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1.  Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma* 
Molecular & Cellular Proteomics : MCP  2014;13(10):2661-2672.
The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.
PMCID: PMC4188994  PMID: 25034945
2.  The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator 
PLoS Biology  2013;11(8):e1001645.
The CK2 kinase plays diverse roles in the Drosophila circadian clock by controlling stability of the CLOCK transcription factor itself, besides its known role in degrading CLOCK repressors.
Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic α subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory β subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of CK2α activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2.
Author Summary
The CK2 kinase is an ancient enzyme known to be at the heart of self-sustaining circadian clocks in animals, plants, and fungi. Circadian clocks are responsible for daily circadian rhythms in molecular, physiological, and behavioral processes. Their mechanism relies on transcriptional activators and repressors that constitute a feedback loop. The CLOCK (CLK) activator is required to initiate the transcription of the period (per) and timeless (tim) genes in the late day. The PER and TIM repressors accumulate in a delayed manner and translocate to the nucleus to repress their own gene. Degradation of these repressors allows the activator to start a new cycle. In the fruit fly Drosophila melanogaster, CK2-mediated phosphorylation of the PER and TIM repressors targets them for degradation. Here we find that the CLK activator is also regulated by CK2. In contrast to PER and TIM, CLK is stabilized by CK2α phosphorylation; we also show that PER and TIM are dispensable for this stabilization of CLK. The identification of CK2–CLK protein complexes and the in vitro phosphorylation of CLK by CK2 hint at a direct action of the kinase on the activator. Although transcription factor stabilization is generally expected to be associated with increased activity, genetic analysis indicates that CK2 represses CLK. Hence CK2 phosphorylation seems to be a key signal for the transcriptional activator complex to adopt a proper activation state at a given point of the circadian cycle.
PMCID: PMC3754892  PMID: 24013921
3.  Individual Profiling of Circulating Tumor Cell Composition and Therapeutic Outcome in Patients with Hepatocellular Carcinoma1 
Translational Oncology  2013;6(4):420-428.
BACKGROUND AND AIMS: Circulating tumor cells (CTCs) have been proposed as a monitoring tool in patients with solid tumors. So far, automated approaches are challenged by the cellular heterogeneity of CTC, especially the epithelial-mesenchymal transition. Recently, Yu and colleagues showed that shifts in these cell populations correlated with response and progression, respectively, to chemotherapy in patients with breast cancer. In this study, we assessed which non-hematopoietic cell types were identifiable in the peripheral blood of hepatocellular carcinoma (HCC) patients and whether their distribution during treatment courses is associated with clinical characteristics. METHODS: Subsequent to few enrichment steps, cell suspensions were spun onto glass slides and further characterized using multi-immunofluorescence staining. All non-hematopoietic cells were counted and individual cell profiles were analyzed per patient and treatment. RESULTS: We detected a remarkable variation of cells with epithelial, mesenchymal, liver-specific, and mixed characteristics and different size ranges. The distribution of these subgroups varied significantly between different patient groups and was associated with therapeutic outcome. Kaplan-Meier log-rank test showed that a change in the ratio of epithelial to mesenchymal cells was associated with longer median time to progression (1 vs 15 months; P = .03; hazard ratio = 0.18; 95% confidence interval = 0.01–2.75). CONCLUSIONS: Our data suggest that different CTC populations are identifiable in peripheral blood of HCC patients and, for the first time in HCC, that these individual cell type profiles may have distinct clinical implications. The further characterization and analysis of patients in this ongoing study seems to be warranted.
PMCID: PMC3730017  PMID: 23908685
4.  Carriage of Mycoplasma pneumoniae in the Upper Respiratory Tract of Symptomatic and Asymptomatic Children: An Observational Study 
PLoS Medicine  2013;10(5):e1001444.
In order to determine the possible asymptomatic carriage of Mycoplasma pneumoniae in the upper respiratory tracts of children, Emiel Spuesens and colleagues investigate the prevalence of M. pneumoniae in symptomatic and asymptomatic children at a hospital in The Netherlands.
Please see later in the article for the Editors' Summary
Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods.
Methods and Findings
This study was conducted at the Erasmus MC–Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (n = 405) and children with RTI symptoms (n = 321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%–25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%–20.2%) of the symptomatic children (p = 0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo.
Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.
Please see later in the article for the Editors' Summary
Editors' Summary
Pneumonia (a form of acute respiratory infection) is the single largest cause of death in children worldwide, killing an estimated 1.2 million children aged five and under every year, particularly in South Asia and sub-Saharan Africa. In these settings, bacterial infections with Streptococcus pneumoniae and Haemophilus influenzae are the most common causes of bacterial pneumonia. However, in high-income settings, bacterial infection with Mycoplasma pneumoniae is a major cause of upper and lower respiratory tract infections in children: over one-third of childhood cases of community-acquired pneumonia that require admission to a hospital are caused by M. pneumoniae. Currently, diagnosis of M. pneumoniae infections relies on the detection of antibodies against M. pneumoniae in the blood or detection of bacterial DNA in samples from the upper respiratory tract through polymerase chain reaction (PCR) tests.
Why Was This Study Done?
Other bacteria, such as Streptococcus pneumoniae, are commonly present in children without causing infection, a situation known as asymptomatic carriage. However, to date, it is unknown whether M. pneumoniae is also commonly carried in the upper respiratory tract of children without causing symptoms or leading to infection. The possibility of asymptomatic carriage of M. pneumoniae could have major implications for the interpretation of the results of diagnostic tests and also for clinical management. So in this study conducted in The Netherlands, the researchers investigated whether asymptomatic carriage of M. pneumoniae exists and also whether symptomatic infection could be differentiated from asymptomatic carriage by current diagnostic methods.
What Did the Researchers Do and Find?
Between 2008 and 2011, the researchers recruited children aged between three months and 16 years attending a hospital in Rotterdam for an elective surgical procedure (asymptomatic group) or admitted with a respiratory tract infection (symptomatic group). All children had blood tests and respiratory samples (nasopharyngeal swab) taken on admission and were tested for other pathogens. The researchers invited children who tested positive for M. pneumoniae by PCR to attend for further follow-up and tested them monthly for the presence of M. pneumoniae DNA in the upper respiratory tract until the test was negative on two occasions. Using these methods, the researchers recruited 726 children over the study period—405 in the asymptomatic group and 321 in the symptomatic group. The researchers found that the prevalence of M. pneumoniae did not differ between the asymptomatic group and the symptomatic group, with prevalences of 21.2% and 16.2%, respectively (the prevalence of M. pneumoniae also did not differ significantly between those with lower versus upper respiratory infection). There were also no differences in prevalence in the asymptomatic and symptomatic groups when diagnosed using blood tests. The researchers found a high rate of multiple, coexisting bacterial and viral pathogens in both asymptomatic and symptomatic children: two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Furthermore, season and the year of enrollment affected the prevalence of M. pneumoniae in the asymptomatic group, ranging from 3% during the spring of 2009 to 58% during the summer of 2010. Finally, of the 21 children from the asymptomatic group who participated in the follow-up study, 15 (71%) tested negative within one month, and in the symptomatic group, 19 of 22 children (86%) tested negative after the first visit.
What Do These Findings Mean?
These findings show that M. pneumoniae is carried at high rates in the upper respiratory tracts of healthy children, and that this asymptomatic carriage cannot be differentiated from symptomatic respiratory tract infection by diagnostic tests (serology or PCR). As the prevalence of M. pneumoniae varied between year and season, carriage of M. pneumoniae may follow a cyclic epidemic pattern. This study is from a single study site in one city in The Netherlands, with a relatively small number of children, and so these findings may not be generalizable to other populations. However, as this study suggests that current diagnostic tests do not discriminate between carriage and infection, clinicians may need to reconsider the clinical significance of a positive test result. Future studies are needed to address this diagnostic challenge and also to investigate possible factors that may affect the progression of asymptomatic carriage of M. pneumoniae to symptomatic infection.
Additional Information
Please access these websites via the online version of this summary at
MicrobeWiki has more information on M. pneumoniae
Lab Tests Online explains current tests for M. pneumoniae
PMCID: PMC3653782  PMID: 23690754
5.  The foot in multistage ultra-marathon runners: experience in a cohort study of 22 participants of the Trans Europe Footrace Project with mobile MRI 
BMJ Open  2012;2(3):e001118.
67 runners participated in the Trans Europe FootRace 2009 (TEFR09), a 4487 km (2789 mi) multistage ultra-marathon covering the south of Europe (Bari, Italy) to the North Cape. Reports on ultra-marathons are lacking, but the literature reports overuse injuries in athletes, especially to the Achilles tendon (AT), ankle or hind foot. Bone oedema may be related to exposure and is present in fatigue fractures. Therefore, the aim of this study was to determine prospectively if sustained maximal load during an ultra-marathon leads to damage to the foot.
Design and participants
In a cohort study, repeated scanning of the 22 athletes participating in the study was performed before and during (approximately every 1000 km) the race. Using the obtained fat saturated inversion recovery sequence, two experienced readers blinded to the clinical data rated the images regarding foot lesions. Statistical analysis included regression analysis and computation of the inter-rater reliability.
The TEFR09 course. MRI scanning was performed according to prearranged schedules for every participant, using a mobile 1.5 Tesla MRI unit on a trailer following the race.
Primary outcome measures
MRI data such as AT diameter, bone or tendon lesions, subcutaneous, plantar fascia or intraosseous oedema.
The 22 study participants did not differ significantly from the total of the 67 TEFR09 runners regarding height, weight and age. The AT diameter increased significantly from 6.8 to 7.8 mm as did intraosseous signal, bone lesions and subcutaneous oedema. However, finishers differed only regarding plantar aponeurosis and subcutaneous oedema from participants aborting the TEFR09. Inter-rater reliability was 0.88–0.98.
Under the extreme stress of the TEFR09, an increase of the AT diameter as well as bone signal are thought to be adaptive since only subcutaneous oedema and plantar fascia oedema were related to abortion of the race.
Trial registration number
University of Ulm, Germany Ethics Committee Number 78/08-UBB/se.
Article summary
Article focus
A study on effects of ultra-marathon running, in this case, the multistage Trans Europe FootRace covering a distance of 4487 km from Bari (Italy) to the North Cape.
Observational cohort study using MRI to look for possible lesions to the foot.
Key messages
During sustained maximal load, AT diameter and bone MRI short τ inversion recovery signal (hinting at subtle oedema) increases. This is thought to be adaptive.
Subcutaneous oedema and plantar fascia signal were related to abortion of the race. These measurements seem to be related to relevant changes leading to discontinuation of the run.
No relevant new foot joint or tendon lesions were detected during the race over 4487 km.
Strengths and limitations of this study
Repeated measurement prospectively during the run was possible only because of the mobile MRI unit used for this research project.
The number of included runners (22) is high compared with other MRI-based studies but may have been too small to detect less frequent lesions.
PMCID: PMC3364457  PMID: 22619270
6.  EPIBLASTER-fast exhaustive two-locus epistasis detection strategy using graphical processing units 
Detection of epistatic interaction between loci has been postulated to provide a more in-depth understanding of the complex biological and biochemical pathways underlying human diseases. Studying the interaction between two loci is the natural progression following traditional and well-established single locus analysis. However, the added costs and time duration required for the computation involved have thus far deterred researchers from pursuing a genome-wide analysis of epistasis. In this paper, we propose a method allowing such analysis to be conducted very rapidly. The method, dubbed EPIBLASTER, is applicable to case–control studies and consists of a two-step process in which the difference in Pearson's correlation coefficients is computed between controls and cases across all possible SNP pairs as an indication of significant interaction warranting further analysis. For the subset of interactions deemed potentially significant, a second-stage analysis is performed using the likelihood ratio test from the logistic regression to obtain the P-value for the estimated coefficients of the individual effects and the interaction term. The algorithm is implemented using the parallel computational capability of commercially available graphical processing units to greatly reduce the computation time involved. In the current setup and example data sets (211 cases, 222 controls, 299468 SNPs; and 601 cases, 825 controls, 291095 SNPs), this coefficient evaluation stage can be completed in roughly 1 day. Our method allows for exhaustive and rapid detection of significant SNP pair interactions without imposing significant marginal effects of the single loci involved in the pair.
PMCID: PMC3060319  PMID: 21150885
Epistasis; genome-wide interaction analysis; graphical processing unit
7.  Establishment of a standard operating procedure for predicting the time of calving in cattle 
Journal of Veterinary Science  2011;12(2):177-185.
Precise calving monitoring is essential for minimizing the effects of dystocia in cows and calves. We conducted two studies in healthy cows that compared seven clinical signs (broad pelvic ligaments relaxation, vaginal secretion, udder hyperplasia, udder edema, teat filling, tail relaxation, and vulva edema) alone and in combination in order to predict the time of parturition. The relaxation of the broad pelvic ligaments combined with teat filling gave the best values for predicting either calving or no calving within 12 h. For the proposed parturition score (PS), a threshold of 4 PS points was identified below which calving within the next 12 h could be ruled out with a probability of 99.3% in cows (95.5% in heifers). Above this threshold, intermitted calving monitoring every 3 h and a progesterone rapid blood test (PRBT) would be recommended. By combining the PS and PRBT (if PS ≥ 4), the prediction of calving within the next 12 h improved from 14.9% to 53.1%, and the probability of ruling out calving was 96.8%. The PRBT was compared to the results of an enzyme immunoassay (sensitivity, 90.2%; specificity, 74.9%). The standard operating procedure developed in this study that combines the PS and PRBT will enable veterinarians to rule out or predict calving within a 12 h period in cows with high accuracy under field conditions.
PMCID: PMC3104173  PMID: 21586878
cattle; forecasting; parturition; progesterone
8.  Caudate Nucleus and Insular Activation During a Pain Suppression Paradigm Comparing Thermal and Electrical Stimulation 
Pain modulation is an integral function of the nervous system. It is needed to adapt to chronic stimuli. To gain insights into pain suppression mechanisms, two studies concerning the suppression of the feeling of pain with different stimulation modalities (heat vs. electrical stimuli) but using the same stimulation paradigms were compared: 15 subjects each had been stimulated on both hands under the instruction to suppress the feeling of pain.
Anterior insula and DLPFC activation was seen in both single modality studies and seems to be a common feature of pain suppression, as it is absent in the interaction analyses presented here.
During the task to suppress the feeling of pain, there were no consistent activations stronger under thermostimulation. But during electrostimulation, there was significantly stronger activation than during thermal stimulation in the caudate nucleus bilaterally and in the contralateral posterior insula. This may be attributed to the higher sensory-discriminative content and more demand on subjective rating and suppression of the painful electrical stimulus, compared to thermostimulation. The caudate nucleus seems to play an important role not only in the motor system but also in the modulation of the pain experience.
PMCID: PMC3106353  PMID: 21643502
Functional MRI; pain; caudate; insula; electrical; thermal.
9.  Role of microRNA-199a-5p and discoidin domain receptor 1 in human hepatocellular carcinoma invasion 
Molecular Cancer  2010;9:227.
Micro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion.
Mature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRT-PCR and western blotting analysis.
A significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells.
Decreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.
PMCID: PMC2939569  PMID: 20799954
10.  Incidental findings on brain magnetic resonance imaging: systematic review and meta-analysis 
Objective To quantify the prevalence of incidental findings on magnetic resonance imaging (MRI) of the brain.
Design Systematic review and meta-analysis of observational studies.
Data sources Ovid Medline (1950 to May 2008), Embase (1980 to May 2008), and bibliographies of relevant articles.
Review methods Two reviewers sought and assessed studies of people without neurological symptoms who underwent MRI of the brain with or without intravenous contrast for research purposes or for occupational, clinical, or commercial screening.
Main outcome measures Overall disease specific and age specific prevalence of incidental brain findings, calculated by meta-analysis of pooled proportions using DerSimonian-Laird weights in a random effects model.
Results In 16 studies, 135 of 19 559 people had neoplastic incidental brain findings (prevalence 0.70%, 95% confidence interval 0.47% to 0.98%), and prevalence increased with age (χ2 for linear trend, P=0.003). In 15 studies, 375 of 15 559 people had non-neoplastic incidental brain findings (prevalence 2.0%, 1.1% to 3.1%, excluding white matter hyperintensities, silent infarcts, and microbleeds). The number of asymptomatic people needed to scan to detect any incidental brain finding was 37. The prevalence of incidental brain findings was higher in studies using high resolution MRI sequences than in those using standard resolution sequences (4.3% v 1.7%, P<0.001). The prevalence of neoplastic incidental brain findings increased with age.
Conclusions Incidental findings on brain MRI are common, prevalence increases with age, and detection is more likely using high resolution MRI sequences than standard resolution sequences. These findings deserve to be mentioned when obtaining informed consent for brain MRI in research and clinical practice but are not sufficient to justify screening healthy asymptomatic people.
PMCID: PMC2728201  PMID: 19687093
11.  Human Cytomegalovirus Infection Alters the Expression of Cellular MicroRNA Species That Affect Its Replication▿  
Journal of Virology  2008;82(18):9065-9074.
The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3′ untranslated regions (3′-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3′-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.
PMCID: PMC2546905  PMID: 18596100
12.  Monthly intravenous methylprednisolone in relapsing-remitting multiple sclerosis - reduction of enhancing lesions, T2 lesion volume and plasma prolactin concentrations 
BMC Neurology  2006;6:19.
Intravenous methylprednisolone (IV-MP) is an established treatment for multiple sclerosis (MS) relapses, accompanied by rapid, though transient reduction of gadolinium enhancing (Gd+) lesions on brain MRI. Intermittent IV-MP, alone or with immunomodulators, has been suggested but insufficiently studied as a strategy to prevent relapses.
In an open, single-cross-over study, nine patients with relapsing-remitting MS (RR-MS) underwent cranial Gd-MRI once monthly for twelve months. From month six on, they received a single i.v.-infusion of 500 mg methylprednisolone (and oral tapering for three days) after the MRI. Primary outcome measure was the mean number of Gd+ lesions during treatment vs. baseline periods; T2 lesion volume and monthly plasma concentrations of cortisol, ACTH and prolactin were secondary outcome measures. Safety was assessed clinically, by routine laboratory and bone mineral density measurements. Soluble immune parameters (sTNF-RI, sTNF-RII, IL1-ra and sVCAM-1) and neuroendocrine tests (ACTH test, combined dexamethasone/CRH test) were additionally analyzed.
Comparing treatment to baseline periods, the number of Gd+ lesions/scan was reduced in eight of the nine patients, by a median of 43.8% (p = 0.013, Wilcoxon). In comparison, a pooled dataset of 83 untreated RR-MS patients from several studies, selected by the same clinical and MRI criteria, showed a non-significant decrease by a median of 14% (p = 0.32). T2 lesion volume decreased by 21% during treatment (p = 0.001). Monthly plasma prolactin showed a parallel decline (p = 0.027), with significant cross-correlation with the number of Gd+ lesions. Other hormones and immune system variables were unchanged, as were ACTH test and dexamethasone-CRH test. Treatment was well tolerated; routine laboratory and bone mineral density were unchanged.
Monthly IV-MP reduces inflammatory activity and T2 lesion volume in RR-MS.
PMCID: PMC1501038  PMID: 16719908
13.  Determination of Mercury in an Assortment of Dietary Supplements Using an Inexpensive Combustion Atomic Absorption Spectrometry Technique 
The concentrations of mercury in forty, commercially available dietary supplements, were determined using a new, inexpensive analysis technique. The method involves thermal decomposition, amalgamation, and detection of mercury by atomic absorption spectrometry with an analysis time of approximately six minutes per sample. The primary cost savings from this approach is that labor-intensive sample digestion is not required prior to analysis, further automating the analytical procedure. As a result, manufacturers and regulatory agencies concerned with monitoring lot-to-lot product quality may find this approach an attractive alternative to the more classical acid-decomposition, cold vapor atomic absorption methodology. Dietary supplement samples analyzed included astragalus, calcium, chromium picolinate, echinacea, ephedra, fish oil, ginger, ginkgo biloba, ginseng, goldenseal, guggul, senna, St John's wort, and yohimbe products. Quality control samples analyzed with the dietary supplements indicated a high level of method accuracy and precision. Ten replicate preparations of a standard reference material (NIST 1573a, tomato leaves) were analyzed, and the average mercury recovery was 109% (2.0% RSD). The method quantitation limit was 0.3 ng, which corresponded to 1.5 ng/g sample. The highest found mercury concentration (123 ng/g) was measured in a concentrated salmon oil sample. When taken as directed by an adult, this product would result in an approximate mercury ingestion of 7 μg per week.
PMCID: PMC2562971  PMID: 18924735
14.  Molecular Cloning and Expression of Major Structural Protein VP1 of the Human Polyomavirus JC Virus: Formation of Virus-Like Particles Useful for Immunological and Therapeutic Studies 
Journal of Virology  1999;73(5):4465-4469.
The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.
PMCID: PMC104235  PMID: 10196348

Results 1-14 (14)