SEARCH · Search PubMed

Results for “next generation sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources

PubMed1993

Distribution of VP4 gene alleles in human rotaviruses by using probes to the hyperdivergent region of the VP4 gene.

The rotavirus VP4 protein elicits the production of neutralizing antibodies and is known to play a role in inducing resistance to disease. At least five human rotavirus VP4 gene alleles have been described on the basis of antigenic polymorphism and/or nucleotide sequence differences. In the present study, we developed cDNA probes directed at the hyperdivergent region of the VP4 gene of the five described human rotavirus VP4 alleles (Wa, DS1, M37, AU228, and 69M) and used them in hybridization assays with human rotavirus strains from Latin America and Europe to determine the distribution of the VP4 gene alleles in nature. The Wa-like allele was detected most frequently, occurring in 57% of the 402 rotavirus strains tested, and the DS1-like allele was the next most common, occurring in 14% of the strains tested. The M37- and AU228-like alleles were detected in only 4 and 3% of the rotavirus strains tested, respectively, whereas the 69M-like VP4 gene allele was not detected. Several rotavirus strains from Europe did not react with any of the VP4 gene probes, although they did hybridize to a probe generated from a representative strain from the group. These data indicate the global distribution of various VP4 gene alleles and raise the possibility that other, unrecognized human VP4 alleles exist in nature because almost one-fourth of the strains could not be classified into any of the established VP4 groups.

Alleles↗

PubMed1985

Swimming activity in marine fish.

Marine fish are capable of swimming long distances in annual migrations; they are also capable of high-speed dashes of short duration, and they can occupy small home territories for long periods with little activity. There is a large effect of fish size on the distance fish migrate at slow swimming speeds. When chased by a fishing trawl the effect of fish size on swimming performance can decide their fate. The identity and thickness of muscle used at each speed and evidence for the timing of myotomes used during the body movement cycle can be detected using electromyogram (EMG) electrodes. The cross-sectional area of muscle needed to maintain different swimming speeds can be predicted by relating the swimming drag force to the muscle force. At maximum swimming speed one completed cycle of swimming force is derived in sequence from the whole cross-sectional area of the muscles along the two sides of the fish. This and other aspects of the swimming cycle suggest that each myotome might be responsible for generating forces involved in particular stages of the tail sweep. The thick myotomes at the head end shorten during the peak thrust of the tail blade whereas the thinner myotomes nearer the tail generate stiffness appropriate for transmission of these forces and reposition the tail for the next cycle.

Action Potentials↗

PubMed2002

Designer microarrays: from soup to nuts.

The recognition that multigene mechanisms control the pathways determining the aging process renders gene screening a necessary skill for biogerontologists. In the past few years, this task has become much more accessible, with the advent of DNA chip technology. Most commercially available microarrays are designed with prefixed templates of genes of general interest, allowing investigators little freedom of choice in attempting to focus gene screening on a particular thematic pathway of interest. This report describes our "designer microarray" approach as a next generation of DNA chips, allowing individual investigators to engage in gene screening with a user friendly, do-it-yourself approach, from designing the probe templates to data mining. The end result is the ability to use microarrays as a platform for versatile gene discovery.

Gene Expression Profiling↗

PubMed2003

The effects of digital anesthesia on force control using a precision grip.

A total of 20 right-handed subjects were asked to perform a grasp-lift-and-hold task using a precision grip. The grasped object was a one-degree-of-freedom manipuladum consisting of a vertically mounted linear motor capable of generating resistive forces to simulate a range of object weights. In the initial study, seven subjects (6 women, 1 man; ages 24-56 yr) were first asked to lift and hold the object stationary for 4 s. The object presented a metal tab with two different surface textures and offered one of four resistive forces (0.5, 1.0, 1.5, and 2.0 N). The lifts were performed both with and without visual feedback. Next, the subjects were asked to perform the same grasping sequence again after ring block anesthesia of the thumb and index finger with mepivacaine. The objective was to determine the degree to which an internal model obtained through prior familiarity might compensate for the loss of cutaneous sensation. In agreement with previous studies, it was found that all subjects applied significantly greater grip force after digital anesthesia, and the coordination between grip and load forces was disrupted. It appears from these data, that the internal model alone is insufficient to completely compensate for the loss of cutaneous sensation. Moreover, the results suggest that the internal model must have either continuous tonic excitation from cutaneous receptors or at least frequent intermittent reiteration to function optimally. A subsequent study performed with 10 additional subjects (9 women, 1 man; ages 24-49 yr) indicated that with unimpaired cutaneous feedback, the grasping and lifting forces were applied together with negligible forces and torques in other directions. In contrast, after digital anesthesia, significant additional linear and torsional forces appeared, particularly in the horizontal and frontal planes. These torques were thought to arise partially from the application of excessive grip force and partially from a misalignment of the two grasping fingers. These torques were further increased by an imbalance in the pressure exerted by the two opposing fingers. Vision of the grasping hand did not significantly correct the finger misalignment after digital anesthesia. Taken together, these results suggest that mechanoreceptors in the fingertips signal the source and direction of pressure applied to the skin. The nervous system uses this information to adjust the fingers and direct the pinch forces optimally for grasping and object manipulation.

Adult↗

PubMed2003

Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents.

Biological warfare agents are the most problematic of the weapons of mass destruction and terror. Both civilian and military sources predict that over the next decade the threat from proliferation of these agents will increase significantly. In this review we summarize the state of the art in detection and identification of biological threat agents based on PCR technology with emphasis on the new technology of microarrays. The advantages and limitations of real-time PCR technology and a review of the literature as it applies to pathogen and virus detection are presented. The paper covers a number of issues related to the challenges facing biological threat agent detection technologies and identifies critical components that must be overcome for the emergence of reliable PCR-based DNA technologies as bioterrorism countermeasures and for environmental applications. The review evaluates various system components developed for an integrated DNA microchip and the potential applications of the next generation of fully automated DNA analyzers with integrated sample preparation and biosensing elements. The article also reviews promising devices and technologies that are near to being, or have been, commercialized.

Biological Warfare↗

PubMed2003

Synthesis of protected 2-amino-2-deoxy-D-xylothionolactam derivatives and some aspects of their reactivity.

The synthesis of polyfunctionalized delta-lactams as key intermediates of glycomimetics in the 2-acetamido-2-deoxy sugar series is presented. Starting from a chiral gamma-amino vinylic ester synthesized from Garner's aldehyde and after regioselective reduction, 1-azido-3-(N-tert-butyloxycarbonyl-2,2-dimethyloxazolidin-4-yl)-2-propene was obtained. Next, a cis-dihydroxylation reaction provided the protected D-xylitol and L-arabinitol azides. A simple protection-deprotection sequence, followed by an oxidation and a reductive cyclization, led to protected 2-amino-delta-lactams bearing a tert-butyloxycarbonyl group on the amine functionality. To explore the reactivity of such compounds, activation of the lactam into the corresponding thionolactam was performed. The resulting 2-amino-D-xylothionolactam derivative, a versatile intermediate, allowed access to a first generation of protected 2-amino-D-xylosamidoxime derivatives which are of interest as precursors of N-acetylhexosaminidase and N-acetylglucosaminyltransferase inhibitors. In this series of compounds, epimerization at C-2 was observed. AM(1) calculations performed on these analogs showed that they adopted a B(2,5) conformation and that the axial epimer was favored in the protected series whereas the equatorial epimer was preferred in the unprotected series.

Lactams↗

PubMed2002

Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells.

Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L-transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.

Animals↗

PubMed1984

Murine leukemia virus sequences are encoded in the murine major histocompatibility complex.

The studies reported here localize murine leukemia viral sequences to the TL region of the major histocompatibility complex, H-2. We examined a battery of 38 cosmids, isolated from two large genomic libraries constructed from C57BL/10 spleen DNA, that define 25 class I gene sequences. The viral probes used hybridized with only four cosmids, containing overlapping mouse sequences, that define four class I gene-related sequences in a region of 90 kilobases of DNA. The data show that two distinct viral envelope sequences are contained in the cluster. One of these sequences is situated with its 3' end next to the 3' end of a class I sequence. The other sequence, which does not contain the entire viral envelope, is proximal to the 3' end of a different class I sequence. Hybridization of the viral probes with the H-2 cosmid clones does not appear to be due to homology between viral and H-2 sequences. Rather, the viral sequences detected appear to be linked to or inserted amid class I genes. These findings may be significant in understanding molecular mechanisms involved in the generation of H-2 class I gene diversity.

Animals↗

PubMed2002

Biosynthesis of the cyclitol moiety of pyralomicin 1a in Nonomuraea spiralis MI178-34F18.

The biosynthetic pathway leading to the cyclitol moiety of pyralomicin 1a (1) in Nonomuraea spiralis MI178-34F18 has been studied using a series of 2H-labeled potential precursors. The results demonstrate that 2-epi-5-epi-valiolone (7), a common precursor for acarbose (4) and validamycin A (5) biosynthesis, is an immediate precursor of pyralomicin 1a. 5-epi-Valiolone (8) was also incorporated into 1, albeit less efficiently than 7. Other potential intermediates, such as valiolone (9), valienone (10), valienol (11), 1-epi-valienol (12), 5-epi-valiolol (13), and 1-epi-5-epi-valiolol (14) were not incorporated into pyralomicin 1a. To explain this surprising observation, it is proposed that either 2-epi-5-epi-valiolone (7) is specifically activated (e.g., to its phosphate) and that the further transformations take place on activated intermediates (which can not be generated directly from their unactivated counterparts), or that the transformation of 7 into 1 involves a substrate-channeling mechanism in which enzyme-bound intermediates are directly transferred from one enzyme active site to the next in a multi-enzyme complex.

Actinomycetales↗

PubMed2001

[Growth and development of plants in a sequence of generations under the conditions of space flight (experiment Greenhouse-3)].

The purpose was to study characteristic features of growth and development of several plant generations in space flight in experiment GREENHOUSE-3 as a part of the Russian-US space research program MIR/NASA in 1997. The experiment consisted of cultivation of Brassica rapa L. in board greenhouse Svet. Two vegetative cycles were fully completed and the third vegetation was terminated on day 13 on the phase of budding. The total duration of the space experiment was 122 days, i.e. same as in the ground controls. In the experiment with Brassica rapa L. viable seeds produced by the first crop were planted in space flight and yielded next crop. Crops raised from the ground and space seeds were found to differ in height and number of buds. Both parameters were lowered in the plants grown from the space seeds. The prime course for smaller size and reduced organogenic potential of plantTs reproductive system seems to be a less content of nutrients in seeds that had matured in the space flight. Experiment GREENHOUSE-3 demonstrated principle feasibility of plant reproduction in space greenhouse from seeds developed in microgravity.

Brassica rapa↗

PubMed1995

Evidence that Spitzenkörper behavior determines the shape of a fungal hypha: a test of the hyphoid model.

Hyphae of the fungus Rhizoctonia solani have a characteristic Spitzenkörper in their growing tips and a cell shape described by the mathematical hyphoid equation. A mild disturbance of hyphae growing in a slide culture chamber on a microscope stage caused the Spitzenkörper to move away from its usual position next to the apical pole and wander briefly inside the apical dome. Hyphal elongation rate declined abruptly, and the apex became rounded and increased in diameter. As the Spitzenkörper migrated back to its polar position, rapid cell elongation resumed, and the contour of the growing hyphal tip returned to the typical hyphoid shape. The brief dislocation of the Spitzenkörper left a permanent bulge in the hyphal profile. This morphogenetic sequence was mimicked by computer simulation, based on the hyphoid equation which relates the generation of hyphal shape to the linear displacement of a vesicle supply center (VSC). The VSC was programmed to retrace the observed movements of the Spitzenkörper during the above sequence. The resulting similarity of shape between real and computer-simulated cells reinforces the mathematical prediction that the Spitzenkörper acts as a VSC and that its continuous linear advancement generates a typical hyphal tube with the characteristic hyphoid shape. Accordingly, the hyphoid model and its VSC concept provide a plausible hypothesis to explain the cellular basis of polarized growth of fungal hyphae.

Computer Simulation↗

PubMed2001

From chicken coops to genome maps: generating phenotype from the molecular blueprint.

The tools of molecular and cellular biology can be used to precisely describe traits in terms of a sequence of nucleic acids when their molecular and cellular bases are well understood. The entire genome of elite production birds, however, cannot be written as a series of A's, T's, C's, and G's because the interaction between alleles at the same and different loci is too large and there is likely to be many genotypes that encode the same production trait phenotype. A first draft of the genetic map of the chicken is anticipated within the next few years, but a complete molecular description of the genome of birds with elite production characteristics is not anticipated in the near future. Quantitative genetics will remain the cornerstone of breeding programs for production traits. Novel sequences encoding traits such as enhanced nutritional capability (e.g., expression of phytase) and resistance to specific diseases could be introduced into lines of chickens using the tools of molecular and cellular biology. Cloning could be used by the poultry industry to disperse highly desirable genotypes without the need for grandparent and parent flocks for multiplication.

Animals↗

PubMed2002

Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment.

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.

Base Pair Mismatch↗

PubMed2003

Two-step regulation and continuous retrotransposition of the rice LINE-type retrotransposon Karma.

Here, we report the identification of Karma, a LINE-type retrotransposon of plants for which continuous retrotransposition was observed in consecutive generations. The transcription of Karma is activated in cultured cells of rice upon DNA hypomethylation. However, transcription is insufficient for retrotransposition, because no increase in the copy number was observed in cultured cells or in the first generation of plants regenerated from them. Despite that finding, copy number increase was detected in the next generation of regenerated plants as well as in later generations, suggesting that the post-transcriptional regulation of Karma retrotransposition is development dependent. Our results indicate that two different mechanisms, one transcriptional and the other developmental, control the mobilization of KARMA: In addition, unlike other known active plant retrotransposons, Karma is not subject to de novo methylation, and retrotransposition persists through several generations.

Amino Acid Sequence↗

PubMed2000

Genomics and proteomics tools for the clinic.

The sequencing of the human genome was only made possible by the massively parallel use of automated high-throughput technologies. These technologies have required the development and interfacing of new hardware and software in a way which would have been hardly conceivable only ten years ago. As a consequence, an unbroken trend to more 'industrialized science' is apparent. The wealth of information generated by intensive sequencing efforts is now being further exploited by using complex tools to comprehensively analyze complex systems at the DNA, RNA and protein level. A landmark innovation was the introduction of the biochip principle, best exemplified with the development of the DNA chip, mainly used for RNA expression profiling. The chip principle, together with miniaturization, has now become the dominating theme for a number of new genomics and proteomics technologies, culminating in the lab-on-a-chip concept which, in the next five to ten years, could advance at a comparable rate to that of computers over the last 50 years. Some of the new technologies are already used for comprehensive analysis of clinical samples in an attempt to describe disease and disease risk at the molecular level. However, all of these technologies are far from routine in clinical use and it is also too early to decide whether molecular fingerprints or signature profiles will have the diagnostic and prognostic power currently predicted.

Gene Expression Profiling↗

PubMed2006

The 13C4 monoclonal antibody that neutralizes Shiga toxin Type 1 (Stx1) recognizes three regions on the Stx1 B subunit and prevents Stx1 from binding to its eukaryotic receptor globotriaosylceramide.

The 13C4 monoclonal antibody (MAb) recognizes the B subunit of Stx1 (StxB1) and neutralizes the cytotoxic and lethal activities of Stx1. However, this MAb does not bind to the B polypeptide of Stx2, despite the 73% amino acid sequence similarity between StxB1 and StxB2. When we compared the amino acid sequences of StxB1 and StxB2, we noted three regions of dissimilarity (amino acids 1 to 6, 25 to 32, and 54 to 61) located near each other on the crystal structure of StxB1. To identify the 13C4 epitope, we generated seven Stx1/Stx2 B chimeric polypeptides that contained one, two, or three of the dissimilar StxB1 regions. The 13C4 MAb reacted strongly with StxB1 and the triple-chimeric B subunit but not with the other chimeras. Mice immunized with the triple-chimeric B subunit survived a lethal challenge with Stx1 but not Stx2, substantiating the identified regions as the 13C4 MAb epitope and suggesting that the incorporation of this epitope into StxB2 altered sites necessary for anti-Stx2-neutralizing Ab production. Next, single amino acid substitutions were made in StxB1 to mimic Stx1d, a variant not recognized by the 13C4 MAb. The 13C4 MAb reacted strongly to StxB1 with the T1A or G25A mutations but not with the N55T change. Finally, we found that the 13C4 MAb blocked the binding of Stx1 to its receptor, globotriaosyl ceramide. Taken together, these results indicate that the 13C4 MAb prevents the interaction of Stx1 with its receptor by binding three nonlinear regions of the molecule that span receptor recognition sites on StxB1, one of which includes the essential residue 55N.

Amino Acid Sequence↗

PubMed2005

Update to pulse sequences for interventional MR imaging.

The motivations for developing MR-guided minimally invasive therapy include its excellent soft tissue contrast, tomographic imaging in any direction (as opposed to projection imaging as in fluoroscopy), the absence of ionizing radiation,the abundance of contrast mechanisms (including bright blood pulse sequences that lead to excellent vessel conspicuity without exogenous contrast agent injection), the ability to obtain physiologic information such as perfusion, and an overall excellent safety profile. The main pulse sequences used today for interventional MR imaging are T1/T2-weighted FISP and TrueFISP, T2-weighted turbo spin-echo, and T1-weighted FLASH. The specific clinical question, the underlying pathophysiology,and the procedure to be performed dictate which sequence is used. Each of these sequences has been written to acquire data in conventional rectilinear trajectories, radial k-space paths, or even spirals. In many ways, the questions being researched in interventional MR imaging have been dictated by the primary issues in greatest need of resolution or that most directly facilitate new clinical development. A decade ago, research focused on exploration of new scan strategies for contrast and temporal resolution. Advancements in the last decade have made it possible to acquire and display greater than 10 images per second in realtime with millimeter resolution in all three directions. This temporal and spatial resolution is considered high enough to guide most interventions. With this capability, other research has focused on instrument tracking. The field has gone from the capability to track a single coil and superimpose it on a previously acquired roadmap to systems that follow, adapt, and provide high-resolution images due to the advent of multichannel receiver systems, improved graphics, higher processor speeds, and increases in speed and quantity of memory. Hence, instruments can be reliably identified and tracked and the information can be used to update pulse sequence parameters in real time, thereby opening new opportunities for interventional MR imaging that extend from biopsy and thermal therapy to image-guided vascular and cardiac procedures. Today, we see such issues as RF heating of wires used for device localization and the noise generated by rapid switching of MR gradients being significant obstacles yet to overcome to allow the full strength of MR-guided interventions to be realized clinically. It is anticipated that these topics will emerge as critical concepts in the next decade of interventional MR imaging research.

Algorithms↗

PubMed1999

Identification of soluble interleukin-4 receptor in rat glomerular epithelial cells(1).

Interleukin (IL)-4, a pleiotropic cytokine involved in many glomerular diseases, is regulated positively by membrane-bound IL-4R (mIL-4R) and negatively by soluble IL-4R (sIL-4R). Because natural sIL-4R has been documented only in mice, we undertook this study in rats to determine whether they, too, express sIL-4R, particularly in kidney cells. A pair of IL-4R primers was designed for this purpose and used in the polymerase chain reaction. As a result, sIL-4R was found not only in rats spleen cells but also in their glomerular epithelial cells (GEC). Sequence analysis revealed that the mRNA of rat sIL-4R has a 75-bp insert sequence. This insert generated a termination TGA codon upstream from the transmembrane region, resulting in formation of the sIL-4R. Subsequent screening of the kidney cDNA library enabled us to obtain the whole 3605-bp cDNA of sIL-4R; the full-length 3530-bp mIL-4R cDNA was also identified as a much longer sequence than previously published. Among the total 39 clones positive for IL-4R, two were confirmed as sIL-4R, and 37 clones were positive for mIL-4R. Next, the translated portion of sIL-4R cDNA was constructed into an expression vector, enabling us to obtain a recombinant sIL4R-myc fusion protein. By using this recombinant sIL-4R, we proved that sIL-4R can antagonize the IL-4-induced proliferation of spleen cells. Present study demonstrated that sIL-4R is expressed in kidney cells and antagonistically functional.

Animals↗