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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↗
PubMed2006

Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase.

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1-5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3'-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.

Catalysis↗
PubMed2006

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias.

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.

Animals↗
PubMed2006

Impact of clinical environment on embolus detection: a comparison of automated and manual detection of Doppler embolic signals.

BACKGROUND: Transcranial Doppler ultrasound detection of weak embolic signals is inhibited by intrinsic limitations within the human auditory system. Psychoacoustics effects are likely to be exacerbated in a clinical environment, where automated embolus detection has potential to surpass manual detection. In this study we quantify the impact of clinical environment on manual detection of Doppler embolic signals following carotid surgery. We also discuss the implications of psychoacoustics considerations for the evaluation of automated detection systems. METHOD: Concurrent monitoring by vascular technologists and an automated embolus detection system were performed for 50 consecutive patients during postoperative recovery. Both detection methods were evaluated against a majority decision human expert panel analyzing under ideal conditions. RESULTS: Clinical environment reduced the overall sensitivity of manual monitoring by approximately 23%, mainly due to a approximately 2-dB increase in the lower threshold for detection. Clinical environment was also associated with a reduction in positive predictive value for manual detection of approximately 9% compared to ideal conditions. Automated monitoring, which is not affected by environment, was marginally more sensitive for detection of weaker embolic signals. CONCLUSIONS: One in 4 weak embolic signals was missed during routine clinical monitoring compared to ideal conditions. Automated detection (in this study) performed slightly better than human observers but did not approach the performance of the majority decision panel.

Aged↗
PubMed2006

Mutations in the E9L polymerase gene of cidofovir-resistant vaccinia virus strain WR are associated with the drug resistance phenotype.

Cidofovir (CDV) is an effective drug against viruses of the Orthopoxviridae family and is active in vitro against variola virus, the cause of smallpox. However, CDV-resistant poxviruses can be generated by repeated in vitro passage in the presence of suboptimal concentrations of CDV. To determine if mutations in the E9L polymerase gene could confer resistance to this nucleoside analog, this gene was sequenced from CDV-resistant vaccinia virus and found to encode five amino acid changes, centered on an N-terminal region associated with 3'-->5' exonuclease activity. Transfer of this mutant E9L gene into wild-type vaccinia virus by marker rescue sufficed to confer the resistance phenotype. E9L polymerase mutations occurred sequentially during passage in CDV, and an H296Y/S338F double mutant that conferred an intermediate CDV resistance phenotype was identified. In vitro, the marker-rescued CDV-resistant vaccinia virus containing all five mutations grew nearly as well as wild-type vaccinia virus. However, the virulence of this virus for mice was reduced, as 10- to 30-fold more CDV-resistant virus than wild-type virus was required for lethality following intranasal challenge. Cidofovir and hexadecyloxypropyl-cidofovir gave partial protection to mice infected with the virus when used at 50 and 100 mg/kg of body weight given as single treatments 24 h after virus exposure, whereas 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) was completely protective at 25, 50, and 100 mg/kg/day when given daily for 5 days. These findings suggest that drug therapy for poxviruses may be complicated by drug resistance but that treatment of the infection with currently known compounds is possible.

Administration, Intranasal↗
PubMed2006

Neuronal nitric oxide synthase in the gill of the killifish, Fundulus heteroclitus.

Neuronal NOS (nNOS) is a constitutively expressed enzyme that catalyzes the oxidation of L-arginine and water to L-citrulline and the gas nitric oxide (NO). Nitric oxide is involved in regulation of a variety of processes, including: vascular tone, neurotransmission, and ion balance in mammals and fishes. In this study, we have cloned and characterized a putative NOS homologue from the brain of the euryhaline killifish, Fundulus heteroclitus. Killifish NOS has 75% amino acid identity to human nNOS, and phylogenetic analysis groups the killifish sequence with the mammalian nNOS, suggesting that it is a mammalian orthologue. Relative quantitative reverse transcriptase-PCR demonstrated that killifish nNOS mRNA is highly expressed in the brain and gill followed by the stomach, kidney, opercular epithelium, intestine and heart. Immunohistochemistry localized nNOS to nerve fibers and epithelial cells adjacent to mitochondrion-rich cells (ion transporting cell) in the gill, suggesting that nNOS production of NO may contribute to regulation of vascular tone and/or MRC function in the teleost gill.

Amino Acid Sequence↗
PubMed2006

Characteristics of Doppler embolic signals observed following carotid endarterectomy.

Postoperative Doppler embolic signals following carotid endarterectomy (CEA) are associated with an increased risk of stroke, but the characteristics of these signals are rarely reported. In this study, we survey signals from 1485 emboli, assumed to consist predominantly of thrombus. Data were obtained by monitoring the middle cerebral arteries of 100 consecutive CEA patients during postoperative recovery. The distribution of embolic signal frequencies, intensities and durations revealed that embolic signals do not occur randomly in the sonogram. In particular, we find that the signals possess a characteristic distribution of velocities reflecting the preferred path of the embolus through the artery (at approximately 75% of the distance between the centre of the artery and the artery wall). Embolic signals were more likely to be observed at cardiac cycle positions between 35% and 80% from the start of systole than elsewhere. After eliminating other considerations, we hypothesized that this peak in the distribution of signals in the sonogram arose due to the localization of emboli trajectories and a strong tendency for emboli to detach from the carotid bifurcation during systole.

Aged↗
PubMed2006

Automatic detection of emboli in the TCD RF signal using principal component analysis.

The transcranial Doppler (TCD) radio-frequency (RF) signal can provide additional information on events recorded during ultrasonic monitoring. Embolic signals appear as uniform and predictable shapes within the RF signal, enabling pattern recognition and image processing techniques to be used for their automated detection. This paper uses principal component analysis (PCA) to characterise the typical variation in embolic signal shape, within the RF signal, using training sets of in vitro and in vivo data. PCA techniques are then utilised to discriminate between previously unseen embolic and artifact signals. Although the results of this study show that the algorithms described in this paper do not yet have the accuracy required for their use in a clinical setting, it does demonstrate that this novel technique has the potential to be developed further.

Algorithms↗
PubMed2006

Detection of cough signals in continuous audio recordings using hidden Markov models.

Cough is a common symptom of many respiratory diseases. The evaluation of its intensity and frequency of occurrence could provide valuable clinical information in the assessment of patients with chronic cough. In this paper we propose the use of hidden Markov models (HMMs) to automatically detect cough sounds from continuous ambulatory recordings. The recording system consists of a digital sound recorder and a microphone attached to the patient's chest. The recognition algorithm follows a keyword-spotting approach, with cough sounds representing the keywords. It was trained on 821 min selected from 10 ambulatory recordings, including 2473 manually labeled cough events, and tested on a database of nine recordings from separate patients with a total recording time of 3060 min and comprising 2155 cough events. The average detection rate was 82% at a false alarm rate of seven events/h, when considering only events above an energy threshold relative to each recording's average energy. These results suggest that HMMs can be applied to the detection of cough sounds from ambulatory patients. A postprocessing stage to perform a more detailed analysis on the detected events is under development, and could allow the rejection of some of the incorrectly detected events.

Algorithms↗
PubMed2006

Multigate transcranial Doppler ultrasound system with real-time embolic signal identification and archival.

An integrated system for acquisition and processing of intracranial and extracranial Doppler signals and automatic embolic signal detection has been developed. The hardware basis of the system is a purpose-built acquisition/processing board that includes a multigate Doppler unit controlled through a computer. The signal-processing engine of the system contains a fast Fourier transform (FFT)-based, spectral-analysis unit and an embolic signal-detection unit using expert system reasoning theory. The system is designed so that up to four receive gates from a single transducer can be used to provide useful reasoning information to the embolic signal-detection unit. Alternatively, two transducers can be used simultaneously, either for bilateral transcranial Doppler (TCD) investigations or for simultaneous intra- and extracranial investigation of different arteries. The structure of the software will allow the future implementation of embolus detection algorithms that use the information from all four channels when a single transducer is used, or of independent embolus detection in two sets of two channels when two transducers are used. The user-friendly system has been tested in-vitro, and it has demonstrated a 93.6% sensitivity for micro-embolic signal (MES) identification. Preliminary in-vivo results also are encouraging.

Adult↗
PubMed2006

Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice.

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3'-5' exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

Alleles↗
PubMed2006

COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation.

In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription-PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish (Fundulus heteroclitus). The 2,738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70% identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na(+)/K(+)/2Cl(-) cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence, the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

Acclimatization↗
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↗