SEARCH · PubMed Health

Results for “Next generation sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 703 records · Page 39Linked to original sources

PubMed1991

A computer management system for patient simulations.

A series of interactive videodisc patient simulations is being used to teach clinical problem-solving skills, including diagnosis and management, to dental students. This series is called Oral Disease Simulations for Diagnosis and Management (ODSDM). A computer management system has been developed in response to the following needs. First, the sequence in which students perform simulations is critical. Second, maintaining records of completed simulations and student performance on each simulation is a time-consuming task for faculty. Third, the simulations require ongoing evaluation to ensure high quality instruction. The primary objective of the management system is to ensure that each student masters diagnosis. Mastery must be obtained at a specific level before advancing to the next level. The management system does this by individualizing the sequence of the simulations to adapt to the needs of each student. The management system generates reports which provide information about students or the simulations. Student reports contain demographic and performance information. System reports include information about individual patient simulations and act as a quality control mechanism for the simulations.

Computer Simulation↗

PubMed1988

Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPs).

In this paper we present a systematic approach to gene mapping and genotyping based on the simultaneous analysis of multiple amplified sequence polymorphisms (ASPs). These genetic markers measure variation in DNA sequences which have been amplified by a polymerase and/or a ligase. The amplified sequence lengths are determined by appropriate choice of oligonucleotides used in the amplification reaction. We describe three classes of ASPs: restriction site polymorphisms, sequence length polymorphisms, and DNA base pair changes not associated with restriction sites. Simultaneous analysis of multiple ASPs using a modified automated DNA sequencing apparatus should be possible because amplification with oligonucleotides provides control over the fragment lengths generated. Development of an automated ASP technology is therefore the next logical step for efficient gene mapping and genotyping of individuals. With this technology, one gel would be sufficient to indicate the most probable locations of a gene and a second gel would permit the selection of the correct location while simultaneously providing a fine structure map.

Chromosome Mapping↗

PubMed1993

Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector.

In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1. However, this construct did not generate detectable expression of RBP in E. coli. When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm. Deletion of the 3' untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx. 20-fold). After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin. The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e. it interacted with retinol, transthyretin and its cell-surface receptor. Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher. This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.

Autoradiography↗

PubMed1987

Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression.

Ferritin stores iron within a protein shell consisting of 24 subunits of two types, heavy (H) and light (L). According to Southern blotting, the rat genome contains four copies homologous to the H-subunit cDNA (H cDNA). To determine whether only one of these is expressed, H cDNAs isolated from rat liver and heart mRNAs were compared and found to share identical nucleotide sequences. Next, genomic clones for three of the four rat H-subunit loci were isolated. Two were classical processed pseudogenes, whereas the third contained an expressed gene. RNase intron mapping of this expressed gene generated the same exon protection pattern when total RNA from rat liver or heart was used, indicating that this gene accounts for most or all of the H-subunit mRNAs (H mRNAs) in these tissues. Comparison of the expressed rat H-subunit gene (H gene) structure with published sequences for other species displays considerable conservation. The coding sequence of the rat H gene predicts 95% similarity to the human amino acid sequence, thus being more highly conserved than the L-subunit sequence of these species. Near the cap region of the 5' untranslated region, the rat H mRNA displays a 28-nucleotide sequence that is almost totally conserved in the corresponding region of the human, bullfrog, and chicken H mRNA and is also faithfully represented in the rat and human L-subunit mRNAs (L mRNAs), thus making this sequence a prime candidate for involvement in the known translational regulation of both subunits by iron. In the 5' flanking region, partially conserved sequences common to H gene and L-subunit gene (L gene) of the rat may be involved in transcriptional regulation by iron, whereas those conserved only in the H gene of man and the rat imply that other factors may independently control H-subunit regulation.

Amino Acid Sequence↗

PubMed1986

A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus.

In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene. Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2-terminal amino acids from SV40 VP1, followed by poliovirus VP1. Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum. The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus. Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated. Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted. Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus.

Antigens, Polyomavirus Transforming↗

PubMed2001

Human antibodies as next generation therapeutics.

Antibodies and antibody derivatives constitute twenty five percent of therapeutics currently in development, and a number of therapeutic monoclonal antibodies have recently reached the market. All antibodies approved by the US Food and Drug Administration, however, contain mouse protein sequences. These partially murine antibodies, therefore, have the potential to elicit allergic or other complications when used in human patients. Recent developments aim to reduce or eliminate murine components, and fully human antibodies are rapidly becoming the norm. A number of technologies exist which enable the development of 100% human antibodies.

Animals↗

PubMed1986

Age differences in male drivers' perception of accident risk: the role of perceived driving ability.

Young (18-25) and older (35-50) male drivers were compared in their perception of driving risk and confidence in driving ability. Both groups provided responses a questionnaire on accident risk and driving ability and further generated subjective ratings of risk to a series of videotaped sequences depicting various elements of driving behavior. Although young drivers' estimates of accident involvement in the next year were higher than those of older drivers, young drivers gave lower ratings of accident risk for specific driving situations which demanded fast driving reflexes or substantial vehicle-handling skills. Young drivers rated their own risk of an accident and driving abilities as being the same as for older drivers. However, they saw their peers as being significantly higher at risk and having poorer abilities than themselves. Young drivers were more confident in their driving abilities than the older drivers. Evidence is provided to suggest that perceived risk and self-perceived driving abilities are interrelated. Further, the data from young drivers showed two disturbing characteristics: first, a notable dissociation between perceived and actual ability and, second, a tendency to view themselves as immune from the effects of higher levels of risk, which they are prepared to ascribe to their peers but not to themselves.

Accidents, Traffic↗

PubMed2024

Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes.

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Humans↗

PubMed2006

Antibodies neutralizing feline leukaemia virus (FeLV) in cats immunized with the transmembrane envelope protein p15E.

The feline leukaemia virus (FeLV) vaccines that are currently in wide use are generally poor inducers of virus-neutralizing antibodies, although such antibodies appear after recovering from challenge. However, the presence of neutralizing antibodies in cats recovering from natural FeLV infection clearly correlates with resistance to subsequent infection and passive transfer of antibodies can protect other animals. After demonstrating the induction of neutralizing antibodies in rats and goats immunized with the transmembrane envelope protein p15E of FeLV, cats were immunized with the same antigen. High titres of neutralizing antibodies specific for FeLV were induced and epitope mapping revealed a pattern of recognition similar to that seen following immunization of rats and goats. These epitopes are highly related to epitopes recognized after immunization with porcine endogenous retrovirus (PERV) p15E and to epitopes recognized by neutralizing antibodies in patients infected with human immunodeficiency virus type 1. The ability of p15E to induce neutralizing antibodies in cats suggests that it should be included in the next generation of vaccines. In contrast, sera from FeLV-infected animals usually fail to recognize the neutralization-relevant epitopes in p15E. Since homologous epitope sequences are present in feline endogenous retroviruses, it appears that tolerance against these sequences is not induced.

Amino Acid Sequence↗

PubMed1999

A new approach for studying gene regulation by distant DNA elements in transgenic mice.

Apolipoprotein B (apo-B) plays a crucial role in the assembly of lipoproteins in the liver and the intestine. Here, we review how transgenic mouse expression studies with large genomic clones have been used to define distant cis-acting regulatory DNA sequences that control the expression of the apo-B gene. In early studies, apo-B transgenic mice were generated with approximately 80-kb P1 bacteriophage clones spanning either the human or the mouse apo-B genes. Both the human and mouse clones directed high levels of transgene expression in the liver, but transgene expression was absent in the intestine. The absence of transgene expression in the intestine was surprising because both P1 clones contained more than 11 kb of flanking sequences both 5' and 3' to the gene. Subsequently, we isolated and characterized 145-kb and 207-kb bacterial artificial chromosome (BAC) clones that spanned the human apo-B gene. Each of these BAC's contained extensive 5 and 3' flanking sequences and each directed spatially and physiologically appropriate apo-B gene expression in the intestines of transgenic mice. To define the location of the sequences that control intestinal expression of the apo-B gene, we generated transgenic mice by co-microinjecting the approximately 80-kb P1 bacteriophage clone (which did not confer intestinal expression of apo-B) with either the 5' sequences or the 3' sequences from the 145-kb BAC. Analysis of the apo-B expression pattern in those mice revealed that the DNA sequences controlling intestinal expression were located 5' to the apo-B gene. Next, we used recA-assisted restriction endonuclease (RARE) cleavage to truncate specific segments of the 5' and 3' flanking sequences from the 145-kb BAC. A series of the truncated BAC's containing different lengths of 5' and 3' sequences was used to generate more than 40 additional lines of human apo-B transgenic mice. Analysis of human apo-B gene expression in those mice demonstrated that the sequences controlling the expression of the apo-B gene in the intestine are located more than 50 kb 5' to the apo-B gene. Our studies demonstrate that the RARE cleavage/transgenic expression strategy is a powerful approach for examining gene regulation by distant gene-regulatory elements.

Animals↗

PubMed1987

Biochemical and genetic characterization of type I familial amyloidotic polyneuropathy.

Type I familial amyloidotic polyneuropathy is an autosomal dominant, inherited systemic amyloidosis characterized initially by dissociated sensory disturbance and autonomic dysfunction. The amyloid fibril protein seen in patients of Portuguese, Japanese, and Swedish descent in the U.S. mainly consists of a variant form of transthyretin (also called prealbumin) with the substitution of methionine for valine at position 30. Methods have been developed to detect this variant transthyretin in the serum, and to detect a base change in a mutated transthyretin gene. The biochemical and genetic abnormalities in transthyretin are completely linked to the clinical diagnosis of type I familial amyloidotic polyneuropathy. These diagnostic methods may allow early diagnosis and genetic counseling to avoid transmission of this intractable disorder to the next generation.

Amyloidosis↗

PubMed2006

Whole-genome microarray in Arabidopsis facilitates global analysis of retained introns.

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Different types of AS have been observed; these include exon skipping, alternative donor or acceptor site and intron retention. In humans, exon skipping is the most common type while intron retention is rare. In contrast, in Arabidopsis, intron retention is the most prevalent AS type (approximately 40%). Here we show that direct transcript expression analysis using high-density oligonucleotide-based whole-genome microarrays (WGAs) is particularly amenable for assessing global intron retention in Arabidopsis. By applying a novel algorithm retained introns are detected in 8% of the transcripts examined. A sampling of 14 transcripts showed that 86% can be confirmed by RT-PCR. This rate of detection predicts an overall total AS rate of 20% for Arabidopsis compared with 10-22% based on EST/cDNA-based analysis. These findings will facilitate monitoring constitutive and dynamic whole-genome splicing on the next generation WGA slides.

Algorithms↗

PubMed1995

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.

Bacteriophage T4↗

PubMed2003

Methylation dynamics of repetitive DNA elements in the mouse germ cell lineage.

Repetitive DNA elements account for a substantial fraction of the mammalian genome. Many are subject to DNA methylation, which is known to undergo dynamic change during mouse germ cell development. We found that repeat sequences of three different classes retain high levels of methylation at E12.5, when methylation is erased from many single-copy genes. Maximal demethylation of repeats was seen later in development and at different times in male and female germ cells. At none of the time points examined (E12.5, E15.5, and E17.5) did we see complete demethylation, suggesting that methylation patterns on repeats may be passed on from one generation to the next. In male germ cells, we observed a de novo methylation event resulting in complete methylation of all the repeats in the interval between E15.5 and E17.5, which was not seen in females. These results suggest that repeat sequences undergo coordinate changes in methylation during germ cell development and give further insights into germ cell reprogramming in mice.

Animals↗

PubMed1994

Relationship between submandibular gland epidermal growth factor and spermatogenesis in C3H mice.

Epidermal growth factor (EGF), a potent mitogen produced primarily in the submandibular gland of adult male mice, has been implicated in modulating processes known to be of vital importance in the regulation of spermatogenesis. In the present investigation we demonstrate that submandibular gland EGF from adult male mice is indeed capable of displacing radiolabeled EGF from testicular membranes. Scatchard analysis of this binding site reveals that it is of high affinity (Kd = 0.77 nM) and low capacity (Bmax = 8.15 fmol/mg protein). Cross-linking of 125I-EGF to the identical membrane preparation resulted in the SDS-PAGE/autoradiography identification of a single band at approximately 170 kDa. Next, we examined the cellular distribution of the EGF receptor in the testis using biotin-streptavidin immunoperoxidase and employing different antisera probes generated to a conserved sequence of the EGF receptor. The Scatchard and cross-linking data described above, along with the immunocytochemistry results, suggest strongly that there is only one functional binding site for EGF in the adult testis and that this receptor is present in Sertoli and Leydig cells.

Animals↗

PubMed2002

First and next generation native rFVIII in the treatment of hemophilia A. What has been achieved? Can patients be switched safely?

The introduction of plasma-derived human factor VIII (FVIII) and later human recombinant FVIII (rFVIII) has potentially allowed patients suffering from hemophilia A to have a quality of life and life expectancy similar to the population at large. One of the major achievements in molecular biology over the past 15 years was the sequencing of the gene coding for FVIII, leading eventually to the ability to isolate the human gene for FVIII and transfect cells to produce human rFVIII. The first rFVIII products, which are native full-length FVIII molecules, have proved to have an excellent efficacy and safety profile in patients with hemophilia A. Initial concerns about a potential increased inhibitor formation have not been confirmed so far but long-term pharmacovigilance of inhibitor formation is still ongoing. To date, no transmission of hepatitis or human immunodeficiency virus (HIV) attributable to rFVIII products has been reported. However, a theoretical risk of transmission of infectious disease does exist as long as nonsynthetic proteins are used during the production process. The next-generation native rFVIII has been developed to minimize the exposure of patients to animal or human plasma-derived proteins. This has been achieved through major changes to the process of production of rFVIII from baby hamster kidney cells (BHK). This change has included the introduction of a solvent/detergent step and, of more importance, the introduction of a purification procedure without using albumin as a stabilizer. Finally, the rFVIII (BHK) is formulated using sucrose as the final stabilizer to produce the sucrose formulated rFVIII referred to as rFVIII-FS. This article summarizes the recently published pharmacokinetic, safety, and efficacy data for the native rFVIII-FS and compares its clinical profile with that of the first-generation rFVIII.

Animals↗

PubMed1999

Expression and molecular characterization of the growth hormone receptor in canine mammary tissue and mammary tumors.

GH synthesis has been documented in canine mammary tissue and mammary tumors. In the present report, the characteristics of the GH receptor (GHR) are studied in these tissues. First, using immunohistochemistry, GHR was found to be present throughout normal and tumorous mammary tissues, being localized in epithelial and myoepithelial/spindle cell components and in the activated fibroblasts of desmoplastic tumor stroma. GHR expression seemed to be down-regulated only in terminally differentiated alveoli in normal tissue. GHR immunoreactivity in particular mammary (adeno)carcinomas was heterogenous. Second, the canine GHR was characterized at the molecular level. Northern blot analysis revealed a major GHR transcript of approximately 4.2 kb. The coding sequence of the canine GHR shows extensive homology with the GHR of several species. Seminested RT-PCR (using primers annealing in exons 4-5, exon 6, and exon 9) generated, next to the primary product, four different products in mammary tissues and the canine mammary tumor cell line CMT-U335, which seemed to be alternative GHR transcripts. These alternative GHR transcripts were characterized by exon 8 skipping, exon 7 skipping, and use of alternative splice donor and acceptor sites. Especially, the transcript that is missing exon 8 may encode a GH binding protein. In most malignant mammary samples, only the primary transcript was present; and alternative transcripts could not be detected. The absence of alternative GHR transcripts in mammary carcinomas, and thus putative inhibitors of GH-induced signal transduction, may contribute to enhanced sensitivity of malignant tumors to GH.

Alternative Splicing↗

PubMed2004

Development of biometric DNA ink for authentication security.

Among the various types of biometric personal identification systems, DNA provides the most reliable personal identification. It is intrinsically digital and unchangeable while the person is alive, and even after his/her death. Increasing the number of DNA loci examined can enhance the power of discrimination. This report describes the development of DNA ink, which contains synthetic DNA mixed with printing inks. Single-stranded DNA fragments encoding a personalized set of short tandem repeats (STR) were synthesized. The sequence was defined as follows. First, a decimal DNA personal identification (DNA-ID) was established based on the number of STRs in the locus. Next, this DNA-ID was encrypted using a binary, 160-bit algorithm, using a hashing function to protect privacy. Since this function is irreversible, no one can recover the original information from the encrypted code. Finally, the bit series generated above is transformed into base sequences, and double-stranded DNA fragments are amplified by the polymerase chain reaction (PCR) to protect against physical attacks. Synthesized DNA was detected successfully after samples printed in DNA ink were subjected to several resistance tests used to assess the stability of printing inks. Endurance test results showed that this DNA ink would be suitable for practical use as a printing ink and was resistant to 40 hours of ultraviolet exposure, performance commensurate with that of photogravure ink.

Algorithms↗