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J G Hall

Publications and source records attributed to J G Hall.

At least 19 recordsLinked to original sources
PubMed2007

Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy.

Attempts to investigate changes in various forms of intrahepatic hepatitis B virus (HBV) DNA during antiviral therapy have been hampered by limitations in technologies and scarcity of adequate tissue for analysis. We used a sensitive, specific assay to detect and quantitate covalently closed circular DNA (cccDNA) from total intrahepatic HBV DNA in clinical liver specimens. Total HBV DNA and cccDNA from 21 needle-biopsy specimens were quantified, with levels ranging from 0.1 to 9.8 copies/cell and 0.3 to 491.0 copies/cell, respectively. Then, we performed the same determinations on baseline and week-52 liver needle-biopsy specimens from eight patients enrolled in a clinical trial and evaluated the association between intrahepatic HBV DNA levels and serological and virological endpoints. In most patients, levels of intrahepatic HBV DNA, including cccDNA, decreased over the 52-week study, regardless of therapy or serological outcome. Higher ratios of cccDNA to total HBV DNA were detected at week 52 than at baseline indicating a shift in predominance of nonreplicating virus in posttreatment specimens. In patients who achieved treatment-related or spontaneous hepatitis B e antigen (HBeAg) responses, including those harbouring tyrosine-methionine-aspartate-aspartate-mutant HBV, levels of intrahepatic and serum HBV DNA suppression were greater than those in patients without HBeAg responses. In conclusion, this pilot study of intrahepatic HBV replicative forms in patients with chronic hepatitis B indicated that total intrahepatic and, specifically, cccDNA levels are not static but change as a reflection of serological and virological events.

Alanine Transaminase↗
PubMed2006

Clinical and molecular cytogenetic characterisation of a newly recognised microdeletion syndrome involving 2p15-16.1.

BACKGROUND: During whole genome microarray-based comparative genomic hybridisation (array CGH) screening of subjects with idiopathic intellectual disability, we identified two unrelated individuals with a similar de novo interstitial microdeletion at 2p15-2p16.1. Both individuals share a similar clinical phenotype including moderate to severe intellectual disability, autism/autistic features, microcephaly, structural brain anomalies including cortical dysplasia/pachygyria, renal anomalies (multicystic kidney, hydronephrosis), digital camptodactyly, visual impairment, strabismus, neuromotor deficits, communication and attention impairments, and a distinctive pattern of craniofacial features. Dysmorphic craniofacial features include progressive microcephaly, flat occiput, widened inner canthal distance, small palpebral fissures, ptosis, long and straight eyelashes, broad and high nasal root extending to a widened, prominent nasal tip with elongated, smooth philtrum, rounding of the upper vermillion border and everted lower lips. METHODS: Clinical assessments, and cytogenetic, array CGH and fluorescence in situ hybridisation (FISH) analyses were performed. RESULTS: The microdeletions discovered in each individual measured 4.5 Mb and 5.7 Mb, spanning the chromosome 2p region from 57.2 to 61.7 Mb and from 56 to 61.7 Mb, respectively. Each deleted clone in this range demonstrated a dosage reduction from two to one copy in each proband except for clone RP11-79K21, which was present in three copies in each proband and in four copies in their respective parents (two per each chromosome 2 homologue). DISCUSSION: The common constellation of features found in the two affected subjects indicates that they have a newly recognised microdeletion syndrome involving haploinsufficiency of one or more genes deleted within at least a 4.5-Mb segment of the 2p15-16.1 region.

Abnormalities, Multiple↗
PubMed2003

Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis.

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.

Apolipoproteins E↗
PubMed2001

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

Alleles↗
PubMed2001

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

Base Sequence↗
PubMed2001

A DNA computing readout operation based on structure-specific cleavage.

We describe a structure-specific cleavage-based READOUT strategy for surface-based DNA computing. The strategy was demonstrated in the solution of a 4-variable/3-satisfiability (SAT) problem. The READOUT step identifies the DNA molecules present at the end of the computational process. The specificity of the sequence detection used here derives from the sequence specificity of DNA hybridization coupled with the structure specificity of the enzymatic cleavage. The process is linear, yielding a higher uniformity of detection of the DNA computing products compared to that obtained with PCR amplification. The structure-specific cleavage-based readout is simple, accurate, and compatible with multiple-word DNA computing.

Animals↗
PubMed2000

Experimental and theoretical analysis of the invasive signal amplification reaction.

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.

3' Untranslated Regions↗
PubMed2000

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

Base Sequence↗
PubMed2000

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor.

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days.

Biosensing Techniques↗
PubMed2000

Poor postnatal weight gain: a risk factor for severe retinopathy of prematurity.

PURPOSE: This study aimed to investigate the relationship between rate of postnatal weight gain and severity of retinopathy of prematurity (ROP). METHODS: All infants (n = 111) screened for ROP at a single tertiary intensive care nursery over a 2-year period with an estimated gestational age of 30 weeks or less and follow-up to at least 42 weeks' postconception were included. The authors performed a retrospective review of records and statistical analysis of risk factors for ROP using multivariate analysis. RESULTS: Infants with severe (stage 3 or greater) ROP gained an average 10.9 g/kg per day in the first 6 weeks of life, compared to a mean of 9.6 g/kg per day for those with mild or no ROP (P =.04). By multiple regression, which included birth weight, gestational age, and 9 other reported risk factors, there was an association between rate of postnatal weight gain and severity of ROP (P =.02). By stepwise regression, 4 variables were associated with ROP severity: estimated gestational age at birth (P =.002), rate of postnatal weight gain (P = .0002), volume of transfused erythrocytes (P =.0001), and culture-proven sepsis (P = .02). CONCLUSION: Poor postnatal weight gain is a risk factor for the development of severe (stage 3 or greater) ROP. Ophthalmologists should take note of those infants who gain less than 50% of their birth weight in the first 6 weeks of life.

Disease Progression↗
PubMed1999

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry.

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Base Sequence↗
PubMed1999

Human diseases and genomic imprinting.

In summary, there are a number of conditions where genomic imprinting effects are recognized to be associated clinical disorders of importance in humans. There may be many more. Genomic imprinting should be suspected in any disorder with overgrowth, undergrowth, or behavior abnormalities. Disorders with unusual pattern of inheritance should be studied for the possibility that genomically imprinted gene(s) are involved. Understanding the mechanisms of genomic imprinting has major ramifications in terms of recurrence risk, prediction of whether offspring will be affected, and risk of malignancy. Of particular concern is the potential for uniparental disomy when trisomy is found during prenatal diagnosis.

Aneuploidy↗
PubMed1999

U-P-what?

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Aneuploidy↗
PubMed1999

Lack of competition of substrates for P-glycoprotein in MCF-7 breast cancer cells overexpressing MDR1.

The MCF-7/Adr cells overexpress MDR-1 which contributes to the drug-resistant phenotype. Our studies show: 1. The retention of daunomycin in the MCF-7/Adr cells relates to a temperature-dependent and energy-dependent process. 2. The MCF-7/Adr cells retain less rhodamine-123 than the parental MCF-7 cells. 3. The MCF-7/Adr cells retain less daunomycin than the parental MCF-7 cells as measured by mean fluorescence or radioactive daunomycin. 4. Cyclosporin A and verapamil effectively block the effluxes of rhodamine-123 and daunomycin from the MCF-7/Adr cells. 5. On short-term incubation, 2-deoxyglucose lowers the NTP levels to a greater extent than sodium azide, showing the importance of glycolysis in the MCF-7 cell lines. 6. Although the MCF-7/Adr cells show cross-resistance to VP-16, actinomycin D and vinblastine, these drugs do not compete with daunomycin for the efflux pump. 7. These data suggest that either there must be multiple MDR-1 pumps that differ in substrate specificity or that there are distinct substrate sites on MDR-1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The importance of fluorescein angiography in planning laser treatment of diabetic macular edema.

OBJECTIVE: To test the hypothesis that pretreatment fluorescein angiography (FA) is not necessary for effective laser treatment of patients with clinically significant diabetic macular edema (CSME). DESIGN: Prospective, randomized, controlled treatment simulation. PARTICIPANTS: Six fellowship trained retina specialists. INTERVENTION: The authors compared the ability of four retina specialists (observers) to plan laser treatment with and without the use of FA. One hundred consecutive cases of CSME were selected, each case consisting of a stereo pair of color photographs and a corresponding fluorescein angiogram. These cases were first read by two retina specialists who reached consensus on a treatment plan for each case (standard map). Each of the 4 observers reviewed 50 of these cases on 2 occasions and plotted 2 sets of treatment maps, 1 set created with and 1 without the aid of FA. Each observer's 100 treatment maps were graded for accuracy by comparing them to the corresponding standard maps. The role of FA in improving the accuracy of treatment maps was evaluated using logistic regression analysis to control for different observers, different cases, and different posterior pole characteristics. MAIN OUTCOME MEASURES: Accuracy was defined as the proportion of standard treatment correctly treated by the observer. RESULTS: For the observers as a group, the use of FA improved treatment planning accuracy from 49% to 54.5% (P = 0.02); however, there was significant interobserver variation in performance (P < 0.001). Treatment planning accuracy without and with FA was as follows: observer 1, 40.8% and 40.2%; observer 2, 49.8% and 72%; observer 3, 56.1% and 59.5%; and observer 4, 49.2% and 46.4%. CONCLUSION: The use of FA improves the accuracy of treatment planning for CSME. The authors' study supports the use of FA in laser treatment of patients with CSME.

Diabetic Retinopathy↗