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PubMed2001

Association among neuromuscular and anatomic measures for patients with knee osteoarthritis.

OBJECTIVE: To investigate neuromuscular and anatomic factors involved in varus gonarthrosis by identifying measures associated with degenerative changes. DESIGN: Descriptive study. Individual measures that explained substantial portions of the variability in ratings of knee joint-degenerative changes in patients with knee medial compartment osteoarthritis. SETTING: Outpatient orthopedic clinic and biomechanics and muscular assessment laboratory. PATIENTS: Volunteer sample of 20 subjects (age range, 59 +/- 9 yr) with no history of neurologic disease. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: We assessed degenerative changes, varus alignment, standing balance, and knee proprioception. Weight-bearing radiographs were used to assess the extent of degenerative changes and the degree of varus alignment. Single-limb standing balance control was assessed through tests performed on a force platform. Knee proprioception was assessed with an isokinetic dynamometer, using a joint angle replication test. RESULTS: Forward-stepwise multiple linear regression indicated that the extent of degenerative changes could be best predicted from a linear combination of the independent variables, varus alignment, and standing balance (R =.80, F(2,17) = 14.81, p =.0002). Sixty-four percent of the variability in ratings of degenerative changes was explained by alignment and standing balance measures (37% by varus alignment, 27% by standing balance). Alignment and balance measures were poorly correlated (r =.12, p =.63), further suggesting that they provided different information about gonarthrosis. CONCLUSIONS: Although varus alignment is widely accepted as a clinically important factor in gonarthrosis, and is the focus of many treatment efforts, our results suggest that objective measures of standing balance are also important. As a result, the potential impact of rehabilitation to improve the control of standing balance should be further evaluated in this patient population.

Biomechanical Phenomena↗

PubMed2004

Familial hypercholesterolemia in Spain: case-finding program, clinical and genetic aspects.

A case-finding program for the identification of patients with familial hypercholesterolemia (FH) has been established in Spain. The program is based on family investigation and molecular genetic testing for mutations in the low-density lipoprotein receptor gene. To assist this program, intensive research into the molecular basis of FH and genotype/phenotype relations is performed. To optimize DNA testing, a DNA-diagnostic platform has been constructed that is composed of systematic mutation screening by single-strand conformation polymorphism (SSCP) analysis, DNA-sequencing, Southern blotting, and the use of microarrays for high-throughput analysis. To date, 161 different mutations leading to inherited hypercholesterolemia have been identified in Spanish patients with FH. In addition, a patient organization was founded to ensure patient support and follow-up. To further facilitate FH case-finding and patient follow-up, we initiated the publication of a set of guidelines for diagnosis and clinical management of FH that can be applied internationally.

DNA Mutational Analysis↗

PubMedDate not supplied

Open magnetic and electric graphic analysis.

The OMEGA software provides an analysis platform for user-independent, fast, and reproducible multimodal data analysis in one single software environment. Synergetic interactions pursued between the two functional imaging techniques fMRI and MEG use the morphological MRI recording as a basis for a common coordinate frame. In this way, direct interchange, comparison, and integration among the results of the different modalities have become feasible. The fMRI data analysis provides information about the localization of functional activity with low temporal resolution, whereas the MEG recording complements the corresponding time evolution with a high temporal resolution. The implementation of OMEGA allows the analyst to receive comprehensive MEG/fMRI results in a matter of minutes after the measurements have been completed. With OMEGA, the clinical researcher gets comprehensive information in a quick and standardized approach about the sites and the time course of neurological activation, which is useful for clinical applications and diagnostics.

Algorithms↗

PubMed2025

Novel antibodies for identification, selection, and manipulation of T cells expressing Whitlow linker-containing CARs.

BACKGROUND: The translational study of chimeric antigen receptor (CAR) T-cell function, persistence, immunophenotype, and spatial localization after infusion is crucial for understanding factors that influence clinical outcomes. However, research has been limited by a lack of optimized tools to reliably detect CAR-engineered cells. To address this, we developed a novel platform to generate monoclonal antibodies (mAbs) targeting a linker peptide incorporated in single-chain variable fragments (scFvs) of most CAR constructs. METHODS: Using recombinant proteins and scFv linker peptides as immunogens, we generated murine mAbs against the Whitlow linker peptide, capable of binding cells expressing Whitlow linker-containing CARs in both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. We evaluated these antibodies in multiple in vitro translational applications relevant to CAR T-cell research and manufacturing. RESULTS: We identified five unique mAbs reactive against the Whitlow linker and characterized their binding properties and three-dimensional structural conformation. One clone was evaluated in depth, demonstrating comparable capacity to identify CAR T cells in peripheral blood relative to other methods using anti-idiotype antibodies or recombinant CAR-target proteins. In contrast to these reagents, the anti-Whitlow mAb detects cells expressing Whitlow linker-containing CARs with different antigen specificities, including those harboring the widely employed anti-CD19 FMC63-derived scFv as well as other scFvs, such as those targeting B-cell maturation antigen (BCMA) or CD33. Importantly, the anti-Whitlow mAb identified CAR T cells in situ in archival FFPE tissues, and a DNA-barcoded format enabled their spatial characterization and immunophenotyping in highly multiplexed immunohistochemistry. We also assessed the functional consequences of antibody binding on CAR T cells in vitro and demonstrated the feasibility of anti-Whitlow mAb-mediated selective enrichment of CAR-expressing T cells for potential utility in manufacturing workflows. CONCLUSIONS: Anti-Whitlow mAb clones exhibited distinct structural and functional properties that can be leveraged for multiple applications, providing versatile tools for detection, selection and manipulation of a broad range of clinical and preclinical CAR T-cell products.

Humans↗

PubMed2026

A dual-reporter mouse for therapeutic discovery in Angelman syndrome.

Angelman syndrome is a neurodevelopmental disorder caused by loss of the maternal UBE3A allele, the sole source of UBE3A in mature neurons owing to epigenetic silencing of the paternal allele. Although emerging therapies are being developed to restore UBE3A expression by activating the dormant paternal UBE3A allele, existing mouse models for such preclinical studies have limited throughput and utility, creating bottlenecks for both in vitro therapeutic screening and in vivo characterization. To address this, we developed the Ube3a-INSG dual-reporter knockin mouse, in which an IRES-Nanoluciferase-T2A-Sun1-sfGFP (INSG) cassette was inserted downstream of the endogenous Ube3a stop codon. The INSG model preserves UBE3A protein levels and function while enabling 2 complementary allele-specific readouts: Sun1-sfGFP and Nanoluciferase. We show that Sun1-sfGFP, a nuclear envelope-localized reporter, enables single-cell fluorescence analysis, whole-brain light-sheet imaging, and nuclear quantification by flow cytometry. Further, Nanoluciferase supports high-throughput luminescence assays for sensitive pharmacological profiling in cultured neurons and noninvasive in vivo bioluminescence imaging for pharmacodynamic assessment. By combining scalable screening, cellular analysis, and real-time in vivo monitoring in a single model, the Ube3a-INSG dual-reporter mouse provides a powerful platform to accelerate therapeutic development centered on UBE3A.

Animals↗

PubMed2004

Walking and running in the red-legged running frog, Kassina maculata.

Although most frog species are specialized for jumping or swimming, Kassina maculata (red-legged running frog) primarily uses a third type of locomotion during which the hindlimbs alternate. In the present study, we examined Kassina's distinct locomotory mode to determine whether these frogs walk or run and how their gait may change with speed. We used multiple methods to distinguish between terrestrial gaits: the existence or absence of an aerial phase, duty factor, relative footfall patterns and the mechanics of the animal's center of mass (COM). To measure kinematic and kinetic variables, we recorded digital video as the animals moved over a miniature force platform (N=12 individuals). With respect to footfall patterns, the frogs used a single gait and walked at all speeds examined. Duty factor always exceeded 0.59. Based on COM mechanics, however, the frogs used both walking and running gaits. At slower speeds, the fluctuations in the horizontal kinetic energy (E(k)) and gravitational potential energy (E(p)) of the COM were largely out of phase, indicating a vaulting or walking gait. In most of the trials, Kassina used a combined gait at intermediate speeds, unlike cursorial animals with distinct gait transitions. This combined gait, much like a mammalian gallop, exhibited the mechanics of both vaulting and bouncing gaits. At faster speeds, the E(k) and E(p) of Kassina's COM were more in phase, indicating the use of a bouncing or running gait. Depending on the definition used to distinguish between walking and running, Kassina either only used a walking gait at all speeds or used a walking gait at slower speeds but then switched to a running gait as speed increased.

Animals↗

PubMed2006

Two-dimensional difference gel electrophoresis.

The two-dimensional (2D) polyacrylamide gel-based approach to protein profiling has been successful because it is an accessible, inexpensive, and powerful tool for the analysis of global patterns of protein expression. All protein spots that are resolved and detected within the 104 to 105 dynamic range of gel capacity can be studied qualitatively and quantitatively in relation to each other, and viewed as a single image. Two-dimensional difference gel electrophoresis (DIGE) has strengthened the 2D platform by allowing the detection and quantitation of differences between samples resolved on the same gel, or across multiple gels, when linked by an internal standard. The technology is based on modification of protein extracts with fluorescent cyanine dyes, which have distinct excitation and emission spectra and are migration (charge and size) matched so that the same protein labeled with any of the dyes (Cy2, Cy3, Cy5) will migrate to the same position within a 2D gel, greatly enhancing reproducibility. It is becoming clear that each technology that is currently available for quantifying differential protein expression has its own weaknesses and strengths, and that multiplatform, integrated approaches will be necessary to provide the most complete analysis of any given proteome. We believe that DIGE is, and will remain in the future, a key front-end proteomic tool that will strongly complement other protein-profiling technologies.

Animals↗

PubMed2005

Protein microarrays as a discovery tool for studying protein-protein interactions.

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein-protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein-protein interaction studies.

DNA Replication↗

PubMed2004

Using ANOVA to analyze microarray data.

ANOVA provides a general approach to the analysis of single and multiple factor experiments on both one- and two-color microarray platforms. Mixed model ANOVA is important because in many microarray experiments there are multiple sources of variation that must be taken into consideration when constructing tests for differential expression of a gene. The genome is large, and the signals of expression change can be small, so we must rely on rigorous statistical methods to distinguish signal from noise. We apply statistical tests to ensure that we are not just making up stories based on seeing patterns where there may be none.

Algorithms↗

PubMed1999

A mitogen-activated protein kinase cascade in the CA1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory.

Behavioral, biophysical, and pharmacological studies have implicated the hippocampus in the formation and storage of spatial memory. However, the molecular mechanisms underlying long-term spatial memory are poorly understood. In this study, we show that mitogen-activated protein kinase (MAPK, also called ERK) is activated in the dorsal, but not the ventral, hippocampus of rats after training in a spatial memory task, the Morris water maze. The activation was expressed as enhanced phosphorylation of MAPK in the pyramidal neurons of the CA1/CA2 subfield. In contrast, no increase in the percentage of phospho-MAPK-positive cells was detected in either the CA3 subfield or the dentate gyrus. The enhanced phosphorylation was observed only after multiple training trials but not after a single trial or after multiple trials in which the location of the target platform was randomly changed between each trial. Inhibition of the MAPK/ERK cascade in dorsal hippocampi did not impair acquisition, but blocked the formation of long-term spatial memory. In contrast, intrahippocampal infusion of SB203580, a specific inhibitor of the stress-activated MAPK (p38 MAPK), did not interfere with memory storage. These results demonstrate a MAPK-mediated cellular event in the CA1/CA2 subfields of the dorsal hippocampus that is critical for long-term spatial memory.

Amino Acid Sequence↗

PubMed2001

Candidate genes and single nucleotide polymorphisms (SNPs) in the study of human disease.

The genomic revolution has generated an extraordinary resource, the catalog of variation within the human genome, for investigating biological, evolutionary and medical questions. Together with new, more efficient platforms for high-throughput genotyping, it is possible to begin to dissect genetic contributions to complex trait diseases, specifically examining common variants, such as the single nucleotide polymorphism (SNP). At the same time, these tools will make it possible to identify determinants of disease with the expectation of eventually, tailoring therapies based upon specific profiles. However, a number of methodological, practical and ethical issues must be addressed before the analysis of genetic variation becomes a standard of clinical medicine. The currents of variation in human biology are reviewed here, with a specific emphasis on future challenges and directions.

Base Sequence↗

PubMed2025

Charting Postnatal Heart Development Using In Vivo Single-Cell Functional Genomics.

The transition at birth, marked by increased circulatory demands and rapid growth, necessitates extensive remodeling of the heart's structure, function, and metabolism. This transformation requires precise spatial and temporal coordination among diverse cardiac cell types; central to this process is cardiomyocyte maturation, yet the regulatory mechanisms driving these changes remain poorly understood. Here, we present a temporal and spatial atlas of postnatal hearts by integrating single-nucleus transcriptomics with image-based spatial transcriptomics, which uncovers the dynamic regulatory networks of cardiomyocyte maturation. To functionally interrogate candidate regulators in vivo , we developed Probe-based Indel-detectable Perturb-seq (PIP-seq), a high-throughput platform that uses probe-based chemistry to directly capture sgRNA expression, perturbation status, and transcriptomic profiles at single-nucleus resolution. Applying PIP-seq to postnatal cardiac development identified 21 novel regulators of cardiomyocyte maturation, highlighting critical nodal points in this process. Our study establishes a high-resolution framework for dissecting postnatal heart development, underscoring the integrative and highly ordered roles of microenvironment and intercellular communication in cardiomyocyte maturation. Importantly, PIP-seq enables systematic, high-throughput exploration of gene function and networks underlying complex biological processes in their native in vivo context.

Journal Article↗

PubMed2006

Hematopoietic gene promoters subjected to a group-combinatorial study of DNA samples: identification of a megakaryocytic selective DNA signature.

Identification of common sub-sequences for a group of functionally related DNA sequences can shed light on the role of such elements in cell-specific gene expression. In the megakaryocytic lineage, no one single unique transcription factor was described as linage specific, raising the possibility that a cluster of gene promoter sequences presents a unique signature. Here, the megakaryocytic gene promoter group, which consists of both human and mouse 5' non-coding regions, served as a case study. A methodology for group-combinatorial search has been implemented as a customized software platform. It extracts the longest common sequences for a group of related DNA sequences and allows for single gaps of varying length, as well as double- and multiple-gap sequences. The results point to common DNA sequences in a group of genes that is selectively expressed in megakaryocytes, and which does not appear in a large group of control, random and specific sequences. This suggests a role for a combination of these sequences in cell-specific gene expression in the megakaryocytic lineage. The data also point to an intrinsic cross-species difference in the organization of 5' non-coding sequences within the mammalian genomes. This methodology may be used for the identification of regulatory sequences in other lineages.

Animals↗

PubMed2002

An update on conformation sensitive gel electrophoresis.

Conformation-sensitive gel electrophoresis (CSGE) was developed as a method of heteroduplex analysis to screen large multi-exon genes for sequence variation. The novelty of the method was in the use of a non-proprietary acrylamide gel matrix that used 1,4-bis (acrolyl) piperazine (BAP) as a cross linker with ethylene glycol and formamide as mildly denaturing solvents. The denaturing environment enhances the conformation polymorphism present in DNA heteroduplexes containing variations as small as single nucleotide polymorphisms (SNPs). CSGE has also been adapted for use on a fluorescent platform (F-CSGE) that resulted in higher throughput and sensitivity. Variation in sensitivity of CSGE has been studied extensively. The results demonstrate that the nature of the mismatched base in a defined sequence context has the most profound effect on the conformation of the heteroduplex. Additionally, the size of the PCR product, as well as the location of the mismatch within the PCR product, are two important parameters that determine the resolution of the mismatch-containing heteroduplexes during CSGE. Like any other mutation scanning technique, CSGE can have limited resolution of two closely linked sequence variations. For specific genes, like BRCA1 and BRCA2 where multiple SNPs are present in the coding sequence, each CSGE shift has to be sequenced to define the exact nature of the sequence change. In conclusion, CSGE scanning provides a powerful, cost-efficient way to scan genes with high sensitivity and specificity.

Base Sequence↗

PubMed2024

Nanopore sequencing to detect A-to-I editing sites.

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.

Inosine↗

PubMed1997

High-performance computing and networking as tools for accurate emission computed tomography reconstruction.

It is well known that the quantitative potential of emission computed tomography (ECT) relies on the ability to compensate for resolution, attenuation and scatter effects. Reconstruction algorithms which are able to take these effects into account are highly demanding in terms of computing resources. The reported work aimed to investigate the use of a parallel high-performance computing platform for ECT reconstruction taking into account an accurate model of the acquisition of single-photon emission tomographic (SPET) data. An iterative algorithm with an accurate model of the variable system response was ported on the MIMD (Multiple Instruction Multiple Data) parallel architecture of a 64-node Cray T3D massively parallel computer. The system was organized to make it easily accessible even from low-cost PC-based workstations through standard TCP/IP networking. A complete brain study of 30 (64x64) slices could be reconstructed from a set of 90 (64x64) projections with ten iterations of the conjugate gradients algorithm in 9 s, corresponding to an actual speed-up factor of 135. This work demonstrated the possibility of exploiting remote high-performance computing and networking resources from hospital sites by means of low-cost workstations using standard communication protocols without particular problems for routine use. The achievable speed-up factors allow the assessment of the clinical benefit of advanced reconstruction techniques which require a heavy computational burden for the compensation effects such as variable spatial resolution, scatter and attenuation. The possibility of using the same software on the same hardware platform with data acquired in different laboratories with various kinds of SPET instrumentation is appealing for software quality control and for the evaluation of the clinical impact of the reconstruction methods.

Algorithms↗

PubMed2006

Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous.

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.

Cell Membrane Permeability↗

PubMed1999

Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands.

The effect of testing surface on the rat hind paw withdrawal threshold in the von Frey hair test is investigated in this study. The data indicate that wire mesh, which is typically used to apply von Frey hairs, may have an effect on the paw withdrawal threshold. For example, in control rats tested on the wire mesh, variability in the withdrawal threshold was observed between the left and the right hind paws (51.04+/-12.29 and 64.31+/-9.37 g, respectively) and on different days of testing (35.24+/-9.54 and 45.83+/-12.97 g for the left and right hind paws, respectively, 7 days later). In an attempt to reduce this variability, a customized platform was used to measure the von Frey hair-induced paw withdrawal in the rat. It consists of an opaque, flat-surfaced plastic platform with holes through which von Frey hairs are inserted and applied to the plantar surface of the paw. In control rats tested with von Frey hairs using this customized platform, variability in the paw withdrawal thresholds between the left and right hind paws in single rats over time as well as between different rats was reduced (49.86+/-6.97 and 49.29+/-6.56 g for the left and right hind paws, respectively, on day 0; 48.29+/-5.82 and 53.00+/-4.59 g for the left and right hind paws, respectively, 7 days later). Furthermore, in rats in which a 2 mm polyethylene cuff was used to constrict the left common sciatic nerve, the ipsilateral as well as the contralateral hind paw withdrawal thresholds were decreased (2.45+/-0.65 and 26.09+/-5.86 g, respectively, 7 days later). In similar rats tested on the wire mesh, the ipsilateral but not the contralateral paw withdrawal threshold decreased (12.80+/-2.21 and 65.00+/-10.28 g, respectively, at 7 days). The data suggest that the flat surface and opaque properties of the customized platform enable accurate, reliable and repeatable measurements of ipsilateral and contralateral paw withdrawal threshold using von Frey hairs in normal and nerve-injured rats.

Animals↗