Method of RNA purification from endothelial cells for DNA array experiments.
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Animals↗
Biomedical subjects
C K Brown
Publications and source records attributed to C K Brown.
Structure of neurolysin reveals a deep channel that limits substrate access.
The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded beta-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.
Binding Sites↗
Targeted therapy for malignant melanoma.
There has been much development in the field of targeted therapy for melanoma stemming from efforts to decrease treatment-related toxicities and enhance specific cytotoxicity. This review focuses on three modalities of targeted melanoma therapy based on the biology of the targeting mechanism. The first of these modalities is immunotherapy, which functions to generate a specific antimelanoma immunity. A second modality utilizes metabolic pathways of melanin synthesis to target melanoma cells specifically. A third modality ensues from recent advances in molecular biology and the identification of genes responsible for the malignant transformation of normal melanocytes to melanomas. This work has furthered our understanding of the basis of malignancy, as well as the development of novel strategies aimed at targeting aberrant growth in melanoma.
Humans↗
Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas.
PURPOSE: To provide ophthalmologists and pathologists with expanded criteria for separating patients at high risk of metastatic melanoma from those at low risk on the basis of microcirculation patterns in choroidal and ciliary body melanomas. DESIGN: Tissue culture studies and observational case series. PARTICIPANTS: The pattern-forming ability of four uveal melanoma cell lines of varying degrees of aggressive behavior was studied in vitro. Histologic sections of 234 eyes removed for choroidal or ciliary body melanoma were studied for the presence of microcirculation patterns. METHODS: The study was divided into two phases: the study of histologic sections of eyes removed for choroidal and ciliary body melanomas and observations on the in vitro behavior of cultured melanoma cells of varying degrees of invasive behavior. The presence or absence of each of nine microcirculation patterns was recorded from tissue sections, and interrelationships between different patterns were explored statistically. In vitro reconstitution of patterns and a study of the interrelationships of patterns in histologic sections was carried out. In the in vitro studies, uveal melanoma cell lines of varying degrees of aggressive potential were cultured to observe the development of architectural patterns other than loops and networks. MAIN OUTCOME MEASURES: In histologic studies, the outcome measure was the conditional probability of detecting loops or networks given the presence or absence of other patterns positive for periodic acid-SCHIFF: For tissue culture studies, the outcome measure was either the development or lack of development of patterns of different shapes in vitro. RESULTS: Histologic studies disclosed that given the presence of arcs without or with branching in a tissue section, it is likely that loops or networks will be detected in the same section plane, suggesting that the production of these patterns by aggressive tumor cells reflects a spectrum of architectural potential. In vitro studies confirmed this hypothesis by revealing that highly aggressive and metastatic uveal melanoma cell lines, but not poorly aggressive tumor cell lines, generated parallel channels with and without crosslinking and arcs with and without branching as well as loops and networks. CONCLUSIONS: The criteria for separating patients into low- and high-risk categories for metastasis from uveal melanoma should be expanded to include patterns other than loops or networks. In both the pathology laboratory as well as in a clinical setting, the detection of arcs or arcs with branching and parallel channels should prompt a careful search for loops and networks and for crosslinking parallel channels, respectively.
Choroid Neoplasms↗
Factors affecting injury severity to rear-seated occupants in rural motor vehicle crashes.
Injury to rear-seat occupants (RSOs) has received little systematic study in the literature. Past studies have focused on patterns of injury presented to the emergency department, various aspects of restraint usage and injury, or specific populations of RSOs. This is the first systematic analysis of injury severity to RSOs. RSOs involved in motor vehicle crashes (MVCs) sustain injuries of equal severity as do front-seated occupants (FSOs) involved in the same crash. The setting was a rural North Carolina emergency department where patients were evaluated at the sole hospital for the county. The design was a 2-year retrospective review of all MVCs with RSOs occurring in Pitt County North Carolina in calendar years 1988 and 1989. The Wilcoxon Rank-Sum Test and Logistic Regression Analysis with Injury Severity Score (ISS) as the dependent variable were performed. Three hundred forty-six crashes involving 367 vehicles with RSOs and 1,273 occupants that sent 222 patients to the hospital. ISS was higher for unrestrained occupants (1.87 versus 0.51), occupants of vehicles driven by legally intoxicated drivers (4.04 versus 0.73), occupants between the ages of 30 and 59 years (1.06 versus 0.65) and FSOs (1.04 versus 0.85) (Wilcoxon Rank-Sum test P <.0002). Logistic regression analysis confirmed these findings with the exception of more severe injuries for the 30 to 59 age range and impact speed. The analysis failed in these 2 cases. Restraint usage offered the greatest protective effect (OR 0.37). Lesser protective effects were noted with rear seat occupancy (OR 0.43) and age < 13 years (OR 0.77). More severe injuries were predicted by driver intoxication (OR 2.5), and age > 60 years (OR 1.25). In our rural population, RSOs sustain less severe injuries than FSOs. Restraint usage and sober drivers provide a greater protective effect however. Seat location does not replace restraint usage or sobriety.
Accidents, Traffic↗
Joint Council of State and Local Health Officials: workforce development--principles for action.
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Competency-Based Education↗
Childhood Guillain-Barré syndrome: clinical and electrophysiologic features predictive of outcome.
The hospital records of 27 children with the diagnosis of Guillain-Barré syndrome were retrospectively reviewed. Based on the time to recovery, patients were divided into two groups: group 1 consisted of children whose times to complete or partial recovery extended beyond 2 months from onset of the disease; group 2 consisted of children who attained a full recovery within 2 months from onset of the disease. The clinical and electrophysiologic features of the two groups were statistically compared for their predictive value of outcome. Three clinical features (maximum disability score at presentation, intubation, and cranial nerve involvement) were predictive of poor outcome, whereas one electrophysiologic feature (conduction block) was predictive of favorable outcome. There was also a significant correlation between maximum disability score at presentation and the probability of intubation.
Adolescent↗
Late effects in children treated with radiation therapy for Wilms' tumor.
PURPOSE: To determine the frequency and types of late effects in children receiving radiation therapy (RT) for Wilms' tumor. MATERIALS AND METHODS: From 1968 to 1994, 55 children received megavoltage RT at our institution as part of treatment for Wilms' tumor. A total of 42 (76.4%) have survived and have a minimum follow-up of 5 years. There were 25 female and 17 male patients with a median age at diagnosis of 48 months (range, 7-126 months). There were 12 Stage I, eight Stage II, 15 Stage III, six Stage IV, and one Stage V patient. RT was delivered to the hemiabdomen in 36 and whole abdomen in six patients. RT dose was 1000-1200 cGy (Group A) in 12, 1201-2399 cGy (Group B) in 11, and 2400-4000 cGy (Group C) in 19. Whole-lung RT was delivered to 13 patients either at diagnosis or pulmonary relapse. All patients received chemotherapy; the most common agents were actinomycin-D/vincristine/adriamycin in 13 and actinomycin-D/vincristine in 18. Median follow-up was 181 months (range, 60-306 months). RESULTS: Of 42 patients, 13 (31.0%) did not have late effects of treatment. The number of patients who developed muscular hypoplasia, limb length inequality, kyphosis, and iliac wing hypoplasia were seven (16.7%), five (11.9%), three (7.1%), and three (7.1%), respectively. Scoliosis was seen in 18 (42.9%) with only one patient requiring orthopedic intervention. Median time to development of scoliosis was 102 months, with a range of 16-146 months. The actuarial incidence of scoliosis at 5, 10, and 15 years after RT was 4.8 +/- 3.3%, 51.8 +/- 9.0%, and 56.7 +/- 9.3%, respectively. Only one of 12 Group A patients developed scoliosis. The 10- and 15-year actuarial incidences of scoliosis for Group A and B patients were 37.7 +/- 12.4% and 37.7 +/- 12.4%, whereas for Group C patients the incidences were 65.8 +/- 12.0% and 74.4 +/- 11. 7% (p = 0.03, log rank test). The actuarial incidence of bowel obstruction at 5, 10, and 15 years was 9.5 +/- 4.5%, 13.0 +/- 5.6%, and 17.0 +/- 6.5%. Of 23 patients, five irradiated within 10 days of surgery and one of 19 irradiated after 10 days developed bowel obstruction (p = 0.09, log rank test). Three patients developed hypertension with normal blood urea nitrogen (BUN) and creatinine levels; another patient had chronic renal insufficiency in a nonirradiated kidney. One patient developed diffuse interstitial pneumonitis. Of the 19 female patients who have reached puberty, three have given birth, and 15 have regular and one has irregular menstrual periods. Four patients developed benign neoplasms; three were in the RT field (two osteochondroma, one lipoma) and one outside (cervical intraepithelial neoplasia II). There were three second malignancies (chronic myelogenous leukemia at 9 years, osteosarcoma at 11 years, and breast cancer at 25 years after initial diagnosis of nephroblastoma); both solid malignancies occurred in the RT field. CONCLUSIONS: Late effects of therapy were seen in more than two thirds of children treated for Wilms' tumor. Children treated with lower doses (<2400 cGy) had a lower incidence of scoliosis compared with those who received more than 2400 cGy. There is also a suggestion that the incidence is lower in patients who received 1000-1200 cGy. Severe physical and functional deformity from RT was uncommon.
Child↗
Validation of partial tissue segmentation of single-channel magnetic resonance images of the brain.
We describe and evaluate a practical, automated algorithm based on local statistical mixture modeling for segmenting single-channel, T1-weighted volumetric magnetic resonance images of the brain into gray matter, white matter, and cerebrospinal fluid. We employed a stereological sampling method to assess, prospectively, the performance of the method with respect to human experts on 10 normal T1-weighted brain scans acquired with a three-dimensional gradient echo pulse sequence. The overall kappa statistic for the concordance of the algorithm with the human experts was 0.806, while that among raters, excluding the algorithm, was 0.802. The algorithm had better agreement with the modal expert decision (kappa = 0.878). The algorithm could not be distinguished from the experts by this measure. We also validated the algorithm on a simulated MR scan of a digital brain phantom with known tissue composition. Global gray matter and white matter errors were 1% and <1%, respectively, and correlation coefficients with the underlying tissue model were 0.95 for gray matter, 0.98 for white matter, and 0.95 for cerebrospinal fluid. In both approaches to validation, we evaluated both local and global performance of the algorithm. Human experts generated slightly higher global gray matter proportion estimates on the test brain scans relative to the algorithm (3.7%) and on the simulated MR scan relative to the true tissue model (4.4%). The algorithm underestimated gray in some subcortical nuclei which contain admixed gray and white matter. We demonstrate the reliability of the method on individual 1 NEX data sets of the test subjects, and its insensitivity to the precise values of initial model parameters. The output of this algorithm is suitable for quantifying cerebral cortical tissue, using a commonly performed commercial pulse sequence.
Algorithms↗
A novel approach for the identification of unique tumor vasculature binding peptides using an E. coli peptide display library.
BACKGROUND: Tumor neovascularization is necessary for continued tumor growth and metastasis. During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface. Targeting these molecular markers would, in effect, allow for specific tumor targeting. Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs. These sequences were identified from in vivo phage peptide display libraries. The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature. METHODS: FliTrx is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E. coli. Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor-derived endothelial cells (TDEC). MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor-containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature. FliTrx cells were incubated with MAGIC at 4 degrees C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets. The non-binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs. The bacterial population was then expanded and this "panning" process was carried out a total of five times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs. RESULTS: Recurring peptide sequences were detected that were 3-mers (13 sequences) and 4-mers (4 sequences). Of the 3-mers, four repeated 3 times, whereas none of the 4-mers repeated more than twice. All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid. A tripeptide basic-basic-nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences). Two test peptides showed TDEC binding specificity, and both conformed to the basic-basic-nonpolar motif. CONCLUSIONS: We report peptide sequences derived from panning an in vitro system designed to detect tumor-EC specific markers. These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.
3T3 Cells↗
Auditory evoked responses and learning and awareness during general anesthesia.
BACKGROUND: There is a major distinction between conscious and unconscious learning. Monitoring the mid-latency auditory evoked responses (AER) has been proposed as a measure to ascertain the adequacy of the hypnotic state during surgery. In the present study, we investigated the presence of explicit and implicit memories after anesthesia and examined the relationships of such memories to the AER. METHODS: We studied 180 patients scheduled for elective surgical procedures. After a thiopental induction, one of four anesthetics were studied: Opioid bolus: 7.5 microg x kg(-1) fentanyl, 70% N2O, with 2.5 microg x kg(-1) supplements as needed (n=100); Opioid infusion: Alfentanil 50 microg x kg(-1) bolus, 1-1.5 microg x kg(-1) x min(-1) infusion, 70% N2O (n=40); Isoflurane 0.3%: Fentanyl 1 microg x kg(-1), 70% N2O, isoflurane 0.3% expired (n=16); Isoflurane 0.7%: Fentanyl 1 microg x kg(-1), 70% N2O, isoflurane 0.7% expired (n=23). AER were recorded before anesthesia, 5 min after surgical incision and then every 30 min until the end of surgery. A tape of either the story of the "Three Little Pigs" or the "Wizard of Oz" was played continuously between the recordings. Explicit memory was assessed postoperatively by tests of recall and recognition, and implicit memory was assessed by the frequency of story-related free associations to target words from the stories, which were solicited twice during a structured interview. RESULTS: Six patients showed explicit recall of intraoperative events: All received the opioid bolus regimen. About 7% of patients reported dreaming during anesthesia. The incidence of picking the correct story that had been presented during anesthesia averaged 49%, i.e., very close to chance level. Overall, priming occurred only at the second association tests for the opioid bolus regimen, for which the frequency of an association to the presented story among those not giving an association to the control story was 26%, which was double the frequency (13%) of an association to the control story among those not giving an association to the presented story. This was significant by McNemar's test, P=0.02. There were significant associations between awareness, priming and AER, e.g., recall was associated with higher Nb amplitudes during anesthesia and priming was associated with shorter wave latencies. CONCLUSIONS: The incidence of awareness in patients anesthetized with nitrous oxide and bolus supplementation was 6%. Thus, this anesthetic technique did not reduce the risk of awareness compared with the use of nitrous oxide alone. Implicit memory occurred during nitrous oxide and bolus supplementation. Recording AER during anesthesia may help to predict awareness and implicit memory, particularly the former. The short contents of most of the dreams which were recalled could hamper future studies in this area.
Adolescent↗
Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.
Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.
Animals↗
Methodological problems of clinical research into spiritual healing: the healer's perspective.
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Attitude of Health Personnel↗
Using the essential services as a foundation for performance measurement and assessment of local public health systems.
Efforts are under way to develop a performance measurement monitoring system for state and local public health systems and to develop a strategic planning tool for local public health systems. The development of these measures is being based on the Essential Public Health Services. This article provides the rationale for why the Essential Services offer a good framework for identifying, analyzing, and evaluating public health activities. The article also reviews the history of local public health and the development and application of the Essential Public Health Services and their predecessor frameworks such as the core functions, the organizational practices, and the essential elements.
Community Health Services↗
Crystallization and preliminary analysis of neurolysin.
Neuropeptidases inactivate or modify the activity of peptide neurotransmitters and neurohormones. The neuropeptidase neurolysin acts only on short peptides and accepts a variety of cleavage-site sequences. Structures of the enzyme and enzyme-substrate complexes will help to determine the mechanisms of substrate selectivity used by this enzyme. Crystals of recombinant neurolysin have been grown in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 157.8, b = 88.0, c = 58.4 A. Data have been collected to 2.3 A at 110 K with observed diffraction to 1.8 A. Circular dichroism measurements suggest that the enzyme is primarily alpha-helical, with little beta-strand secondary structure. Sequence-based secondary-structure prediction supports this conclusion.
Animals↗
Diagnostic evaluation of patients with blunt abdominal trauma: a decision analysis.
OBJECTIVES: Using decision analysis, to compare the expected utility (EU) of diagnostic peritoneal lavage (DPL), computed tomography (CT), and ultrasonography (US) to determine the optimal modality for the evaluation of blunt abdominal trauma (BAT) in hemodynamically stable adults. METHODS: Data points for the decision analysis were obtained from three sources: 1) prevalence of BAT and the sensitivity and specificity of each diagnostic modality were determined through a criteria-based review of the literature; 2) rate of BAT necessitating immediate intervention, perioperative complication rate, and operative mortality rate were calculated using data from the authors' institution's trauma registry; and 3) outcome utilities were determined by telephone survey of adults in a random sample of households in the region. The decision tree was constructed and evaluated in standard fashion. For each diagnostic modality, the authors calculated the EU using the minimum, mean, and maximum sensitivity and specificity across a range of prevalence. Mean outcome utilities were used for each branch of the tree when calculating the EU. RESULTS: The EU of CT was consistently lower than the EUs of DPL and US at all levels of prevalence. However, the rank order of the EUs of US and DPL varied with the prevalence of BAT. When the prevalence was <30%, the EU of US was higher than that for DPL. When the prevalence was 30-40%, the EUs were similar. When the prevalence was >40%, the EU of US was less than that of DPL. CONCLUSIONS: Among institutions operating under constraints similar to those used in this model, the optimal diagnostic modality for the evaluation of BAT can be determined based on the sensitivity and specificity of the modality at their institution and the prevalence of BAT in their patient population.
Abdominal Injuries↗
Augmentation of melanoma-specific gene expression using a tandem melanocyte-specific enhancer results in increased cytotoxicity of the purine nucleoside phosphorylase gene in melanoma.
The lineage-specific human tyrosinase promoter has been used to successfully target gene expression at the transcriptional level to melanoma cells. The tyrosinase promoter, alone and in combination with a single, or a dual, tandem melanocyte-specific enhancer, was used to regulate expression of the firefly luciferase reporter gene. Transient transfections of these tissue-specific luciferase constructs in human and murine melanoma (Pmel, B16mel) and colon carcinoma (WiDr, MC38) cell lines resulted in melanoma-specific luciferase expression that was amplified 5- and 500-fold with the addition of a single or double enhancer, respectively, to the tyrosinase promoter. When the double enhancer-promoter construct expressed the highly toxic Escherichia coli purine nucleoside phosphorylase (PNP) gene, transfection of the same cell lines followed by administration of the prodrug 6-methyl purine deoxyriboside (6-MPDR) at a concentration of 50 microM caused melanoma-specific in vitro cell killing. Within 5 days after prodrug administration methylthiazol-tetrazolium (MTT) cytotoxicity assays showed that only 15 and 9% of Pmel and B16mel cells, respectively, remained viable compared with controls. This effect was highly specific, as 90 and 96% of WiDr and MC38 colon carcinoma cells remained viable 5 days after identical treatment. This effect was a direct result of increased tissue-specific conversion of 6-MPDR to the toxic metabolite 6-methylpurine (6-MP), as documented by HPLC analysis of culture supernatants. These results show that the dual tandem melanocyte-specific enhancer provides powerful amplification of the transcriptional targeting of gene expression afforded by use of the tyrosinase promoter. This amplification translates into increased, highly specific cytotoxicity to melanoma by the PNP/6-MPDR enzyme/prodrug system and, therefore, has potential efficacy in the use of gene therapy for the treatment of metastatic melanoma.
Animals↗
Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy.
Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome. A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine. In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV [365 nm]) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days. Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin. The potency of such a system was similar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.
Animals↗