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PubMed2004

Using solution-phase nanoparticles, surface-confined nanoparticle arrays and single nanoparticles as biological sensing platforms.

The intense colors of noble metal nanoparticles have inspired artists and fascinated scientists for hundreds of years. In this review, we describe three sensing platforms based on the tunability of the localized surface plasmon resonance (LSPR) of gold and silver nanoparticles. Specifically, the color associated with solution-phase nanoparticles, surface-confined nanoparticle arrays, and single nanoparticles will be shown to be tunable and useful as platforms for biological sensing.

Algorithms↗

PubMed1994

Distinction between places and paths in rats' spatial representations.

Rats were trained on a 3-dimensional, 4-arm radial maze. In Experiment 1, Ss trained to climb to the single goal platform chose fewer novel routes to the goal than Ss trained to climb to the 4 spatially distinct platforms. In Experiment 2 a reinforcement contingency was imposed, requiring a novel route choice on each trial to receive reinforcement. Learning to associate route choice with reinforcement outcome was much more difficult for Ss tested with the single goal than for Ss tested with the 4 distinct goals. In Experiment 3 a partitioned central platform group learned the reinforcement contingency as quickly as the Ss given 4 spatially distinct platforms. In Experiment 4, distinctive floor inserts did not affect performance relative to no inserts.

Animals↗

PubMed1995

Medial prefrontal lesions in the rat and spatial navigation: evidence for impaired planning.

Rats with medial prefrontal cortical lesions were tested in a modified water maze navigation task. In Stage 1, the rats were trained to locate a hidden platform from a single start location. They were then subjected to a series of trials during which a second start position was used (Stage 2). In Stage 3, the rats had to navigate to a new goal location from the 2 experienced start positions. Stage 4 required the rats to navigate to the same goal as in Stage 3, starting from 4 distinct positions. Finally, a single probe trial with no platform was conducted. Rats with prefrontal lesions were impaired only during Stage 4. This deficit was specific to the 2 start positions newly introduced during this stage, suggesting a dysfunction of planning processes. This impairment might result from a working memory deficit, precluding the animal from forming an adequate representation of the whole course of movements required to reach the platform.

Animals↗

PubMed2006

Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts.

BACKGROUND: Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed) system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way. RESULTS: The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli-derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFalpha1/IL-6 fusion was performed in a faithful way. CONCLUSION: rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired.

Journal Article↗

PubMed2005

Sleep deprivation-induced alterations in excitatory synaptic transmission in the CA1 region of the rat hippocampus.

Although the function of sleep remains elusive, there is compelling evidence to suggest that sleep plays an important role in learning and memory. A number of studies have now shown that sleep deprivation (SD) results in significant impairment of long-term potentiation (LTP) in the hippocampus. In this study, we have attempted to determine the mechanisms responsible for this impairment. After 72 h SD using the multiple-platform technique, we observed a reduction in the whole-cell recorded NMDA/AMPA ratio of CA1 pyramidal cells in response to Schaffer collateral stimulation. This impairment was specific to sleep deprivation as rats placed over a single large platform, which allowed sleep, had a normal NMDA/AMPA ratio. mEPSCs evoked by local application of a high osmolarity solution revealed no differences in the AMPA receptor function. NMDA currents recorded from outside-out patches excised from the distal dendrites of CA1 cells displayed a reduction in amplitude after SD. While there were no alterations in the glutamate sensitivity, channel open probability or the single channel conductance of the receptor, a crosslinking assay demonstrated that the NR1 and NR2A subunits of NMDA receptors were preferentially retained in the cytoplasm after SD, indicating that SD alters NMDAR surface expression. In summary, we have identified a potential mechanism underlying SD-induced LTP impairment. This synaptic alteration may underlie the cognitive deficits seen following sleep deprivation and could represent a target for future intervention studies.

Animals↗

PubMed2002

Changes in weight-bearing following injury or surgical reconstruction of the ACL: relationship to quadriceps strength and function.

PURPOSE: Changes in weight-bearing an average of 1.5 months after ACL injury (ACLD) and up to 3 months after surgical reconstruction (ACLR) were investigated using a force platform. Correlations between force platform test variables, quadriceps strength and functional self-report scores were also examined. RESULTS: Each force platform test revealed impaired weight-bearing by either ACLD and/or ACLR subjects. Many variables were correlated to quadriceps strength; therefore, improved strength in ACLR subjects by 6 weeks post-operative negated any weight-bearing impairment. No single force platform variable correlated with the subjects' perception of function. CONCLUSION: Only the step up/over test provided insight into disability in these patients. Modification of force platform analysis programs may allow for other clinical questions to be answered.

Adult↗

PubMed2005

Sequencing sit-to-stand and upright posture for mobility limitation assessment: determination of the timing of the task phases from force platform data.

The identification of quantitative tools to assess an individual's mobility limitation is a complex and challenging task. Several motor tasks have been designated as potential indicators of mobility limitation. In this study, a multiple motor task obtained by sequencing sit-to-stand and upright posture was used. Algorithms based on data obtained exclusively from a single force platform were developed to detect the timing of the motor task phases (sit-to-stand, preparation to the upright posture and upright posture). To test these algorithms, an experimental protocol inducing predictable changes in the acquired signals was designed. Twenty-two young, able-bodied subjects performed the task in four different conditions: self-selected natural and high speed with feet kept together, and self-selected natural and high speed with feet pelvis-width apart. The proposed algorithms effectively detected the timing of the task phases, the duration of which was sensitive to the four different experimental conditions. As expected, the duration of the sit-to-stand was sensitive to the speed of the task and not to the foot position, while the duration of the preparation to the upright posture was sensitive to foot position but not to speed. In addition to providing a simple and effective description of the execution of the motor task, the correct timing of the studied multiple task could facilitate the accurate determination of variables descriptive of the single isolated phases, allowing for a more thorough description of the motor task and therefore could contribute to the development of effective quantitative functional evaluation tests.

Adult↗

PubMed2005

GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors.

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.

Animals↗

PubMedDate not supplied

The relationship between limb function and radiographic osteoarthrosis in dogs with stifle osteoarthrosis.

OBJECTIVE: To evaluate the relationship between limb function and radiographic evidence of stifle osteoarthrosis (OA) in dogs. STUDY DESIGN: The relationship between force platform data and radiographic OA score was evaluated on 2 separate days using regression analysis. Interday variation was also assessed. ANIMALS: Forty-one dogs with visible lameness and radiographic evidence of stifle OA. METHODS: Force platform data were collected at a velocity of 1.7 to 2.0 m/s for 5 trials on day 1 and day 8. Radiographs taken on day 1 were scored using a previously reported OA scoring system. RESULTS: No significant relationship was found between force platform data and OA score. No significant differences were found between any day 1 and day 8 force platform values. CONCLUSIONS: Although radiographic evidence of stifle OA provides evidence of pathology, it does a poor job of representing limb function. In addition, the absence of significant differences between day 1 and day 8 values in this population of dogs supports use of only a single force platform evaluation before measuring a treatment effect. CLINICAL RELEVANCE: The presence of OA in the stifle joint does not correlate with clinical function; radiographic outcome should be used cautiously as a predictor of clinical outcome.

Animals↗

PubMed2026

Mapping convergent regulators of melanoma drug resistance by PerturbFate.

High-throughput genomic studies have uncovered associations between diverse genetic alterations and disease phenotypes. However, elucidating how perturbations in functionally disparate genes give rise to convergent cellular states remains challenging. Here we present PerturbFate, a high-throughput, cost-effective, combinatorial-indexing single-cell platform that enables systematic interrogation of massively parallel CRISPR interference1 perturbations across the full spectrum of gene regulation, from chromatin remodelling and nascent transcription to steady-state transcriptomic phenotypes. Using PerturbFate, we profiled more than 300,000 cultured melanoma cells to characterize multimodal phenotypic and gene regulatory responses to perturbations in more than 140 vemurafenib resistance-associated genes. We uncovered a shared dedifferentiated cell state marked by convergent cooperative transcription factor activities across diverse genetic perturbations. We further dissected phenotypic responses to perturbations in Mediator complex components, linking module-specific biochemical properties to convergent transcriptional activations. We identified common regulatory nodes that drive similar phenotypic outcomes across distinct genetic perturbations. We also delineated how perturbations in functionally unrelated genes reshape cell state. Thus, PerturbFate establishes a versatile platform for identifying key molecular regulators by anchoring multimodal regulatory dynamics to disease-relevant phenotypes.

Humans↗

PubMed2007

Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms.

The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of alpha(S1)-casein, beta-casein, kappa-casein, and beta-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the kappa-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr(136) and Ala(148). This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.

Animals↗

PubMed2004

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis.

Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.

Alleles↗

PubMed2002

Commercial molecular diagnostics in the U.S.: The Human Genome Project to the clinical laboratory.

Molecular diagnosis is the detection of pathogenic mutations in DNA and RNA samples to aid in detection, diagnosis, subclassification, prognosis, and monitoring response to therapy. Principles underlying nucleic-based diagnosis originate from localization, identification, and characterization of genes responsible for human disease. Clinical molecular genetics is now part of the mainstream of medical care in the United States. All commercial clinical reference laboratories now have a molecular genetic diagnostic unit, many of which are in contractual agreement with third party payers to provide services. Gene discovery provides valuable insight into the mechanisms of disease processes and gene-based markers will enable clinicians to study disease predisposition, as well as improved methods for diagnoses, prognosis, and monitoring of therapy. The broad range of mutation spectrum and type performed in the clinical laboratory requires the use of multiple technologies rather than a single typing platform. Platform choice depends on such diverse factors as local expertise, test volume, economies of scale, R&D budget, and royalties. Test validation is a major hurdle and positive control samples are often not readily available. Oversight and the regulatory environment for clinical molecular genetics laboratories in the United States are evolving rapidly. Several government agencies and private organizations are currently involved in revision of specific laboratory standards, including the Secretary's Advisory Committee on Genetic Testing (SACGT), Food and Drug Administration (FDA), Center for Disease Control (CDC), College of American Pathologists (CAP), American College of Medical Genetics (ACMG), and the individual states.

DNA Mutational Analysis↗

PubMed2005

The influence of external ankle braces on subjective and objective parameters of performance in a sports-related agility course.

With lateral ankle sprains being the most frequent sports-related injury, there is an evident demand for the preventive measures in active individuals with chronic ankle instability. Braces are commonly used for prevention and treatment of ankle injuries. Various investigations-mostly performed with healthy subjects-focused on this problem, yet they often compared only a few models or used only limited testing procedures. However, controversy exists whether braces affect sports performance. The purpose of the present study was to compare the effects of ten different ankle braces-one rigid, five semirigid, four soft models-in a comprehensive evaluation with multiple testing procedures in 34 subjects with self-reported chronic ankle instability. The multiple testing procedures evaluated objective performance-related parameters and subjective parameters related to comfort and stability. The subjects performed an agility course with maximal effort. The course included a vertical jump and a cutting maneuver, both on a force platform, a single leg hopping test on level and inclined plates, a combined straight and curve sprint and sidesteps. Three valid trials were measured and averaged for each brace and every subject. Subjective aspects were evaluated with a questionnaire about handling, perceived restrictions, support and comfort; it was completed after each brace was worn and tried. With regard to the objective parameters, no significant differences were found between the braces except for the rigid brace which showed decreased values for the vertical jump and longer times for the other tests compared to all other braces. The subjective evaluation of the braces revealed significant differences with respect to comfort and handling and therefore, permitted a distinction between semirigid and soft braces. Although significant differences between braces were found in subjective performance restriction, no significant differences were revealed in the objective evaluation. From that point of view, patients could choose a brace model according to their individual needs. A comfortable brace might have a positive influence on the athlete's state of mind although other aspects like the brace's stabilizing effect play an additional role and should also be taken into account for recommendation of braces.

Adolescent↗

PubMed2004

Postural control in athletes participating in an ironman triathlon.

We studied the degree of dependence on vision of static postural control among ten male adult ironmen and ten healthy subjects (firemen, control group) who took part in regular physical activity, and the perturbations of equilibrium after prolonged exercise in ironmen. Static postural stability was measured during standing on a single-force platform alternating between eyes open and eyes closed. First, body sway was analysed on a force plate in both groups, and the athletes then took part in an ironman triathlon. The measurement was repeated after the race. The sway in both directions was subjected to spectral analysis. The frequency spectrum of the platform oscillations was calculated by fast Fourier transformation in the intervals 0-0.3, 0.3-1 and 1-3 Hz. The sway path in both directions and the total path were significantly lower in the ironmen than in the control group without vision, and the absence of visual control caused a significant increase in sway in both directions in the control group, but not in the ironmen. The frequency analysis revealed a higher level of stability in the medio-lateral direction with closed eyes. The endurance race caused increases in both the total sway path only with closed eyes, and these changes were significant at higher frequency bands. These results indicate that ironmen are more stable and less dependent on vision for postural control than the control subjects, and the prolonged stimulation of the proprioceptive, vestibular and visual inputs in the endurance race causes a significant disturbance in postural control.

Adult↗

PubMed2026

ONT-only genome assembly of a Korean male individual using a semen sample.

BACKGROUND: Long-read sequencing has enabled the generation of high-quality human genome assemblies, but many previous assemblies were based on blood-derived DNA and often relied on limited data types from a single sequencing strategy. OBJECTIVE: This study aimed to generate high-quality phased genome assemblies of a Korean individual using multiple independent long-read datasets produced from a single sequencing platform and to evaluate their utility for chromosome-scale assembly and variant detection. METHODS: Genomic DNA was extracted from a semen sample of a Korean male. Long-read, ultra-long-read, and chromatin conformation capture sequencing data were generated using Oxford Nanopore Technologies. These datasets were integrated to construct phased genome assemblies, followed by correction of noticeable phasing errors and assessment of assembly continuity, chromosomal representation, telomeric repeat recovery, and variant detection performance. RESULTS: The final phased assemblies spanned approximately 2.9 Gb and represented 23 pairs of chromosomes with an NG50 of 150 Mb. Telomeric repeats were detected at 36 and 37 of the 48 chromosomal ends in the two assemblies, indicating high end-to-end completeness. In addition, we successfully identified structural variants, including small variants. These results demonstrate that combining multiple Oxford Nanopore data types can produce highly continuous and informative phased human genome assemblies. CONCLUSIONS: We generated high-quality phased genome assemblies of a Korean individual using Oxford Nanopore long-read sequencing data derived from semen DNA. This publicly available genome resource will support broader applications of long-read sequencing in human genomics and variant analysis.

Humans↗

PubMed2004

Nanogold-plasmon-resonance-based glucose sensing.

Noble metal nanoparticles are well known for their strong interactions with light through the resonant excitations of the collective oscillations of the conduction electrons on the particles, the so-called surface plasmon resonances. The close proximity of two nanoparticles is known to result in a red-shifted resonance wavelength peak, due to near-field coupling. We have subsequently employed this phenomenon and developed a new approach to glucose sensing, which is based on the aggregation and disassociation of 20-nm gold particles and the changes in plasmon absorption induced by the presence of glucose. High-molecular-weight dextran-coated nanoparticles are aggregated with concanavalin A (Con A), which results in a significant shift and broadening of the gold plasmon absorption. The addition of glucose competitively binds to Con A, reducing gold nanoparticle aggregation and therefore the plasmon absorption when monitored at a near-red arbitrary wavelength. We have optimized our plasmonic-type glucose nanosensors with regard to particle stability, pH effects, the dynamic range for glucose sensing, and the observation wavelength to be compatible with clinical glucose requirements and measurements. In addition, by modifying the amount of dextran or Con A used in nanoparticle fabrication, we can to some extent tune the glucose response range, which means that a single sensing platform could potentially be used to monitor microM --> mM glucose levels in many physiological fluids, such as tears, blood, and urine, where the glucose concentrations are significantly different.

Biosensing Techniques↗

PubMed2006

High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D(7)-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.

Cholesterol↗