Biomedical subjects

David H Evans

Publications and source records attributed to David H Evans.

At least 19 recordsLinked to original sources

PubMed2025

Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.

The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.

CRISPR-Cas Systems↗

PubMed2005

Cough frequency, cough sensitivity and health status in patients with chronic cough.

BACKGROUND: Little is known about the frequency of cough in health and in patients with chronic cough. METHODS: We measured cough frequency and its relationship with other markers of cough severity in 20 patients with chronic cough and 9 healthy subjects using the Leicester Cough Monitor (LCM), which is an automated ambulatory digital cough monitor that records sound only. All subjects had a 6-h recording and recordings were manually counted. A subgroup of 6 normals and 6 patients with a stable chronic cough had repeat measurements up to 6 months apart. RESULTS: Mean (sem) cough counts/hour were 43(8) in patients with chronic cough and 2(1) in normals (mean difference 41; 95% confidence interval 24-59; P<0.001). The cough counts were repeatable (within subject standard deviation: 23 coughs/hour; intraclass correlation coefficient 0.8). Cough counts correlated significantly with physical (r=-0.6, P=0.03), social (r=-0.7, P=0.01) and total Leicester Cough Questionnaire (LCQ) health status scores (r=-0.6, P=0.03) and cough sensitivity (concentration of capsaicin causing 5 coughs: r=0.9, P=0.008). CONCLUSION: We have shown that there are marked differences in cough frequency between patients with chronic cough and healthy subjects, that these measurements are repeatable, and that they correlate with cough-specific health status.

Adult↗

PubMed2005

Characterization of the major capsid proteins of myxoma virus particles using MALDI-TOF mass spectrometry.

The protein composition of poxvirus particles remains uncertain because of their large size and structural complexity. This has complicated the characterization of even well-studied Orthopoxviruses, like vaccinia virus, and little or nothing is known about the capsid composition of viruses belonging to other poxvirus genera. This paper describes methods that address this problem and have been used to identify 17 different Leporipoxvirus capsid proteins. Myxoma virus particles were purified using sucrose and Nicodenz gradient centrifugation and subfractionated into membrane and core fractions by thiol and detergent treatment. These materials were further fractionated using reverse-phase chromatography and SDS-PAGE and the resulting proteins identified by mass spectroscopy. Most of the myxoma proteins identified in this manner were homologs of either vaccinia virus structural proteins (F17R, L4R, J1R, H3L, A3L, A10L, A27L, and A45R) or virion-associated enzymes (I7L, H4L, D11L, A7L, and A22R). However, the myxoma homolog of the vaccinia P4a/A10L protein (M099L) differs from P4a protein in being proteolytically cleaved only once. M095L and M151R were also detected in core fractions. M095L and M151R are homologs of vaccinia A6L and B13R proteins, respectively, and poxvirus proteins not previously known to be capsid components. M093L, a protein of unknown function and having no certain Orthopoxvirus homolog associates with membrane fractions. These studies illustrate the conservation of Chordopoxvirion architecture and the methods that can be used to elucidate the proteins comprising these structures.

Amino Acid Sequence↗

PubMed2005

NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills.

In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules, where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine whether epithelial NHE homologs exist in elasmobranchs and, if so, to localize their expression in gills and determine whether their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologs from the gills of the euryhaline Atlantic stingray (Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homolog increased when stingrays were transferred to low salinities but not during hypercapnia. Expression of the NHE2 homolog did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2,744-base pair cDNA includes a coding region for a 2,511-amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to freshwater caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.

Amino Acid Sequence↗

PubMed2005

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase.

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3'-unpaired overhang, a 5' overhang, or both 3' and 5' overhangs. The sequence of the 5' overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3' strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5' strand, but since such molecules cannot be processed into nicked intermediates, the 3'-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5' overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.

DNA↗

PubMed2005

Limits of uncertainty in measured values of embolus-to-blood ratios in dual-frequency TCD recordings due to nonidentical sample volume shapes.

Transcranial Doppler (TCD) ultrasound (US) is widely used to detect cerebral embolisation. A drawback of the technique is that it is difficult to distinguish between small gaseous emboli and large solid emboli. One potential solution is to make measurements from blood and emboli at two different frequencies, because the frequency-dependence of ultrasonic backscatter will be different for the two types of emboli. Unfortunately, it is not possible to produce two identical sample volumes at two different frequencies, and this will introduce an uncertainty into any such measurements. Experimental measurements have been made of US fields distorted by temporal bone at 2.0 MHz and 2.5 MHz, and a mathematical model has been developed to examine the uncertainty introduced by the different field shapes at these frequencies. The results suggest that the uncertainties are sufficiently large (on the order of 2 to 4 dB) that a significant percentage of emboli are likely to be misclassified by the dual-frequency method.

Blood↗

PubMed2005

RF signals provide additional information on embolic events recorded during TCD monitoring.

Transcranial Doppler ultrasound (TCD) is commonly used to detect embolic signals in the cerebral circulation. However, current techniques to discriminate between signals from emboli and artifacts are subjective and ambiguous. The radiofrequency (RF) signal provides an extra dimension to the information available from conventional TCD systems that may help to interpret complex events. Artifacts generated by healthy volunteers and embolic signals recorded from a flow phantom were used to characterize the appearance of the two types of event. Characteristics of events, recorded during and immediately after carotid endarterectomy surgery, were compared with those from known sources. Additional information was provided by the RF signal on events recorded during TCD monitoring thus aiding classification. The RF signal may have a role as a "gold standard" for embolus detection.

Carotid Arteries↗

PubMed2005

Detection of Doppler embolic signals: psychoacoustic considerations.

The purpose of this study was to improve reliability in the identification of Doppler embolic signals by determining the decibel threshold for reproducible detection of simulated "emboli" as a function of signal duration, frequency and cardiac-cycle position. The auditory sensitivity of 16 participants to 574 simulated "emboli" was examined using psychoacoustic techniques to assess how the probability of detection varies with embolic signal parameters. Detailed measurements of the threshold for detection of simulated embolic signals are presented. These provide evidence that the measured embolus-to-blood threshold ranges between 2 dB and 14 dB as a continuous function of signal duration and frequency. The level of the threshold is closely linked to both embolic signal parameters and the properties of the blood flow signal. We conclude that the current fixed choice of threshold does not provide a good approximation to the true threshold of detection across the full range of embolic signal parameters.

Auditory Perception↗

PubMed2005

Mechanism of inhibition of vaccinia virus DNA polymerase by cidofovir diphosphate.

Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primer-template pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV + 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3' terminus by the 3'-to-5' proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3' residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.

Antiviral Agents↗

PubMed2005

Tumorigenic poxviruses up-regulate intracellular superoxide to inhibit apoptosis and promote cell proliferation.

Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RDelta virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RDelta virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death.

Animals↗

PubMed2005

Targeted modification of the complete chicken lysozyme gene by poxvirus-mediated recombination.

We have developed a novel ex vivo system for the rapid one-step targeted modification of large eucaryotic DNA sequences. The highly recombinant environment resulting from infection of rabbit cornea cells with the Shope fibroma virus was exploited to mediate precise modifications of the complete chicken lysozyme gene domain (21.5 kb). Homologous recombination was designed to occur between target DNA (containing the complete lysozyme gene domain) maintained in a lambda bacteriophage vector and modified targeting DNA maintained in a plasmid. The targeting plasmids were designed to transfer exogenous sequences (for example, beta-galactosidase alpha-complement, green fluorescent protein, and hydrophobic tail coding sequences) to specific sites within the lysozyme gene domain. Cotransfection of the target phage and a targeting plasmid into Shope fibroma virus infected cells resulted in the poxvirus-mediated transfer of the modified sequences from plasmid to phage. Phage DNA (recombinant and nonrecombinant) was then harvested from the total cellular DNA by packaging into lambda phage particles and correct recombinants were identified. Four different gene-targeting pairings were carried out, and from 3% to 11% of the recovered phages were recombinant. Using this poxvirus-mediated targeting system, four different regions of the chicken lysozyme gene domain have been modified precisely by our research group overall with a variety of inserts (6-971 bp), deletions (584-3000 bp), and replacements. We have never failed to obtain the desired recombinant. Poxvirus-mediated recombination thus constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise modification of large eucaryotic gene domains when compared with traditional practices.

Animals↗

PubMed2005

The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste.

The fish gill is a multipurpose organ that, in addition to providing for aquatic gas exchange, plays dominant roles in osmotic and ionic regulation, acid-base regulation, and excretion of nitrogenous wastes. Thus, despite the fact that all fish groups have functional kidneys, the gill epithelium is the site of many processes that are mediated by renal epithelia in terrestrial vertebrates. Indeed, many of the pathways that mediate these processes in mammalian renal epithelial are expressed in the gill, and many of the extrinsic and intrinsic modulators of these processes are also found in fish endocrine tissues and the gill itself. The basic patterns of gill physiology were outlined over a half century ago, but modern immunological and molecular techniques are bringing new insights into this complicated system. Nevertheless, substantial questions about the evolution of these mechanisms and control remain.

Acid-Base Equilibrium↗

PubMed2004

MALDI-TOF mass spectroscopy detects the capsid structural instabilities created by deleting the myxoma virus cupro-zinc SOD1 homolog M131R.

The myxoma virus M131R gene encodes a catalytically inactive homolog of cellular Cu-Zn superoxide dismutase (SOD1) and this 17,786 Da protein is a major virion component. We have used matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS) to study the effect(s) of deleting the gene on virion composition and structure. This approach confirmed that the M131R gene product is an abundant virion component. This conclusion was based upon the ready detection of a 1805.3 Da peptide released from the N-terminus of the myxoma SOD1 protein by mild trypsin treatment, as well as the detection of a 17,790 Da protein in HPLC fractionated virus extracts, which subsequently yielded M131R-encoded tryptic peptides. Neither peptide nor protein was detected in particles bearing a genome encoding an M131RDelta deletion mutation. Curiously, more proteins and tryptic peptides were detected when M131RDelta mutant virions were subjected to MALDI-TOF MS analysis compared with wild-type virus particles. This suggested that particles assembled in the absence of myxoma SOD protein are structurally unstable. Plaque analysis confirmed this conjecture by showing that SOD-deficient MYX particles are unusually heat labile and trypsin sensitive. Mutant Shope fibroma virus exhibited the same phenotype. Thus a previously unappreciated feature of MALDI-TOF MS is that the method can sometimes detect alterations in virion stability.

Capsid Proteins↗

PubMed2004

Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis.

As adults, anadromous lampreys migrate from seawater into freshwater rivers, where they require branchial ion (NaCl) absorption for osmoregulation. In teleosts and elasmobranchs, pharmological, immunohistochemical, and molecular data support roles for Na+/K+-ATPase (NPPase), carbonic anhydrase II (CAII), and vacuolar H+-ATPase (V-ATPase) in two different models of branchial ion absorption. To our knowledge, these transport-related proteins have not been studied in adult freshwater lampreys, and therefore it is not known if they are expressed, or have similar functions, in lampreys. The purpose of this study was to localize NPPase, CAII, and V-ATPase in the gills of adult freshwater lampreys and determine if any of these transport-related proteins are expressed in the same cells. Heterologous antibodies were used to localize the three proteins in gill tissue from pouched lamprey (Geotria australis). Immunoreactivity (IR) for all three proteins occurred between, and at the base of, lamellae in cells that match previous descriptions of mitochondrion-rich-cells (MRCs). NPPase-IR was always on the basolateral side of cells that did not stain for CAII or V-ATPase. In contrast, CAII-IR was always on the apical side of cells that also contained diffuse V-ATPase-IR. Therefore, we have identified two types of MRC in adult freshwater lamprey gills based on immunohistochemical staining for three transport proteins. A model of ion transport, based on our results, is proposed for adult freshwater lampreys.

Animals↗

PubMed2004

A putative H+-K+-ATPase in the Atlantic stingray, Dasyatis sabina: primary sequence and expression in gills.

In mammals, the gastric H(+)-K(+)-ATPase (HKalpha1) mediates acid secretion in the stomach and kidneys. Like mammals, elasmobranchs also secrete acid from their stomachs, but unlike mammals they primarily use their gills for systemic acid excretion instead of their kidneys. The purpose of this study was to determine if an HKalpha1 orthologue exists in an elasmobranch (Atlantic stingray, Dasyatis sabina), to determine if it is expressed in gills and, if so, to localize its expression and determine if its expression is regulated during hypercapnia or freshwater acclimation. A polyclonal antibody made against an HKalpha1 peptide detected HKalpha1 immunoreactivity in protein isolates and tissue sections of stingray stomachs and gills. Immunohistochemistry demonstrated that HKalpha1 immunoreactivity was present in a subpopulation of epithelial cells in both organs. Double-labeling experiments in the gills showed that HKalpha1 immunoreactivity occurred in Na(+)-K(+)-ATPase-rich cells and not in V-type H(+)-ATPase-rich cells. RT-PCRs were used to deduce the primary sequence of a putative H(+)-K(+)-ATPase from the stomach of Atlantic stingrays. The 3,421-base pair cDNA includes a coding region for a 1,025-amino acid protein that is over 80% identical to HKalpha1 of mammals. RT-PCRs were then used to demonstrate that this transcript is also expressed in the gills. To our knowledge, this is the first H(+)-K(+)-ATPase sequence reported for any elasmobranch and the first full-length sequence for any fish. We also provide the first evidence for its expression in the gills of any fish and demonstrate that its expression increased during freshwater acclimation but not exposure to hypercapnia.

Amino Acid Sequence↗

PubMed2004

Assessing blood flow control through a bootstrap method.

In order to assess blood flow control, the relationship between blood pressure and blood flow can be modeled by linear filters. We present a bootstrap method, which allows the statistical analysis of an index of blood flow control that is obtained from constrained system identification using an established set of pre-defined filters.

Algorithms↗

PubMed2004

Construction of recombinant vaccinia viruses using leporipoxvirus-catalyzed recombination and reactivation of orthopoxvirus DNA.

Poxvirus DNA is not infectious because the initiation of the infective process requires proteins encapsidated along with the virus genome. However, infectious virus can be produced if purified poxvirus DNA is transfected into cells previously infected with another poxvirus. This process is termed heterologous reactivation if the infecting virus is different from the transfected virus. We describe a method in which the high-frequency recombination and replication reactions catalyzed by the Leporipoxvirus, Shope fibroma virus (SFV), can be coupled with SFV-promoted reactivation reactions to rapidly construct recombinant vaccinia viruses in high yields (25-100% recombinant progeny). The reactivated vaccinia viruses are easily purified free of the SFV helper virus by plating mixed populations of virus on cells that support only the growth of vaccinia virus. These heterologous reactivation reactions can be used to manipulate the structure of virus genomes and produce viruses that express recombinant proteins at high levels. We illustrate the method by polymerase chain reaction (PCR) cloning the gene encoding green fluorescent protein (GFP), then using double-strand break repair reactions to produce a recombinant virus that expresses high levels of GFP.

Animals↗

PubMed2003

Tissue Doppler imaging of carotid plaque wall motion: a pilot study.

BACKGROUND: Studies suggest the physical and mechanical properties of vessel walls and plaque may be of clinical value in the diagnosis and treatment of cardiovascular atherosclerotic disease. The purpose of this pilot study was to investigate the potential clinical application of ultrasound Tissue Doppler Imaging (TDI) of Arterial Wall Motion (AWM) and to quantify simple wall motion indices in normal and diseased carotid arteries. METHODS: 224 normal and diseased carotid arteries (0-100% stenoses) were imaged in 126 patients (age 25-88 years, mean 68 +/- 11). Longitudinal sections of the carotid bifurcation were imaged using a Philips HDI5000 scanner and L12-5 probe under optimized TDI settings. Temporal and spatial AWMs were analyzed to evaluate the vessel wall displacements and spatial gradients at peak systole averaged over 5 cardiac cycles. RESULTS: AWM data were successfully extracted in 91% of cases. Within the carotid bifurcation/plaque region, the maximum wall dilation at peak systole ranged from -100 to 750 microns, mean 335 +/- 138 microns. Maximum wall dilation spatial gradients ranged 0-0.49, mean 0.14 +/- 0.08. The AWM parameters showed a wide variation and had poor correlation with stenoses severity. Case studies illustrated a variety of pertinent qualitative and quantitative wall motion features related to the biophysics of arterial disease. CONCLUSION: Our clinical experience, using a challenging but realistic imaging protocol, suggests the use of simple quantitative AWM measures may have limitations due to high variability. Despite this, pertinent features of AWM in normal and diseased arteries demonstrate the potential clinical benefit of the biomechanical information provided by TDI.

Adult↗