Biomedical subjects

Paul R Young

Publications and source records attributed to Paul R Young.

At least 19 recordsLinked to original sources

PubMed2006

Generation and characterization of proteolytically active and highly stable truncated and full-length recombinant West Nile virus NS3.

West Nile virus is a medically significant emerging pathogen for which there is no effective antiviral therapy. The viral protease encoded by NS2B and NS3 is an attractive target for development of an inhibitor and has been the focus of numerous studies. Most have employed recombinant proteases based on an expression strategy we developed which links the essential hydrophilic cofactor domain within NS2B to the NS3 protease domain by a flexible glycine linker. However, autoproteolysis has been a significant problem associated with this construct. The recently resolved crystal structure of the cofactor bound WNV NS3 protease for example, was found to be truncated by 18 residues at its N-terminus. In this study, the autocatalytic cleavage site was identified and removed along with nonessential regions of the glycine linker and cofactor domain. In addition, the optimal size of the NS3 protease was defined. Based on this optimized construct, a recombinant protease incorporating the full length of NS3 was also successfully expressed and purified. Somewhat surprisingly, comparative analysis of the proteolytic activity of this recombinant with that of the protease domain alone revealed little influence of the C-terminal two thirds of NS3 on substrate binding. These modifications have yielded highly stable and constrained recombinant proteases, which are more suitable than existing constructs for both activity and structural studies.

Amino Acid Sequence↗

PubMed2006

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies.

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

Amino Acid Sequence↗

PubMed2006

Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus.

A 13-residue peptide sequence from a respiratory syncitial virus fusion protein was constrained in an alpha-helical conformation by fusing two back-to-back cyclic alpha-turn mimetics. The resulting peptide, Ac-(3-->7; 8-->12)-bicyclo-FP[KDEFD][KSIRD]V-NH(2), was highly alpha-helical in water by CD and NMR spectroscopy, correctly positioning crucial binding residues (F488, I491, V493) on one face of the helix and side chain-side chain linkers on a noninteracting face of the helix. This compound displayed potent activity in both a recombinant fusion assay and an RSV antiviral assay (IC(50) = 36 nM) and demonstrates for the first time that back-to-back modular alpha-helix mimetics can produce functional antagonists of important protein-protein interactions.

Amino Acid Sequence↗

PubMed2006

Retroviral invasion of the koala genome.

Endogenous retroviruses are a common ancestral feature of mammalian genomes with most having been inactivated over time through mutation and deletion. A group of more intact endogenous retroviruses are considered to have entered the genomes of some species more recently, through infection by exogenous viruses, but this event has never been directly proved. We have previously reported koala retrovirus (KoRV) to be a functional virus that is associated with neoplasia. Here we show that KoRV also shows features of a recently inserted endogenous retrovirus that is vertically transmitted. The finding that some isolated koala populations have not yet incorporated KoRV into their genomes, combined with its high level of activity and variability in individual koalas, suggests that KoRV is a virus in transition between an exogenous and endogenous element. This ongoing dynamic interaction with a wild species provides an exciting opportunity to study the process and consequences of retroviral endogenization in action, and is an attractive model for studying the evolutionary event in which a retrovirus invades a mammalian genome.

Animals↗

PubMed2006

SwarmPS: rapid, semi-automated single particle selection software.

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.

Algorithms↗

PubMed2006

The discriminative bilateral filter: an enhanced denoising filter for electron microscopy data.

Advances in three-dimensional (3D) electron microscopy (EM) and image processing are providing considerable improvements in the resolution of subcellular volumes, macromolecular assemblies and individual proteins. However, the recovery of high-frequency information from biological samples is hindered by specimen sensitivity to beam damage. Low dose electron cryo-microscopy conditions afford reduced beam damage but typically yield images with reduced contrast and low signal-to-noise ratios (SNRs). Here, we describe the properties of a new discriminative bilateral (DBL) filter that is based upon the bilateral filter implementation of Jiang et al. (Jiang, W., Baker, M.L., Wu, Q., Bajaj, C., Chiu, W., 2003. Applications of a bilateral denoising filter in biological electron microscopy. J. Struc. Biol. 128, 82-97.). In contrast to the latter, the DBL filter can distinguish between object edges and high-frequency noise pixels through the use of an additional photometric exclusion function. As a result, high frequency noise pixels are smoothed, yet object edge detail is preserved. In the present study, we show that the DBL filter effectively reduces noise in low SNR single particle data as well as cellular tomograms of stained plastic sections. The properties of the DBL filter are discussed in terms of its usefulness for single particle analysis and for pre-processing cellular tomograms ahead of image segmentation.

Animals↗

PubMed2006

The Role of histidine residues in low-pH-mediated viral membrane fusion.

A central event in the invasion of a host cell by an enveloped virus is the fusion of viral and cell membranes. For many viruses, membrane fusion is driven by specific viral surface proteins that undergo large-scale conformational rearrangements, triggered by exposure to low pH in the endosome upon internalization. Here, we present evidence suggesting that in both class I (helical hairpin proteins) and class II (beta-structure-rich proteins) pH-dependent fusion proteins the protonation of specific histidine residues triggers fusion via an analogous molecular mechanism. These histidines are located in the vicinity of positively charged residues in the prefusion conformation, and they subsequently form salt bridges with negatively charged residues in the postfusion conformation. The molecular surfaces involved in the corresponding structural rearrangements leading to fusion are highly conserved and thus might provide a suitable common target for the design of antivirals, which could be active against a diverse range of pathogenic viruses.

Amino Acid Sequence↗

PubMed2006

Current status of short synthetic peptides as vaccines.

Preventative medicine in the form of vaccination had a huge impact on human health in the 20th Century. Vaccines are now recognized as the most effective line of defence against infectious agents that cause disease and death, and in some cases vaccines have enabled complete eradication of disease from the globe (e.g. smallpox). Nevertheless there are still many human diseases (e.g. viral and parasitic infections, cancers) for which there are no effective vaccines. Current vaccines are mainly live and attenuated viruses or viral, bacterial or recombinant proteins and polypeptides. By virtue of their natural amino acid composition, polypeptides and proteins are relatively safe materials for vaccination, but they are expensive to manufacture making them inaccessible to the most vulnerable and needy human populations that cannot afford such medicines. This review will focus on shorter synthetic peptides that are cheaper to manufacture, conceivably even safer for human use because of increased specificity, but they also suffer from problems that have presumably resulted in their lack of progress in clinical trials. Since 1990, over 100 chemically synthesized short peptide vaccines have entered Phase I clinical trials, less than 20 have progressed into Phase II, but none have entered Phase III clinical trials. In this review we discuss reasons why vaccines based on short peptides may not have succeeded in the clinic, identify problems such as insufficient immunogenicity, structural/conformational instability, chemical instability due to degradation, and describe possible solutions to some of these problems that have been investigated in recent years.

Clinical Trials as Topic↗

PubMed2006

Human metapneumovirus, Australia, 2001-2004.

We examined 10,025 respiratory samples collected for 4 years (2001-2004) and found a 7.1% average annual incidence of human metapneumovirus. The epidemic peak of infection was late winter to spring, and genotyping showed a change in predominant viral genotype in 3 of the 4 years.

Adolescent↗

PubMed2006

Does indigo carmine prevent early artificial urinary sphincter cuff erosion?

INTRODUCTION: Urethral erosion following artificial urinary sphincter (AUS) placement is hypothesized to be secondary to unrecognized intra-operative urethral injury. Intra-urethral indigo carmine solution (ICS), a blue dye, following urethral mobilization should identify intra-operative urethrotomy and prevent early post-operative cuff erosion. METHODS: Retrospective review was completed of all men undergoing AUS (AMS 800 device) insertion between January 2000 and January 2005 for post prostatectomy stress incontinence at one institution. Operative reports were examined for use of intra-operative injection of ICS as well as documentation of urethral injury. Post-operative course was reviewed for evidence of early cuff erosion. All patients were followed a minimum of 6 months post-operatively. RESULTS: Seventy-eight men underwent AUS placement during the investigative period. Forty-one men received intra-operative ICS injection following urethral mobilization and 37 men did not. ICS identified one intra-operative urethral injury. No urethral injuries were noted in the non-injection group. The ICS group suffered 3 (7.3%) early urethral erosions; the control group had one early urethral erosion (2.7%). CONCLUSION: Intra-operative ICS use is easy, safe, and able to identify urethral injury. However, its use did not preclude the incidence of early cuff erosion. This may postulate the existence of early urethral cuff erosion as a separate entity not dependent on intra-operative urethrotomy.

Aged↗

PubMed2005

Systematic transperineal ultrasound-guided template biopsy of the prostate: three-year experience.

OBJECTIVES: To review the technique of transperineal saturation prostate biopsy and to update our results on patients at high risk of prostate cancer. METHODS: A total of 210 men who met the study inclusion criteria underwent systematic transperineal ultrasound-guided template biopsy of the prostate. All patients had previously undergone at least one set of transrectal prostate biopsies and 170 (81%) had undergone two or more. The mean number of prostate cores obtained before the template biopsy was 17.4. A mean of 21.2 cores (range 12 to 41) were obtained at the template biopsy, depending on prostate size. The study inclusion criteria included prostate-specific antigen level of 10 ng/mL or greater, prostate-specific antigen velocity of 0.75 ng/mL per year or greater, or the presence of prostatic intraepithelial neoplasia and/or atypical small cell acinar proliferation on the previous biopsy. RESULTS: Adenocarcinoma was detected in 78 men (37%). Cancer was detected in the transition zone in 60 (77%) of these 78 men, including 36 (46%) in whom cancer was identified uniquely in the transition zone. Gleason sum 6 (range 3 to 9) was the most common biopsy grade. Thirty patients underwent radical prostatectomy, and 27 (90%) had pathologic Stage pT2 disease as the final pathologic stage. Complications from biopsy were limited to postprocedural urinary retention in 11% of patients. CONCLUSIONS: A systematic transperineal template biopsy provides uniform sampling of the entire prostate. This technique appears to enhance the identification of transition zone cancers not detected by previous transrectal prostate biopsy in patients at high risk of prostate adenocarcinoma.

Adenocarcinoma↗

PubMed2004

Site-directed mutagenesis and kinetic studies of the West Nile Virus NS3 protease identify key enzyme-substrate interactions.

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

Amino Acid Sequence↗

PubMed2004

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease.

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I <0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 approximately 1 microm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Amino Acid Sequence↗

PubMed2004

Acute urinary retention after transperineal template-guided prostate biopsy.

PURPOSE: Urinary retention occurs in 5%-36% of patients with prostate cancer after implantation of radioactive seeds for brachytherapy. We used transperineal biopsy as a model to determine the influence of needle trauma on urinary retention. METHODS AND MATERIAL: We retrospectively reviewed medical records of 157 men with high risk of prostate cancer who underwent systematic ultrasound-guided biopsy of the prostate with the transperineal template technique and an 18-gauge automated biopsy device. RESULTS: Eighteen of 157 patients (11.5%; 95% confidence interval, 6.9%-17.5%) had urinary retention within 48 hours after biopsy. Median age was 68.5 years in patients with retention vs. 67.0 years in patients without (p = 0.319); median calculated prostate volume, 76.5 vs. 51.5 mL (p = 0.015); and median number of biopsy cores, 22.0 vs. 20.0 (p = 0.038). Age distribution differed between groups (p = 0.047), with more younger men in the no-retention group. On multivariate analysis, only number of biopsy cores significantly predicted urinary retention (p = 0.003). Four patients required transurethral resection; 1 had an indwelling catheter until radical prostatectomy; and 13 were catheter-free within 1-5 days. CONCLUSIONS: Needle trauma alone may cause urinary retention in men undergoing transperineal procedures. The number of needle incursions and prostate size are predictors of postprocedure urinary retention.

Acute Disease↗

PubMed2004

Determination of the disulfide bond arrangement of dengue virus NS1 protein.

The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models.

Amino Acid Sequence↗

PubMed2004

Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic-like stem cells.

Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self-renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. When implanted in vivo, undifferentiated noninduced embryonic stem cells formed teratomas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle and derived by repetitive single-cell clonogenic analysis. In the undifferentiated state it consists of very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecular and immunological markers for embryonic stem cells. It exhibits telomerase activity, which is consistent with its extended capability for self-renewal. When induced to differentiate, it expressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. The clone was designated as a postnatal pluripotent epiblastic-like stem cell (PPELSC). The undifferentiated clone was transfected with a genomic marker and assayed for alterations in stem cell characteristics. No alterations were noted. The labeled clone, when implanted into heart after injury, incorporated into myocardial tissues undergoing repair. The labeled clone was subjected to directed lineage induction in vitro, resulting in the formation of islet-like structures (ILSs) that secreted insulin in response to a glucose challenge. This study suggests that embryonic-like stem cells are retained within postnatal mammals and have the potential for use in gene therapy and tissue engineering.

Animals↗

PubMed2004

Conservative management of incidental contrast-enhancing renal masses as safe alternative to invasive therapy.

OBJECTIVES: To describe small contrast-enhancing renal masses suggestive of cancer that were managed conservatively with watchful waiting and serial computed tomography scans. Advanced diagnostic imaging has led to the increased incidental detection of renal masses in patients whose multiple comorbid conditions preclude invasive management. METHODS: A retrospective review was conducted of 29 consecutive patients with 29 incidentally detected asymptomatic renal masses less than 3.5 cm in diameter that were managed conservatively with watchful waiting (because of patient wishes or multiple comorbid conditions) and serial computed tomography scans. RESULTS: The average patient age was 70 years (range 51 to 88), and the average duration of follow-up imaging was 32 months (range 10 to 89). The average number of follow-up computed tomography scans was 4.9 per patient (range 1 to 11). The average width of the renal masses at diagnosis was 1.83 cm (range 0.4 to 3.5), and the average change in size per year was 0.12 cm for all patients. Four patients underwent radical nephrectomy because of growth of the renal mass (n = 1) or patient wishes (n = 3). The histologic findings in 3 of these 4 patients were consistent with renal cell carcinoma. Two patients underwent radiofrequency ablation of the masses. At last follow-up, metastatic disease had not developed in any patient, and no patient had died of renal cell carcinoma. Two patients had died of other causes. CONCLUSIONS: The results of our study showed that when comorbid conditions or patient wishes preclude invasive treatment, contrast-enhancing renal masses less than 3.5 cm wide that are suggestive of cancer can be safely managed with watchful waiting and serial computed tomography scans.

Adenoma, Oxyphilic↗

PubMed2004

Prediction of genitourinary tract morbidity after brachytherapy for prostate adenocarcinoma.

OBJECTIVE: To investigate whether preoperative genitourinary variables in patients undergoing brachytherapy for localized prostate adenocarcinoma could predict postoperative genitourinary tract morbidity. PATIENTS AND METHODS: We retrospectively reviewed medical records of 105 men who received either iodine 125 or palladium 103 radioactive seed implants with or without external beam radiotherapy or hormone blockade from January 1, 1998, through December 31, 2000, at the Mayo Clinic in Jacksonville, Fla. Patients with one or more of the following were classified as having a high risk of postoperative genitourinary tract morbidity: American Urological Association symptom scores greater than 15, maximum urinary flow rate less than 10 mL/s, postvoid residual urinary volume greater than 100 mL, or prostate volume greater than 40 cm3. Of the 105 men, 59 (56%) were classified as high risk and 46 (44%) as low risk. Mean follow-up after brachytherapy was 23.6 months. Modified Radiation Therapy Oncology Group scores were used to assess postoperative genitourinary tract morbidity. The term significant genitourinary tract morbidity was applied to patients with a Radiation Therapy Oncology Group grade of 3 or 4 after at least 6 months of follow-up. RESULTS: Significant morbidity occurred in 37% of high-risk vs 15% of low-risk patients (P = .006). In high-risk patients, transurethral resection or incision of the prostate was required in 5 patients, urethral dilation in 4, direct-vision internal urethrotomy in 1, and placement of a suprapubic catheter in 1. In low-risk patients, transurethral incision of the prostate was required in only 1 patient. Urinary flow rate was a significant individual predictor of postoperative morbidity (P = .03). CONCLUSIONS: A combination of urinary flow rate, prostate volume, postvoid residual urinary volume, and American Urological Association symptom score can help identify patients with underlying voiding dysfunction. Urinary flow rate was a statistically significant predictor of genitourinary tract morbidity after brachytherapy for localized prostate adenocarcinoma. Patients and physicians should consider these factors before a patient decides to undergo brachytherapy.

Adenocarcinoma↗