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PubMed2026

Promises and pitfalls of long-read sequencing for resolving microbial complexity.

Long-read sequencing (LRS) has driven a transition in microbial genomics, overcoming the assembly fragmentation inherent to short-read sequencing. This review elucidates the impact of LRS across isolate genomics, metagenomics, and multi-omics domains. By spanning extensive repetitive regions, LRS facilitates the reconstruction of circular chromosomes and precisely resolves mobile genetic elements (MGEs). In metagenomics, LRS enables strain-level resolution, the recovery of circular metagenome-assembled genomes, and the precise localization of MGEs within host replicons. Furthermore, the single-molecule, amplification-free properties of LRS provide enhanced resolution of native epigenetic modifications and full-length transcriptomes. Despite these advancements, widespread implementation remains constrained by multidimensional challenges, including stringent high-molecular-weight DNA requirements, depth deficits, and computational overhead. Nevertheless, LRS is increasingly becoming the method of choice for isolate genomics and metagenomics. As detection technologies and algorithms progress, LRS will further improve our ability to decipher the structural and functional diversity of microbial ecosystems.

Metagenomics↗

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↗

PubMed1998

Social and behavioral correlates of pelvic inflammatory disease.

OBJECTIVES: To review the social and behavioral correlates of pelvic inflammatory disease (PID) in the light of the renewed interest in the transmission dynamics of communicable diseases, the new emphasis on health care seeking and health service provision, and increased attention to contextual and population level factors affecting morbidity. METHODS: Social and behavioral correlates of PID are reviewed using a conceptual scheme that matrixes the differences among risk factors for exposure, acquisition, and development of sequelae with the differences between individual-level risk factors and population-level determinants. RESULTS: Two major factors contribute to the development of PID: recurrent (or persistent) chlamydial infection of the cervix, which are geographically concentrated and associated with contextual variables, and critical delays in detection and treatment of cervical infection, which are amenable to interventions. CONCLUSIONS: Widespread screening for cervical infection followed by timely and appropriate treatment is key for prevention of PID. Health care seeking, provider training, and availability of detection technologies and drugs need to be improved.

Chlamydia Infections↗

PubMed1999

Screening for genital human papillomavirus: results from an international validation study on human papillomavirus sampling techniques.

The objective of this study was to determine the validity of human papillomavirus (HPV) detection using exfoliated cervical cells compared with cervical biopsy specimens in women with normal cervix and to assess whether HPV detection rates using exfoliated cells is dependent on the number and order in which cervical scrapes are taken. Women undergoing hysterectomy for reasons other than cervical cancer were recruited in three hospitals in countries with varying risks of cervical cancer. After informed consent and at the time of surgery, three consecutive cervical scrapes were taken as well as four biopsy specimens, one in each of the quadrants around the cervical os. In this study, 331 women were recruited and provided 992 cell samples and 1324 biopsy samples. All scrapes and a sample of biopsy specimens (n = 103) were tested by polymerase chain reaction enzyme immunoassay using a general primer (GP5+/ bio6+). Type-specific tests were performed for 14 HPV types at the subpicogram level in one test and individually. Positive samples were verified using Southern blot hybridization. The prevalence of HPV DNA was 6.3% in cervical cells. Of 19 HPV positive samples in the scrapes, 17 were confirmed in the biopsy specimens. The agreement, as measured by the Kappa statistic, was 0.90 (P < 0.0001). The concordance in detecting HPV infection between scrapes and biopsy specimens was 97.5%, and the concordance in categorizing the samples as negatives was 94.4%. These values were unchanged when the order in which scrapes were taken was compared. Among women without cervical cancer, HPV DNA detection rates do not vary if exfoliated cells or random biopsy specimens are taken as the primary testing specimen. Screening programs based on highly sensitive HPV DNA detection technology in cell scrapes should expect a minimal underdetection.

Biopsy↗

PubMed2004

Where optics meets electronics: recent progress in decreasing the terahertz gap.

The terahertz region of the electromagnetic spectrum offers considerable opportunities for exploitation. Recent advances in source and coherent-detection technology have enabled advances to be made in a wide range of applications. This article reviews the historical development of the field, summarizes the current state of system technology and outlines a number of current system applications, including medical imaging. The limitations of present-day technology are discussed and some future possible applications are considered.

Journal Article↗

PubMed2005

Physical landscapes in biological membranes: physico-chemical terrains for spatio-temporal control of biomolecular interactions and behaviour.

The evolving complexities of biological membranes are discussed from the point of view of potential roles of the physical constitution of the membrane. These include features of the surface and dipole potentials and membrane 'rafts'. These properties are outlined; they emphasize that protein-lipid and specific lipid environments are influential parameters in how biomolecular interactions may take place with and within membranes. Several fluorescence detection technologies directed towards measurement of these properties are also outlined that permit high-resolution experimental determination of intermolecular interactions with membranes by measuring small changes of these potentials. These point to the possibility that the membrane dipole potential in particular is enormously influential in determining the behaviour of receptor and signalling systems within membrane rafts, and offers the means of a novel mechanism for biological control.

Cell Membrane↗

PubMed2000

Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing.

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

Alleles↗

PubMed1995

An infrared fluorescent dATP for labeling DNA.

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Adenosine Triphosphate↗

PubMed1992

Bacillus anthracis but not always anthrax.

Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B. cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B. anthracis which lack the plasmid carrying the capsule gene (pXO2). While these techniques cannot, of course, be used to confirm the identities of strains resembling B. anthracis but which also lack the plasmid carrying the toxin genes (pXO1), the likelihood that these also are bona fide B. anthracis becomes more acceptable. (As yet no naturally occurring pXO1-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.

Animals↗

PubMed1997

Trinucleotide repeat polymorphism within exon 5 of the MICA gene (MHC class I chain-related gene A): allele frequency data in the nine population groups Japanese, Northern Han, Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian, Greek and Italian.

We recently identified a trinucleotide repeat polymorphism, (GCT)n, within the transmembrane (TM) segment of the human MHC class I MICA gene (MHC class I chain-related gene A). Five distinct alleles (A4, A5, A5.1, A6, A9) corresponding to 4, 5, 5 with one nucleotide insertion, 6 and 9 repetitions, respectively, have been detected in various HLA-homozygous B cell lines. Here we present allele frequencies for this trimeric short tandem repeat (STR) in 604 unrelated individuals collected from nine human populations (Japanese, Northern Han, Hui, Uygur, Kazakhstan, Iranian, Saudi Arabian, Greek and Italian) determined using the polymerase chain reaction (PCR) combined with fluorescent-based automated fragment detection technology. All alleles were present in each population, but allelic distributions varied from one population to another. No new alleles (such as A7 or A8) were identified. The evolutionary and structural significance of these data as well as the potential application to forensic medicine is discussed.

Alleles↗

PubMed2006

A cost-benefit analysis of alternative device configurations for aviation-checked baggage security screening.

The terrorist attacks of September 11, 2001 have resulted in dramatic changes in aviation security. As of early 2003, an estimated 1,100 explosive detection systems (EDS) and 6,000 explosive trace detection machines (ETD) have been deployed to ensure 100% checked baggage screening at all commercial airports throughout the United States. The prohibitive costs associated with deploying and operating such devices is a serious issue for the Transportation Security Administration. This article evaluates the cost effectiveness of the explosive detection technologies currently deployed to screen checked baggage as well as new technologies that could be used in the future. Both single-device and two-device systems are considered. In particular, the expected annual direct cost of using these devices for 100% checked baggage screening under various scenarios is obtained and the tradeoffs between using single- and two-device strategies are studied. The expected number of successful threats under the different checked baggage screening scenarios with 100% checked baggage screening is also obtained. Lastly, a risk-based screening strategy proposed in the literature is analyzed. The results reported suggest that for the existing security setup, with current device costs and probability parameters, single-device systems are less costly and have fewer expected number of successful threats than two-device systems due to the way the second device affects the alarm or clear decision. The risk-based approach is found to have the potential to significantly improve security. The cost model introduced provides an effective tool for the execution of cost-benefit analyses of alternative device configurations for aviation-checked baggage security screening.

Journal Article↗

PubMed1998

Color Kinesis: Principles of Operation and Technical Guidelines.

Color kinesis is a new echocardiographic technique that aids in the assessment of global and regional left ventricular performance during either systole or diastole. Color kinesis uses automated border detection technology based on backscatter data to display both the magnitude and timing of endocardial motion in real time. The color kinesis display superimposes a color overlay on the two-dimensional echocardiographic image; the number of color pixels represents the magnitude of endocardial motion, while the different colors represent the timing of endocardial motion according to a predefined color scheme. Because color kinesis is an operator-dependent technique, the steps involved in performing a technically adequate study will be reviewed as well as the pitfalls and technical limitations. The potential clinical applications of color kinesis will also be discussed.

Journal Article↗

PubMed2003

Background noise in piezoresistive, electret condenser, and ceramic microphones.

Background noise studies have been extended from air condenser microphones to piezoresistive, electret condenser, and ceramic microphones. Theoretical models of the respective noise sources within each microphone are developed and are used to derive analytical expressions for the noise power spectral density for each type. Several additional noise sources for the piezoresistive and electret microphones, beyond what had previously been considered, were applied to the models and were found to contribute significantly to the total noise power spectral density. Experimental background noise measurements were taken using an upgraded acoustic isolation vessel and data acquisition system, and the results were compared to the theoretically obtained expressions. The models were found to yield power spectral densities consistent with the experimental results. The measurements reveal that the 1/f noise coefficient is strongly correlated with the diaphragm damping resistance, irrespective of the detection technology, i.e., air condenser, piezoresistive, etc. This conclusion has profound implications upon the expected 1/f noise component of micromachined (MEMS) microphones.

Journal Article↗

PubMed2002

A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-microl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.

Animals↗

PubMed2003

Statistical and graphical methods for quality control determination of high-throughput screening data.

High-throughput screening (HTS) is used in modern drug discovery to screen hundreds of thousands to millions of compounds on selected protein targets. It is an industrial-scale process relying on sophisticated automation and state-of-the-art detection technologies. Quality control (QC) is an integral part of the process and is used to ensure good quality data and mini mize assay variability while maintaining assay sensitivity. The authors describe new QC methods and show numerous real examples from their biologist-friendly Stat Server HTS application, a custom-developed software tool built from the commercially available S-PLUS and Stat Server statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables. It allows users to visualize HTS data and examine assay performance during the HTS campaign to quickly react to or avoid quality problems.

Computer Graphics↗

PubMed2003

Improved statistical methods for hit selection in high-throughput screening.

High-throughput screening (HTS) plays a central role in modern drug discovery, allowing the rapid screening of large compound collections against a variety of putative drug targets. HTS is an industrial-scale process, relying on sophisticated automation, control, and state-of-the art detection technologies to organize, test, and measure hundreds of thousands to millions of compounds in nano- to microliter volumes. Despite this high technology, hit selection for HTS is still typically done using simple data analysis and basic statistical methods. The authors discuss in this article some shortcomings of these methods and present alternatives based on modern methods of statistical data analysis. Most important, they describe and show numerous real examples from the biologist-friendly Stat Server HTS application (SHS), a custom-developed software tool built on the commercially available S-PLUS and StatServer statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables.

Algorithms↗

PubMed2005

Adopting a practical statistical approach for evaluating assay agreement in drug discovery.

The authors assess the equivalence of 2 assays and put forward a general approach for assay agreement analysis that can be applied during drug discovery. Data sets generated by different assays are routinely compared to each other during the process of drug discovery. For a given target, the assays used for high-throughput screening and structure-activity relationship studies will most likely differ in their assay reagents, assay conditions, and/or detection technology, which makes the interpretation of data between assays difficult, particularly as most assays are used to measure quantitative changes in compound potency against the target. To better quantify the relationship of data sets from different assays for the same target, the authors evaluated the agreement between results generated by 2 different assays that measure the activity of compounds against the same protein, ALK5. The authors show that the agreement between data sets can be quantified using correlation and Bland-Altman plots, and the precision of the assays can be used to define the expectations of agreement between 2 assays. They propose a scheme for addressing issues of assay data equivalence, which can be applied to address questions of how data sets compare during the lead identification and lead optimization processes in which assays are frequently added and changed.

Activin Receptors, Type I↗

PubMed2005

Probing the primary screening efficiency by multiple replicate testing: a quantitative analysis of hit confirmation and false screening results of a biochemical assay.

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65% or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary screening.

DNA-Binding Proteins↗