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PubMed2005

SNPNB: analyzing neighboring-nucleotide biases on single nucleotide polymorphisms (SNPs).

UNLABELLED: SNPNB is a user-friendly and platform-independent application for analyzing Single Nucleotide Polymorphism NeighBoring sequence context and nucleotide bias patterns, and subsequently evaluating the effective SNP size for the bias patterns observed from the whole data. It was implemented by Java and Perl. SNPNB can efficiently handle genome-wide or chromosome-wide SNP data analysis in a PC or a workstation. It provides visualizations of the bias patterns for SNPs or each type of SNPs. AVAILABILITY: SNPNB and its full description are freely available at http://bioinfo.vipbg.vcu.edu/SNPNB/

Algorithms↗

PubMed2007

Micropallet arrays for the separation of single, adherent cells.

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.

Animals↗

PubMed2002

Precise CD4 T-cell counting using red diode laser excitation: for richer, for poorer.

BACKGROUND: Measuring CD4 T-cell counts at low cost is relevant in dealing with the human immunodeficiency virus (HIV) epidemic throughout the developing world. The recently introduced novel concepts in gating strategies and sample stabilization facilitate affordable immunophenotyping by flow cytometry. However, the impact of these developments is still limited by the high cost of currently available flow cytometers. METHODS: Diode lasers emitting 10-15 mW at 635 nm are one-tenth the size and cost and require one thousandth the power of an equivalent 488-nm argon ion laser. We used the available 635-nm diode-based flow cytometers, including PA-II, Luminex 100, SuperMot, and FACSCalibur, to investigate whether these instruments can generate reliable CD4 counts when used with allophycocyanin (APC) and cyanin-5 (Cy5)-labeled CD4 antibodies. RESULTS: We document the feasibility of obtaining leucocyte differential counts using orthogonal side scatter (SSC) without the need for forward scatter (FSC). Accurate CD4% values among lymphocytes and leucocytes can be obtained by primary CD4 gating using a single CD4 monoclonal antibody conjugated to APC or Cy5. Double immunofluorescence (IF) staining with CD4-APC (FL1) and CD45-APC-Cy7 (FL2) introduces pan-leucogating for a convenient assessment of absolute CD4 counts on double platforms. We demonstrate that small flow cytometers with laser diodes are capable of delivering absolute CD4 T-cell counts with a precision similar to the performance of the current state-of-the-art single-platform instruments (e.g., the CytoronAbsolute; R(2) = 0.961). In this respect, they appear to be superior to the nonflow CD4 counting techniques. CONCLUSIONS: Accurate CD4 counts can be generated at minimal cost on red diode laser-operated flow cytometers, retaining the potential for high throughput capacity without compromising precision. With further improvements in volumetric technology and clinical software, these cytometers may develop into a new generation of inexpensive battery-operated laboratory hardware that combines cellular phenotyping with bead-based multiplexing immunoassays for (HIV) serology.

CD4 Antigens↗

PubMed2004

Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms.

BACKGROUND: We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs). METHODS: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers. The products were then sorted on the Tag-It array and detected by use of the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Empirical validation of the universal array showed that the highest nonspecific signal was 3.7% of the specific signal. Patient genotypes showed 100% concordance with direct DNA sequencing data for 736 SNP determinations. CONCLUSIONS: The Tag-It microsphere-based universal array platform is a highly accurate, multiplexed, high-throughput SNP-detection platform.

Genotype↗

PubMed1989

Hippocampal place cells: stereotypy and plasticity.

Hippocampal complex spike cells were recorded during exploration for water delivered to cups located in various regions of an elevated platform. Place fields were recorded with a video monitoring system that recorded movements as the animal explored each of the 5 cup locations where water was delivered on the platform. Plasticity of place cell firing as a function of selective water delivery to specific cup locations on the platform was also examined. Several characteristics of place cell firing were studied that indicated a high degree of control by factors such as relative direction of movement and trajectory through the field. Time-shift analyses indicated cell firing was most representative of the place field at the time of spike occurrence. It was demonstrated that place fields possess borders in which firing was increased or decreased upon entering or leaving a particular region of the platform. The most important finding from this investigation was the pronounced degree of plasticity exhibited by place cells. Selective delivery of water to a single location on the platform was sufficient in most of the cases tested to shift the location of the field to the location where water was available. These findings suggest hippocampal place cell firing, although highly influenced by spatial and directional features of the environment, can readily change under conditions in which significant stimuli are added or removed from those locations.

Animals↗

PubMed2004

Microfabricated system for parallel single-cell capillary electrophoresis.

Performing single-cell electrophoresis separations using multiple parallel microchannels offers the possibility of both increasing throughput and eliminating cross-contamination between different separations. The instrumentation for such a system requires spatial and temporal control of both single-cell selection and lysis. To address these problems, a compact platform is presented for single-cell capillary electrophoresis in parallel microchannels that combines optical tweezers for cell selection and electromechanical lysis. Calcein-labeled acute myloid leukemia (AML) cells were selected from an on-chip reservoir and transported by optical tweezers to one of four parallel microfluidic channels. Each channel entrance was manufactured by F2-laser ablation to form a 20- to 10-microm tapered lysis reservoir, creating an injector geometry effective in confining the cellular contents during mechanical shearing of the cell at the 10-microm capillary entrance. The contents of individual cells were simultaneously injected into parallel channels resulting in electrophoretic separation as recorded by laser-induced fluorescence of the labeled cellular contents.

Comet Assay↗

PubMed2006

A fast and reliable route integrating calibration and analysis protocols for water-soluble vitamin determination on microchip-electrochemistry platforms.

A novel analytical route to determine water-soluble vitamins (B group and C) using single channel microchip-electrochemistry platforms is presented. The electrochemical detection protocol was carefully optimized, and it was shown that it was crucial to use 1 M nitric acid in the detector compartment to detect folic acid. A phosphate buffer (pH 6, 10 mM) and a separation voltage of 2 kV gave the complete separation of vitamins in less than 130 s, with good reproducibility (RSDs less than 10%) and accuracy (error less than 9%). In addition, a methodological innovation integrating calibration and analysis of water-soluble vitamins on the chip is also proposed. The strategy consisted in sequentially using both reservoirs (named calibration and analysis reservoirs) as well as a calibration factor (defined as signal/concentration of analyte). The analytical route required 350 s in the overall protocol (employing 130 s in calibration plus 130 s in analysis), an improvement over the times used in both conventional and microchip protocols.

Ascorbic Acid↗

PubMed2003

Cellular genomics: which genes are transcribed, when and where?

Gene expression involves a cascade of events, from transcription factor transactivation and RNA polymerase binding to RNA processing and maturation. mRNA profiling has yielded a vast amount of information about the total RNA content in different tissues physiological states. However, it has been unclear how the variable amounts of mRNA might correlate with transcriptional events. Recent advances in single-cell imaging offer a platform for 'cellular genomics', the high throughput analysis of transcriptional activity in single cells.

Animals↗

PubMed2026

Barcoded oligonucleotide system (BOLT) for targeted organ delivery.

The therapeutic potential of oligonucleotides (oligos) is limited by insufficient delivery to extrahepatic tissues. In vitro assays often fail to accurately predict in vivo behavior, while testing each oligo candidate in animals remains inherently low throughput. Here, we conceive a barcoded oligonucleotide system (BOLT), a platform that enables high-throughput in vivo evaluations of small-molecule ligands and identifies tissue-specific oligo delivery. BOLT integrates rational design of oligo barcodes, modular conjugation chemistry, and next-generation sequencing (NGS)-based quantification, allowing simultaneous evaluation of many chemically diverse ligand-oligo conjugates within a single animal. Notably, this platform is applicable in both mice and nonhuman primates (NHPs). Using BOLT, we discovered ligands with tropism for tissues such as the brain, lung, and muscle. Collectively, these results indicate that the BOLT platform can accelerate the discovery of tissue-targeting ligands for broad oligo therapeutics.

Journal Article↗

PubMed1999

Adaptation of the circular platform spatial memory task for mice: use in detecting cognitive impairment in the APP(SW) transgenic mouse model for Alzheimer's disease.

A methodology is described for use of a 16-hole circular platform task to test spatial memory in mice. Both bright light and a fan were used to motivate mice to escape the platform surface through a single hole containing an attached escape box. For each daily trial, three correlated measures (escape latency, number of errors, and error rating) comprehensively evaluated cognitive performance. In an initial study, the 'spatial' nature of this task was demonstrated by the much poorer performance of non-transgenic mice when visual cues are removed. Behavioral sensitivity of the circular platform task was then shown through its ability to discern cognitive impairment in 7-month-old transgenic mice, carrying the mutant APP(SW) gene for early-onset Alzheimer's disease in humans, from non-transgenic litter-mates. Since there are currently only a few tasks available to definitively test cognitive performance in mice, the circular platform task offers a versatile, multiple-measure option with numerous advantages. Particularly in view of the increasing number of genetically manipulated mouse models being produced, the circular platform task should be most useful in providing a sensitive evaluation of cognition in mice.

Alzheimer Disease↗

PubMed2006

Balance in single-limb stance after surgically treated ankle fractures: a 14-month follow-up.

BACKGROUND: The maintenance of postural control is fundamental for different types of physical activity. This can be measured by having subjects stand on one leg on a force plate. Many studies assessing standing balance have previously been carried out in patients with ankle ligament injuries but not in patients with ankle fractures. The aim of this study was to evaluate whether patients operated on because of an ankle fracture had impaired postural control compared to an uninjured age- and gender-matched control group. METHODS: Fifty-four individuals (patients) operated on because of an ankle fracture were examined 14 months postoperatively. Muscle strength, ankle mobility, and single-limb stance on a force-platform were measured. Average speed of centre of pressure movements and number of movements exceeding 10 mm from the mean value of centre of pressure were registered in the frontal and sagittal planes on a force-platform. Fifty-four age- and gender-matched uninjured individuals (controls) were examined in the single-limb stance test only. The paired Student t-test was used for comparisons between patients' injured and uninjured legs and between side-matched legs within the controls. The independent Student t-test was used for comparisons between patients and controls. The Chi-square test, and when applicable, Fisher's exact test were used for comparisons between groups. Multiple logistic regression was performed to identify factors associated with belonging to the group unable to complete the single-limb stance test on the force-platform. RESULTS: Fourteen of the 54 patients (26%) did not manage to complete the single-limb stance test on the force-platform, whereas all controls managed this (p < 0.001). Age over 45 years was the only factor significantly associated with not managing the test. When not adjusted for age, decreased strength in the ankle plantar flexors and dorsiflexors was significantly associated with not managing the test. In the 40 patients who managed to complete the single-limb stance test no differences were found between the results of patients' injured leg and the side-matched leg of the controls regarding average speed and the number of centre of pressure movements. CONCLUSION: One in four patients operated on because of an ankle fracture had impaired postural control compared to an age- and gender-matched control group. Age over 45 years and decreased strength in the ankle plantar flexors and dorsiflexors were found to be associated with decreased balance performance. Further, longitudinal studies are required to evaluate whether muscle and balance training in the rehabilitation phase may improve postural control.

Adult↗

PubMed2006

Performance-based measures of physical function for high-function populations.

OBJECTIVES: To improve and broaden the applicability of performance-based measures of function for use in clinical and research settings. DESIGN: Cross-sectional repeated-measures study. SETTING: Research clinic. PARTICIPANTS: Population-based sample of Japanese Americans without significant functional impairments aged 35 to 55 and 56 to 71 (N=203). MEASUREMENTS: Performance-based measures of physical function, including range of standard tests, newer automated measures of balance and strength and data on cognitive function, lifestyle, medical history, and physical activity. RESULTS: Of the nonplatform balance measures, only the one-leg stand was reliable (reliability coefficient (rc) =0.69) and able to discriminate between functional levels. Combining the Fourth National Health and Nutrition Examination Survey protocol of folded arm position while standing on a foam pad with the more-sophisticated balance platform test had the highest reliability and discrimination. With the strength chair, high rcs (0.88-0.96) were found for upper and lower extremity tests. Timed chair stands, a test of lower extremity and central strength were reliable. The 6-minute walk had a high rc (0.90). CONCLUSION: Many performance tests used today are not reliable. Only a few discriminate between the most highly functioning individuals and individuals with good function. Thus, a new recommended battery includes unassisted single-leg stand, balance platform "foam pad, eyes closed," elbow flexion and knee extension strength (strength chair), grip strength, timed chair stands, and the 6-minute walk. These simple performance-based tests have good reliability and discrimination across the range of function and can be used in most clinical and research settings to quickly assess global functional level.

Adult↗

PubMed2003

The future of clinical cancer management: one tumor, one chip.

Recent advances in gene expression profiling technology have now made it feasible to consider using microarray technology in the routine management of the cancer patient. Microarray chips are now capable of interrogating up to 48,000 or more different genes in a single experiment using multiple platforms. Sophisticated data analysis has already demonstrated that multiple tumor types can be distinguished on the basis of their gene expression patterns. These analyses have led to the detection of new tumor markers and markers of tumor progression. Gene expression arrays have also been demonstrated to be capable of predicting the survival of patients with breast cancer, lung cancer, brain cancer, and acute lymphocytic leukemia. The future holds great promise for the rapid development of molecular medicine with diagnosis, prognosis, and even therapy being based on a single microarray chip. These developments signal a significant paradigm shift in the clinical management of human cancer.

Gene Expression Profiling↗

PubMed2001

High-throughput genomic and proteomic analysis using microarray technology.

BACKGROUND: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. METHODS: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000-24,000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. RESULTS: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82-0.99 (depending on analyte) compared with ELISAs. CONCLUSIONS: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

Arylamine N-Acetyltransferase↗

PubMed2001

Knee bracing after ACL reconstruction: effects on postural control and proprioception.

PURPOSE: To evaluate the effects an anterior cruciate ligament (ACL) brace has on various measures of knee proprioception and postural control. METHODS: Thirty subjects (mean age 27 +/- 11 yr) having undergone unilateral ACL reconstruction were tested with and without wearing their own custom-fit brace on their involved limb. Proprioception was assessed using joint angle replication tests completed on an isokinetic dynamometer. Postural control was assessed using a series of single-limb standing balance tests completed on a force platform. The balance tests included: 1) standing on the stable platform with eyes open, 2) standing on a foam mat placed over the platform with eyes open, 3) standing on the platform with eyes closed, and 4) standing on the platform after landing from a maximal single-limb forward hop. RESULTS: The brace provided a small but statistically significant improvement in proprioception (mean reduction in error scores between target and reproduced angles = 0.64 +/- 1.4 degrees, P = 0.02). For the postural control tests, there was a significant brace condition by test situation interaction (P = 0.02), with the brace providing a small but statistically significant improvement during the test completed on the stable platform with eyes open (mean reduction in center of pressure path length = 4.2 +/- 8.4 cm, P = 0.02) but not during the other more challenging test situations. Additional post hoc analyses indicated that the relationship between knee proprioception and postural control measures were low and not significant (r = 0.003 to 0.19, P > 0.32), consistent with the suggestion that changes in knee proprioception can occur in the absence of substantial changes in postural control. Also, standing balance tests that challenged the somatosensory contribution to postural control (i.e., those completed on foam, or with eyes closed) were significantly related to single-limb forward hop distances (r = -0.4, P < 0.05), whereas performance during the proprioception test was not (r = 0.1, P > 0.50). CONCLUSIONS: In general, bracing appears to improve performance during tasks characterized by relatively limited somatosensory input but not during tasks characterized by increased somatosenory input. The small magnitude of the improvements, coupled with their apparent lack of carry over to more difficult and functionally relevant tasks, questions the clinical benefit of the present effects of bracing.

Adult↗

PubMed2002

Terahertz polariton propagation in patterned materials.

Generation and control of pulsed terahertz-frequency radiation have received extensive attention, with applications in terahertz spectroscopy, imaging and ultrahigh-bandwidth electro-optic signal processing. Terahertz 'polaritonics', in which terahertz lattice waves called phonon-polaritons are generated, manipulated and visualized with femtosecond optical pulses, offers prospects for an integrated solid-state platform for terahertz signal generation and guidance. Here, we extend terahertz polaritonics methods to patterned structures. We demonstrate femtosecond laser fabrication of polaritonic waveguide structures in lithium tantalate and lithium niobate crystals, and illustrate polariton focusing into, and propagation within, the fabricated waveguide structures. We also demonstrate a 90 degrees turn within a structure consisting of two waveguides and a reflecting face, as well as a structure consisting of splitting and recombining elements that can be used as a terahertz Mach-Zehnder interferometer. The structures permit integrated terahertz signal generation, propagation through waveguide-based devices, and readout within a single solid-state platform.

Computer Simulation↗

PubMed1993

Balance improvements in older women: effects of exercise training.

BACKGROUND AND PURPOSE: Loss of lower-extremity strength increases the risk of falls in older persons. The purpose of this study was to test the hypothesis that a vigorous program of lower-extremity strengthening, walking, and postural control exercises would improve the single-stance balance of healthy older women and lower their risk of falls and fall-associated injuries. SUBJECTS: From a total of 38 respondents, 21 women were randomly assigned to either a treatment group (combined training, n = 12) or a control group (flexibility training, n = 9). The subjects ranged in age from 62 to 75 years (mean = 68, SD = 3.5). METHODS: A randomized control trial compared the effects of two exercise programs on static balance. The combined training group exercised three times per week on knee extension and sitting leg press machines, walked briskly for 20 minutes, and performed postural control exercises, which included simple tai chi movements. The flexibility training group performed postural control exercises weekly. Measurements of balance were obtained on a force platform in double and single stance, at baseline and following 6 months of exercise training. RESULTS: Double-stance measurements were unchanged after training. The mean displacement of the center of pressure in single stance improved 17% in the combined training group and did not change in the flexibility training group. A repeated-measures analysis of variance revealed that the difference in improvement between the combined training and flexibility training groups was not significant. DISCUSSION AND CONCLUSION: This is the first intervention trial to demonstrate improvements in single-stance postural sway in older women with exercise training. Additional studies with more subjects will be needed to determine whether a combined training program of resistance training, walking, and postural exercises can improve balance more than a program of postural control exercises alone.

Accidental Falls↗

PubMed2026

VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature.

The human cerebrovasculature is a critical yet historically understudied component of neurological health. Dysfunction of the diverse endothelial, mural, and perivascular cells that comprise cerebral vessels is central to diseases ranging from stroke to Alzheimer's disease. However, characterizing these cell populations at a molecular level has proven exceptionally challenging. Encased within a robust basement membrane, vascular cells resist standard dissociation methods, leading to their systematic depletion and underrepresentation in existing single-nucleus genomic atlases. This has created a major blind spot in neuroscience. To overcome this barrier, we developed vessel isolation and nucleus extraction for sequencing (VINE-seq) and its advanced iteration, MultiVINE-seq. The protocol provides a robust, reproducible workflow for the enrichment and high-resolution profiling of vascular, perivascular, and immune cells from fresh or frozen human and mouse brain tissue. First, intact vessels (predominantly capillaries and small arterioles/venules, 100 µm in diameter) are isolated from homogenized brain tissue via dextran-based density-gradient centrifugation, separating the vascular pellet from myelin and the parenchymal fraction. Second, the collected vessels are rigorously washed over a cell strainer to remove trapped contaminants. A critical innovation lies in the third stage: the optimized extraction of nuclei from purified vessels using enzymatic digestion. After extraction, the protocol uses fluorescence-activated cell sorting (FACS) to ensure collection of high-purity nuclei suitable for widely used droplet-based sequencing platforms (e.g., 10x Genomics single cell 3' or multiome). This protocol requires 4-5 h to complete and can be carried out by researchers with single-cell and flow cytometry training.

Journal Article↗