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PubMed2000

Large-scale SNP scoring from unamplified genomic DNA.

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.

DNA↗

PubMed2006

Microelectronic array system for molecular diagnostic genotyping: Nanogen NanoChip 400 and molecular biology workstation.

Hundreds of gene mutations responsible for Mendelian disorders are currently tested in the clinical laboratory for pre- and postnatal diagnosis, carrier screening and presymptomatic testing. Since human genetic research is currently focused on determining the etiology of complex diseases, including heart disease, diabetes and neuropsychiatric traits, laboratorians will genotype increasing numbers of clinically relevant loci in the future. This will require accurate, high-throughput and cost-effective genotyping platforms, such as the DNA microarray. The Nanogen NanoChip platforms employ hybridization-based technology, using fluorescent detection and electronic control of the target or probe, to obtain clear genotype signal relative to background, and increased flexibility relative to similar chip-based single nucleotide polymorphism genotyping platforms. The scope of this review is intended to describe the operating principle, chips and instrumentation, analyte-specific reagents, published assay protocols, assay development, and clinical use of the NanoChip platforms. It is beyond the scope of this review to describe the use of NanoChip platforms in basic research, and to compare it against all available clinical single nucleotide polymorphism genotyping applications and platforms.

Computational Biology↗

PubMed2025

MitoTracer facilitates the identification of informative mitochondrial mutations for precise lineage reconstruction.

Mitochondrial (MT) mutations serve as natural genetic markers for inferring clonal relationships using single cell sequencing data. However, the fundamental challenge of MT mutation-based lineage tracing is automated identification of informative MT mutations. Here, we introduced an open-source computational algorithm called "MitoTracer", which accurately identified clonally informative MT mutations and inferred evolutionary lineage from scRNA-seq or scATAC-seq samples. We benchmarked MitoTracer using the ground-truth experimental lineage sequencing data and demonstrated its superior performance over the existing methods measured by high sensitivity and specificity. MitoTracer is compatible with multiple single cell sequencing platforms. Its application to a cancer evolution dataset revealed the genes related to primary BRAF-inhibitor resistance from scRNA-seq data of BRAF-mutated cancer cells. Overall, our work provided a valuable tool for capturing real informative MT mutations and tracing the lineages among cells.

Humans↗

PubMed1995

Selective depression of medium-latency leg and foot muscle responses to stretch by an alpha 2-agonist in humans.

1. In standing humans, toe-up rotation of a platform induces a short-latency (SLR) and a medium-latency response (MLR) in both soleus (Sol) and flexor digitorum brevis (FDB) muscles. Toe-down rotation evokes a MLR in the tibialis anterior (TA). The SLR is the counterpart of the monosynaptic stretch reflex, but the origin of the MLR is still debated. By means of tizanidine (an alpha 2-adrenergic receptor agonist) we tested the hypothesis that the MLR is relayed by group II afferent fibres, since animal data indicate that tizanidine or stimulation of monoaminergic brainstem centres decrease the excitability of spinal interneurones supplied by those fibres. In addition, we compared the effect of the drug on these responses with that induced by stabilization of posture. 2. Eight subjects received tizanidine (150 micrograms kg-1 orally) or placebo, in a single-blind design. Platform rotations were delivered prior to administration and for 3 h afterwards. Both TA- and FDB-MLRs decreased in size, starting from about 1 h after tizanidine administration. Sol-SLR was unaffected. Response latencies were unchanged. Placebo induced no changes in any response. In each subject, the extent of TA-MLR depression induced by holding onto a frame and by tizanidine was superimposable. 3. The selective effect of tizanidine on MLR supports the notion that it is relayed through group II afferent fibres. The similar effects of holding and tizanidine on the response suggests that it is modulated by monoaminergic centres.

Adrenergic alpha-Agonists↗

PubMed2005

High-throughput automated platform for nuclear magnetic resonance-based structural proteomics.

The development of new systems and strategies capable of synthesizing any desired soluble, labeled protein or protein fragment on a preparative scale is one of the most important tasks in biotechnology today. The Center for Eukaryotic Structural Genomics (WI, USA), in co-operation with Ehime University (Matsuyama, Japan) and CellFree Sciences Co., Ltd, has developed an automated platform for nuclear magnetic resonance-based structural proteomics that employs wheat germ extracts for cell-free production of labeled protein. The platform utilizes a single construct for all targets without any redesign of the DNA or RNA. Therefore, it offers advantages over commercial cell-free methods utilizing Escherichia coli extracts that require multiple constructs or redesign of the open reading frame. The protein production and labeling protocol is no more costly than E. coli cell-based approaches, is robust and scalable for high-throughput applications. This protocol has been used in the authors center to screen eukaryotic open reading frames from the Arabidopsis thaliana and human genomes and for the determination of nuclear magnetic resonance structures. With the recent addition of the GeneDecoder 1000 (CellFree Sciences Co., Ltd) robotic system, the Center for Eukaryotic Structural Genomics is able to carry out as many as 384 small-scale (50 microl) screening reactions per week. Furthermore, the Protemist (CellFree Sciences Co., Ltd) robotic system enables the Center for Eukaryotic Structural Genomics to carry out 16 production-scale (4 ml) reactions per week. Utilization of this automated platform technology to screen targets for expression and solubility and to produce stable isotope-labeled samples for nuclear magnetic resonance structure determinations is discussed.

Automation↗

PubMed2005

A single nucleotide polymorphism based approach for the identification and characterization of gene expression modulation using MassARRAY.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. Their abundance and the ease with which they can be assayed have lead to their use in applications beyond simple genotyping. One such application is the quantitative determination of transcript levels associated with distinct alleles or haplotypes found in promoters and coding regions of genes. These changes in expression due to allelic variation are often associated with additional genomic or transcript modifications such as DNA methylation or RNA editing. Here, we describe the use of an integrated genetic analysis platform, based on matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to first, discover coding SNPs (cSNPs); second, use these cSNPs to identify and analyze allele-specific expression; and third, from this knowledge to further analyze methylation patterns as a putative cause for the allele-specific expression. An established model involving allele-specific expression profiles of the human tumor protein 73 (TP73) gene is presented as an example to outline and validate data obtained from the MassARRAY platform. The availability of a single integrated platform to assay stable and dynamic variation at the genomic and transcript level greatly simplifies complex functional genomic studies.

5' Untranslated Regions↗

PubMed2002

Optical imaging fiber-based single live cell arrays: a high-density cell assay platform.

A high-density, ordered array containing thousands of microwells is fabricated on an optical imaging fiber. Each individually addressable microwell is used to accommodate a single living cell. A charged coupled device (CCD) detector is employed to monitor and spatially resolve the fluorescence signals obtained from each individual cell, allowing simultaneous monitoring of cellular responses of all the cells in the array using reporter genes (lacZ, EGFP, ECFP, DsRed) or fluorescent indicators. Yeast and bacteria cell arrays were fabricated and used to perform multiplexed cell assays with resolution at the single-cell level. Monitoring gene expression in single yeast cells carrying a two-hybrid system was used to detect in vivo protein-protein interactions. The single-cell array technology provides a new platform for monitoring the unique multiple responses of large populations of individual cells from different strains or cell lines. The rich data acquired by the cell array has the potential to be employed as a new tool for cell biology research as well as to improve cell-based high-throughput screening (HTS) applications, such as the validation of new disease-associated cellular targets and the early-stage evaluation of potential drug candidates.

Escherichia coli↗

PubMed2005

Evaluation of a new single-parameter volumetric flow cytometer (CyFlow(green)) for enumeration of absolute CD4+ T lymphocytes in human immunodeficiency virus type 1-infected Thai patients.

Use of the standard dual-platform flow cytometric method for determination of CD4(+) T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4(+) T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlow(green) (Partec), a single-parameter SP volumetric FCM. The performance of CyFlow(green) was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4(+) and CD8(+) T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlow(green) were compared with those obtained with the bead-based three-color TruCOUNT system (R(2)=0.96; mean bias, -69.1 cells/microl; 95% confidence interval [CI], -225.7 to+87.5 cells/microl) and the FACSCount system (R(2)=0.97; mean bias, -40.0 cells/microl; 95% CI, -165.1 to+85.1 cells/microl). The correlation of the CD4(+) T-lymphocyte counts obtained by the two bead-based systems was high (R(2)=0.98). Interestingly, CyFlow(green) yielded CD4(+) T-lymphocyte counts that were 21.8 and 7.2 cells/microl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4(+) T-lymphocyte counts were <250 CD4(+) T-lymphocyte counts/microl range or 17.3 and 5.8 cells/microl less, respectively, when CD4(+) T-lymphocyte counts were <200 cells/microl. The single-parameter CyFlow(green) volumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings.

Adult↗

PubMed2025

BOGO: A Proteome-Wide Gene Overexpression Platform for Discovering Rational Cancer Combination Therapies.

Cancer drug resistance remains a major barrier to durable treatment success, often leading to relapse despite advances in precision oncology. While combination therapies are being increasingly investigated, such as chemotherapy with small molecule inhibitors, predicting drug response and identifying rational drug combinations based on resistance mechanisms remain major challenges. Therefore, a proteome-wide, single-gene overexpression screening platform is essential for guiding rational therapy selection. Here, we present BOGO (Bxb1-landing pad human ORFeome-integrated system for a proteome-wide Gene Overexpression), a robust, scalable, and reproducible screening platform that enables single-copy, site-specific integration and overexpression of ~19,000 human open across cancer cell models. Using BOGO, we identified drug-specific response drivers for 16 chemotherapeutic agents and integrated clinical datasets to uncover proliferation and resistance-associated genes with prognostic potential. Drug response similarity networks revealed both shared and unique mechanisms, highlighting key pathways such as autophagy, apoptosis, and Wnt signaling, and notable resistance-associated genes including BCL2, POLD2, and TRADD. In particular, we proposed a synergistic combination of the BCL2 family inhibitor ABT-263 (Navitoclax®) and the DNA analog TAS-102 (Lonsurf®), which revealed that lysosomal modulation is a key mechanism driving DNA analog resistance. This combination therapy selectively enhanced cytotoxicity in colorectal and pancreatic cancer cells in vitro, and demonstrated therapeutic benefit in vivo in both cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Together, these findings establish BOGO as a powerful gene overexpression perturbation platform for systematically identifying chemoresistance and chemosensitization drivers, and for discovering rational combination therapies. Its scalability and reproducibility position BOGO as a broadly applicable tool for functional genomics and therapeutic discovery beyond cancer resistance.

Journal Article↗

PubMed2026

Exploratory single-nucleus multiomics analysis of myeloid cell states associated with neoadjuvant chemotherapy response in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most lethal human malignancies, with the vast majority of patients ineligible for immunotherapy. Tumour-associated macrophages (TAMs) are key regulators of the PDAC tumour microenvironment (TME), yet their transcriptional and epigenomic heterogeneity in the context of chemotherapy response is poorly understood. Therefore, we performed an exploratory single nucleus multiomics analysis of PDAC tumors stratified by histopathologic response to neoadjuvant chemotherapy. METHODS: Surgical resection specimens from PDAC patients were classified as responders or non-responders using the American College of Pathologists (CAP) histopathologic criteria. Frozen tissue underwent simultaneous snRNA-seq and snATAC-seq on the 10x Genomics Chromium Single Cell Multiome platform, followed by downstream analyses such as differential gene expression, GO and hallmark pathway enrichment, pseudotime trajectory inference and ChromVAR transcription factor motif analysis. RESULTS: Multiomics profiling of 30 840 high-quality nuclei revealed a myeloid compartment that differed in composition and transcriptional state between CAP-defined responders and non-responders in this small cohort. We observed a trend toward higher LAM-like state proportions in the responders than non-responders (38.4% vs. 26.7%), although this disparity did not achieve statistical significance. The transcriptional programs of the responder myeloid cells are associated with phagocytosis and lipid handling. Chromatin accessibility analysis further suggested candidate response-associated transcription factor motif accessibility patterns. CONCLUSIONS: Neoadjuvant-treated PDAC tumours from CAP-defined responders in this cohort myeloid landscape with apparent enrichment of LAM-like states and immune-activating transcriptional/epigenetic programs. However, these findings are preliminary and hypothesis-generating because of the small cohort size, heterogeneous treatment regimens, absence of matched pre-treatment biopsies, and lack of knockout validation. Larger treatment cohorts and functional/mechanistic studies are needed to determine whether LAM-like myeloid programs contribute to chemotherapy response or reflect a consequence of chemotherapy treatment.

Humans↗

PubMed2025

Tracking Somatic Mutations for Lineage Reconstruction.

The human genome is composed of distinct genomic regions that are susceptible to various types of somatic mutations. Among these, Short Tandem Repeats (STRs) stand out as the most mutable genetic elements. STRs are short repetitive polymorphic sequences, predominantly situated within noncoding sectors of the genome. The intrinsic repetition characterizing these sequences makes them highly mutable in vivo. Consequently, this characteristic provides the chance to unravel the natural developmental history of human viable cells retrospectively. However, STRs also introduce stutter noise in vitro amplification, which makes their analysis challenging. Here we describe our integrated biochemical-computational platform for single-cell lineage analysis. It consists of a pipeline whose inputs are single cells and whose output is a lineage tree of input cells.

Humans↗

PubMed1978

Visuomotor restriction of one eye in kittens reared with alternate monocular deprivation.

It is known that kittens reared in ways that restrict movement while visual stimulation is received exhibit deficits in visually guided behavior. Presumably, the behavioral dysfunction is due to a lack of sensorimotor coordination during visual exposure. The current investigation was undertaken to study this effect both physiologically and behaviorally. Two groups of kittens were normally reared until they were nearly 4 weeks old. They were then placed in a darkroom and exposed daily for 1--2 hours while one eye was occluded. On alternate days, alternate eyes were covered. One group was restrained in a body cast while a given eye was exposed, but the kittens were free to move about while the other eye received visual experience. A second control group was alternately occluded, but freely mobile during all exposures. An additional cat was also unrestrained during daily rearing periods and neither eye was ever occluded. Behavioral tests showed clear deficits when the "passive" eye of the restrained-unrestrained group was used. Deficiencies were found in visually guided paw placing, pursuit eye movements, and jumping behavior to a platform. Responses of single cells were studied in area 17 of the visual cortex. Ocular dominance distributions showed marked reductions in binocularity for alternated occluder cats. The eye that had received exposure while animals were active in the restrained-unrestrained group also tended to predominate but the difference was not statistically significant.

Animals↗

PubMed2004

Anxiogenic effect of sleep deprivation in the elevated plus-maze test in mice.

RATIONALE: Several clinical studies demonstrate that the absence of periods of sleep is closely related to occurrence of anxiety symptoms. However, the basis of these interactions is poorly understood. Studies performed with animal models of sleep deprivation and anxiety would be helpful in the understanding of the mechanisms underlying this relationship, but some animal studies have not corroborated clinical data, reporting anxiolytic effects of sleep deprivation. OBJECTIVES: The aim of the present study was to verify the effects of different protocols of sleep deprivation in mice tested in the elevated plus-maze and to assess the effect of chlordiazepoxide and clonidine. METHODS: Three-month-old male mice were sleep-deprived for 24 or 72 h using the methods of single or multiple platforms in water tanks. Mice kept in their home cages were used as controls. Plus-maze behavior was observed immediately after the deprivation period. RESULTS: Mice that were sleep-deprived for 72 h spent a lower percent time in the open arms of the apparatus than control animals. This sleep deprivation-induced anxiety-like behavior was unaffected by treatment with chlordiazepoxide (5.0 and 7.5 mg/kg IP), but reversed by an administration of 5 or 10 microg/kg IP clonidine. CONCLUSION: The results indicate that under specific methodological conditions sleep deprivation causes an increase in anxiety-like behavior in mice exposed to the elevated plus-maze.

Animals↗

PubMed2026

CRISPR screens for the discovery of novel ferroptosis targets: progress and perspectives.

Ferroptosis is a distinct, iron-dependent form of regulated cell death characterized by lipid peroxidation. Despite its growing significance in physiology and disease, the molecular networks that govern ferroptosis are not yet fully understood. Genome-wide CRISPR screens have broadened the regulatory landscape of ferroptosis by revealing both conserved and context-dependent mechanisms. In this review, we summarize recent advances in CRISPR-based ferroptosis screens, highlighting a transition from in vitro CRISPR screens to in vivo platforms and single-cell CRISPR screens. We also discuss the potential translation of key targets, focusing on their structural druggability and therapeutic potential. By outlining objective-driven screening strategies, this review seeks to provide options for exploring the distinct mechanisms of ferroptosis and to accelerate its translation into therapeutic opportunities for various diseases.

CRISPR screens↗

PubMed2005

Does head extension and flexion increase postural instability in elderly subjects when visual information is kept constant?

The present study determined the effects of flexing and extending the head on the postural stability and mean anterior-posterior (A-P) center of mass (CM) position during upright stance in the elderly. To ensure visual input to stability was not a confounding variable, visual information was kept as constant as possible for all head positions. Twelve healthy elderly subjects (72.3 +/- 4.7 years) were asked to stand stationary on a single force-platform. Postural stability (assessed using the rms A-P excursion of the center of pressure (CP)) was determined for standing with the head erect, and with the head flexed and extended. The vestibular contribution to postural stability becomes increasingly important under challenging conditions, so to highlight the effects of vestibular system input, measurements of postural stability under conditions where visual and somatosensory inputs were disrupted were included. Changes in the mean A-P CM position when tilting the head were assessed by determining changes in the mean A-P location of the CP from standing with the head erect. Compared to standing with the head erect and looking straight ahead, postural stability was reduced when the head was flexed or extended (P < 0.01). Changes in mean A-P CM position were only significant when standing with the head flexed (P < 0.05). This suggests that increases in postural instability with the head tilted from the erect position may be in part due to mechanical perturbation rather than solely vestibular disruption.

Aged↗

PubMed2001

Variability in vocal and behavioural responses to visual isolation between full-sibling families of beef calves.

In order to assess the value of vocal response as an indicator of welfare in cattle, it is necessary to investigate factors which influence vocal behaviour of individuals, that are independent of the specific environmental conditions to which they are exposed. The effect of parental differences, sex, age and weight on vocal responses of calves to visual isolation were examined. The relationship between vocal response and the amount of body movement during isolation were also evaluated.Seventeen full-sibling families of beef calves (N=130) were created by breeding five sires with 13 superovulated dams. Embryos were transferred into unrelated cows, which reared the calves to weaning age. Vocal response of calves and body movement were measured on four occasions, while they were visually isolated for 1min on a single animal scale platform. The first two observations were made on the day of weaning (mean age 166 days) and the following day. At the third and fourth observations their average ages were 278 and 350 days, respectively. On the four occasions 33.3, 34.8, 27.8 and 38.6% of calves, respectively, vocalized. Both sire and family had a significant influence on the number of vocalizations and acoustic properties of the sounds. Electronically-measured movement while on the scale was also influenced by sire and family, but there was no relationship between vocal responses and body movement. There was a tendency (P=0.08) for older and therefore heavier calves to produce longer vocalizations. In the final observing session, age and weight were positively correlated with fundamental frequency and the sound pressure level at the frequency of greatest intensity, and negatively correlated with the loudest harmonic. At the last two observations, significantly more heifer calves than bull calves vocalized. There was little difference between the acoustic properties of heifers' and bulls' vocalizations.The considerable individual variability in vocal response within a group of identically-treated calves is partly due to parental genetic influences that are independent of rearing and early experience. Other endogenous characteristics such as sex, age and weight also influence vocal response. But there is no clear relationship between individual temperament (as assessed by the amount of body movement during isolation) and characteristics of vocal response.

Journal Article↗

PubMed1999

An integrated microfabricated device for dual microdialysis and on-line ESI-ion trap mass spectrometry for analysis of complex biological samples.

A microfabricated dual-microdialysis device in a single integrated microfabricated platform was constructed using laser micromachining techniques for the rapid fractionation and cleanup of complex biological samples. On-line dual microdialysis and ESI-MS of biological samples was demonstrated using an ion trap mass spectrometer. The mass spectra obtained demonstrated the efficiency of dual microdialysis for removing both high-molecular-weight and low-molecular-weight species that interfere with effective ESI-MS analysis of target biopolymers. Signal-to-noise ratios were also greatly improved compared to direct sample infusion. In addition to its compactness, negligible dead volume, and robustness, the device can be used at a flow rate of only 200 nL/min, an order of magnitude lower than that obtained previously. This reduced sample consumption and improved sensitivity with ESI-MS. The results suggest the potential for integration of such microfabricated devices with other sample manipulations for the rapid ESI-MS analysis of complex biological samples.

Escherichia coli↗

PubMed2006

Inorganic nanotubes: a novel platform for nanofluidics.

Templating approaches are being developed for the synthesis of inorganic nanotubes, a novel platform for nanofluidics. Single crystalline semiconductor GaN nanotubes have been synthesized using an epitaxial casting method. The partial thermal oxidation of silicon nanowires leads to the synthesis of silica nanotubes. The dimension of these nanotubes can be precisely controlled during the templating process. These inorganic nanotubes can be integrated into metal-oxide solution field effect transistors (MOSolFETs), which exhibit rapid field effect modulation of ionic conductance. These nanofluidic devices have been further demonstrated to be useful for single-molecule sensing, as single DNA molecules can be readily detected either by charge effect or by geometry effect. These inorganic nanotubes will have great implications in subfemtoliter analytical technology and large-scale nanofluidic integration.

Chemistry, Inorganic↗