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1.  Bladder Cancer Diagnosis and Identification of Clinically Significant Disease by Combined Urinary Detection of Mcm5 and Nuclear Matrix Protein 22 
PLoS ONE  2012;7(7):e40305.
Urinary biomarkers for bladder cancer detection are constrained by inadequate sensitivity or specificity. Here we evaluate the diagnostic accuracy of Mcm5, a novel cell cycle biomarker of aberrant growth, alone and in combination with NMP22.
1677 consecutive patients under investigation for urinary tract malignancy were recruited to a prospective blinded observational study. All patients underwent ultrasound, intravenous urography, cystoscopy, urine culture and cytologic analysis. An immunofluorometric assay was used to measure Mcm5 levels in urine cell sediments. NMP22 urinary levels were determined with the FDA-approved NMP22® Test Kit.
Genito-urinary tract cancers were identified in 210/1564 (13%) patients with an Mcm5 result and in 195/1396 (14%) patients with an NMP22 result. At the assay cut-point where sensitivity and specificity were equal, the Mcm5 test detected primary and recurrent bladder cancers with 69% sensitivity (95% confidence interval = 62–75%) and 93% negative predictive value (95% CI = 92–95%). The area under the receiver operating characteristic curve for Mcm5 was 0.75 (95% CI = 0.71–0.79) and 0.72 (95% CI = 0.67–0.77) for NMP22. Importantly, Mcm5 combined with NMP22 identified 95% (79/83; 95% CI = 88–99%) of potentially life threatening diagnoses (i.e. grade 3 or carcinoma in situ or stage ≥pT1) with high specificity (72%, 95% CI = 69–74%).
The Mcm5 immunoassay is a non-invasive test for identifying patients with urothelial cancers with similar accuracy to the FDA-approved NMP22 ELISA Test Kit. The combination of Mcm5 plus NMP22 improves the detection of UCC and identifies 95% of clinically significant disease. Trials of a commercially developed Mcm5 assay suitable for an end-user laboratory alongside NMP22 are required to assess their potential clinical utility in improving diagnostic and surveillance care pathways.
PMCID: PMC3392249  PMID: 22792272
2.  Usefulness of the NMP22BladderChek Test for Screening and Follow-up of Bladder Cancer 
Korean Journal of Urology  2010;51(2):88-93.
We evaluated the usefulness of the nuclear matrix protein 22 BladderChek (NMP22BC) test for the screening and follow-up of bladder cancer.
Materials and Methods
From February 2006 to September 2009, we enrolled 1,070 patients who had hematuria or who were being followed up for bladder cancer. We compared the sensitivity and specificity of the NMP22BC test with those of urine cytology.
The sensitivity of the NMP22BC test (77.5%) was significantly higher than that of urine cytology (46.3%). The specificity of the NMP22BC test was 88.8%, compared with 97.9% for urine cytology. The sensitivity of the NMP22BC test (81.8%) in non-muscle-invasive bladder cancer was higher than that of cytology (36.4%). However, the sensitivity of the NMP22BC test and of urine cytology in invasive bladder cancer were 57.1% and 92.9%, respectively. The sensitivity of the NMP22BC test was higher for low-grade bladder cancer (83.9%) than for high-grade (62.5%), and the sensitivity of cytology was higher for high-grade bladder cancer (66.7%) than for low-grade (37.5%). Follow-up bladder cancer was detected in 262 patients. The sensitivity of the NMP22BC test in that group (72.7%) was decreased and the specificity (91.7%) was increased. The sensitivity of cytology (54.5%) in the follow-up group was increased and the specificity (95.6%) was decreased. The presence of pyuria was significantly associated with the lower specificity of the NMP22BC test.
The greater sensitivity of the NMP22BC test may be more useful for the diagnosis of non-muscle-invasive bladder cancer and low-grade bladder cancer than for the diagnosis of invasive or high-grade bladder cancer. If the NMP22BC test is performed in the absence of pyuria, it may play a compensatory role for urine cytology.
PMCID: PMC2855476  PMID: 20414419
Nuclear matrix protein 22; Cytology; Urinary bladder neoplasms
3.  Galactosyl Human Serum Albumin-NMP1 Conjugate: A Near Infrared (NIR)-Activatable Fluorescence Imaging Agent to Detect Peritoneal Ovarian Cancer Metastases 
Bioconjugate chemistry  2012;23(8):1671-1679.
Patient survival depends on the completeness of resection of peritoneal ovarian cancer metastases (POCM) and therefore, it is important to develop methods to enhance detection. Previous probe designs based on activatable galactosyl human serum albumin (hGSA)-fluorophore pairs, which target lectin receptors expressed on POCM, have used only visible range dyes conjugated to hGSA. However, imaging probes emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and result in lower background autofluorescence than those emitting in the visible range. A NIR-activatable hGSA fluorophore was synthesized using a bacteriochlorin-based dye, NMP1. NMP1 has two unique absorption peaks, one in the green range and the other in the NIR range, but emits at a NIR peak of 780 nm. NMP1, thus, has two different Stokes shifts that have the potential to allow imaging of POCM both at the peritoneal surface and just below it.
hGSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (hGSA-NMP1). The activation ratio of hGSA-NMP1 was measured by the fluorescence intensity before and after exposure to 10% SDS. The activation ratio of hGSA-NMP1 was ~100-fold in vitro. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare hGSA-NMP1 with hGSA-IR800 and hGSA-ICG (two always-on control agents with similar emission to NMP1) in terms of comparative fluorescence signal and the ability to detect POCM in mice models. The sensitivity and specificity of hGSA-NMP1 for POCM implant detection were determined by co-localizing NMP1 emission spectra with red fluorescent protein (RFP) expressed constitutively in SHIN3 tumor implants at different depths below the peritoneal surface. In vitro, SHIN3 cells were easily detectable after 3 hours of incubation with hGSA-NMP1. In vivo submillimeter POCM foci were clearly detectable with spectral fluorescence imaging using hGSA-NMP1. Among 555 peritoneal lesions, hGSA-NMP, using NIR and green excitation light, respectively, detect 75% of all lesions and 91% of lesions ~0.8 mm or greater in diameter. Few false positives were encountered. Nodules located at a depth below the small bowel surface were only depicted with hGSA-NMP1.
We conclude that hGSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity.
PMCID: PMC3432315  PMID: 22799539
fluorescence imaging; activatable; near infrared; multiple excitations
4.  Biomarkers for detection and surveillance of bladder cancer 
Bladder cancer is the fourth most common cancer in men and the ninth most common cancer in women in Canada. Early detection of tumours is essential for improved prognosis and long-term survival. The standard method for detection and surveillance is cystoscopy together with urine cytology. Cystoscopy is relatively sensitive but is expensive and invasive. Urinary cytology is a noninvasive method that has poor sensitivity but high specificity; it is relied on for the detection of carcinoma in situ. Currently, several urinary-based bladder tumour biomarkers with USFDA/Health Canada approval are available commercially, but none have been widely adopted by urologists despite their offering high sensitivity and/or specificity. We present here a review of recent studies evaluating 7 commercial biomarker assays for the detection and/or surveillance of bladder cancer.
Sensitivity and specificity ranges, respectively, for each marker were reported as follows: BTA Stat (Polymedco), 52.5%–78.0% and 69.0%–87.1%; BTA Trak (Polymedco), 51%–100% and 73%–92.5%; cytology, 12.1%–84.6% and 78.0%–100%; hematuria dipstick, 47.0%–92.6% and 51.0%–84.0%; NMP22 Bladder Cancer Test (Matritech), 34.6%–100% and 60.0%–95.0%; NMP22 BladderChek (Matritech), 49.5%–65.0% and 40.0%–89.8%; ImmunoCyt/uCyt+ (DiagnoCure), 63.3%–84.9% and 62.0%–78.1%; ImmunoCyt/uCyt+ and cytology, 81.0%–89.3% and 61.0%–77.7%; and UroVysion (Abbott Molecular)/florescence in situ hybridization, 68.6%–100% and 65.0%–96.0%.
We find that no currently available bladder cancer urinary marker is sensitive enough to eliminate the need for cystoscopy. In addition, cytology remains integral to the detection of occult cancer. However, owing to their relatively high sensitivities, these markers may be used to extend the period between cystoscopies in the surveillance of patients with transitional cell carcinoma. Further study is required to determine which markers, alone or in panel, would best accomplish this.
PMCID: PMC2494897  PMID: 18682775
5.  Assessing the clinical benefit of NMP22 in the surveillance of patients with non–muscle-invasive bladder cancer and negative cytology: a decision-curve analysis 
Cancer  2011;117(13):2892-2897.
Several studies have shown that abnormal levels of nuclear matrix protein 22 (NMP22) are associated with bladder cancer, leading to NMP22 being approved as a urinary biomarker by the FDA. Nonetheless, the clinical significance of NMP22 remains unclear.
To use decision analysis to determine whether NMP22 improves medical decision-making.
Design, Setting, and Participants
The study included 2,222 patients with a history of non–muscle-invasive bladder cancer and current negative cytology. We developed models to predict cancer recurrence or progression to muscle-invasive disease using NMP22 levels, age, and gender.
Voided NMP22 and cystoscopy.
Clinical net benefit was calculated by summing the benefits (true positives) and subtracting the harms (false positives) and weighting these by the threshold probability at which a patient or clinician would opt for cytoscopy.
Results and limitations
After cystoscopy, 581 (26%) patients were found to have cancer. NMP22 level was significantly associated with bladder cancer recurrence and progression (p<0.001 for both). Using NMP22 in a model with age and gender was associated with better patient outcomes than performing cystoscopy on everyone for threshold probabilities above 8% for recurrence and above 3% for progression. Only offering cystoscopy to those with a 15% or greater risk would reduce the number of cystoscopies by 229, while missing only 25 cancer recurrences per 1000 men with a negative cytology. The study was limited by its multicenter design.
For clinicians who would perform a cystoscopy at a threshold of 5% for recurrence or 1% for progression, NMP22 will not aid clinical decision-making. For less risk-averse clinicians who would only perform a cystoscopy at a threshold probability >8% for recurrence or >3% for progression, NMP22 can help determine which patients require cystoscopy and which can be spared this procedure.
PMCID: PMC3334293  PMID: 21692050
nuclear matrix protein 22; bladder cancer; urothelial carcinoma; detection; surveillance
6.  NMP22 is predictive of recurrence in high-risk superficial bladder cancer patients 
The nuclear matrix protein 22 (NMP22) assay has been shown to have greater sensitivity for the diagnosis and detection of recurrent urothelial carcinoma of the bladder (UCB) over that of traditional urine cytology. We assessed the use of NMP22 to predict which high-risk superficial UCB patients will have recurrence, progression or disease-related death; we compared these results to standard urine cytology.
One hundred consecutive patients with high-risk superficial UCB were enrolled. During surveillance, urine was collected for cytology and NMP22 testing. Patients were followed for at least 6 months. Retrospective chart review was undertaken to collect data on previous tumour history, tumour characteristics, disease recurrences, progression and death. Kaplan-Meier analyses were performed to determine the significance between NMP22-positive and -negative patients in terms of recurrence-free, progression-free and overall survival. Similar analyses were performed for urine cytology.
From 94 eligible patients, 15 and 79 were NMP22 positive and negative, respectively. The baseline characteristics between the 2 groups were not significantly different in terms of patient characteristics, prior tumour history or intravesical therapies received. Mean recurrence-free survival time was significantly lower in the NMP22 positive group (p = 0.038); however, mean progression-free and overall survival were not significantly different between the 2 groups (p = 0.297 and 0.519, respectively). Urine cytology demonstrated no significant predictive power for disease recurrence, progression or survival.
The nuclear matrix protein 22 assay appears to have predictive value for future tumour recurrences, but not progression or overall survival in patients with high-risk superficial UCB.
PMCID: PMC2792415  PMID: 20019971
7.  Prospective evaluation of fluorescence-guided cystoscopy to detect bladder cancer in a high-risk population: results from the UroScreen-Study 
SpringerPlus  2014;3:24.
To prospectively evaluate the role of fluorescence-guided cystoscopy in a high-risk bladder cancer population undergoing screening based on a multi-marker panel of urine-tests (UroScreen-study).
Patients and methods
UroScreen was conducted as a validation study for tumor markers within the frame of a health surveillance program of workers with occupational exposure to aromatic amines. Voluntary annual screens were done in 1,609 men. Cytology, quantitative NMP22® assay, and UroVysion (FISH) were applied to 7091 urine samples. Subjects with at least one positive urine-based tumor marker and/or persisting microscopic hematuria were offered fluorescence-guided (PDD) instead of white light cystoscopy. In case of suspicious findings histopathological evaluation by transurethral biopsy was performed. Data were statistically summarized and compared to tumors found by the standard algorithm of the screening study.
Twenty-two subjects with a mean age of 58 years (39–72) underwent PDD cystoscopy. Of those 3 had positive NMP22 tests, 14 positive FISH tests and 9 suspicious cytologies. Two had persisting microscopic hematuria only. PDD cystoscopy revealed enhanced unifocal fluorescence in 14. All had subsequent transurethral biopsy or resection. In total, 1 urothelial carcinoma (pTaG1, low grade) was diagnosed. In the other participants urothelial cancer of the bladder was ruled out. Chronic cystitis was revealed in 8 of 14 biopsies. No higher detection rate was found using PDD than with the standard algorithm of the UroScreen study in which 17 tumors were detected by white light cystoscopy.
The use of PDD does not lead to a higher cancer detection rate in a high-risk screening population. Larger sample sizes may be needed to ultimately asses the value of PDD for bladder cancer screening.
PMCID: PMC3905106  PMID: 24478941
Urothelial cancer of the bladder; Urine based tumor marker; Bladder cancer screening; NMP22; UroVysion; UroScreen; Cytology; Photodynamic diagnostics; Cystoscopy
8.  Experimental exposure of male volunteers to N-methyl-2-pyrrolidone (NMP): acute effects and pharmacokinetics of NMP in plasma and urine. 
OBJECTIVES: To study the acute effects of exposure to the increasingly used solvent, N-methyl-2-pyrrolidone (NMP) in male volunteers. Further, to determine the NMP concentration in plasma and urine during and after the exposure. METHODS: Six male volunteers were exposed for eight hours on four different days to 0, 10, 25, and 50 mg/m3 NMP. Plasma was collected and urine was sampled during and after the exposure. Changes in nasal volume were measured by acoustic rhinometry and in airway resistance by spirometry. RESULTS: The eight-hour experimental exposure to 10, 25, and 50 mg/m3 did not induce discomfort to eyes or upper airways. Acute changes in nasal volume were not found, and no changes in the spirometric data could be registered. The elimination curves suggested a non-linear pattern and at the end of exposure showed mean (range) half lifes of NMP in plasma of about 4.0 (2.9-5.8) hours and in urine 4.5 (3.5-6.6) hours. The unmetabolised NMP found in urine samples collected during exposure and at the subsequent 44 hours corresponded to about 2% of the calculated absorbed dose. At the end of the exposure there was a close correlation between exposures and the plasma concentration and urinary excretion of NMP. CONCLUSIONS: NMP was absorbed through the respiratory tract and readily eliminated from the body, mainly by biotransformation to other compounds. Exposure to 10, 25, or 50 mg/m3 NMP did not cause nose, eye, or airway irritation. Thus, NMP is a mild irritant.
PMCID: PMC1128696  PMID: 9166128
9.  Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist 
Molecular Pain  2013;9:32.
Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181.
NMP-181 action on CB1 and CB2 receptors was characterized in radioligand binding and in vitro GTPγ[35S] functional assays, and block of transiently expressed human Cav3.2 T-type channels by NMP-181 was analyzed by patch clamp. The analgesic effects and in vivo mechanism of action of NMP-181 delivered spinally or systemically were analyzed in formalin and CFA mouse models of pain. NMP-181 inhibited peak CaV3.2 currents with IC50 values in the low micromolar range and acted as a CB2 agonist. Inactivated state dependence further augmented the inhibitory action of NMP-181. NMP-181 produced a dose-dependent antinociceptive effect when administered either spinally or systemically in both phases of the formalin test. Both i.t. and i.p. treatment of mice with NMP-181 reversed the mechanical hyperalgesia induced by CFA injection. NMP-181 showed no antinocieptive effect in CaV3.2 null mice. The antinociceptive effect of intrathecally delivered NMP-181 in the formalin test was reversed by i.t. treatment of mice with AM-630 (CB2 antagonist). In contrast, the NMP-181-induced antinociception was not affected by treatment of mice with AM-281 (CB1 antagonist).
Our work shows that both T-type channels as well as CB2 receptors play a role in the antinociceptive action of NMP-181, and also provides a novel avenue for suppressing chronic pain through novel mixed T-type/cannabinoid receptor ligands.
PMCID: PMC3703287  PMID: 23815854
T-type Channels; Cannabinoid Receptors; Pain; Patch-Clamp; Mice
10.  TNF-α Promotes Caspase Activation and Apoptosis in Human Fetal Membranes 
Purpose: Increased amniotic fluid tumor necrosis factor (TNF) is a marker of infection when associated with preterm labor and preterm premature rupture of the amniochorionic membranes (PROM). We have noted increased apoptosis in membranes derived from women with PROM. This study examines the role of TNF in promoting fetal membrane apoptosis. Methods: Amniochorion (n = 8), collected at the time of elective repeat cesarean section prior to labor from normal term gestation, were placed in an organ explant system. After 48 h in culture, the membranes were stimulated with recombinant TNF-α (20 ng/mL) for 24 h. Tissue frozen after stimulation was subjected to RT-PCR to study the expression of TNF-induced caspase genes. ELISA assayed the levels of proapoptotic p53 in tissues and cell death related nuclear matrix protein (NMP) in tissue culture supernatants. The activity of caspases in tissue homogenates was measured using substrates specific for caspase 2, 3, 6, 8, and 9. Results were analyzed by using the Wilcoxon nonparametric test for paired samples. A p < 0.05 was considered significant. Results: RT-PCR showed induction of caspases 2, 8, and 9 (caspase cascade initiators) in human fetal membranes after TNF stimulation. Caspases 3 and 6 (effector caspases) expression was constitutive in both TNF stimulated- and control membranes. Caspases, 2, 3, 8, and 9 activity was significantly higher in TNF-stimulated tissues compared with control, whereas, no significant change in caspase 6 activity was noticed. TNF-stimulated tissues released increased levels of NMP (24.03 U/mL) compared with control (13.5U/mL) (p = 0.03). TNF also increased p53 levels in the tissues (0.05 ng/mL) compared with control cultures (0.03 ng/mL; p = 0.02). Conclusions: TNF increases proapoptotic p53 levels and caspase activities in fetal membranes. Increased NMP reflects cell death.
PMCID: PMC3455651  PMID: 12036088
amniochorion; caspases; preterm labor; p53; PROM; TNF
11.  Identification of Second Messenger Mediating Signal Transduction in the Olfactory Receptor Cell 
The Journal of General Physiology  2003;122(5):557-567.
One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed “InsP3 odorants”). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells (∼2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.
PMCID: PMC2229575  PMID: 14581582
olfactory receptor cell; signal transduction; second messenger; cAMP; caged compound
12.  Down-Regulation of Bax-Interacting Factor-1 (Bif-1) in Human Pancreatic Ductal Adenocarcinoma 
Pancreas  2011;40(3):433-437.
Bax-Interacting Factor-1 (Bif-1) protein plays a critical role in apoptosis, mitochondrial morphogenesis and autophagy, and its loss promotes tumorigenesis. The role of Bif-1 in pancreatic cancer has not been studied.
We determined Bif-1 expression in 82 human pancreatic ductal adenocarcinomas (PDC) and in 82 non-malignant pancreatic specimens (NMP), using immunohistochemistry and tissue microarray. Bif-1 immunostain was semiquantitatively scored on a scale of 0 to 9.
Bif-1 scores in NMP were either 6 or 9, with lower scores in only 19 of 82 NMP (23.2%). Low Bif-1 expression (score < 6) was found in 37 of 82 PDC (45.1%), a proportion significantly greater than that found in NMP (p = 0.005). Bif-1 expression was twice as likely to be low in PDC as in NMP (RR = 1.95, 95% CI: 1.23, 3.09). Kaplan-Meier survival estimates showed no difference in survival between patients with low and high Bif-1 expression (p = 0.21, log-rank test).
Bif-1 expression is down-regulated in a subset of PDC. This novel finding is in agreement with the tumor suppressor function of Bif-1. The lack of association with Bif-1 expression with patient survival may be best explained by the complexity of carcinogenesis.
PMCID: PMC3063470  PMID: 21283040
Bif-1; pancreatic ductal adenocarcinoma; tissue microarray; immunohistochemistry; outcomes
13.  Nmp4/CIZ inhibits mechanically-induced β-catenin signaling activity in osteoblasts 
Journal of cellular physiology  2010;223(2):435-441.
Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) and β-catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria-derived osteoblasts from Nmp4-deficient and wild-type mice, we found that the normal translocation of β-catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS-induced mechanotransduction generally associated with the β-catenin signaling pathway, including ERK, Akt and GSK3β activity, as well as expression of the β-catenin-responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS-induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS.
PMCID: PMC2872931  PMID: 20112285
Akt; bone; ERK; fluid shear mechanosome; mechanotransduction
14.  Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT) 
Journal of Cellular Physiology  2012;227(5):1873-1882.
Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, ALK3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism.
PMCID: PMC3209493  PMID: 21732358
adipogenesis; BMP2; differentiation; osteoporosis; PTH; proliferation
15.  Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ε 
DNA repair  2011;10(5):476-482.
During DNA synthesis in vitro using dNTP and rNTP concentrations present in vivo, yeast replicative DNA polymerases α, δ and ε (Pols α, δ and ε) stably incorporate rNTPs into DNA. rNTPs are also incorporated during replication in vivo, and they are repaired in an RNase H2-dependent manner. In strains encoding a mutator allele of Pol ε (pol2-M644G), failure to remove rNMPs from DNA due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2, results in deletion of 2-5 base pairs in short repetitive sequences. Deletion rates depend on the orientation of the reporter gene relative to a nearby replication origin, suggesting that mutations result from rNMPs incorporated during replication. Here we demonstrate that 2-5 base pair deletion mutagenesis also strongly increases in rnh201Δ strains encoding wild type DNA polymerases. As in the pol2-M644G strains, the deletions occur at repetitive sequences and are orientation-dependent, suggesting that mismatches involving misaligned strands arise that could be subject to mismatch repair. Unexpectedly however, 2-5 base pair deletion rates resulting from loss of RNH201 in the pol2-M644G strain are unaffected by concomitant loss of MSH3, MSH6, or both. It could be that the mismatch repair machinery is unable to repair mismatches resulting from unrepaired rNMPs incorporated into DNA by M644G Pol ε, but this possibility is belied by the observation that Msh2-Msh6 can bind to a ribonucleotide-containing mismatch. Alternatively, following incorporation of rNMPs by M644G Pol ε during replication, the conversion of unrepaired rNMPs into mutations may occur outside the context of replication, e.g., during the repair of nicks resulting from rNMPs in DNA. The results make interesting predictions that can be tested.
PMCID: PMC3652408  PMID: 21414850
ribonucleotide incorporation; RNase H2; DNA mismatch repair; deletions; genome instability
16.  Investigation of CCL18 and A1AT as potential urinary biomarkers for bladder cancer detection 
BMC Urology  2013;13:42.
In this study, we further investigated the association of two biomarkers, CCL18 and A1AT, with bladder cancer (BCa) and evaluated the influence of potentially confounding factors in an experimental model.
In a cohort of 308 subjects (102 with BCa), urinary concentrations of CCL18 and A1AT were assessed by enzyme-linked immunosorbent assay (ELISA). In an experimental model, benign or cancerous cells, in addition to blood, were added to urines from healthy controls and analyzed by ELISA. Lastly, immunohistochemical staining for CCL18 and A1AT in human bladder tumors was performed.
Median urinary protein concentrations of CCL18 (52.84 pg/ml vs. 11.13 pg/ml, p < 0.0001) and A1AT (606.4 ng/ml vs. 120.0 ng/ml, p < 0.0001) were significantly elevated in BCa subjects compared to controls. Furthermore, the addition of whole blood to pooled normal urine resulted in a significant increase in both CCL18 and A1AT. IHC staining of bladder tumors revealed CCL18 immunoreactivity in inflammatory cells only, and there was no significant increase in these immunoreactive cells within benign and cancerous tissue and no association with BCa grade nor stage was noted. A1AT immunoreactivity was observed in the cytoplasm of epithelia cells and intensity of immunostaining increased with tumor grade, but not tumor stage.
Further development of A1AT as a diagnostic biomarker for BCa is warranted.
PMCID: PMC3846766  PMID: 24011266
Biomarkers; Bladder cancer; Specificity; Urine
17.  Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain 
PLoS ONE  2013;8(10):e78384.
The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology.
PMCID: PMC3813627  PMID: 24205218
18.  Transdermal delivery of zidovudine (AZT): The effects of vehicles, enhancers, and polymer membranes on permeation across cadaver pig skin 
AAPS PharmSciTech  2004;5(3):82-89.
The purpose of this study was to investigate the effects of vehicles, enhancers, and polymer membranes on 3-azido-3-deoxythymidine (AZT) permeation across cadaver pig skin. Four binary vehicles (ethanol/water, isopropyl alcohol/water, polyethylene glycol 400/water, and ethanol/isopropyl myristate [IPM] were tested for AZT solubility and permeability across pig skin; ethanol/IPM (50/50, vol/vol) demonstrated the highest AZT flux (185.23 μ/cm2/h). Next, the addition of various concentrations of different enhancers (N-methyl-2-pyrrolidone [NMP], oleic acid, and lauric acid) to different volume ratios of ethanol/IPM was investigated for their effect on AZT solubility and permeability across pig skin. The use of 2 conbinations (ethanol/IPM [20/80] plus 10% NMP and ethanol/IPM [30/70] plus 10% NMP) resulted in increased AZT solubility (42.6 and 56.27 mg/mL, respectively) and also high AZT flux values (284.92 and 460.34 μg/cm2/h, respectively) without appreciable changes in lag times (6.25 and 7.49 hours, respectively) when compared with formulations using only ethanol/IPM at 20/80 and 30/70 volume ratios without addition of the enhancer NMP. Finally, AZT permeation across pig skin covered with a microporous polyethylene (PE) membrane was investigated. The addition of the PE membrane to the pig skin reduced AZT flux values to ∼50% of that seen with pig skin alone. However, the AZT flux value attained with ethanol/IPM (30/70) plus 10% NMP was 215.30 μg/cm2/h, which was greater than the target flux (208 μg/cm2/h) needed to maintain the steady-state plasma concentration in humans. The results obtained from this study will be helpful in the development of an AZT transdermal drug delivery system.
PMCID: PMC2750270  PMID: 15760081
Zidovudine permeation; enhancer; binary vehicles; polymer membrane; transdermal delivery system
19.  Characterization of Periplasmic Protein BP26 Epitopes of Brucella melitensis Reacting with Murine Monoclonal and Sheep Antibodies 
PLoS ONE  2012;7(3):e34246.
More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.
PMCID: PMC3311636  PMID: 22457830
20.  Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase. 
Journal of Bacteriology  1980;143(1):151-157.
Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.
PMCID: PMC294200  PMID: 6995425
21.  Methylation Markers for Urine-Based Detection of Bladder Cancer: The Next Generation of Urinary Markers for Diagnosis and Surveillance of Bladder Cancer 
Advances in Urology  2012;2012:503271.
Cancer of the urinary bladder is the fifth most common neoplasm in the industrialized countries. Diagnosis and surveillance are dependent on invasive evaluation with cystoscopy and to some degree cytology as an adjunct analysis. Nomuscle invasive bladder cancer is characterized by frequent recurrences after resection, and up to 30% will develop an aggressive phenotype. The journey towards a noninvasive test for diagnosing bladder cancer, in order to replace or extend time between cystoscopy, has been ongoing for more than a decade. However, only a handful of tests that aid in clinical decision making are commercially available. Recent reports of DNA methylation in urine specimens highlight a possible clinical use of this marker type, as high sensitivities and specificities have been shown. This paper will focus on the currently available markers NMP22, ImmunoCyt, and UroVysion as well as novel DNA methylation markers for diagnosis and surveillance of bladder cancer.
PMCID: PMC3385670  PMID: 22761614
22.  The current role of telomerase in the diagnosis of bladder cancer 
Bladder cancer has an incidence of 15 cases per 100,000 persons in the global population and is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are either invasive or not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers still remains. Among the markers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Relevant papers on the etiology, diagnosis, and evaluation of bladder cancer using telomerase in urine were searched for and considered. The PubMed search was performed using the text terms “bladder cancer”, “diagnosis”, and “telomerase”. Previous studies have shown that the quantitative Telomerase Repeat Amplification Protocol (TRAP) assay performed in voided urine is an important non-invasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low grade tumors. The main limitation of this test is the rate of false positive results due to the presence of inflammatory or non-tumor cells (i.e., epithelial cells from the lower genital tract), which express telomerase activity (TA). Consequently, an in situ analysis would seem to be important to identify the nature of telomerase-positive cells. Immunocytochemical detection of the hTERT subunit by a specific antibody seemed to open up the possibility to identify different cellular components of urine. However, the lack of a strict relationship between hTERT protein expression and telomerase activity has, to a certain extent, made this approach less relevant. In conclusion, telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost/benefit terms when used in selected symptomatic patients or professionally high-risk subgroups.
PMCID: PMC2684324  PMID: 19468427
Bladder cancer; early diagnosis; telomerase; telomerase repeat amplification protocol
23.  Hyaluronic Acid-Paclitaxel: Antitumor Efficacy against CD44(+) Human Ovarian Carcinoma Xenografts1 
Neoplasia (New York, N.Y.)  2007;9(6):479-486.
Numerous human tumor types, including ovarian cancer, display a significant expression of the CD44 family of cell surface proteoglycans. To develop tumor-targeted drugs, we have initially evaluated whether the CD44 ligand hyaluronic acid (HA) could serve as a backbone for paclitaxel (TXL) prodrugs. HA-TXL was prepared by modification of previous techniques. The in vitro cytotoxicity of HA-TXL against the CD44(+) human ovarian carcinoma cell lines SKOV-3ip and NMP-1 could be significantly blocked by preincubation with a molar excess of free HA. Female nude mice bearing intraperitoneal implants of NMP-1 cells were treated intraperitoneally with a single sub-maximum tolerated dose dose of HA-TXL or with multiple-dose regimens of paclitaxel (Taxol; Mead Johnson, Princeton, NJ) to determine the effects of these regimens on host survival and intraperitoneal tumor burden, with the latter being assessed by magnetic resonance imaging. NMP-1 xenografts were highly resistant to Taxol regimens, as host survival was only nominally improved compared to controls (T//C ∼ 120), whereas single-dose HA-TXL treatment significantly improved survival in this model (T//C ∼ 140; P = .004). In both NMP-1 and SKOV-3ip models, MR images of abdomens of HA-TXL-treated mice obtained shortly before controls required humane sacrifice revealed markedly reduced tumor burdens compared to control mice. This study is among the first to demonstrate that HA-based prodrugs administered locoregionally have antitumor activity in vivo.
PMCID: PMC1899257  PMID: 17603630
Hyaluronic acid; CD44; paclitaxel; prodrug; human ovarian carcinoma
24.  Non-medical prescribing of chemotherapy: engaging stakeholders to maximise success? 
ecancermedicalscience  2014;8:417.
This study report examines the views and experiences of professional stakeholders about non-medical prescribing (NMP) of chemotherapy.
The introduction of open formulary NMP has created opportunities to radically change health-care delivery. For chemotherapy services, the most recent advice from the National Chemotherapy Advisory Group [Department of Health (2009) Chemotherapy Services in England, ensuring quality and safety: a report from the National Chemotherapy Advisory Group, London Her Majesty’s Stationary Office] clearly endorses the development of nurse- or pharmacist-led chemotherapy clinics. This is very much welcomed but is based on very limited evidence as to their effectiveness.
A fourth-generation evaluation study.
A purposeful sample of 23 stakeholders connected with the chemotherapy service was used. A serial data collection technique with individual interviews followed by uni-professional focus groups was adopted. Finally, a multi-professional focus group was held to determine the strategic way forward. Data were collected in 2009–2010.
The study illuminated the key features necessary to maximise success of NMP in chemotherapy clinics and captures the importance of good working relationships. Whilst different practice models will emerge, fundamental and core to services is the need for good team working, established and effective communication strategies, and most importantly avoiding isolation in practice. This study additionally reinforced any evaluation takes place within preexisting political contexts and in particular medical dominance. Not all medical colleagues agreed with or wanted NMP for their patients, highlighting difficulties of developing new models of working within a resisting culture.
No objections to NMP of chemotherapy were found, but, clearly, the context of practice needs to be agreed and supportedby all professional stakeholders.
What is already known about this topic
Open formulary non-medical prescribing has been rapidly introduced over the past decade.
Little research has been conducted in acute care and none in the chemotherapy setting.
Cancer policy recommends the introduction of nurse-led chemotherapy clinics.
What this paper adds
Non-medical prescribing (NMP) in chemotherapy is appropriate with the right model of practice.
Well-established professional relationships are a key to success.
NMP is not appropriate in isolation of the multidisciplinary team (MDT).
Implications for practice and/or policy
Nurses need to demonstrate the value of non-medical prescribing in chemotherapy using available metrics.
Models of practice need to ensure good communication channels, MDT working, and transparency of prescribing.
PMCID: PMC3990663  PMID: 24761158
non-medical prescribing; chemotherapy; nurse-led clinic
25.  A Candida albicans Mannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells and Induces T-Cell Activation without Stimulating Proinflammatory Cytokine Production▿  
Infection and Immunity  2008;76(9):4359-4367.
Mannoproteins are cell wall components of pathogenic fungi and play major virulence and immunogenic roles with both their mannan and protein moieties. The 65-kDa mannoprotein (MP65) of Candida albicans is a β-glucanase adhesin recognized as a major target of the human immune response against this fungus, and its recombinant product (rMP65; devoid of the mannan moiety) is presently under consideration as a vaccine candidate. Here we investigated cellular and molecular aspects of the interaction of rMP65 with human antigen-presenting cells. We also assessed the ability of rMP65 to initiate a T-cell response. Both the native mannosylated MP65 (nMP65) and the recombinant product were efficiently bound and taken up by macrophages and dendritic cells. However, contrarily to nMP65, rMP65 did not induce tumor necrosis factor alpha and interleukin-6 release from these cells. On the other hand, rMP65 was rapidly endocytosed by both macrophages and dendritic cells, in a process involving both clathrin-dependent and clathrin-independent mechanisms. Moreover, the RGD sequence inhibited rMP65 uptake to some extent. After internalization, rMP65 partially colocalized with lysosomal membrane-associated glycoproteins 1 and 2. This possibly resulted in efficient protein degradation and presentation to CD4+ T cells, which proliferated and produced gamma interferon. Collectively, these results demonstrate that the absence of the mannan moiety does not deprive MP65 of the capacity to initiate the pattern of cellular and molecular events leading to antigen presentation and T-cell activation, which are essential features for further consideration of MP65 as a potential vaccine candidate.
PMCID: PMC2519437  PMID: 18591233

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