inherited homozygous deletion of the GSTT1 gene are at increased risk of acute myeloid leukemia (AML). The GSTT1 and GST mu 1 (GSTM1) genotypes were determined by PCR using lymphocyte or bone marrow DNA from 297 AML patients and 152 controls. AML patients were selected from Southwest Oncology Group clinical studies, and controls were identified by random digit dialing in Washington state. No association was observed between the GSTT1 gene deletion and AML [race-adjusted odds ratio (OR), 0.94; 95% confidence interval (CI), 0.55-1.60] or between the GSTM1 gene deletion and AML (race-adjusted OR, 1.26; 95% CI, 0.85-1.88). Patients with secondary AML had a slightly higher prevalence of the GSTT1 and GSTM1 gene deletions compared with de novo AML patients or controls, but this was consistent with chance. Exploratory analyses of AML cytogenetics suggested a few associations, i.e., between the GSTT1 gene deletion and trisomy 8, and between the GSTM1 gene deletion and non-8 trisomies or inv(16). These results do not support the hypothesis that the GSTT1 gene deletion is related to the incidence of AML.

Acute Disease↗

PubMed2000

Study on DNA polymorphism at D1S8 locus in Hebei Han population.

OBJECTIVE: To study the polymorphism at D1S8 locus and to provide basic information for the construction of DNA fingerprint database and the application in forensic medicine. METHODS: Minisatellite variant repeat-polymerase chain reaction (MVR-PCR) and polyacrylamide gradient gel electrophoresis followed by silver staining were used to detect the variant repeat sequences at D1S8 locus of 240 unrelated individuals in Hebei Han population, and digital codes were obtained. RESULTS: Each individual obtained about 30 digital codes, in which none of two unrelated individuals had the same code. The probability of identity of 30 digital codes was 3.55x10(-11). The percentages of three repeat units, a-type, t-type and o-type were 54.77%, 42.54% and 2.69% respectively. The heterozygosity (H) was 0.9837. The excluding probability of paternity(EPP) and polymorphism information content(PIC) were 0.9669 and 0.9833 respectively. CONCLUSION: Because of its high polymorphic nature, D1S8 locus is a valuable marker for forensic identification and paternity testing.

Chromosome Mapping↗

PubMed1993

Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates.

DNA typing systems currently used in parasitology involve either hybridising Southern blots with repetitive sequence probes or amplifying genomic sequences using the polymerase chain reaction (PCR). Both such approaches assay allelic length variation, usually in unexpressed tandemly repeated DNA sequences. Where an appropriate target locus exists, an alternative PCR-based strategy which reveals allelic sequence variation in tandemly repeated DNA offers a more accurate and internally controlled assay. We describe such a strategy for the rapid extraction of information on tandem repeat sequence variation from hypervariable alleles, and apply it to the Plasmodium falciparum CS gene. The extreme variability of such DNA 'barcodes' can be used to identify parasite stocks and lineages. This system is also potentially useful for population genetic and epidemiological studies since it offers the possibility of following the spread of distinctively marked parasite genotypes in samples taken from infected individuals.

Animals↗

PubMed2006

Saturation screening for p53 target genes by digital fluorescent differential display.

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shotgun" approach of identifying one gene at a time, with limited polymerase chain reaction (PCR) reactions set up manually, giving DD a low-technology and low-throughput image. With our newly created DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. We are using this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets.

Animals↗

PubMed2003

Genetic profiling of noncultivated bacteria from the rhizospheres of sugar beet (Beta vulgaris) reveal field and annual variability but no effect of a transgenic herbicide resistance.

In this field study, we compared the bacterial communities inhabiting the rhizosphere of a transgenic, herbicide-resistant sugar beet (Beta vulgaris) cultivar with those of its nonengineered counterpart, using a genetic profiling technique based on PCR amplifications of partial 16S rRNA gene sequences and single-strand conformation polymorphism (SSCP). As a control for the plasticity of the bacterial community, we also analyzed the influence of herbicides, the field heterogeneity, and the annual variation. DNA was isolated from bacterial cell consortia that were directly collected from root material. PCR was carried out with primers that hybridized to evolutionarily conserved regions flanking variable regions 4 and 5 of the 16S rRNA gene. SSCP patterns of these PCR products were composed of approximately 50 distinguishable bands, as detected by silver staining of the gels after electrophoresis. Patterns of the replicates and the different treatments were highly similar, but digital image and similarity analyses revealed differences that corresponded to the positions of the replicates in the field. In addition, communities collected from sugar beet in two successive growing seasons could be distinguished. In contrast, no effect of the transgenic herbicide resistance was detectable. Sequencing of 24 dominant products of the SSCP profiles indicated the presence of bacteria from different phylogenetic groups, with Proteobacteria and members of the Cytophaga-Flavobacterium-Bacteroides group being most abundant.

Bacteria↗

PubMed1998

Digital cloning: identification of human cDNAs homologous to novel kinases through expressed sequence tag database searching.

Identification of novel kinases based on their sequence conservation within kinase catalytic domain has relied so far on two major approaches, low-stringency hybridization of cDNA libraries, and PCR method using degenerate primers. Both of these approaches at times are technically difficult and time-consuming. We have developed a procedure that can significantly reduce the time and effort involved in searching for novel kinases and increase the sensitivity of the analysis. This procedure exploits the computer analysis of a vast resource of human cDNA sequences represented in the expressed sequence tag (EST) database. Seventeen novel human cDNA clones showing significant homology to serine/threonine kinases, including STE-20, CDK- and YAK-related family kinases, were identified by searching EST database. Further sequence analysis of these novel kinases obtained either directly from EST clones or from PCR-RACE products confirmed their identity as protein kinases. Given the rapid accumulation of the EST database and the advent of powerful computer analysis software, this approach provides a fast, sensitive, and economical way to identify novel kinases as well as other genes from EST database.

Amino Acid Sequence↗

PubMed2003

Renal mRNA levels as prognostic tools in kidney diseases.

Molecular biologic techniques are currently considered as new diagnostic and prognostic parameters with a sensitivity and specificity exceeding those of histologic and functional data currently used in clinical practice. The results in various clinical settings have been of limited value up to now. This study is an investigation of the use of tissue levels of RNA determined in routine clinical kidney biopsies as prognostic tools. The focus was on RNA encoding for molecules known to be involved in the pathogenesis of renal disorders. Fresh kidney biopsy tissue was obtained from 52 patients with various renal diseases. The GFR was followed for 12 mo. The extent of glomerulosclerosis and interstitial fibrosis in the biopsies was determined with quantitative digital image analysis. Glomerular and tubulointerstitial compartments from each biopsy specimen were separated, and mRNA levels of TGF-beta, collagen I, collagen IV, and fibronectin were quantitated by real-time PCR. Correlations, along with 95% confidence intervals (CI), between all variables tested at time biopsy were determined. To assess their prognostic value, these variables were correlated with the slope of GFR within several time intervals after biopsy. In addition, to evaluate the predictive value of the variables for outcome in individual patients, differences for each variable were tested between patients showing progressive decline in renal function (slope GFR < 0) and patients showing stable or improving renal function over time (slope GFR >or= 0). In chronic renal diseases, the extent of histologic damage correlated with the GFR at the time of biopsy (r = -0.44; CI -0.68 to -0.11), but it did not correlate with the slope expressing a change in GFR after the biopsy. Tubulointerstitial TGF-beta mRNA levels correlated with the rate of change in GFR between time of biopsy and 1 mo later (r = 0.41; CI, 0.07 to 0.67). The GFR at the time of biopsy correlated with the slope of change in GFR between time of biopsy and 12 mo later (r = -0.50; CI, -0.73 to -0.18). In chronic renal diseases, glomerular fibronectin mRNA levels, in comparison with the GFR at time of biopsy, correlated relatively strongly with the slope of change in GFR between 3 and 12 mo (r = 0.50; CI, 0.16 to 0.74). Patients with favorable renal outcome after 12 mo showed significantly higher TGF-beta mRNA levels and lower proteinuria levels at time of biopsy (P < 0.05) than patients with a progressive decline in renal function. This study shows that mRNA levels measured in kidney biopsies can function as prognostic tools in human renal diseases. In particular, relatively high levels of tubulointerstitial TGF-beta mRNA and glomerular fibronectin mRNA are associated with less deterioration in renal function.

Adult↗

PubMed2005

Differences in tumor necrosis factor (TNF)alpha and TNF receptor-1-mediated intracellular signaling factors in normal, inflamed and scar-formed horse tendons.

Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R1 in tendinocytes. In this study, we focused on four signaling factors, TNFalpha, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and some samples were also subjected to reverse transcription-polymerase chain reaction (RT-PCR), PCR-Southern blot analysis and in situ hybridization to detect the expression of TNFalpha mRNA. Distribution of the four factors differed depending on the tendon condition, normal, inflamed or scar-formed. In the normal tendon, large amounts of TRAF2 were found in tendinocytes, but the amounts of TNF-R1 were small. TNFalpha mRNA was expressed most highly in the inflamed tendon. TNF-R1, which was only faintly detected in the normal tendon, was detected at a high level in the inflamed tendon, and the amounts of TRAF2 and caspase-3 also increased. Activated caspase-3 was only detected in the inflamed tendon. TNFalpha mRNA was also expressed in the scar-formed tendon, though it showed weak signals, and the expression levels of TNF-R1, TRAF2 and caspase-3 proteins were very low. Two distinct intracellular signaling pathways of TNFalpha, which lead to cell survival and apoptosis, might be present in tendinocytes mediated through TNF-R1. These results, which reflect the dynamism of TNFalpha, provide important clues for means to prevent tendinopathy.

Animals↗

PubMed2001

High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer.

Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.

Electrophoresis↗

PubMed2005

[Molecular cloning and character analysis of the mouse zinc finger protein gene Zfp474 exclusively expressed in testis and ovary].

A novel mouse zinc finger protein gene that contains C2HC/C3H domain was first isolated by using a data-mining tool called Digital Differential Display (DDD) from the National Center for Biotechnology Information. The full-length cDNA of this transcript was deduced and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Total four exons of the mouse gene spaning a 29 869 bp genomic DNA sequence was mapped to chromosome 18D1. The cDNA encodes a novel protein of 347 amino acids and the protein contains four C2HC/C3H domains. Northern blot analyses revealed that Zfp474 mRNA was exclusively expressed in testis and ovary and had one transcript. We hypothesize that Zfp474 functions as a germ cell specific transcription factor that plays important roles in spermatid differentiation and oocyte development.

Amino Acid Sequence↗

PubMed2000

Detecting circulating prostate cells in patients with clinically localized prostate cancer: clinical implications for molecular staging.

OBJECTIVE: To evaluate the clinical utility of using the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect prostate-specific antigen (PSA) mRNA in peripheral blood samples from patients with prostate cancer, as a predictor of extraprostatic disease, and to assess any correlations with known predictive markers of this condition. PATIENTS AND METHODS: Immediately before radical prostatectomy, peripheral blood samples were taken from 25 men with clinically localized prostate cancer and analysed for PSA mRNA using RT-PCR (in 'hot-start' conditions and confirmed using ClaI restriction enzyme). The relationships between PSA mRNA positivity, pathological and clinical features were analysed; PSA mRNA positivity, PSA level and biopsy Gleason score were then compared as predictors of extraprostatic disease. RESULTS: There was no relationship between PSA mRNA positivity and pathological stage (pT2 or pT3), and no association between PSA mRNA positivity and serum PSA level, PSA density, the findings on a digital rectal examination or transrectal ultrasonography, and perineural invasion in the prostatic biopsy. However, there was a significant correlation between the Gleason score of the preoperative biopsy and PSA mRNA positivity. The best predictors of extraprostatic disease were the biopsy Gleason score and the PSA level. CONCLUSION: There was no significant advantage in using the RT-PCR assay of PSA mRNA before surgery to stage prostate cancer and to discriminate between organ-confined and extraprostatic neoplasms.

Adult↗

PubMed1991

Initial experience with a radiology imaging network to newborn and intensive care units.

A digital image network has been installed in the James Whitcomb Riley Hospital for Children on the Indiana University Medical Center to create a limited all digital imaging system. The system is composed of commercial components, Philips/AT&T CommView system, (Philips Medical Systems, Shelton, CT; AT&T Bell Laboratories, West Long Beach, NJ) and connects an existing Philips Computed Radiology (PCR) system to two remote workstations that reside in the intensive care unit and the newborn nursery. The purpose of the system is to display images obtained from the PCR system on the remote workstations for direct viewing by referring clinicians, and to reduce many of their visits to the radiology reading room three floors away. The design criteria includes the ability to centrally control all image management functions on the remote workstations to relieve the clinicians from any image management tasks except for recalling patient images. The principal components of the system are the Philips PCR system, the acquisition module (AM), and the PCR interface to the Data Management Module (DMM). Connected to the DMM are an Enhanced Graphics Display Workstation (EGDW), an optical disk drive, and a network gateway to an ethernet link. The ethernet network is the connection to the two Results Viewing Stations (RVS) and both RVSs are approximately 100 m from the gateway. The DMM acts as an image file server and an image archive device. The DMM manages the image data base and can load images to the EGDW and the two RVSs. The system has met the initial design specifications and can successfully capture images from the PCR and direct them to the RVSs.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Communication Networks↗

PubMed2003

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30-40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Biosensing Techniques↗

PubMed2006

Experimental transmission of Bovine leukemia virus in cattle via rectal palpation.

We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV-negative steers. Three of four steers developed antibodies against BLV as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR 1 to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.

Animals↗

PubMed2003

Pythium regulare sp. nov., isolated from the Canary Islands, its taxonomy, its region of rDNA, and comparison with related species.

Pythium regulare (CI-34) was isolated from some soil samples taken in the Canary Islands (Spain). This new species is very closely related to P. irregulare isolated from pea roots in The Netherlands by Buisman in 1927. The species of Pythium are members of the kingdom Chromista. Pythium regulare is characterized by its ornamented oogonia bearing blunt or digitate spines, and its non-sporulating type of sporangia or hyphal bodies, its aplerotic oospores, its monoclinous and diclinous antheridia that at times crowd around the oogonia. The taxonomic description of this oomycete, the PCR of the internal transcribed region (spacers ITS1, ITS2, and the gene 5.8 S) of its ribosomal nuclear DNA as well as the nucleotide sequences, and its comparison with related species are given here.

Base Sequence↗

PubMed2000

The p53 Arg72Pro polymorphism, human papillomavirus, and invasive squamous cell cervical cancer.

A. Storey et al. [Nature (Lond.), 393: 229-234, 1998)] reported a 7-fold increased risk of cervical cancer associated with having an Arg/Arg polymorphism at codon 72 of p53 compared with the Pro/Arg heterozygotes (odds ratio, 7.4; 95% confidence interval, 2.1-29.4). Complementary in vitro studies suggested that the HPV E6 oncoprotein more readily targets the arginine form, as opposed to the proline form, of p53 for degradation. We investigated the impact of this polymorphism in a population-based case-control study of invasive cervical cancer. Using a PCR assay to detect the p53 codon 72 polymorphism, we tested blood samples from 111 women with invasive squamous cell cancer of the cervix identified by a population-based registry and 164 random-digit telephone-dialed controls. The distribution of the genotype among control women was 38% heterozygous, 7% proline homozygous, and 55% arginine homozygous, and among the cases was 38%, 6%, and 56%, respectively. There was no increased risk of squamous cell invasive cervical cancer associated with homozygosity for the arginine allele (odds ratio, 1.0; 95% confidence interval, 0.6-1.7). Furthermore, there was no modification of this result by human papillomavirus (HPV) DNA status of the tumor, age, or smoking status. Among controls, there was no association between the polymorphism and HPV-16 L1 seropositivity. However, among case subjects, the codon 72 polymorphism may be related to HPV 16L1 seropositivity status.

Adult↗

PubMed2025

Integration of Gene Expression and Digital Histology to Predict Treatment-Specific Responses in Breast Cancer.

Deep learning models applied to digital histology can predict gene expression signatures (GES) and offer a low-cost, rapidly available alternative to molecular testing at the time of diagnosis. We optimized transformer-based models to infer GES results and applied this approach to pre-treatment H&E-stained biopsies from 1,940 breast cancer patients treated with neoadjuvant chemotherapy in clinical trial and real-world cohorts. The most predictive histology-derived GES for pathologic complete response (pCR) in the I-SPY2 trial was validated in four external cohorts: CALGB 40601, CALGB 40603, a trial of durvalumab plus CT, and standard-of-care CT-treated patients from the University of Chicago. Among HER2-negative patients, a transformer-based model trained using a signature composed of estrogen-regulated genes, proliferation, apoptosis, and interferon response genes predicted pCR with an AUC of 0.794, outperforming models based on clinical features alone (AUC 0.704, p = 0.001), pathologist TIL assessment, and a model trained directly to predict response from I-SPY2 cases. Tertiles of this signature stratify patients into clinically relevant groups with increasing likelihood of complete response, with pCR rates ≥50% in the top tertile regardless of treatment or hormone receptor status. Additional transformer-based signature models predicted response to specific therapies (but not chemotherapy alone), including a HER2 signaling signature in IO-treated patients, and a claudin-low signature in bevacizumab treated patients. In HER2- cohorts with available gene expression data and histology, models trained on expression data performed similarly to digital histology predictions, but the combination of gene expression and histology outperformed histology alone. These findings suggest that histology-based GES provides additive information to RNA sequencing data and can inform precision treatment selection across breast cancer subtypes.

Journal Article↗

PubMed1993

Verrucous cysts: histopathologic characterization and molecular detection of human papillomavirus-specific DNA.

Verrucous cyst is an uncommon newly described benign epithelial cyst which may be associated with human papillomavirus infection. The purpose of this project was to study the clinicopathological features of verrucous cysts and to demonstrate human papillomavirus (HPV) by polymerase chain reaction (PCR) in these lesions. We have analyzed 5 examples of verrucous cysts: the age range of the patients was between 37-82 years and the most common locations were face and extremities. Clinically the lesions were interpreted as cyst (2), dermatofibroma (2) and basal-cell carcinoma (1). Histopathologic examination of the biopsy specimens showed in all cases an intradermal infundibular cyst lined by a papillated and/or digitated epithelium with focal prominent hypergranulosis and irregular keratohyalin granules. Furthermore, numerous whorls of keratinocytes (so-called squamous eddies) were present within the squamous areas of the hyperplastic epithelium of the cyst, morphologically reminiscent of inverted follicular keratosis. Architectural and cytological features of malignancy were not present. Sections of paraffin-embedded tissue of the 5 verrucous cysts and of 5 "conventional" follicular cysts, infundibular type, were investigated for the presence of HPV DNA sequences by PCR using consensus primers. In all 5 cases of verrucous cysts, HPV-specific DNA sequences were detected, whereas all follicular cysts, infundibular type, were negative. These results support the concept that verrucous cysts can be added to the large spectrum of HPV-induced cutaneous neoplasms. Further studies are needed to identify the specified HPV-type in these distinctive benign cystic proliferations.

Adult↗