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PubMed2004

Anti-Stokes Raman spectrometry with 1064-nm excitation: an effective instrumental approach for field detection of explosives.

Anti-Stokes Raman spectra of 28 explosive materials were obtained with 1064-nm excitation using fiber-optic sampling and a dispersive spectrograph equipped with a charge-coupled device (CCD) array detector. By using a silicon CCD detector, anti-Stokes features could clearly be observed for the majority of samples from -250 to -1650 cm(-1). Using the fiber-optic probe, spectra were routinely obtained from samples positioned up to twelve meters from the spectrograph within 240 s. The utility of an anti-Stokes correction routine is demonstrated, which routine allowed anti-Stokes spectra measured with 1064-nm excitation to be successfully searched and identified against libraries of Stokes spectra obtained using a Fourier transform (FT) Raman system equipped with a 1064-nm Nd:YAG laser.

Journal Article↗
PubMed1999

[Isolating candidate inserted fragment from positive fused phage clones using quantitative PCR].

OBJECTIVE: To isolate quickly and exactly the specific inserted fragment from fused phage clones which were obtained from cDNA library by hybridization. METHODS: According to the amplification principle of quantitative polymerase chain reaction (PCR) and based on the difference of original template quantity,target cDNA fragment was isolated and identified by two PCRs. RESULTS: A positive clone with specific cDNA fragment of HumGT-H1 gene was obtained from a two-phage fused clone by using this method,and the inserted fragment was verified to be the 5' cDNA sequence of HumGT-H1,1.9kb in length. So another hybridization screening is not necessary. CONCLUSION: The method presented is effective and rapid in gene cloning and can greatly save time and materials.

Bacteriophages↗
PubMed1998

Cystoscopic findings consistent with interstitial cystitis in normal women undergoing tubal ligation.

PURPOSE: We test the hypothesis that women without chronic pelvic pain or irritative voiding symptoms do not demonstrate petechial hemorrhages known as glomerulations that are characteristic of patients with interstitial cystitis. MATERIALS AND METHODS: A prospective cohort design was used for examination with cystoscopy and bladder distention of 20 asymptomatic women undergoing tubal ligation. Cystoscopy with the patient under general anesthesia was performed to inspect the bladder mucosa before and after distention at 70 cm. of water pressure for 2 or 6 minutes. Photographs of the right, posterior and left of the bladder surfaces taken before and after the distention were scored on a scale of 1 to 5 using a panel of standards. Five urologists blinded to the source of individual photographs independently evaluated 120 research images interspersed with 46 other pictures from a library containing images from 19 symptomatic patients with and without interstitial cystitis. RESULTS: A total of 20 normal women with a mean age plus or minus standard deviation of 29+/-6 years consented to participate in this trial during laparoscopic tubal ligation. Photographs of bladder sites before and after distention with 890+/-140 ml. were scored as 1.4+/-0.3 (before distention) and 3.1+/-1.1 (after distention) on the scale of 1 to 5. The increase in scores following distention in normal subjects was seen to the same degree and in the same proportion as in patients with symptoms of interstitial cystitis (8 of 19 symptomatic patients in this series met current diagnostic criteria for interstitial cystitis). Slight but significant differences were seen among sites in the bladder but not between 2 and 6-minute distention durations. CONCLUSIONS: Bladder mucosal lesions characteristically associated with irritative voiding symptoms and pelvic pain in patients diagnosed with interstitial cystitis were observed in asymptomatic women.

Adult↗
PubMed1984

Fair use versus fair return: copyright legislation and its consequences.

The purpose of this article is to examine the ramifications of legislative recognition of the concept of fair use in the Copyright Act of 1976. The fair use concept, while of small consequence in its normative origins, has turned out to be the foundation of the most perplexing and divisive issues in the new legislative guidelines governing copyright. Legislative recognition of the concept of fair use, coupled with enormous growth of a new technology--extending from xerography to on-line database systems--creates de facto exemptions to both the intent and content of new copyright guidelines. The issue is not one of limiting use or suppressing information, but of mechanisms for safeguarding the rights of copyright holders, be they authors or publishers, and insuring the free flow of information by providing a proper return on both intellectual creativity and capital expenditures. The authors argue that the elimination, or at least curtailment of fair use doctrine, coupled with an increase in technological approaches to reporting of secondary use of copyrighted material, will benefit all sections of the knowledge industry. Authors will receive proper royalties on use; publishers will be able to sell more books and journals at lower prices; and librarians will be liberated from extensive chores such as monitoring usage or determining fee schedules and transferences. The issue is one of fair return--an issue obscured and ultimately subverted by fair use.

Copyright↗
PubMed2004

Optimized RNA amplification using T7-RNA-polymerase based in vitro transcription.

The use of expression profiling to explore a cell's transcriptional landscape has exploded in recent years. In many cases, however, the very limited amount of starting material poses a major problem, making the amplification of the isolated RNA obligatory. The most prominent amplification method used was developed by the Eberwine lab in 1990: cDNA synthesis is started with an oligo(dT) primer containing a T7 RNA polymerase promoter. After second-strand synthesis RNA is transcribed in vitro using T7 RNA polymerase. It has been demonstrated that antisense RNA amplification not only preserves the fidelity of RNA-based microarray analysis but even improves the sensitivity. In our aim to improve the yield of in vitro transcription reactions and to facilitate the use of amplified RNA for the construction of cDNA libraries we tested a series of T7 primers with different 3' flanking sequences containing restriction sites. In addition we tested the impact of different DNA polymerases used for synthesizing the templates on the efficiency of the in vitro transcription reaction. A total of 28 different oligo(dT)-T7 promoter primers were tested. Two of them showed a dramatically increased yield of RNA from the in vitro transcription reaction. The combination of the improved second-strand synthesis with the new T7 primer increased the RNA yield 60-fold compared to the yield of standard procedures.

3' Flanking Region↗
PubMed1999

Gas chromatographic flow method for the preconcentration and simultaneous determination of antioxidant and preservative additives in fatty foods.

A partially automated gas chromatographic method for the simultaneous determination of antioxidants (tert.-butyl-4-hydroxyanisole, 2,6-di-tert.-butyl-p-hydroxytoluene, tert.-butylhydroquinone, alpha-tocopherol and alpha-tocopheryl acetate) and preservatives (sorbic acid, benzoic acid and their esters) in fatty foods without derivatization is reported. About 95% of triglycerides from lipid material are avoided by manually extracting the samples with a mixture of solvents and the remaining by using a continuous solid-phase extraction system. By using aqueous sample solutions (in 0.1 M HNO3), XAD-2 sorbent and selective elution with 200 microliters of 2-propanol, only ca. 0.03% of total triglycerides present in the original sample remains in the final extract. Manual injection of the extract into a GC-flame ionization detection allows the additives to be determine at concentrations in the range 0.5-100 micrograms/ml with a high precision (ca. 3%). Mass spectrometry in the conventional electron impact mode, in conjunction with library search, permits the unequivocal identification of all the additives. Margarine, oil, cheese, pâté and sauce samples were analysed in this way, all with good results.

Antioxidants↗
PubMed1987

Semisynthetic human [[3H2]Phe1]proinsulin.

Biosynthetic human proinsulin (obtained by recombinant DNA techniques) was used as the starting material for the preparation, by semisynthetic methods, of [3H]proinsulin with the label at the N-terminal phenylalanine residue. The labelled proinsulin was characterized by its retention time on reversed-phase h.p.l.c., by polyacrylamide-gel electrophoresis, by the time course of its enzymic conversion into insulin and by chromatographic analysis after extensive proteolytic degradation. The specific radioactivity of the product was 5 Ci/mmol. Experimental details of the preparation of human [[3H]Phe1]proinsulin, the isolation of this product by isocratic h.p.l.c. and gel filtration, and further characterization of protein intermediates have been deposited as supplement SUP 50138 (12 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on prepayment [see Biochem. J. (1987) 241, 5].

Chromatography, High Pressure Liquid↗
PubMed2005

[Screening of HBeAgTP interacting proteins in hepatocytes with yeast-two hybrid technique].

OBJECTIVE: To screen proteins in hepatocytes interacting with HBeAg transactivated protein (HBeAgTP) with yeast-two hybrid technique for investigating the biological functions of HBeAgTP. METHODS: Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HBeAg. The HBeAgTP gene was amplified by polymerase chain reaction (PCR) and HBeAgTP bait plasmid was constructed with yeast-two hybrid system 3, and then transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, the results were analyzed by bioinformatics. RESULTS: HBeAgTP gene was successfully cloned and expressed in yeast cells. Fifteen genes in twenty-four positive colonies were obtained using yeast-two hybrid technique. CONCLUSION: HBeAgTP conjugated protein genes were successfully cloned, along with the genes involved in transcription and translation of proteins, immunoloregulation, materials and energy metabolism in vivo.

Hepatitis B e Antigens↗
PubMed2004

A problem-based e-Learning prototype system for clinical medical education.

The purpose of this system is to establish virtual medical school (VMS) as the platform of e-learning center, which integrates collaborative and self-directed learning environment by virtual group, classroom and library, and automatically converts valuable clinical case from Hospital Information System (HIS) database into virtual patient by online authoring tools for problem-based e-learning. In this system, the VMS provides a problem-based e-learning environment, and utilizes HIS to capture and store valuable clinical cases. All medical students and residents now have the opportunity to learn from these typical cases online. The VMS at National Taiwan University has the potential to develop into a national medical education network for the meditation and provision of comprehensive medical resources. The system will use the international standard SCORM 1.2 to develop teaching material and assist with the HL7 v2.4, CDA v1.0 standards to connect Electronic Medical Record (EMR) systems in the hospital. It can provide resources sharing among medical centers by using high transportation ability of Grid Computing integrated with the broadband video platform, Access Grid, and personal multiple point videoconference platform, Multi-video, to popularize the application of e-learning in clinical medical education.

Clinical Medicine↗
PubMedDate not supplied

Identification of chromosomal rearrangements in the human myeloid leukemia cell line GF-D8 by dual-colour fluorescence in situ hybridization.

Fluorescence In Situ Hybridization (FISH) studies with chromosome-specific libraries and repetitive probes were performed on the human acute myeloid leukemia cell line GF-D8 in order to define the complex chromosomal rearrangements observed by conventional cytogenetic analysis. Two-colour FISH with whole chromosome painting probes 8 and 11 showed that the add(8) chromosome had an 11-derived region inserted at q24, whereas the add(11) chromosome had an 8-derived region translocated onto q23. It also demonstrated that no normal chromosome 11 is present in GF-D8 cells, since a translocation involving chromosomes 11 and 17q was detected in addition to the add(11). The der(7) chromosome with extra material in its long arm, identified by QFQ and GTG banding, turned out to have a chromosome 15-derived segment translocated to q22. The deletion of 7q was proved to be interstitial, as the 7q-specific telomere as well as a tiny 7-specific band were observed on an unknown chromosome. Fine mapping of the breakpoints involved in the multiple chromosomal rearrangements of the GF-D8 cell line might provide insights into the mechanisms of myeloid leukaemogenesis.

Chromosome Aberrations↗
PubMed2000

Preservation of gene expression ratios among multiple complex cDNAs after PCR amplification: application to differential gene expression studies.

Comparative gene expression studies are often limited by low availability of tissue and poor quality of extractable mRNA. Collective PCR amplification of minute quantities of mRNA has great potential for overcoming these limitations. However, there remains significant concern about the effects of amplification on the absolute and relative abundance of individual mRNAs that could complicate subsequent gene expression studies. To address this problem, we systematically compared the relative abundance of many specific mRNAs from complex cDNA preparations (from tissue and cultured cells) both before and after amplification by PCR. Our results demonstrated that, as expected, the absolute abundance of different mRNAs in a cDNA library is altered in an unpredictable manner by PCR amplification. However, we found that the concentration ratios of specific mRNAs among different cDNA preparations were routinely well conserved after PCR amplification. Thus, for the purpose of comparative expression studies for specific mRNAs in two (or more) complex cDNAs, PCR-amplified cDNA is equally useful as unamplified cDNA. These results provide a rigorous experimental validation and offer a theoretical treatment to support the utility of PCR amplified cDNA for differential gene expression studies. We conclude that the inherent difficulties in performing differential screening studies such as gene chip and array analyses on limited amounts of biological materials can be overcome by a PCR amplification step without compromising data quality.

Base Sequence↗
PubMed2002

Potential microbiological hazards in the production of refined paper products for food applications.

This study sought to investigate the significance of raw materials (starch-based glues, raw material papers) at different microbiologically critical stages in the manufacturing process of refined paper products. The study examined the occurrence of microorganisms in the process and in end-product samples. Microbiological surveys verified that the production and use of pasteurized starch-based glue was the most important factor threatening the process hygiene and product safety. Subsequently, the production and use of starch-based glue was changed, and a follow-up programme targeting the microbiological quality of glue was developed as part of a hygiene and safety management system. A total of 33 spore-forming bacterial and 15 enterobacterial isolates were ribotyped, and 22 and 10 different ribogroups (ribotypes), respectively, were generated. These isolates from starch-based glue, raw material paper and end products were atypical and, thus, in many cases physiological, chemotaxonomic (FAME) and molecular (partial 16S rDNA) results did not correspond. The most common spore-forming bacteria (55% of the isolates) were Paenibacillus sp. and within this genus several new species were also proposed. The most common enterobacteria (87%) were Enterobacter cloacae and Citrobacter freundii belonging to bacteria in hazard group 2, or species closely related to them. It was demonstrated that the same spore-forming bacteria (ribotypes) were present in both the glue samples and the end products (45% of isolates). All RiboPrint patterns were saved at the VTT identification library for future use.

Adhesives↗
PubMed1998

Broadsheet number 46: Internet for pathologists.

The Internet, and its most user-friendly manifestation, the World Wide Web (WWW), has undergone an exponential growth in the last few years. The volume of medical and pathology related information available has kept pace, making the Internet invaluable tool for the practising pathologist, supplementing traditional hard-copy textbooks and professional journals. E-mail, newsgroups, mailing lists, access to libraries, Medline, atlases, case studies and tutorials are only some of the additional features to which the net offers rapid access from your work-station. To make optimal use of electronic resources, a basic understanding of the hardware, software, communication protocols and information retrieval tools underlying the Internet is desirable. This paper is intended only as a brief introduction to what the Net has to offer to pathology in particular, and medicine in general, it is not meant to supplement the many excellent and more detailed reviews already available (see references). Parallel with the information explosion on the Net, concerns for the quality of posted material and possibility of misinformation have arisen. These issues will be addressed. Website addresses are given in parentheses in the text [e.g.: the website address of the Royal College of Pathologists of Australasia (RCPA) is given as (http:/(/)www.rcpa.edu.au/)] for easy retrieval on your Web browser. Because of the constantly evolving nature of the Web, I apologise in advance if some of the listed sites are no longer current.

Australia↗
PubMed2005

Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes.

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.

Amino Acid Sequence↗
PubMed2003

Novel selective inhibitors of the interaction of individual nuclear hormone receptors with a mutually shared steroid receptor coactivator 2.

Nuclear hormone receptor (NR) signaling, currently a therapeutic target in multiple diseases, involves an ordered series of protein interactions to regulate transcription in response to changing hormone levels. Later steps in the process of ligand-dependent signaling are driven by a highly conserved interaction between the NRs and the steroid receptor coactivators (SRCs) that is effected by a conserved interaction motif (L1XXL2L3), known as an NR box. Using computational design and combinatorial chemistry, we have produced novel alpha-helical proteomimetics of the second NR box of SRC2 that exploit structural differences between human estrogen receptor alpha (hERalpha), human estrogen receptor beta (hERbeta), and human thyroid hormone receptor beta (hTRbeta). The resulting library sequentially replaced each leucine with non-natural side chains. Screening this library using a quantitative competition assay revealed compounds that selectively inhibit the interaction of SRC2-2 with each individual NR in preference to its interaction with the other NR. This approach generated highly selective compounds from one that had no specificity for a particular family member. These compounds represent the first family-member-selective competitive inhibitors of the protein interactions of transcription factors.

Biomimetic Materials↗
PubMed1984

Structure of extrachromosomal circular DNAs containing both the Alu family of dispersed repetitive sequences and other regions of chromosomal DNA.

Small polydisperse circular DNA (spcDNA) isolated from the BSC-1 line of African Green monkey kidney cells was digested with the restriction endonuclease BamHI and cloned in bacteriophage lambda. The resulting library of 25,000 phage was then screened for the presence of the Alu family of short interspersed nucleotide sequences, and four of the 100 Alu-positive clones were characterized. In summary: (1) all four clones contained regions other than Alu that were homologous to the BSC-1 chromosome. Two contained Alu plus unique chromosomal DNA, one contained Alu plus an uncharacterized repetitive chromosomal DNA, and one contained Alu plus both unique and a specific tandemly repeated chromosomal DNA (alpha-satellite); (2) all four clones were derived from extrachromosomal circular DNAs and not from the accidental cloning of a very small amount of contaminating chromosomal material assumed to be present in spcDNA preparations; and (3) one clone represented an intact circular DNA with a restriction endonuclease cleavage map that was a circularly permuted version of its chromosomal homologue.

Animals↗
PubMed2006

[Rational management of antibiotherapy in ORL infections in children: critical review of the best scientific evidences].

INTRODUCTION: Upper respiratory tract infections are the more prevalent infections in childhood and it is important to know the use and overuse of antibiotics. The objective of this article is to make a systematic and critical review of the best scientific evidence in bibliography in order to use antibiotics rationally in otorhinolaryngology (ORL) infections. MATERIAL AND METHODS: Systematic and structured review of the articles regarding antibiotherapy in ORL infections (pharyngitis, tonsillitis, otitis, sinusitis and laryngitis) in childhood published in secondary (Cochrane Collaboration, clinical practice guidelines, health technology assessment database, etc) and primary (bibliographic databases, biomedical journals, books, etc.) publications and critical appraisal by means of methodology of the Evidence-Based Medicine Working Group. We selected the publications with the main scientific evidence in therapeutical articles (clinical trial, systematic review, meta-analysis and clinical practice guideline). DATA SELECTION: study design, population, intervention, outcomes, main results and applicability in clinical practice. RESULTS: The main secondary information is found in The Cochrane Library and in Clinical practice guideline clearinghouses. The documents in Cochrane Library are, basically, clinical trials, systematic reviews and/or meta-analysis, mainly about otitis (13 documents), sinusitis/rhinosinusitis (6), pharyngitis/tonsillitis (3) and nasopharyngitis (3). We found 17 guidelines, mainly for otitis (8 guidelines), pharyngitis/tonsillitis (5), sinusitis (3) and laryngitis (1). Also, we found some relevant articles in Pubmed database, complementary to secondary publications. CONCLUSIONS: There are a high number of quality scientific studies who support an evidence-based decision making in clinical practice about the rational use of antibiotics in ORL infections in childhood, mainly in otitis, tonsillitis and sinusitis. Every decision in this field will have to be based on a systematic appraisal of the best evidence available in the context of the prevailing values and resources available. Doctor's knowledge (mainly in otorhinolaryngologists and pediatricians) of systematic and critical review of the current literature can help to modify prescribing habits about overuse and misuse of antibiotics in ORL infections.

Anti-Bacterial Agents↗
PubMed2006

Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway.

CONCLUSION: Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. OBJECTIVE: To gain understanding of the gene pathways involved in the development of the normal cochlea. MATERIALS AND METHODS: Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. RESULTS: Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.

Animals↗