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At least 649 records · Page 36Linked to original sources

PubMed2005

Integron cassette insertion: a recombination process involving a folded single strand substrate.

Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the 'bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed.

Attachment Sites, Microbiological↗

PubMed2006

Strong electronic coupling between single C60 molecules and gold electrodes prepared by quench condensation at 4 K. A single molecule three terminal device study.

We report the first measurements of single C60 molecules trapped in three terminal devices prepared by quench condensation of a gold source and drain electrode on top of an aluminium gate electrode covered with a thin oxide. Our experimental platform allows source and drain electrodes to be fabricated on the gate oxide at low temperatures and high vacuum. In a subsequent step, single molecules are evaporated in situ onto the surface and caught in the gap between a source and a drain electrode. This fabrication method ensures a clean contact between the molecule and the gold electrode due to the unbroken vacuum. Our measurements reveal a strong interaction between the C60 molecule and the gold electrodes resulting in the absence of the Coulomb blockade effects observed by others. In addition, we observe an insignificant gate dependence but a pronounced negative differential resistance (NDR) at bias voltages from 20-50 meV. The position of the peak in the NDR shows a pronounced and universal temperature dependence for all six devices included in the study. The results are related to previous measurements in such devices which focus on the detailed nature of the contact region between the molecule the gold electrode.

Journal Article↗

PubMed2026

Artificial intelligence-assisted clinical exome sequencing: Insights and outcomes from 822 pediatric diagnoses.

PURPOSE: This retrospective study examined the clinical and genetic characteristics of pediatric patients undergoing clinical exome sequencing (ES) and evaluated the performance of a commercially available artificial intelligence (AI) platform that was integrated into our analysis pipeline. METHODS: ES was performed in 822 consecutive patients at a single clinical laboratory. AI-based tools were used to jointly assess genetic information and the proband's Human Phenotype Ontology terms to support variant prioritization during the initial case review. RESULTS: A definitive molecular diagnosis was established in 22% (181 of 822) of index cases, while 40% (325 of 822) had variants of uncertain significance. Among those with a definitive diagnosis, 93% (168 of 181) had a single finding and 7% (13 of 181) had multiple findings. Of the 152 reported pathogenic/likely pathogenic variants in the fully resolved cases, 98.7% were successfully flagged by AI, and 75.0% ranked among the top 10 "most likely" variants. CONCLUSION: Clinical ES provides a substantial diagnostic yield in complex pediatric disorders. Integration of AI-powered platforms can accelerate phenotype-driven variant prioritization and facilitate rare disease diagnostics, but underscores the need for careful validation and optimization in clinical workflows.

Artificial intelligence↗

PubMed2005

Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

Chromatography, Gel↗

PubMed2005

A structure of the human apoptosome at 12.8 A resolution provides insights into this cell death platform.

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of beta propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.

Apoptosis↗

PubMed2001

Evaluation of book backpack load during walking.

This investigation evaluated accumulated mean and peak impact forces per stride and per metre associated with two book backpack loads and two cadences during single and double support phases of walking. Thirty college participants randomly performed three trials while either walking a self-selected cadence or fixed cadence without (empty pack) or with a load (15% body mass) carried in a bookbag. The fixed cadence (55.5 steps/min) was regulated by a metronome. A computerized Kistler force platform system (phase-locked timing device) recorded (200 Hz) three-dimensional reaction forces, impulses, and time in single and double support phases. A Panasonic video camera AG-450 was set perpendicular to the plane of walking motion to film (60 Hz) the walking pattern from which stride length and selected kinematic data were determined. Repeated measure ANOVA (p<0.05) determined differences of loads and cadences in walking. Accumulated force was evaluated as impulses per stride and impulses per metre (stress index). When carrying the 15% load, there was a decrease in speed, a decrease in single support time (SST), and an increase in double support time (DST). The impulses per stride significantly increased in DST, and significantly decrease in SST. When impulses were analysed per metre, the stress index signficantly increased in DST, but not during SST. These differences in SST may be important when load stress is applied to the single support leg/foot in any given distance of walking. While stride analysis identifies accumulative forces resulting from varying stride lengths, the stress index provides a standardized measure per metre of the accumulative forces that negate the variances of individual stride lengths, and the index measure can easily represent data for any given distance traversed.

Adult↗

PubMed2007

Automated laser scanning cytometry: a powerful tool for multi-parameter analysis of drug-induced apoptosis.

BACKGROUND: Simultaneous analysis of multiple intracellular events is critical for assessing the effect of biological response modifiers, including the efficacy of chemotherapy. Here we used the automated laser scanning cytometry (LSC) for multi-parameter analysis of drug-induced tumor cell apoptosis. MATERIALS: Using 2-mercaptopyridine-N-oxide-hydrate sodium salt, or the commonly used chemotherapeutic agents etoposide and camptothecin, we performed simultaneous analyses of apoptosis-related morphological features as well as fluorescence-based biochemical changes in a 96-well format. RESULTS: We demonstrate the scope of LSC as a platform for comparing multiple variables between different cell populations, distinguishing unique events at a single cell level within a sample population, and enabling simultaneous screenings in a single assay at multiple dosages and time-points. CONCLUSION: These data underscore the power of LSC for simultaneous multi-parameter analysis, which could have implications for screening or assessing the efficacy of drug responses in heterogeneous cell populations and at the single cell level.

Animals↗

PubMed1995

View-responsive neurons in the primate hippocampal complex.

Recordings were made from single neurons in the hippocampus and parahippocampal gyrus while macaques were moved on a platform mounted on a free-moving robot or on wheels in a cue-controlled 2 m x 2 m x 2 m environment, in order to investigate the representation of space and of spatial memory in the primate hippocampus. The test conditions allowed factors that might account for spatial firing of the cells, including the spatial location where the monkey looked, the place were the monkey was, and the head direction of the monkey, to be identified. The responses of some ("view") neurons depended on where the monkey was looking in the environment, but not on the place of the monkey in the environment. The responses of one other neuron depended on a combination of where the monkey was facing and his place in the test chamber. The response of view-dependent neurons was affected by occlusion of the visual field. It was possible to show for one neuron that its "view" response rotated with rotation of the test chamber. Some neurons responded to a combination of whole-body motion and view or place, and one neuron responded in relation to whole-body movement to a particular place. One neuron responded depending on the place where the monkey was in the environment and relatively independently of view. The representations of space provided by hippocampal view-responsive neurons may be useful in forming memories of spatial environments (for example, of where an object has been seen and of where the monkey is as defined by seen views) and, together with whole-body motion cells, in remembering trajectories through environments, which is of use, for example, in short range spatial navigation.

Animals↗

PubMed2000

Chronic propranolol induces deficits in retention but not acquisition performance in the water maze in mice.

Agents that alter adrenergic receptors, such as "beta-blockers," also alter memory storage. However, reports suggest that beta-adrenergic receptor antagonists, such as propranolol, have conflicting behavioral effects with acute vs chronic dosing. This study was designed to evaluate the effects of chronic propranolol on retention for a spatial learning task. Adult male ICR mice were given daily injections of propranolol (2, 4, 8, or 12 mg/kg ip) or 0. 9% NaCl for 15 days prior to, and during, trials in a Morris water maze. Mice received five massed acquisition (escape) trials in each of three daily sessions, followed by a single 60-s probe trial on the fourth day. The location of the submerged platform was constant for each animal over acquisition trials, but varied across animals; starting position varied across trials. A 5 (dose) x 3 (trial blocks) mixed factorial ANOVA for escape time yielded a significant trial blocks effect only (p <.001), showing performance improving over sessions. Time spent in the target quadrant on the probe trial was shorter under all doses of propranolol when compared to vehicle group (all p <.001), indicating poorer retention of prior platform location. This effect, however, was not dose-related. Swim speed was not significantly affected by propranolol. These data demonstrate that chronic dosing with propranolol can impair retention of spatial learning, which cannot be attributed to reduced arousal or motor function.

Adrenergic beta-Antagonists↗

PubMed2006

Preventive and therapeutic vaccination with PAP-3, a novel human prostate cancer peptide, inhibits carcinoma development in HLA transgenic mice.

Conventional treatment of recurrent and metastasized prostate cancer (CaP) remains inadequate; this fact mandates development of alternative therapeutic modalities, such as specific active or passive immunotherapy. Previously, we reported the identification of a novel highly immunogenic HLA-A*0201-restricted Prostatic Acid Phosphatase-derived peptide (PAP-3) by a two-step in vivo screening in an HLA-transgenic (HHD) mouse system. In the present study we aimed at elucidating the efficiency of PAP-3-based vaccine upon active antitumor immunization. To this end we established preventive and therapeutic carcinoma models in HHD mice. The 3LL murine Lewis lung carcinoma clone D122 transduced to express HLA-A*0201 and PAP served as a platform for these models. The HLA-A*0201-PAP-3 complex specific recombinant single chain scFV-PAP-3 antibodies were generated and used to confirm an endogenous PAP processing resulting in PAP-3 presentation by HLA-A*0201. PAP-3 based vaccines significantly decreased tumor incidence in a preventive immunization setting. Therapeutic vaccination of HHD mice with PAP-3 led to rejection of early established tumors and to increase of mouse survival. These results strongly support a therapeutic relevance of the identified CTL epitope upon active antitumor immunization. The newly established carcinoma model presented herein might be a useful tool for cancer vaccine design and optimization.

Acid Phosphatase↗

PubMed1975

Intercellular communication in a positional field. Ultrastructural correlates and tracer analysis of communication between insect epidermal cells.

The junctional membrane in the epidermal cells of the larval beetle (Tenebrio molitor L.) is comprised of macular gap junctions embedded in septate junctions. Ultrastructural and morphometric analysis of the distribution of gap junctions within the segmental epidermis suggests that this junction alone could account for the high electrotonic coupling recorded for the epidermal sheet. Analysis of the lanthanum-impregnated septate junction makes it doubtful that this junction serves as a communicating channel between beetle cells. A new model for the septate junction is presented in which pleated septa, less than 30 A thick, connect adjacent plasma membranes; the septa themselves are interconnected by two interseptal platforms that are coplanar with the plasma membranes. Iontophoretic injection of organic tracers into single epidermal cells suggests that only molecules of less than MW 1000 can transfer between cells through low-resistance junctions.

Animals↗

PubMed2005

Highly parallel microbial diagnostics using oligonucleotide microarrays.

Oligonucleotide microarrays are highly parallel hybridization platforms, allowing rapid and simultaneous identification of many different microorganisms and viruses in a single assay. In the past few years, researchers have been confronted with a dramatic increase in the number of studies reporting development and/or improvement of oligonucleotide microarrays for microbial diagnostics, but use of the technology in routine diagnostics is still constrained by a variety of factors. Careful development of microarray essentials (such as oligonucleotide probes, protocols for target preparation and hybridization, etc.) combined with extensive performance testing are thus mandatory requirements for the maturation of diagnostic microarrays from fancy technological gimmicks to robust and routinely applicable tools.

Bacteria↗

PubMed2004

From atoms to systems: a cross-disciplinary approach to complement-mediated functions.

With an ever-increasing wealth of information made available to researchers from expanding genomic sequence and protein structure databases, traditional experimentation and research are being drastically revisited. The unidirectional study of single molecules and pathways is being replaced by a combinatorial and cross-disciplinary platform that investigates interactive biological systems and dynamic networks. The complement system constitutes an ideal paradigm of how this concept is being applied in the field of contemporary immunology. Our laboratory has adopted such a cross-disciplinary approach in elucidating key aspects of complement functions and determining the role of several complement proteins in both inflammatory and developmental processes. Here we discuss recent findings pertaining to the rational development of complement inhibitors, our studies on protein-protein interactions and our progress in the study of viral immune evasion and complement evolution. Furthermore, we present recent studies implicating complement components in complex developmental processes, such as organ regeneration, hematopoietic development, and stem cell engraftment.

Animals↗

PubMed2002

A label-free optical technique for detecting small molecule interactions.

A novel approach for the label-free detection of molecular interactions is presented in which a colorimetric resonant grating is used as a surface binding platform. The grating, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or radioactive label. The detection technique is capable of detecting the addition and removal of small molecules as they interact with receptor molecules on the sensor surface or enzymes in the solution surrounding the sensor. Two assays are presented to exemplify the detection of small molecule interactions with the biosensor. First, an avidin receptor layer is used to detect 244 Da biotin binding. Second, a protease assay is performed in which a 136 Da p-nitroanilide (pNA) moeity is cleaved from an immobilized substrate. Because the sensor structure can be embedded in the plastic surfaces of microtiter plates or the glass surfaces of microarray slides, it is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. Screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.

Avidin↗

PubMed1997

Fractionating the nonspatial pretraining effect in the water maze task.

Nonspatial pretraining (NSP) enables rats to learn the general strategies of the water maze task (WMT; e.g., learning to swim away from the wall and to climb onto the hidden platform), reduces sensorimotor disturbances, and eliminates acquisition impairments caused by scopolamine hydrobromide, a muscarinic antagonist. To evaluate the contributions of the components of NSP to these effects, NSP was fractionated so that different groups of male rats swam, were placed onto the hidden platform, climbed onto the hidden platform, or were placed into an empty maze before spatial training under scopolamine. No single component of the NSP procedure was sufficient to produce its full effects on sensorimotor disturbances and WMT acquisition. Experience with most or perhaps all of the specific behaviors required in the WMT appears to be important for NSP to produce its full effects.

Animals↗

PubMed1999

Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand.

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2D(d) through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2D(d). The Ly49A dimer interacts extensively with two H-2D(d) molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2D(d) alpha2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.

Antigens, Ly↗

PubMed2006

Combo: a whole genome comparative browser.

SUMMARY: Combo is a comparative genome browser that provides a dynamic view of whole genome alignments along with their associated annotations. Combo provides two different visualization perspectives. The perpendicular (dot plot) view provides a dot plot of genome alignments synchronized with a display of genome annotations along each axis. The parallel view displays two genome annotations horizontally, synchronized through a panel displaying local alignments as trapezoids. Users can zoom to any resolution, from whole chromosomes to individual bases. They can select, highlight and view detailed information from specific alignments and annotations. Combo is an organism agnostic and can import data from a variety of file formats. AVAILABILITY: Combo is integrated as part of the Argo Genome Browser which also provides single-genome browsing and editing capabilities. Argo is written in Java, runs on multiple platforms and is freely available for download at http://www.broad.mit.edu/annotation/argo/.

Algorithms↗

PubMed2003

Neuberger's double-pass algorithm.

We analyze Neuberger's double-pass algorithm for the matrix-vector multiplication R(H) x Y [where R(H) is (n-1,n)th degree rational polynomial of positive definite operator H], and show that the number of floating-point operations is independent of the degree n, provided that the number of sites is much larger than the number of iterations in the conjugate gradient. This implies that the matrix-vector product (H)(-1/2)Y approximately R((n-1,n))(H).Y can be approximated to very high precision with sufficiently large n, without noticeably extra costs. Further, we show that there exists a threshold n(T) such that the double-pass is faster than the single pass for n>n(T), where n(T) approximately 12-25 for most platforms.

Journal Article↗