Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

Reciprocal subtraction differential RNA display: An efficient and rapid procedure for isolating differentially expressed gene sequences

A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5′ leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Identification of human DNA helicase V with the far upstream element-binding protein

The properties of human DNA helicase V (HDH V) were studied in greater detail following an improved purification procedure. From 450 g of cultured cells, <0.1 mg of pure protein was isolated. HDH V unwinds DNA unidirectionally by moving in the 3′ to 5′ direction along the bound strand in an ATP- and Mg2+-dependent fashion. The enzyme is not processive and can also unwind partial RNA–RNA duplexes such as HDH IV and HDH VIII. The Mr determined by SDS–PAGE (66 kDa) corresponds to that measured under native conditions, suggesting that HDH V exists as a monomer in the nucleus. Microsequencing of the purified HDH V shows that this enzyme is identical to the far upstream element-binding protein (FBP), a protein that stimulates the activity of the c-myc gene by binding specifically to the ‘FUSE’ DNA region localized upstream of its promoter. The sequence of HDH V/FBP contains RGG motifs like HDH IV/nucleolin, HDH VIII/G3BP as well as other human RNA and DNA helicases identified by other laboratories.

Outer Dense Fiber 2 Is a Widespread Centrosome Scaffold Component Preferentially Associated with Mother Centrioles: Its Identification from Isolated Centrosomes

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ∼90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.

Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries.

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.

Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability.

We have developed a modified rhodamine (Rho) staining procedure to study uptake and efflux in murine hematopoietic stem cells. Distinct populations of Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could be discriminated. Sorted Rho- cells were subjected to a second Rho staining procedure with the P-glycoprotein blocking agent verapamil (VP). Most cells became Rho positive [Rho-/Rho(VP)+ cells] and some remained Rho negative [Rho-/Rho(VP)- cells]. These cell fractions were characterized by their marrow-repopulating ability in a syngeneic, sex-mismatch transplantation model. Short-term repopulating ability was determined by recipient survival for at least 6 weeks after lethal irradiation and transplantation--i.e., radioprotection. Long-term repopulating ability at 6 months after transplantation was measured by fluorescence in situ hybridization with a Y-chromosome-specific probe, by graft function and recipient survival. Marrow-repopulating cells were mainly present in the small Rho- cell fraction. Transplantation of 30 Rho- cells resulted in 50% radioprotection and > 80% donor repopulation in marrow, spleen, and thymus 6 months after transplantation. Cotransplantation of cells from both fractions in individual mice directly showed that within this Rho- cell fraction, the Rho-/Rho(VP)+ cells exhibited mainly short-term and the Rho-/Rho(VP)- cells exhibited mainly long-term repopulating ability. Our results indicate that hematopoietic stem cells have relatively high P-glycoprotein expression and that the cells responsible for long-term repopulating ability can be separated from cells exhibiting short-term repopulating ability, probably by a reduced mitochondrial Rho-binding capacity.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

mecA locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone

An emerging public health phenomenon is the increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of mecA in combination with a single nucleotide polymorphism (SNP) defined by the S. aureus multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms-interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of mecA were determined. The CA-MRSA isolates were found to contain a truncated mecA downstream region consisting of mecA-HVR-IS431 mec-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different mecA downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the S. aureus MLST database, and the arcC 2726 polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated mecA downstream region in combination with the interrogation of arcC position 272 provided an unambiguous identification of WSPP isolates.

Re-admission to intensive care: Identification of risk factors

Background and Purpose. The re-admission of patients to intensive care is associated with increased morbidity, mortality, loss of morale for patients and family, and increased health costs. The aim of the present study was to identify factors which place patients at a higher risk of re-admission to intensive care. Method. A prospective study of patients who were re-admitted to a 22-bed tertiary level intensive care facility within a 12-month period. Data were kept on every patient re-admitted to intensive care, including standard demographic data, initial admission diagnosis, co-morbidities, re-admission diagnosis, mobility on discharge, secretions, airway, chest X-ray, PaCO2, PaO2, PaO2/FiO2and time of discharge. Subjects included 74 patients who had been re-admitted to intensive care in a 12-month period and a comparison group of patients who were not re-admitted to intensive care. A cross-tabs procedure was initially used to estimate maximum likelihood. Significant factors with an value of 65 years (p

Am I being treated fairly by my leaders? The roles of group identification and self-other comparisons in judgements of procedural justice

The current experiment focuses on the roles of social identity and social comparison in perceptions of procedural justice. Participants are randomly allocated to conditions in a 2 (whether the participant has the opportunity to voice an opinion), X 2 (whether the comparison other has the opportunity to voice an opinion), X 2 (whether the comparison other is an ingroup or an outgroup member), between subjects design. Participants are then asked to report the extent to which they perceive the procedure they are involved in to be fair. It is predicted that participants will have a strong feeling of procedural unfairness when they are not given an opportunity by the leader to voice their opinion, but learn that their comparison other is given that opportunity. It is also predicted that the feeling of unfairness should be stronger when the comparison other is an outgroup rather than an ingroup member. Additionally, participants receiving a fair treatment may regard the procedure as fair when their outgoup comparison other receives an unfair treatment. Results support these predictions and reveal that how people make judgments of procedural justice through social comparison is qualified by the social identities of the parties involved.

Model of Active Structural Monitoring and Decision-making for Dynamic Identification of Buildings, Monuments and Engineering Facilities

Structural monitoring and dynamic identification of the manmade and natural hazard objects is under consideration. Math model of testing object by set of weak stationary dynamic actions is offered. The response of structures to the set of signals is under processing for getting important information about object condition in high frequency band. Making decision procedure into active monitoring system is discussed as well. As an example the monitoring outcome of pillar-type monument is given.

Individual identification using electrocardiogram morphology

The use of electrocardiogram as biometric has raised attention in the last decade and a wide variety of ECG features were explored to verify the feasibility of such a signal. In this work the authors aim to describe a simple template based approach to the electrocardiographic biometric identification using the morphology of individual's heartbeat. The developed algorithm was tested on different recordings made available in the Physionet public database Fantasia: two different sets of heartbeats were extracted from individual recordings one was used for the template building while the second for the tests. The performances of the algorithm are encouraging with a true acceptance rate of 99.4%, however, the procedure needs to be tested on different recordings of the same individual, or during the course of a whole day or physical activity. © 2013 IEEE.

Improving the identification of high-z Herschel sources with position priors and optical/NIR and FIR/mm photometric redshifts

We present preliminary results about the detection of high redshift (U)LIRGs in the Bullet cluster field by the PACS and SPIRE instruments within the Herschel Lensing Survey (HLS) Program. We describe in detail a photometric procedure designed to recover robust fluxes and deblend faint Herschel sources near the confusion noise. The method is based on the use of the positions of Spitzer/MIPS 24 μm sources as priors. Our catalogs are able to reliably (5σ) recover galaxies with fluxes above 6 and 10 mJy in the PACS 100 and 160 μm channels, respectively, and 12 to 18 mJy in the SPIRE bands. We also obtain spectral energy distributions covering the optical through the far-infrared/millimeter spectral ranges of all the Herschel detected sources, and analyze them to obtain independent estimations of the photometric redshift based on either stellar population or dust emission models. We exemplify the potential of the combined use of Spitzer position priors plus independent optical and IR photometric redshifts to robustly assign optical/NIR counterparts to the sources detected by Herschel and other (sub-)mm instruments.

Footbridge system identification using wireless inertial measurement units for force and response measurements

With the main focus on safety, design of structures for vibration serviceability is often overlooked or mismanaged, resulting in some high profile structures failing publicly to perform adequately under human dynamic loading due to walking, running or jumping. A standard tool to inform better design, prove fitness for purpose before entering service and design retrofits is modal testing, a procedure that typically involves acceleration measurements using an array of wired sensors and force generation using a mechanical shaker. A critical but often overlooked aspect is using input (force) to output (response) relationships to enable estimation of modal mass, which is a key parameter directly controlling vibration levels in service.

This paper describes the use of wireless inertial measurement units (IMUs), designed for biomechanics motion capture applications, for the modal testing of a 109 m footbridge. IMUs were first used for an output-only vibration survey to identify mode frequencies, shapes and damping ratios, then for simultaneous measurement of body accelerations of a human subject jumping to excite specific vibrations modes and build up bridge deck accelerations at the jumping location. Using the mode shapes and the vertical acceleration data from a suitable body landmark scaled by body mass, thus providing jumping force data, it was possible to create frequency response functions and estimate modal masses.

The modal mass estimates for this bridge were checked against estimates obtained using an instrumented hammer and known mass distributions, showing consistency among the experimental estimates. Finally, the method was used in an applied research application on a short span footbridge where the benefits of logistical and operational simplicity afforded by the highly portable and easy to use IMUs proved extremely useful for an efficient evaluation of vibration serviceability, including estimation of modal masses.