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At least 1,099 records · Page 61Linked to original sources

Identification of a new gene product of PKIbeta by HPLC-ESIMS.

An identification method using high-performance liquid chromatography combined with electrospray mass spectrometry (HPLC-ESIMS) has been developed to verify an expressed gene product of kinase inhibitor (PKIbeta). This protein was expressed in this university for the first time from a newly cloned gene in the cDNA library of human fetal brain. The measured MW (8468.9 Da) of PKIbeta-78 was consistent with expectations. The gene product of PKIbeta-78 was monitored by ESIMS to ensure there was no mis-expressed PKIbeta-70 in the process of gene engineering. The peptide mapping of PKIbeta-78 and its partial sequence were, furthermore, determined. By database searching based on the experimental MWs and partial sequences provided, it was verified that this gene product is a new protein. The pseudosubstrate site and leucine-rich site for the function region of PKIbeta-78 are also confirmed.

Amino Acid Sequence↗

Proving the concept of a data broker as an emergent alternative to supra-enterprise EPR systems.

Electronic Patient Records systems configured into large enterprise models have become the assumed best route forward. In England, as in several other countries, this has expanded to a major meta-enterprise procurement programme. However, concerns are raised that such systems lack user ownership, and experience from other sectors shows difficulties with large enterprise systems. At a time of great change and once again shifting organizations, is this move simply building large and ponderous edifices with unstable materials? Latest software engineering research is now demonstrating the potential of an alternative model, enabling trusted information brokers to search out in real time at point of use data held in registered local and departmental systems. If successful, this could enable a new and less cumbersome paradigm. The data could move where needed whatever the service configuration. A concept demonstrator has been built set in the context of health and social care in England. It is important for all technological support to the health sector to be reviewed as new technologies emerge so as to identify and exploit new opportunities, and the results of this 3 year project show that the health record information broker route merits further investigative research.

Efficiency, Organizational↗

Nerve regeneration inside fresh skeletal muscle-enriched synthetic tubes: a laser confocal microscope study in the rat sciatic nerve model.

The search for good conduits for bridging nerve defects is a major challenge of today's tissue engineering research. In this paper we report on a laser confocal microscope study on early nerve regeneration inside a tissue engineered graft made by a poly(DLLA-epsilon-CL) conduit enriched with fresh skeletal muscle. The same biodegradable tubes filled with PBS solution were used as controls. The conduits were placed to bridge unilateral 1-cm-long rat sciatic nerve defects and analysed 10 days after surgery. Results showed that inside the muscle-enriched tubes axon regeneration, labelled by means of anti-neurofilament antibody, was already begun, whilst no axon regeneration was detectable along control tubes. In addition, a-GFAP (glial fibrillar acid protein) immuno-labelling of Schwann cells showed that progression inside muscle-enriched tubes, especially from the distal nerve stump, was much more evident than in control conduits. These results suggest that enrichment of synthetic tubes with fresh skeletal muscle promotes axon regeneration and Schwann cell migration in early nerve repair stages.

Absorbable Implants↗

[Conduct of Bacillus subtilis transformation in mice under conditions of immunologic suppression of exogenous DNAase 1 activity].

Bacillus subtilis transformation was conducted in the abdominal cavity of mice. The frequency of transformation was considerable decreased when bovine DNA-ase 1 (3-- 5microgram) was injected intraperitoneally to these animals. Immune rabbit gamma-globulins containing antibodies to bovine DNA-ase 1 inhibited in vivo the activity of DNA-ase 1, protected transforming DNA from the hydrolyzing effect of this enzyme. This model can be utilized in search for ways to preserve DNA injected into the animal organism for the purpose of genetical engineering.

Animals↗

Structure-based stability engineering of the mouse IgG1 Fab fragment by modifying constant domains.

A semi-rational approach based on structural data was exploited in a search for CH1 and CL domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the CH1 and CL domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the CH1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the CH1 and CL domains and the hydrophobic core regions of both CH1 and CL. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the CH1/CL interface was increased by the replacement of the hydrophilic Thr178 in the CL domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the CH1/CL interface were advantageous for the overall thermodynamic stability of the Fab fragment.

Amino Acid Sequence↗

A systematic approach to standardizing the visual appearance of endometriotic lesions for artificial intelligence recognition.

INTRODUCTION: Numerous studies have shown that the diagnostic performance and reproducibility of visual recognition of endometriosis during laparoscopy are poor. The use of artificial intelligence (AI) seems relevant for exhaustive lesion recognition. Standardization of the visual classification of lesions, in the form of an ontology, is an essential prerequisite to enable medical experts to annotate surgical data consistently and subsequently allow engineers to train and build an artificial intelligence tool for endometriosis recognition. MATERIAL AND METHODS: A systematic search was conducted in the MEDLINE (via PubMed), EMBASE, and the Cochrane Library databases up to May 2022, aiming to identify studies describing the laparoscopic visual appearance of superficial endometriosis, endometriomas, and deep infiltrating endometriosis. The accumulated data in the literature concerning the visual appearance of the different forms of endometriosis were used to create an ontology that could be used for artificial intelligence applications. RESULTS: Out of 932 articles screened, 35 studies were selected based on the inclusion criteria of human subjects with histologically confirmed endometriosis lesions visualized via laparoscopy. The selected studies were reviewed to develop a visual ontology of endometriosis lesions observed via laparoscopy. The lesions were categorized into 4 classes and further subdivided into 11 subclasses: superficial (black, red, white, or subtle), adhesions (dense or filmy), deep (obliteration, retraction, or deformation), and ovarian (endometrioma or chocolate fluid). The positive predictive value (PPV) varied across lesion types: black lesions (PPV 47%-97%), red lesions (PPV 33%-100%), white lesions (PPV 20%-81%), and ovarian endometriosis (PPV 42%-98%). Nonspecific lesions such as adhesions (PPV 16%-50%) and subtle superficial lesions (PPV 0%-67%) presented lower PPVs. Deep endometriosis lesions, often buried within organs, required indirect signs (obliteration, retraction, deformation) for identification. CONCLUSIONS: The visual ontology proposed in this systematic search could facilitate the detection and classification of endometriosis lesions using artificial intelligence. This study highlights the challenges of reaching a consensus on lesion recognition and classification in AI projects due to the diverse visual presentations of endometriosis.

Humans↗

The biological terraforming of Mars: planetary ecosynthesis as ecological succession on a global scale.

Mars is bitterly cold and dry, but robotic spacecraft have returned abundant data that indicate Mars once had a much warmer and wetter climate in the past. These data, the basis of the search for past or present life on Mars, suggest the possibility of returning Mars to its previous climate by global engineering techniques. Greenhouse gases, such as perfluorocarbons, appear to be the best method for warming Mars and increasing its atmospheric density so that liquid water becomes stable. The process of making Mars habitable for terrestrial organisms is called terraforming or planetary ecosynthesis. The process of introducing terrestrial ecosystems to Mars can be compared with a descent down a high mountain. Each drop in elevation results in a warmer, wetter climate and more diverse biological community. Beginning with a polar desert, the sequence of ecosystems passes through tundra, boreal forest, and temperate ecosystems where moisture determines the presence of desert, grassland, or forest. This model suggests a sequence for the introduction of ecosystems to Mars and the communities to search for potential colonizing species for Mars.

Ecosystem↗

Technical development of PubMed interact: an improved interface for MEDLINE/PubMed searches.

BACKGROUND: The project aims to create an alternative search interface for MEDLINE/PubMed that may provide assistance to the novice user and added convenience to the advanced user. An earlier version of the project was the 'Slider Interface for MEDLINE/PubMed searches' (SLIM) which provided JavaScript slider bars to control search parameters. In this new version, recent developments in Web-based technologies were implemented. These changes may prove to be even more valuable in enhancing user interactivity through client-side manipulation and management of results. RESULTS: PubMed Interact is a Web-based MEDLINE/PubMed search application built with HTML, JavaScript and PHP. It is implemented on a Windows Server 2003 with Apache 2.0.52, PHP 4.4.1 and MySQL 4.1.18. PHP scripts provide the backend engine that connects with E-Utilities and parses XML files. JavaScript manages client-side functionalities and converts Web pages into interactive platforms using dynamic HTML (DHTML), Document Object Model (DOM) tree manipulation and Ajax methods. With PubMed Interact, users can limit searches with JavaScript slider bars, preview result counts, delete citations from the list, display and add related articles and create relevance lists. Many interactive features occur at client-side, which allow instant feedback without reloading or refreshing the page resulting in a more efficient user experience. CONCLUSION: PubMed Interact is a highly interactive Web-based search application for MEDLINE/PubMed that explores recent trends in Web technologies like DOM tree manipulation and Ajax. It may become a valuable technical development for online medical search applications.

Consumer Behavior↗

Automated design of degenerate codon libraries.

Degenerate codon libraries are frequently used in protein engineering and evolution studies but are often limited to targeting a small number of positions to adequately limit the search space. To mitigate this, codon degeneracy can be limited using heuristics or previous knowledge of the targeted positions. To automate design of libraries given a set of amino acid sequences, an algorithm (LibDesign) was developed that generates a set of possible degenerate codon libraries, their resulting size, and their score relative to a user-defined scoring function. A gene library of a specified size can then be constructed that is representative of the given amino acid distribution or that includes specific sequences or combinations thereof. LibDesign provides a new tool for automated design of high-quality protein libraries that more effectively harness existing sequence-structure information derived from multiple sequence alignment or computational protein design data.

Algorithms↗

Engineering T-cells with antibody-based chimeric receptors for effective cancer therapy.

The combination of power and specificity inherent within the immune system make its manipulation an attractive prospect for the development of novel anticancer therapies. However, tumors are frequently poorly recognized by the immune system, and intrinsic controls limit the benefit from active immunization. These and other issues have proved major challenges in the search for effective cancer immunotherapy. Recent advances in the understanding of the immune system and the development of methods to manipulate it have led to the point where engineered T-cell therapy can be tested in the clinical setting with a realistic chance of success. These advances are considered and potential future clinical and scientific issues involved in the successful development of effective, engineered T-cell therapy are examined.

Cell Proliferation↗

A search for doxycycline-dependent mutations that increase Drosophila melanogaster life span identifies the VhaSFD, Sugar baby, filamin, fwd and Cctl genes.

BACKGROUND: A P-type transposable element called PdL has been engineered with a doxycycline-inducible promoter directed out through the 3' end of the element. Insertion of PdL near the 5' end of a gene often yields doxycycline-dependent overexpression of that gene and a mutant phenotype. This functional genomics strategy allows for efficient screening of large numbers of genes for overexpression phenotypes. RESULTS: PdL was mobilized to around 10,000 new locations in the Drosophila melanogaster genome and used to search for genes that would extend life span when overexpressed. Six lines were identified in which there was a 5-17% increase in life span in the presence of doxyxcycline. The mutations were molecularly characterized and in each case a gene was found to be overexpressed using northern blots. Two genes did not have previously known phenotypes and are implicated in membrane transport: VhaSFD encodes a regulatory subunit of the vacuolar ATPase proton pump (H+-ATPase), whereas Sugar baby (Sug) is related to a maltose permease from Bacillus. Three PdL mutations identified previously characterized genes: filamin encodes the homolog of an actin-polymerizing protein that interacts with presenilins. four wheel drive (fwd) encodes a phosphatidylinositol-4-kinase (PI 4-kinase) and CTP:phosphocholine cytidylyltransferase-l (Cctl) encodes the rate-limiting enzyme in phosphatidylcholine synthesis. Finally, an apparently novel gene (Red herring, Rdh) was found in the first intron of the encore gene. CONCLUSIONS: Screening for conditional mutations that increase Drosophila life span has identified genes implicated in membrane transport, phospholipid metabolism and signaling, and actin cytoskeleton organization.

1-Phosphatidylinositol 4-Kinase↗

Consequences of molecular engineering enhanced DNA binding in a DNA repair enzyme.

Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.

Base Sequence↗

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.

Amino Acid Sequence↗

Selective pressures on RNA hairpins in vivo and in vitro.

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences.

Evolution, Molecular↗

Eradication of pathogenic beta-catenin by Skp1/Cullin/F box ubiquitination machinery.

The use of Skp1/Cull 1/F box (SCF) ubiquitin-conjugation machinery as a potential knockout tool offers a means of eradicating disease-causing proteins. Here a chimeric F box protein (CFP) was engineered to achieve selective eradication of pathogenic beta-catenin in colorectal cancer. We show that CFP specifically searches for and subsequently links the abnormal beta-catenin to the cellular SCF ubiquitination complex. Introduction of the CFP to colorectal cancer cells induced targeted ubiquitination and proteolytic degradation of nuclear and cytoplasmic free beta-catenin while preserving its normal cellular adhesion counterpart. Elimination of pathogenic beta-catenin suppressed constitutive Wingless/Wnt signaling and inhibited in vitro and in vivo tumor cell growth. This study demonstrates a practical utility of a SCF-based knockout system as a tool in targeting an abnormal protein that affects growth and transformation.

Cell Division↗

Bioelectrical impedance analysis in body composition measurement: National Institutes of Health Technology Assessment Conference Statement.

The objective of this conference was to provide physicians with a responsible assessment of bioelectrical impedance analysis (BIA) technology for body composition measurement. Participants were a non-Federal, nonadvocate, 13-member panel representing the fields of nutrition, pediatrics, surgery, public health, biomedical engineering, epidemiology, and biostatistics. In addition, 20 experts in nutrition, pediatrics, metabolism, biomedical engineering, physiology, and epidemiology presented data to the panel and a conference audience of 220. The literature was searched through Medline and an extensive bibliography of references was provided to the panel and the conference audience. Experts prepared abstracts with relevant citations from the literature. Scientific evidence was given precedence over clinical anecdotal experience. The panel, answering predefined questions, developed their conclusions based on the scientific evidence presented in open forum and the scientific literature. The panel composed a draft statement that was read in its entirety and circulated to the experts and the audience for comment. Thereafter, the panel resolved conflicting recommendations and released a revised statement at the end of the conference. The panel finalized the revisions within a few weeks after the conference. The panel concluded that BIA provides a reliable estimate of total body water under most conditions. It can be a useful technique for body composition analysis in healthy individuals and in those with a number of chronic conditions such as mild-to-moderate obesity, diabetes mellitus, and other medical conditions in which major disturbances of water distribution are not prominent. BIA values are affected by numerous variables including body position, hydration status, consumption of food and beverages, ambient air and skin temperature, recent physical activity, and conductance of the examining table. Reliable BIA requires standardization and control of these variables. A specific, well-defined procedure for performing routine BIA measurements is not practiced. Therefore, the panel recommends that a committee of appropriate scientific experts and instrument manufacturers be formed with the goal of setting instrument standards and procedural methods.

Body Composition↗

Fly models of Huntington's disease.

Can Drosophila models be engineered that accurately reflect Huntington's disease (HD) and other neurological diseases and can they contribute to the search for treatments and cures? A number of publications seem to provide a resounding yes to that question. Here we seek to review some of the salient features of these models.

Animals↗