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PubMed2025

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals↗

PubMed2004

Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.

Adenoviridae↗

PubMed2003

ExpressYourself: A modular platform for processing and visualizing microarray data.

DNA microarrays are widely used in biological research; by analyzing differential hybridization on a single microarray slide, one can detect changes in mRNA expression levels, increases in DNA copy numbers and the location of transcription factor binding sites on a genomic scale. Having performed the experiments, the major challenge is to process large, noisy datasets in order to identify the specific array elements that are significantly differentially hybridized. This normally requires aggregating different, often incompatible programs into a multi-step pipeline. Here we present ExpressYourself, a fully integrated platform for processing microarray data. In completely automated fashion, it will correct the background array signal, normalize the Cy5 and Cy3 signals, score levels of differential hybridization, combine the results of replicate experiments, filter problematic regions of the array and assess the quality of individual and replicate experiments. ExpressYourself is designed with a highly modular architecture so various types of microarray analysis algorithms can readily be incorporated as they are developed; for example, the system currently implements several normalization methods, including those that simultaneously consider signal intensity and slide location. The processed data are presented using a web-based graphical interface to facilitate comparison with the original images of the array slides. In particular, Express Yourself is able to regenerate images of the original microarray after applying various steps of processing, which greatly facilities identification of position-specific artifacts. The program is freely available for use at http://bioinfo.mbb.yale.edu/expressyourself.

Algorithms↗

PubMed2005

Xyloglucan is recognized by carbohydrate-binding modules that interact with beta-glucan chains.

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of beta-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a beta-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated beta-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.

Amino Acid Motifs↗

PubMed2002

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays.

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.

Algorithms↗

PubMed2006

Single-leg hop testing following fatiguing exercise: reliability and biomechanical analysis.

A fatiguing exercise protocol was combined with single-leg hop testing to improve the possibilities of evaluating the effects of training or rehabilitation interventions. In the first test-retest experiment, 11 healthy male subjects performed two trials of single-leg hops under three different test conditions: non-fatigued and following fatiguing exercise, which consisted of unilateral weight machine knee extensions at 80% and 50%, respectively, of 1 repetition maximum (1 RM) strength. Intraclass correlation coefficients ranged from 0.75 to 0.98 for different hop test conditions, indicating that all tests were reliable. For the second experiment, eight healthy male subjects performed the fatiguing exercise protocol to investigate how fatigue influences lower-extremity joint kinematics and kinetics during single-leg hops. Hip, knee and ankle joint angles, moments and powers, as well as ground-reaction forces were recorded with a six-camera, motion-capture system and a force platform. Recovery of hop performance following the fatiguing exercise was also measured. During the take-off for the single-leg hops, hip and knee flexion angles, generated powers for the knee and ankle joints, and ground-reaction forces decreased for the fatigued hop conditions compared with the non-fatigued condition (P<0.05). Compared with landing during the non-fatigued condition, hip moments and ground-reaction forces were lower for the fatigued hop conditions (P<0.05). The negative joint power was two to three times greater for the knee than for the hip and five to 10 times greater for the knee than for the ankle during landing for all test conditions (P<0.05). Most measured variables had recovered three minutes post-exercise. It is concluded that the fatiguing exercise protocol combined with single-leg hop testing was a reliable method for investigating functional performance under fatigued test conditions. Further, subjects utilized an adapted hop strategy, which employed less hip and knee flexion and generated powers for the knee and ankle joints during take-off, and less hip joint moments during landing under fatigued conditions. The large negative power values observed at the knee joint during the landing phase of the single-leg hop, during which the quadriceps muscle activates eccentrically, indicate that not only hop distance but also the ability to perform successful landings should be investigated when assessing dynamic knee function.

Adult↗

PubMed2026

Full-length single-cell spatial transcriptomics reveals spatial and cell-type-specific transcript isoforms in the primate brain.

The primate brain exhibits complex RNA alternative splicing heterogeneity crucial for functional complexity, yet systematic spatial isoform characterization has been lacking. We developed Fullscope-seq, a full-length single-molecule large field-of-view spatial transcriptomics sequencing method at single-cell resolution, based on programmed concatenation cDNA for multiple long-read sequencing platforms. Applying Fullscope-seq to the macaque brain, we uncovered thousands of genes exhibiting differential transcript usage (DTU) across cortical layers, cell types and brain regions. Fullscope-seq resolved hundreds of major isoform switches across distinct brain regions and identified DTUs between superficial and deep cortical layers. Cortical layer-specific DTUs showed cell-composition dependence, whereas regional DTUs were regulated according to both cellular composition and spatial contexts. These isoform variations showed substantial enrichment for neuropsychiatric disorder-associated genes and were conserved across platforms and species. Our study establishes a scalable framework for spatial isoform analysis and provides a resource for understanding transcriptomic diversity in complex tissues.

Animals↗

PubMed1999

Lack of protection of monoamine oxidase B-deficient mice from age-related spatial learning deficits in the Morris water maze.

Monoamine oxidase B (MAO-B) increases in brain in response to aging and neurodegeneration. Whether such increases represent a risk factor to further neuronal damage or simply represent epiphenomena remains unclear. L-deprenyl, an inhibitor of MAO-B, has been shown to improve learning in aged rodents. However, recent data suggests this may occur through mechanisms independent of its enzymatic inhibition. This study investigates visualspatial learning of MAO-B deficient mice and examines what effects absence of MAO-B has on age-related cognitive decline. Learning was tested in the Morris Water Maze in male transgenic MAO-B knockout mice (KO) ages 2 months (n = 9), 7 months (n = 7), and 17 months (n = 8). Performance was compared to that of wild type (WT) littermates. Animals were given four 60 second trials per day with the submerged platform in the "North" position. Animals received 7 days of learning in which they were introduced into the pool facing the wall, alternating between the "East" and "West" positions. A single probe trial followed on day 8, followed by continuation of the original learning paradigm on days 9 and 10. Subsequently, the platform position was changed to the diagonally opposite quadrant and learning continued on days 11-13, followed by a cue phase in which the platform was made visible. Total distance traveled and latency to the platform was increased in 7- and 17- month old mice, most significantly at the beginning of the acquisition phase. This effect reappeared again in 17- month old mice during the reversal phase. No predominant genotypic differences in latency or distance were observed during any phase of the experiment. Our results show that presence or absence of MAO-B does not appear to alter performance in the Morris water maze. Furthermore, presence or absence of MAO-B does not provide protection from the age-dependent deficits in spatial learning.

Aging↗

PubMed2006

Long-term cognitive and neuropsychological symptoms after global cerebral ischemia in Mongolian gerbils.

The objective of this study was to establish a rodent model of vascular dementia that showed long-term cognitive and neuropsychological deficits, and to correlate those behavioral deficits with the patterns of ischemic lesions, thus providing a platform for future testing of potential therapeutic agents. In Mongolian gerbils, either 5-minute single bilateral common carotid artery occlusion (SBCCAO) or repetitive bilateral common carotid artery occlusion (two 7-minute occlusions, RBCCAO) was induced, and the behavioral deficits were evaluated using 2 tests: a modified open-field test with an escape zone to evaluate changes in anxiety and locomotor activity, and a T-maze test to assess cognitive dysfunction. SBCCAO did not induce anxiety changes but caused transient locomotor hyperactivity and mild cognitive deficits. Only pyramidal neuronal death was found in the bilateral CA1 sector of the hippocampus following SBCCAO. In contrast, RBCCAO induced persistent locomotor hyperactivity, reduced anxiety, and caused severe cognitive deficits at 4 weeks post-ischemia. RBCCAO caused significant atrophy associated with diffuse selective neuronal death in the bilateral cerebral cortex and caudate nucleus, as well as the CA1 region. The repetitive ischemia model appears to be a potentially useful platform for the long-term analysis of cognitive and neuropsychological symptoms associated with vascular dementia.

Animals↗

PubMed2000

Thresholds for step initiation induced by support-surface translation: a dynamic center-of-mass model provides much better prediction than a static model.

The need to initiate a step in order to recover balance could, in theory, be predicted by a static model based solely on displacement of the center of mass (COM) with respect to the base of support (BOS), or by a dynamic model based on the interaction between COM displacement and velocity. The purpose of this study was to determine whether the dynamic model provides better prediction than the static model regarding the need to step in response to moving-platform perturbation. The COM phase plane trajectories were determined for 10 healthy young adults for trials where the supporting platform was translated at three different acceleration levels in anterior and posterior directions. These trajectories were compared with the thresholds for step initiation predicted by the static and dynamic COM models. A single-link-plus-foot biomechanical model was employed to mathematically simulate termination of the COM movement, without stepping, using the measured platform acceleration as the input. An optimization routine was used to determine the stability boundaries in COM state space so as to establish the dynamic thresholds where a compensatory step must be initiated in order to recover balance. In the static model, the threshold for step initiation was reached if the COM was displaced beyond the BOS limits. The dynamic model showed substantially better accuracy than the static model in predicting the need to step in order to recover balance: 71% of all stepping responses predicted correctly by the dynamic model versus only 11% by the static model. These results support the proposition that the central nervous system must react to and control dynamic effects, i.e. COM velocity, as well as COM displacement in order to maintain stability with respect to the existing BOS without stepping.

Adult↗

PubMed2004

Enhanced evoked responses after early adversity and repeated platform exposure: the neurobiology of vulnerability?

BACKGROUND: There is a long-standing clinical awareness of the significance of adverse early experiences and subsequent stress in the evolution of psychiatric disorder. METHODS: We investigated the impact of a single episode of preweaning maternal separation on in vivo electrophysiologic responses in the hippocampus of the mature rat after repeated exposure to an open elevated platform. RESULTS: Only rats that had experienced both maternal separation followed by stressful platform exposure when mature had significantly increased granule cell response to perforant path stimulation, compared with control rats. Rats exposed to either maternal separation or the elevated platform in adulthood alone did not differ significantly from control rats. CONCLUSIONS: Adverse early experience seems to induce functional changes in the hippocampus that remain latent until activated by stress in adulthood. Such electrophysiologic changes might represent a neural substrate for vulnerability to stress-associated psychopathology.

Animals↗

PubMed2005

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells.

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.

Adsorption↗

PubMed1999

Rats show unimpaired learning within minutes after recovery from single bolus propofol anesthesia.

PURPOSE: To examine the learning ability of rats shortly after recovery from a bolus dose of propofol by assessing learning on a swim-to-platform task. Also, muscarinic blockade was used as a pharmacological test of whether learning shortly after propofol anesthesia resembles normal learning. METHODS: Propofol anesthetized rats (15-20 mg x kg(-1) i.v.) were trained on a swim-to-platform task five to seven minutes after recovering from surgical anesthesia and tested two to three hours later In addition, the muscarinic antagonist scopolamine hydrobromide (5 mg x kg(-1) s.c.) was given to a subgroup of rats before testing. During 10 trials, the number of times a given rat took 10 sec or longer to locate and climb onto a visible platform was tabulated and counted as errors. RESULTS: When trained shortly after recovery from the anesthetic, propofol anesthetized rats made 3.2 +/- 0.4 compared with 1.0 +/- 0.1 errors in controls (P < 0.0001). Two to three hours later both groups performed equally well. Rats trained after propofol anesthesia and given scopolamine before testing made 0.7 +/- 0.5 errors and performed as well as normal controls, 1.2 +/- 0.2 errors when subjected to the same procedures without propofol anesthesia, and better than scopolamine-treated untrained rats, 5.5 +/- 0.7 errors, (P < 0.05). CONCLUSION: Training five to seven minutes after recovery from propofol anesthesia resulted in normal retention of the swim- to-platform task. It also produced the same resistance to the disruptive effects of scopolamine as did training in rats that were not anesthetized. Thus, the ability to learn recovers rapidly after propofol anesthesia induced by a single intravenous bolus dose.

Anesthesia Recovery Period↗

PubMed2025

RT-RPA Assisted CRISPR/Cas12a Based One-Pot Rapid and Visual Detection of the Pan-Dengue Virus.

Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction- 1, ≥ 1.81 ag reaction-1, ≥ 62.5 fg reaction-1, and ≥ 2.5 pg reaction-1 for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction-1 for DENV-1 and DENV-4, and ≥ 0.5 ng reaction-1 for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.

Dengue Virus↗

PubMed1989

Real-time patient weight monitoring system for chair based renal dialysis.

A system is described for monitoring weight changes in patients undergoing renal dialysis. Relatively fit patients dialyse in easy chairs rather than in bed but no commercial system is currently available to monitor weight changes under these conditions. The authors have considered basic requirements and have developed a cost effective system based on a Single Point Off-Centre load cell, all components being mounted under a platform on which the easy chair is located. There is an electrical output signal which may be used to drive a display or be sampled by a computer system.

Body Weight↗

PubMed1999

Simulated movement termination for balance recovery: can movement strategies be sought to maintain stability in the presence of slipping or forced sliding?

Slipping during various kinds of movement often leads to potentially dangerous incidents of falling. The purpose of this study was to determine whether there was evidence to support the theory that movement strategies could be used by individuals to regain stability during an episode of slipping and whether forced sliding from a moving platform accurately simulated the effect of slipping on stability and balance. A single-link-plus-foot biomechanical model was used to mathematically simulate base of support (BOS) translation and body segment rotation during movement termination in sagittal plane. An optimization routine was used to determine region of stability [defined at given COM locations as the feasible range of horizontal velocities of the center of mass (COM) of human subject that can be reduced to zero with respect to the BOS while still allowing the COM to traverse within the BOS limits]. We found some 30% overlap in the region of stability for slipping and non-slipping conditions. This finding supports the theory that movement strategies can be sought for restoring stability and balance even if slipping unexpectedly occurs. We also found that forced sliding produces effects on stability that are similar to those of slipping, indicated by over 50% overlap in the regions of stability for the two conditions. In addition, forced sliding has distinctive effects on stability, including a "shift" of the region of stability extended beyond the BOS in the direction of sliding. These findings may provide quantifiable guidance for balance training aimed at reducing fall incidents under uncertain floor surface conditions.

Accidental Falls↗

PubMed2001

Trends in biomarker research for cancer detection.

A key challenge in cancer control and prevention is detection of the disease as early as possible, enabling effective interventions and therapies to contribute to reduction in mortality and morbidity. Biomarkers are important as molecular signposts of the physiological state of a cell at a specific time. Active genes, their respective protein products, and other organic chemicals made by the cell create these signposts. As a normal cell progresses through the complex process of transformation to a cancerous state, biomarkers could prove vital for the identification of early cancer and people at risk of developing cancer. We discuss current research into the genetic and molecular signatures of cells, including microsatellite instability, hypermethylation and single-nucleotide polymorphisms. The use of genomic and proteomic high-throughput technology platforms to facilitate detection of early cancer by means of biomarkers, and issues on the analysis, validation, and predictive value of biomarkers based on these technologies are also discussed. We report on recent advances in identifying sources of biomarkers that can be accessed by noninvasive techniques, such as buccal-cell isolates, as well as traditional sources such as serum or plasma. We also focus on the work of the Early Detection Research Network at the National Cancer Institute, harnessing expertise from leading national and international institutions, to identify and validate biomarkers for the detection of precancerous and cancerous cells in assessing risk of cancer. The network also has a role in linking discovery to process development, resulting in early detection tests and clinical assessment.

Biomarkers, Tumor↗

PubMed2002

Rapid prototyping for injection moulded integrated microfluidic devices and diffractive element arrays.

This paper describes two fabrication procedures that makes it possible to design, fabricate and injection mold a microfluidic system with an on board coupling element or an optical array platform in less than four hours. Epoxy masters for the array and a single diffractive element were produced using conventional soft lithography techniques and a commercially available UV curable epoxy. The fabrication of the master for the integrated microfluidic device utilized the surface chemistry of polyester and its interaction with the anionic surfactant sodium dodecyl sulfate (SDS), to selectively inhibit the adhesion between the epoxy and the polyester film during the curing reaction. The transfer of a microfluidic design and the required coupling element (632 nm holographic grating) along the base of the channel was completed in a single step. The turnaround time from design to injection molded device whether a microchannel or array was 3.5 h.

Journal Article↗