PubMed Health⌕ Search

Biomedical subjects

J Herold

Publications and source records attributed to J Herold.

At least 19 recordsLinked to original sources
PubMed2003

Sight and comfort: complex procedures in end-stage Stevens-Johnson syndrome.

BACKGROUND: We describe our complex surgical techniques in the management of a patient with end-stage ocular surface disease from Stevens-Johnson syndrome. METHODS AND RESULTS: Her severe discomfort due to absolute ocular dryness in the right eye was successfully treated with submandibular gland autotransplantation. Impending loss of the left eye due to repeated perforation and infection was prevented with a penetrating corneal graft covered by a new modification of a Cutler-Beard-type full-thickness lower lid skin advancement-flap. Visual rehabilitation was achieved by means of osteo-odonto-keratoprosthesis. CONCLUSION: The procedures described allow the preservation of eyes doomed for enucleation, relief from severe discomfort and rehabilitation from corneal blindness. Due to their complex nature they require the collaborative subspecialist surgical skills of opthalmologists and maxillofacial surgeons.

Adult↗
PubMed2001

Validity and reliability of undergraduate performance assessments in an anesthesia simulator.

PURPOSE: To examine the validity and reliability of performance assessment of undergraduate students using the anesthesia simulator as an evaluation tool. METHODS: After ethics approval and informed consent, 135 final year medical students and 5 elective students participated in a videotaped simulator scenario with a Link-Med Patient Simulator (CAE-Link Corporation). Scenarios were based on published educational objectives of the undergraduate curriculum in anesthesia at the University of Toronto. During the simulator sessions, faculty followed a script guiding student interaction with the mannequin. Two faculty independently viewed and evaluated each videotaped performance with a 25-point criterion-based checklist. Means and standard deviations of simulator-based marks were determined and compared with clinical and written evaluations received during the rotation. Internal consistency of the evaluation protocol was determined using inter-item and item-total correlations and correlations of specific simulator items to existing methods of evaluation. RESULTS: Mean reliability estimates for single and average paired assessments were 0.77 and 0.86 respectively. Means of simulator scores were low and there was minimal correlation between the checklist and clinical marks (r = 0.13), checklist and written marks (r = 0.19) and clinical and written marks (r = 0.23). Inter-item and item-total correlations varied widely and correlation between simulator items and existing evaluation tools was low. CONCLUSIONS: Simulator checklist scoring demonstrated acceptable reliability. Low correlation between different methods of evaluation may reflect reliability problems with the written and clinical marks, or that different aspects are being tested. The performance assessment demonstrated low internal consistency and further work is required.

Anesthesiology↗
PubMed2001

Physician response to surveys. A review of the literature.

OBJECTIVE: Physician-specific surveys are a frequently used tool in health services research, but attempts at ensuring adequate response rates are rarely reported. We reviewed literature of survey methodology specific to physician surveys and report those found to be most effective. DATA SOURCES: Studies were identified by searching MEDLINE and PSYCHInfo from 1967 through February 1999. We included all English-language studies that randomized physician survey respondents to an experimental or control group. The authors independently extracted data from 24 studies examining survey methodology of physician-specific surveys. We included Mantel-Haenszel chi-squares comparing treatment groups, if present. If not, these were calculated from study data. RESULTS: Pre-notification of survey recipients, personalizing the survey mailout package, and nonmonetary incentives were not associated with increased response rates. Monetary incentives, the use of stamps on both outgoing and return envelopes, and short questionnaires did increase response rates. Few differences were reported in response rates of phone surveys compared with mail surveys and between the demographics and practice characteristics of early survey respondents and late respondents. CONCLUSIONS: We report some simple approaches that may significantly increase response rates of mail surveys. Surprisingly, the response rates of mail surveys of physicians compared favorably with those from telephone and personal interview surveys. Nonresponse bias may be of less concern in physician surveys than in surveys of the general public. Future research steps include specifically testing the more compelling results to allow for better control of confounders.

Adult↗
PubMed2001

Poliovirus RNA replication requires genome circularization through a protein-protein bridge.

The mechanisms and factors involved in the replication of positive stranded RNA viruses are still unclear. Using poliovirus as a model, we show that a long-range interaction between ribonucleoprotein (RNP) complexes formed at the ends of the viral genome is necessary for RNA replication. Initiation of negative strand RNA synthesis requires a 3' poly(A) tail. Strikingly, it also requires a cloverleaf-like RNA structure located at the other end of the genome. An RNP complex formed around the 5' cloverleaf RNA structure interacts with the poly(A) binding protein bound to the 3' poly(A) tail, thus linking the ends of the viral RNA and effectively circularizing it. Formation of this circular RNP complex is required for initiation of negative strand RNA synthesis. RNA circularization may be a general replication mechanism for positive stranded RNA viruses.

Base Sequence↗
PubMed2001

Viral replicase gene products suffice for coronavirus discontinuous transcription.

We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5' and 3' ends of the human coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of coronavirus-mediated transcription. These data show that the coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.

Coronavirus↗
PubMed2000

Optical cylinder designs to increase the field of vision in the osteo-odonto-keratoprosthesis.

BACKGROUND: The single optical cylinders used in the osteo-odonto-keratoprosthesis (OOKP) are known to produce very small visual fields. Values of 40 degrees are typically quoted. The purpose of this paper is to present designs for new optical cylinders that significantly increase the field of view and therefore improve the visual rehabilitation of patients having an OOKP. METHODS: Computer ray-tracing techniques were used to design and analyse improved one- and two-piece optical cylinders made from polymethyl methacrylate. All designs were required to have a potential visual acuity of 6/6 before consideration was given to the visual field and optimising off-axis image quality. Aspheric surfaces were used where this significantly improved off-axis image quality. RESULTS: Single optical cylinders, with increased posterior cylinder (intraocular) diameters, gave an increase in the theoretical visual field of 18% (from 76 degrees to 90 degrees) over current designs. Two-piece designs based on an inverted telephoto principle gave theoretical field angles over 120 degrees. Aspheric surfaces were shown to improve the off-axis image quality while maintaining a potential visual acuity of at least 6/6. This may well increase the measured visual field by improving the retinal illuminance off-axis. CONCLUSIONS: Results demonstrate that it is possible to significantly increase the theoretical maximum visual field through OOKP optical cylinders. Such designs will improve the visual rehabilitation of patients undergoing this procedure.

Computer-Aided Design↗
PubMed2000

Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis.

Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5' end, which are deleted during replication to generate authentic viral genomes. We removed those 5'-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5' ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5' end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.

Animals↗
PubMed2000

Abundant defective viral particles budding from microglia in the course of retroviral spongiform encephalopathy.

A pathogenetic hallmark of retroviral neurodegeneration is the affinity of neurovirulent retroviruses for microglia cells, while degenerating neurons are excluded from retroviral infections. Microglia isolated ex vivo from rats peripherally infected with a neurovirulent retrovirus released abundant mature type C virions; however, infectivity associated with microglia was very low. In microglia, viral transcription was unaffected but envelope proteins were insufficiently cleaved into mature viral proteins and were not detected on the microglia cell surface. These microglia-specific defects in envelope protein translocation and processing not only may have prevented formation of infectious virus particles but also may have caused further cellular defects in microglia with the consequence of indirect neuronal damage. It is conceivable that similar events play a role in neuro-AIDS.

Animals↗
PubMed1999

A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold .

A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.

Amino Acid Sequence↗
PubMed1998

Replication and transcription of HCV 229E replicons. p6.

Replicons based upon the human coronavirus 229E (HCV 229E) genome were transfected into HCV 229E infected cells. We demonstrate that a synthetic RNA comprised of 646 nucloetides from the 5' end and 1465 nucloetides from the 3' end of the HCV 229E genome is replication competent. We conclude that the cis-acting elements necessary for replication are located in these 5' and 3' genomic regions. Furthermore, we inserted the intergenic region of the HCV 229E nucleocapsid protein gene into this basic construct and were able to demonstrate the transcription of "subgenomic" RNAs.

Coronavirus↗
PubMed1998

Characterization of a papain-like cysteine-proteinase encoded by gene 1 of the human coronavirus HCV 229E.

Expression of the coronaviral gene 1 polyproteins, pp 1a and pp 1ab, involves a series of proteolytic events that are mediated by virus-encoded proteinases similar to cellular papain-like cysteine-proteinases and the 3C-like proteinases of picornaviruses. In this study, we have characterized, in vitro, the human coronavirus HCV 229E papain-like cysteine-proteinase PCP 1. We show that PCP 1 is able to mediate cleavage of an aminoterminal polypeptide, p9, from in vitro translation products representing the aminoproximal region of pp 1a/pp 1ab. Mutagenesis studies support the prediction of Cys1054 and His1278 as the catalytic amino acids of the HCV 229E PCP 1, since mutation of these residues abolishes the proteolytic activity of the enzyme.

Coronavirus↗
PubMed1998

Long distance RT-PCRs of human coronavirus 229E RNA.

The generation and cloning of cDNA fragments longer than 10 kb is often a difficult and time consuming task. In this study, we have analysed the conditions necessary of produce reverse transcripts longer than 10 kb that can be amplified by polymerase chain reaction. Thus, we isolated poly(A)-RNA from human coronavirus 229E infected MRC-5 cells and did reverse transcription using a sequence-specific primer. Subsequently, we amplified PCR products of varying length upstream of the primer position. Optimisation of the poly(A)-RNA preparation, the reverse transcription protocol and the polymerase chain reaction cycle conditions enabled us to successfully amplify regions of the human coronavirus 229E genome between 11.5 and 20.3 kb in length.

Cell Line↗
PubMed1998

Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate.

Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitro trans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus ppla and pplab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.

Amino Acid Sequence↗
PubMed1997

Effective amplification of 20-kb DNA by reverse transcription PCR.

Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.

Base Sequence↗
PubMed1997

Identification and subcellular localization of a 41 kDa, polyprotein 1ab processing product in human coronavirus 229E-infected cells.

The translation products of the human coronavirus (HCV) 229E open reading frames 1a and 1b, the polyproteins 1a and 1ab, are processed by virus-encoded proteinases. One of the key enzymes in this process is a chymotrypsin-like enzyme, the 3C-like proteinase. In this study we have identified an ORF 1b-encoded, 41 kDa processing product in HCV 229E-infected cells by using a monoclonal antibody with defined specificity. We show that this polypeptide is released from polyprotein 1ab by 3C-like proteinase-mediated cleavage of the peptide bonds Gln-6110/Gly-6111 and Gln-6458/Ser-6459. Also, we have investigated the subcellular localization of the 41 kDa processing product. Immunofluorescence microscopy revealed a punctate, perinuclear distribution of the 41 kDa polypeptide in infected cells and an identical subcellular localization was observed for three additional pp1ab-derived polypeptides. In contrast, the virus nucleocapsid protein showed a homogeneous cytoplasmic localization.

Antibodies, Monoclonal↗