A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

From SARS in 2003 to H1N1 in 2009: lessons learned from Taiwan in preparation for the next pandemic.

In anticipation of a future pandemic potentially arising from H5N1, H7N9 avian influenza or Middle East Respiratory Syndrome, and in large part in response to severe acute respiratory syndrome (SARS) in 2003, the city of Taipei, Taiwan, has developed extensive new strategies to manage pandemics. These strategies were tested during the 2009 H1N1 outbreak. This article assesses pandemic preparedness in Taipei in the wake of recent pandemic experiences in order to draw lessons relevant to the broader international public health community. Drawing on Taiwan and Taipei Centers for Disease Control data on pandemic response and control, we evaluated the effectiveness of the changes in pandemic response policies developed by these governments over time, emphasizing hospital and medical interventions with particular attention paid to Traffic Control Bundling. SARS and H1N1 2009 catalysed the Taiwan and Taipei CDCs to continuously improve and adjust their strategies for a future pandemic. These new strategies for pandemic response and control have been largely effective at providing interim pandemic containment and control, while development and implementation of an effective vaccination programme is underway. As Taipei's experiences with these cases illustrate, in mitigating moderate or severe pandemic influenza, a graduated process including Traffic Control Bundles accompanied by hospital and medical interventions, as well as school- and community-focused interventions, provides an effective interim response while awaiting vaccine development. Once a vaccine is developed, to maximize pandemic control effectiveness, it should be allocated with priority given to vulnerable groups, healthcare workers and school children.

Discrete-time semi-Markov modeling of human papillomavirus persistence.

Multi-state modeling is often employed to describe the progression of a disease process. In epidemiological studies of certain diseases, the disease state is typically only observed at periodic clinical visits, producing incomplete longitudinal data. In this paper we consider fitting semi-Markov models to estimate the persistence of human papillomavirus (HPV) type-specific infection in studies where the status of HPV type(s) is assessed periodically. Simulation study results are presented indicating that the semi-Markov estimator is more accurate than an estimator currently used in the HPV literature. The methods are illustrated using data from the HIV Epidemiology Research Study.

A randomised controlled trial of post-operative radiotherapy following breast-conserving surgery in a minimum-risk population. Quality of life at 5 years in the PRIME trial.

OBJECTIVES: To assess whether omission of post-operative radiotherapy (RT) in women with 'low-risk' axillary node-negative breast cancer [tumour size of less than 5 cm (T0-2) although the eligibility criteria further reduce the eligible size to a maximum of 3 cm] treated by breast-conserving surgery and endocrine therapy improves quality of life and is more cost-effective. DESIGN: A randomised controlled clinical trial, using a method of minimisation balanced by centre, grade of cancer, age, lymphovascular invasion and preoperative endocrine therapy was performed. SETTING: Breast cancer clinics in cancer centres in the UK. PARTICIPANTS: Patients aged ≥ 65 years were eligible provided that their breast cancers were considered to be at low risk of local recurrence, they were suitable for breast conservation surgery, they were receiving endocrine therapy and they were willing and able to give informed consent. INTERVENTIONS: The standard treatment of post-operative whole breast irradiation or the omission of RT. MAIN OUTCOME MEASURES: Quality of life was the primary outcome measure, together with anxiety and depression and cost-effectiveness. Secondary outcome measures were recurrence rates and survival, and treatment-related morbidity. The principal method of data collection was by questionnaire, completed at home with a research nurse on four occasions over 15 months, then by postal questionnaire at 3 and 5 years after surgery. RESULTS: The hypothesised improvement in overall quality of life with the omission of RT was not seen in the summary domains of the European Organisation for Research in the Treatment of Cancer (EORTC) scales. Some differences were apparent within subscales of the EORTC questionnaires, and insights into the impact of treatment were also provided by the qualitative data obtained by open-ended questions added by the trial team. Differences were most apparent shortly after the time of completion of RT. RT was then associated with increased breast symptoms and with greater (self-reported) fatigue, but with lower levels of insomnia and endocrine side effects. These statistically significant differences in breast symptoms persisted for up to 5 years after RT [mean difference, RT was 5.27 units greater than no RT, 95% confidence interval (CI) of 1.46 to 9.07], with similar, though non-significant, trends in insomnia. No significant difference was found in the overall quality of life measure, with the no RT group having 0.36 units greater quality of life than the RT group (95% CI -5.09 to 5.81). CONCLUSIONS: Breast RT is tolerated well by most older breast cancer patients without impairing their overall health-related quality of life (HRQoL). Although HRQoL should always be taken into account when determining treatment, our results show that the addition of RT does not impair overall quality of life. Further economic modelling on the longer-term costs and consequences of omitting RT is required. TRIAL REGISTRATION: Current Controlled Trials ISRCTN14817328. FUNDING: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol 15, No. 12. See the HTA programme website for further project information.

Personalizing Stem Cell Research and Therapy: The Arduous Road Ahead or Missed Opportunity?

The euphoria of stem cell therapy has diminished, allowing scientists, clinicians and the general public to seriously re-examine how and what types of stem cells would effectively repair damaged tissue, prevent further tissue damage and/or replace lost cells. Importantly, there is a growing recognition that there are substantial person-to-person differences in the outcome of stem cell therapy. Even though the small molecule pharmaceuticals have long remained a primary focus of the personalized medicine research, individualized or targeted use of stem cells to suit a particular individual could help forecast potential failures of the therapy or identify, early on, the individuals who might benefit from stem cell interventions. This would however demand collaboration among several specialties such as pharmacology, immunology, genomics and transplantation medicine. Such transdisciplinary work could also inform how best to achieve efficient and predictable stem cell migration to sites of tissue damage, thereby facilitating tissue repair. This paper discusses the possibility of polarizing immune responses to rationalize and individualize therapy with stem cell interventions, since generalized "one-size-fits-all" therapy is difficult to achieve in the face of the diverse complexities posed by stem cell biology. We also present the challenges to stem cell delivery in the context of the host related factors. Although we focus on the mesenchymal stem cells in this paper, the overarching rationale can be extrapolated to other types of stem cells as well. Hence, the broader purpose of this paper is to initiate a dialogue within the personalized medicine community by expanding the scope of inquiry in the field from pharmaceuticals to stem cells and related cell-based health interventions.

Breast radiotherapy: considerations in older patients.

With an ageing population, the number of older women with breast cancer eligible for adjuvant irradiation after breast conserving surgery and mastectomy is rising. There is a dearth of level 1 data on the effect of adjuvant irradiation on local control, quality of life and survival. In large part this reflects the exclusion of patients over the age of 70 years from randomised trials. The prevention of local recurrence may reduce the risks of dissemination. However, older women with early breast cancer and a life expectancy of less than 5 years are unlikely to derive a survival benefit from adjuvant radiotherapy. Rates of access of older patients to adjuvant irradiation are lower than for younger patients. Physician and patient bias and co-morbidities are contributory factors. There are also competing risks of mortality from co-morbidities, particularly in women over the age of 80 years. Postoperative radiotherapy after breast conserving surgery does not seem to compromise overall quality of life of older patients. Although the absolute reduction in local recurrence from adjuvant radiotherapy is modest in lower risk older patients after breast conserving surgery and adjuvant systemic therapy, there has to date been no group of fitter old patients defined from whom radiotherapy can be reasonably omitted. Guidelines for postmastectomy radiotherapy should not differ from younger patients. Adequately powered randomised trials are needed to assess the effect of adjuvant irradiation in older patients on outcomes after breast conserving surgery and mastectomy to provide a more robust basis for evidence-based radiotherapy practice.

Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice.

BACKGROUND: The mechanisms responsible for thrombocytopenia associated with dengue fever (DF) and dengue hemorrhage fever (DHF) remain unclear. OBJECTIVE: In this study, we investigated the pathogenic effects of dengue virus (DENV) non-structural protein 1 (NS1) on the elicitation of platelet cross-reactive antibodies. RESULTS: The results showed that anti-DENV NS1 immunoglobulins (Igs) derived from both patients with DF/DHF and recombinant NS1-immunized rabbits could opsonize normal human platelets and enhance platelet-macrophage engagements in vitro. In addition, treatments with anti-NS1 Igs abnormally activated human platelets and induced thrombocytopenia in mice. These prothrombotic characteristics of anti-NS1 Ig might increase the disease burden of coagulant-aberrant DHF patients. To test this hypothesis, we injected anti-NS1 Igs into C57BL/6J mice that were preconditioned into a hypercoagulable state by warfarin treatments. When given before but not after platelet-lysate pre-adsorption, the anti-NS1 Igs injection treatments significantly increased mortality, fibrin deposition in lung, and plasma D-dimer levels, but significantly decreased anticoagulant proteins C, protein S and antithrombin III. CONCLUSIONS: These results suggest that the platelet-bound antibody fractions of anti-NS1 Ig are prothrombotic, which might exacerbate the severity of disease in hosts with an imbalanced coagulant system.

A randomised controlled trial of postoperative radiotherapy following breast-conserving surgery in a minimum-risk older population. The PRIME trial.

OBJECTIVES: To assess whether omission of postoperative radiotherapy in women with 'low-risk' axillary node negative breast cancer (T0-2) treated by breast-conserving surgery and endocrine therapy improves quality of life and is more cost-effective. DESIGN: A randomised controlled clinical trial, using a method of minimisation balanced by centre, grade of cancer, age, lymphatic/vascular invasion and preoperative endocrine therapy, was performed. A non-randomised cohort was also recruited, in order to complete a comprehensive cohort study. SETTING: The setting was breast cancer clinics in cancer centres in the UK. PARTICIPANTS: Patients aged 65 years or more were eligible provided that their cancers were considered to be at low risk of local recurrence, were suitable for breast-conservation surgery, were receiving endocrine therapy and were able and willing to give informed consent. INTERVENTIONS: The standard treatment of postoperative breast irradiation or the omission of radiotherapy. MAIN OUTCOME MEASURES: Quality of life was the primary outcome measure, together with anxiety and depression and cost-effectiveness. Secondary outcome measures were recurrence rates, functional status, treatment-related morbidity and cosmesis. The principal method of data collection was by questionnaire, completed at home with a research nurse at four times over 15 months. RESULTS: The hypothesised improvement in overall quality of life with the omission of radiotherapy was not seen in the EuroQol assessment or in the functionality and symptoms summary domains of the European Organisation for Research in the Treatment of Cancer (EORTC) scales. Some differences were apparent within subscales of the EORTC questionnaires, and insights into the impact of treatment were also provided by the qualitative data obtained by open-ended questions. Differences were most apparent shortly after the time of completion of radiotherapy. Radiotherapy was then associated with increased breast symptoms and with greater fatigue but with less insomnia and endocrine side-effects. Patients had significant concerns about the delivery of radiotherapy services, such as transport, accommodation and travel costs associated with receiving radiotherapy. By the end of follow-up, patients receiving radiotherapy were expressing less anxiety about recurrence than those who had not received radiotherapy. Functionality was not greatly affected by treatment. Within the randomised controlled trial, the Barthel Index demonstrated a small but significant fall in functionality with radiotherapy compared with the no radiotherapy arm of the trial. Results from the non-randomised patients did not confirm this effect, however. Cosmetic results were better in those not receiving radiotherapy but this did not appear to be an important issue to the patients. The use of home-based assessments by a research nurse proved to be an effective way of obtaining high-quality data. Costs to the NHS associated with postoperative radiotherapy were calculated to be of the order of 2000 pounds per patient. In the follow-up in this study, there were no recurrences, and the quality of life utilities from EuroQol were almost identical. CONCLUSIONS: Although there are no differences in overall quality of life scores between the patients treated with and without radiotherapy, there are several dimensions that exhibit significant advantage to the omission of irradiation. Over the first 15 months, radiotherapy for this population is not a cost-effective treatment. However, the early postoperative outcome does not give a complete answer and the eventual cost-effectiveness will only become clear after long-term follow-up. Extrapolations from these data suggest that radiotherapy may not be a cost-effective treatment unless it results in a recurrence rate that is at least 5% lower in absolute terms than those treated without radiotherapy. Further research is needed into a number of areas including the long-term aspects of quality of life, clinical outcomes, costs and consequences of omitting radiotherapy.

Assessment of intervention measures for the 2003 SARS epidemic in Taiwan by use of a back-projection method.

OBJECTIVES: To reconstruct the infection curve for the 2003 severe acute respiratory syndrome (SARS) epidemic in Taiwan and to ascertain the temporal changes in the daily number of infections that occurred during the course of the outbreak. METHOD: Back-projection method. RESULTS: The peaks of the epidemic correspond well with the occurrence of major infection clusters in the hospitals. The overall downward trend of the infection curve after early May corresponds well to the date (May 10) when changes in the review and classification procedure were implemented by the SARS Prevention and Extrication Committee. CONCLUSION: The major infection control measures taken by the Taiwanese government over the course of the SARS epidemic, particularly those regarding infection control in hospitals, played a crucial role in containing the outbreak.

When may adjuvant radiotherapy be avoided in operable breast cancer?

Randomised trials in which the omission of radiotherapy has been tested after breast-conserving surgery, with or without adjuvant systemic therapy, show a significant four- to five-fold reduction in local recurrence. As yet, no subgroup of women managed by breast-conserving surgery has been identified from whom radiotherapy can be withheld. Few randomised data have been published on the effect of omission of radiotherapy on local control, quality of life and costs, particularly in older women for whom the risk of local recurrence is generally lower. Ongoing trials are evaluating the role of radiotherapy in this population of low risk, older women. Adjuvant radiotherapy after breast-conserving surgery or mastectomy significantly reduces the incidence of local recurrence. In women who have had a mastectomy at high risk of recurrence (> 20% risk of recurrence at 10 years), adjuvant radiotherapy improves survival if combined with adjuvant systemic therapy. Among women with T3 tumours, and those with four or more involved axillary nodes treated by mastectomy, postoperative radiotherapy is the standard of care. For women at intermediate risk of recurrence (i.e. <15% 10-year risk of recurrence after surgery and systemic therapy alone), with one to three involved nodes or node negative with other risk factors, the role of radiotherapy is unclear. Clinical trials to assess the role of postmastectomy radiotherapy (PMRT) in this setting are needed. For pT1-2, pNO tumours without other risk factors, there is no evidence at present that PMRT is needed.

Identification and characterization of a lymphocytic Rho-GTPase effector: rhotekin-2.

Rhotekin belongs to the group of proteins containing a Rho-binding domain that are target peptides (effectors) for the Rho-GTPases. We previously identified a novel cDNA with homology to human rhotekin and in this study we cloned and characterized the coding region of this novel 12-exon gene. The ORF encodes a 609 amino-acid protein comprising a Class I Rho-binding domain and pleckstrin homology (PH) domain. Cellular cDNA expression of this new protein, designated Rhotekin-2 (RTKN2), was shown in the cytosol and nucleus of CHO cells. Using bioinformatics and RTPCR we identified three major splice variants, which vary in both the Rho-binding and PH domains. Real-time PCR studies showed exclusive RTKN2 expression in pooled lymphocytes and further purification indicated sole expression in CD4(pos) T-cells and bone marrow-derived B-cells. Gene expression was increased in quiescent T-cells but negligible in activated proliferating cells. In malignant samples expression was absent in myeloid leukaemias, low in most B-cell malignancies and CD8(pos) T-cell malignancies, but very high in CD4(pos)/CD8(pos) T-lymphoblastic lymphoma. As the Rho family is critical in lymphocyte development and function, RTKN2 may play an important role in lymphopoiesis.

ORP3 splice variants and their expression in human tissues and hematopoietic cells.

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture.

Dengue virus (DV) was detected early in infected mosquito C6/36 cells by using indirect immunofluorescence (IF) in conjunction with flow cytometry. Three fixation-permeabilization methods and three DV serotype 1 (DEN-1)-specific monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti-NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional indirect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformaldehyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization method for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity similar to that of nested reverse transcription-PCR, was more sensitive in detecting DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation at day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Moreover, among 12 positive samples which were detected by conventional culture method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal antibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagnosis of DV by both the IF staining and flow cytometry methods.

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells.

Using differential display polymerase chain reaction, a gene was identified in CD34(+)-enriched populations that had with low or absent expression in CD34(-) populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34(+) cells than in CD34(-) cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34(+)38(-) phenotype and was down-regulated with the proliferation and differentiation of CD34(+) cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Genetic analysis of Asian measles virus strains--new endemic genotype in Nepal.

In many parts of Asia measles virus (MV) continues to be endemic. However, little is known about the genetic characteristics of viruses circulating on this continent. This study reports the molecular epidemiological analysis based on the entire nucleocapsid (N) and hemagglutinin (H) genes of the first isolates from Nepal and Taiwan, as well as of recent MV strains from India, Indonesia, and China. Four isolates collected in various regions in Nepal during 1999 belonged to a new genotype, tentatively called D8. Another Nepalese isolate and one from India belonged to genotype D4. The diversity of the Nepalese strains indicated that measles continues to be endemic in this country. The isolate from Taiwan grouped with D3 viruses and one Chinese strain isolated in The Netherlands was assigned to the previously described clade H, known to be endemic in Mainland China. Molecular characterization emerges as an important tool for monitoring virus endemicity and vaccination efforts.

BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase.

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.

Identification of B-cell epitope of dengue virus type 1 and its application in diagnosis of patients.

Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine(111) (His(111)) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine(111) (Leu(111)). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.