Divergent evolutionary trajectories of influenza B viruses underlie their contemporaneous epidemic activity.

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages ("B/Victoria/2/87-like" and "B/Yamagata/16/88-like," termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment-a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.

Impact of Outpatient Neuraminidase Inhibitor Treatment in Patients Infected With Influenza A(H1N1)pdm09 at High Risk of Hospitalization: An Individual Participant Data Metaanalysis.

BACKGROUND: While evidence exists to support the effectiveness of neuraminidase inhibitors (NAIs) in reducing mortality when given to hospitalized patients with A(H1N1)pdm09 virus infection, the impact of outpatient treatment on hospitalization has not been clearly established. We investigated the impact of outpatient NAI treatment on subsequent hospitalization in patients with A(H1N1)pdm09 virus infection. METHODS: We assembled general community and outpatient data from 9 clinical centers in different countries collected between January 2009 and December 2010. We standardized data from each study center to create a pooled dataset and then used mixed-effects logistic regression modeling to determine the effect of NAI treatment on hospitalization. We adjusted for NAI treatment propensity and preadmission antibiotic use, including "study center" as a random intercept to account for differences in baseline hospitalization rate between centers. RESULTS: We included 3376 patients with influenza A(H1N1)pdm09, of whom 3085 (91.4%) had laboratory-confirmed infection. Eight hundred seventy-three patients (25.8%) received outpatient or community-based NAI treatment, 928 of 2395 (38.8%) with available data had dyspnea or respiratory distress, and hospitalizations occurred in 1705 (50.5%). After adjustment for preadmission antibiotics and NAI treatment propensity, preadmission NAI treatment was associated with decreased odds of hospital admission compared to no NAI treatment (adjusted odds ratio, 0.24; 95% confidence interval, 0.20-0.30). CONCLUSIONS: In a population with confirmed or suspected A(H1N1)pdm09 and at high risk of hospitalization, outpatient or community-based NAI treatment significantly reduced the likelihood of requiring hospital admission. These data suggest that community patients with severe influenza should receive NAI treatment.

Recombination of Globally Circulating Varicella-Zoster Virus.

UNLABELLED: Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpes virus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE: Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Two-step evolution of the hepatitis B drug-resistant mutations in a patient who developed primary entecavir resistance.

AIM: Few cases of primary entecavir resistance in chronic hepatitis B patients have been reported to date. The serial profiling of the HBV polymerase gene mutations from a treatment-naive patient who developed drug resistance after 32 months of entecavir therapy is presented here. DESIGN: Serum samples were collected at multiple time points from before the start of therapy to virological and biochemical breakthrough. The evolution of the hepatitis B virus polymerase gene mutations was analysed with commercial line probe assay and pyrosequencing. RESULTS: Drug resistance mutation analysis by pyrosequencing revealed a two-step process in the selection of drug resistance. The patient had a good initial response to entecavir 0.5 mg/day. A partially resistant HBV strain first emerged as the predominant species from as early as 2 weeks. After a period of non-compliance to therapy, there was virological breakthrough, which resolved on restarting entecavir. Shortly after, there was secondary failure of entecavir therapy, caused by a new resistant strain carrying all three mutations required. CONCLUSION: In this patient, pre-existence of minor population of partially resistant viral strains and treatment non-compliance probably contributed to his development of primary entecavir resistance.

Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.

OBJECTIVE: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing. METHOD: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. RESULTS: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). CONCLUSION: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.

A diagnostic polymerase chain reaction assay for Zika virus.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Infection results in a dengue-like illness with fever, headache, malaise, and a maculopapular rash. Nearly all cases are mild and self-limiting but in 2007, a large outbreak of ZIKV was reported from the island of Yap (in Micronesia, northwest of Indonesia). Singapore is already endemic for dengue, and its impact on public health and economic burden is significant. Other dengue-like infections (e.g., Chikungunya virus) are present. Yet only 10% of reported dengue cases have laboratory confirmation. The identification and control of other dengue-like, mosquito-transmitted infections is thus important for the health of Singapore's population, as well as its economy. Given that ZIKV shares the same Aedes mosquito vector with both dengue and Chikungunya, it is possible that this virus is present in Singapore and causing some of the mild dengue-like illness. A specific and sensitive one-step, reverse transcription polymerase chain reaction (RT-PCR) with an internal control (IC) was designed and tested on 88 archived samples of dengue-negative, Chikungunya-negative sera from patients presenting to our hospital with a dengue-like illness, to determine the presence of ZIKV in Singapore. The assay was specific for detection of ZIKV and displayed a lower limit of detection (LoD) of 140 copies viral RNA/reaction when tested on synthetic RNA standards prepared using pooled negative patient plasma. Of the 88 samples tested, none were positive for ZIKV RNA, however, the vast majority of these were from patients admitted to hospital and further study may be warranted in community-based environments.

Angioimmunoblastic T-cell lymphoma with hyperplastic germinal centres (pattern 1) shows superior survival to patterns 2 and 3: a meta-analysis of 56 cases.

AIMS: Angioimmunoblastic T-cell lymphoma (AITL) may present in patterns 1, 2 or 3, representing those with hyperplastic, regressed or effaced germinal centres (GCs), respectively, but the prognostic utility of this subclassification has not been previously validated. METHODS AND RESULTS: Twenty-five cases of AITL were reviewed immunohistologically and with in-situ hybridization for Epstein-Barr virus-encoded RNA and polymerase chain reaction for T-cell receptor gamma and immunoglobulin heavy chain clonality and followed for up to 120 months. Four cases had conventional hyperplastic GCs, two had floral GCs, and one had progressively transformed GCs, consistent with pattern 1 and one additional case had hyalinized GCs, consistent with pattern 2. The remaining 17 (pattern 3) cases lacked morphologically discernible GCs. The Kaplan-Meier survival distribution of pattern 1 cases (5-year survival 83%) was superior to that of pattern 2 and 3 cases [5-year-survival 36% (P = 0.0417)] only when combined with the 31 cases, seven of which were pattern 1, that Attygalle et al. had followed for up to 247 months and previously published. Furthermore, the development of B-lineage (classical Hodgkin or diffuse large-cell) lymphoma was associated exclusively with pattern 3 (P = 0.0057). CONCLUSIONS: Pattern 1 represents an indolent phase/grade of AITL, unassociated with the development of secondary B-lineage lymphoma and uninfluenced by treatment regimen.

Comparison of pandemic (H1N1) 2009 and seasonal influenza viral loads, Singapore.

Mean viral loads for patients with pandemic (H1N1) 2009 were ≈1 log10 times lower than those for patients with seasonal influenza within the first week after symptom onset. Neither pandemic nor seasonal influenza viral loads correlated with clinical severity of illness. No correlation was found between viral loads and concurrent illness.

Silencing of the PP2A catalytic subunit causes HER-2/neu positive breast cancer cells to undergo apoptosis.

Protein phosphatase 2A (PP2A), in its activated form as a phosphatase, is a tumour suppressor. However, when PP2A is phosphorylated at the tyrosine residue (pY307), it loses its phosphatase activity and becomes inactivated. In our previous study, we found a higher expression of pY307-PP2A in HER-2/neu positive breast tumour samples and significantly correlated to tumour progression, and in this context, it could function as a proto-oncogene. The above and subsequent findings led us to postulate that the critical role of PP2A in maintaining the balance between cell survival and cell death may be linked to its phosphorylation status at its Y307 residue. Hence, we further investigated the effects of knocking down the PP2A catalytic subunit which contains the Y307 amino acid residue in two HER-2/neu positive breast cancer cell lines, BT474 and SKBR3. We showed that this causes the silenced HER-2/neu breast cancer cells to undergo apoptosis and furthermore, that such apoptosis is mediated by p38 MAPK-caspase 3/PARP activation. Understanding the role of PP2A in HER2/neu positive cells might thus provide insight into new targets for breast cancer therapy.

A self-contained all-in-one cartridge for sample preparation and real-time PCR in rapid influenza diagnosis.

Herein we present a fully automated system with pseudo-multiplexing capability for rapid infectious disease diagnosis. The all-in-one system was comprised of a polymer cartridge, a miniaturized thermal cycler, 1-color, 3-chamber fluorescence detectors for real-time reverse transcription polymerase chain reaction (RRT-PCR), and a pneumatic fluidic delivery unit consisting of two pinch-valve manifolds and two pneumatic pumps. The disposable, self-contained cartridge held all the necessary reagents for viral RNA purification and reverse transcription polymerase chain reaction (RT-PCR) detection, which took place all within the completely sealed cartridge. The operator only needed to pipette the patient's sample with lysis buffer into the cartridge, and the system would automatically perform the entire sample preparation and diagnosis within 2.5 h. We have successfully employed this system for seasonal influenza A H1N1 typing and sub-typing, obtaining comparable sensitivity as the experiments conducted using manual RNA extraction and commercial thermal cycler. A minimum detectable virus loading of 100 copies per µl has been determined by serial dilution experiments. This all-in-one desktop system would be suitable for decentralized disease diagnosis at immigration check points and outpatient clinics, and would not require highly skilled operators.

Viral loads of herpes simplex virus in clinical samples--a 5-year retrospective analysis.

Viral loads of herpes simplex virus (HSV) are not monitored usually for the effective clinical management of HSV-related diseases. However, recently, there has been more interest about the typical HSV levels in clinical specimens, and how such data may improve understanding of the behavior of this virus in such clinical presentations, particularly in immunocompromised patients, where more prolonged therapy using higher doses of antiviral drugs may be required. Using an in-house quantitative HSV-1/HSV-2 polymerase chain reaction assay, an observational, retrospective 5-year analysis of diagnostic, quantitative HSV-1 and HSV-2 DNA levels was conducted. The results (all in median log(10) DNA copies/ml), including perhaps the first quantitative comparison of cerebrospinal fluid (CSF) HSV viral loads, were as follows: CSF: HSV-1, 3.40 (range 2.30-8.98) versus HSV-2, 3.60 (range 2.31-6.86) (P=0.559); plasma: HSV-1, 3.20 (range 2.23-5.51) versus HSV-2, 3.20 (range 3.18-3.41) (P=0.905); genital swabs: HSV-1, 6.79 (range 2.28-8.48) versus HSV-2, 6.97 (range 3.40-9.66) (P=0.810); oral swabs: HSV-1, 7.28 (range 2.46-10.04) versus HSV-2, 5.62 (range 4.60-6.63) (P=0.529). Note that with the samples usually collected for HSV testing (i.e., CSF, plasma, oral, and genital swabs) there was no significant difference in the viral loads between HSV-1 and HSV-2 types, nor between immunocompetent and immunocompromised patients for each of these different HSV types. Indeed, even between immunocompromised patients with similar diseases, for these samples, the HSV loads were found to vary considerably. These findings may therefore limit the usefulness of monitoring HSV loads in everyday clinical practice.

Noninvasive prenatal exclusion of haemoglobin Bart's using foetal DNA from maternal plasma.

OBJECTIVE: Prenatal diagnosis of alpha-thalassaemia requires invasive testing associated with a risk of miscarriage. Cell-free foetal DNA in maternal plasma presents an alternative source of foetal genetic material for noninvasive prenatal diagnosis. We aimed to exclude HbBart's noninvasively by detection of unaffected paternal alleles in maternal plasma using quantitative fluorescence PCR (QF-PCR). METHOD: Microsatellite markers (16PTEL05, 16PTEL06) within the breakpoint regions of -(SEA), -(FIL) and -(THAI) deletions were analysed using QF-PCR of maternal plasma from 30 families. In this blinded study, genotypes were confirmed using conventional PCR. Maternal plasma from two known cases of HbBart's were also analysed. RESULTS: HbBart's was excluded in 10 out of 30 (33.3%, 95% CI, 17.3-52.8%) mothers by identifying the presence of nondeleted paternally inherited fetal alleles; either only 16PTEL05 (n = 1) or only 16PTEL06 (n = 4), or both (n = 5), and confirmed through direct analysis of fetal DNA. Paternally inherited foetal alleles of 16PTEL05 and 16PTEL06 were not detected in maternal plasma of the two known HbBarts cases. False negatives were excluded with the detection of paternally inherited fetal control marker, D21S1270 in maternal plasma. CONCLUSION: We show proof-of-principle that such a test can accurately exclude HbBart's in the foetus by identifying the nondeleted paternally inherited fetal alleles in maternal plasma in one out of three pregnancies, avoiding invasive testing in these pregnancies.

A novel diagnostic target in the hepatitis C virus genome.

BACKGROUND: Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays. METHODS AND FINDINGS: In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties. CONCLUSION: This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Tyrosine phosphorylation of PP2A is regulated by HER-2 signalling and correlates with breast cancer progression.

Activation of HER-2/neu leads to multiple signalling cascades and plays a vital role in cell survival and growth. We used a signal transduction antibody array to characterize the tyrosine phosphorylation profiles in heregulin (HRG alpha1)-treated BT474 breast cancer cells, and identified a group of 80 molecules in which tyrosine phosphorylation was highly regulated by HRG-enhanced HER-2/neu signalling. These phosphoproteins included many known HER-2/neu-regulated molecules (e.g., SHC, Akt, Syk and Stat1) and proteins that had not been previously linked to HER-2/neu signalling, such as Fas-associated death domain protein (FADD), apoptosis repressor with CARD domain (ARC), and the tumour suppressor, protein phosphatase type 2A (PP2A). Pharmacological inhibition with HER-2 inhibitor AG825, PI3K inhibitor LY294002, MEK1/2 inhibitor PD98095, and p38MAPK inhibitor SB203580 confirmed that PP2A phosphorylation was modulated by the complicated, HER-2/neu-driven downstream signal network, with the PI3K and MEK1/2 positively, while the p38MAPK negatively regulating its tyrosine phosphorylation. In breast tumour specimens, expression of tyrosine-phosphorylated PP2A (pY307-PP2A) was highly increased in the HER-2/neu positive breast tumours, and significantly correlated to tumour progression, thus enhancing its potential prognostic value. Our data provide meaningful information in the elucidation of the HER-2-driven tyrosine phosphorylation network, and in the development of phosphopeptide-related targets as prognostication indicators.

Lactate, procalcitonin, and amino-terminal pro-B-type natriuretic peptide versus cytokine measurements and clinical severity scores for prognostication in septic shock.

The biomarkers lactate, procalcitonin, and amino-terminal pro-B-type natriuretic peptide (NT-proBNP) are often promoted as being useful for prognostication in septic shock. This study aimed to compare the prognostic utility of these biomarkers with each other and with cytokine measurements and clinical severity scores, and to assess how these biomarkers may be combined to improve their prognostic utility. Seventy-two patients with septic shock were studied. The biomarkers were measured on the first 3 days of stay in the intensive care unit together with serum IL-1beta, IL-6, IL-10, and TNF-alpha levels. Although elevated baseline lactate levels predicted 28-day mortality, elevated procalcitonin and NT-proBNP levels were only predictive from days 2 and 3, respectively. The prognostic utility of baseline lactate levels was poorer than that of baseline cytokine levels, the Acute Physiology and Chronic Health Evaluation II score, and the Sequential Organ Failure Assessment score. However, a rising trend in lactate and procalcitonin levels between days 1 and 2 had superior prognostic utility compared with absolute levels. Indeed, using multivariate analysis, the presence of a concurrent increase in both lactate and procalcitonin levels between days 1 and 2 superseded all cytokine measurements and clinical severity scores as the sole independent predictor of 28-day mortality. In conclusion, elevated baseline lactate levels offer superior prognostic accuracy to baseline procalcitonin levels, which in turn are superior to NT-proBNP levels. To improve their prognostic utility beyond those of cytokine measurements and clinical severity scores, serial lactate and procalcitonin measurements may be combined.

Longitudinal monitoring reveals dynamic changes in circulating tumor cells (CTCs) and CTC-associated miRNAs in response to chemotherapy in metastatic colorectal cancer patients.

We evaluated the changes in CTC count and CTC-associated miRNAs during the course of chemotherapy in patients with metastatic colorectal cancer. Blood samples were collected from 9 metastatic colorectal cancer patients prior to chemotherapy and at every other chemotherapy session during the course of treatment. CTCs were isolated and enumerated using a size-exclusion method (CellSievo, Singapore). CTC-associated miRNAs were isolated using a paper-based, partitioning method, and analyzed using reverse transcription quantitative real-time PCR (MiRXES, Singapore). CTC count trends generally correlated with disease progression defined by radiological measurements and trends in carcinoembryonic antigen (CEA) levels; hence CTC counts may be useful in cases where CEA is not elevated. CTC-associated miRNAs identified were miR-15b, miR-16, miR-19a, miR-21, miR-25, miR-30d, miR-126, miR-185, miR-221, miR-222, and miR-324-5p. The expression of CTC-associated miRNAs did not appear to correlate with CTC count and exhibited inter-individual heterogeneity. This pilot study suggests that analysis of CTC changes during the course of treatment may be useful in monitoring response to therapy in metastatic colorectal cancer.

Phosphorylation of Ser78 of Hsp27 correlated with HER-2/neu status and lymph node positivity in breast cancer.

BACKGROUND: Abnormal amplification/expression of HER-2/neu oncogene has been causally linked with tumorigenesis and metastasis in breast cancer and associated with shortened overall survival of patients. Recently, heat shock protein 27 (Hsp27) was reported to be highly expressed in HER-2/neu positive tumors and cell lines. However, putative functional links between phosphorylation of Hsp27 with HER-2/neu status and other clinicopathological features remain to be elucidated. RESULTS: Comparative phosphoproteomic studies of HER-2/neu positive and -negative breast tumors revealed that Hsp27, one of the identified phosphoproteins, was highly phosphorylated in HER-2/neu positive tumors. The extent of Hsp27 phosphorylation at its Ser15, Ser78 and Ser82 residues were further evaluated with site-specific antibodies in tumor samples by tissue lysate array- and tissue microarray-based analyses, and in the BT474 breast cancer cell line treated with heregulin alpha1 (HRG alpha1) or the p38 MAPK inhibitor, SB203580. The tissue lysate array study indicated that only the level of pSer78 in HER-2/neu positive tumors was more than 2-fold that in HER-2/neu negative tumors. Treatment of BT474 cells with HRG alpha1 and SB203580 indicated that Ser78 phosphorylation was mainly regulated by the HER-2/neu-p38 MAPK pathway. Immunohistochemical staining of sections from a tissue microarray with 97 breast tumors showed that positive staining of pSer78 significantly correlated with HER-2/neu (p = 0.004) and lymph node positivity (p = 0.026). CONCLUSION: This investigation demonstrated the significant correlation of enhanced phosphorylation of the Ser78 residue of Hsp27 with HER-2/neu and lymph node positivity in breast cancer.

Proteomic identification of down-regulation of oncoprotein DJ-1 and proteasome activator subunit 1 in hepatitis B virus-infected well-differentiated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumour. Development of HCC is a multi-step process from well-differentiated (G1), moderately differentiated (G2) to poorly differentiated (G3) phenotype. The early molecular modulators causing the onset of hepatocarcinogenesis are not fully understood. In the present study, we conducted comparative proteomics to analyze the differential proteome of G1 tumour and adjacent non-tumour tissues, with aims to identify the molecules as early tumour markers and to understand the early molecular events involved in initiation of tumorigenesis in hepatitis B virus (HBV)-infected G1 tumour. Differentially expressed proteins were identified by MALDI-TOF/TOF tandem mass spectrometry and NCBInr database interrogation. A total of 15 differentially expressed proteins with diverse biological functions were identified. Among these, 4 proteins were down-regulated, whereas the other 11 proteins were up-regulated in the G1 tumours. Two proteins, Proteasome activator subunit 1 (PA28alpha) and DJ-1, were firstly found to be down-regulated in HBV-infected G1 tumours. Down-regulations of these two proteins were further validated by Western blotting and immunohistochemistry in a panel of clinical specimens. These findings elucidate, at least in part, the molecular events underlying the mechanism and the potential roles of DJ-1 and PA28alpha in the onset of hepatocarcinogenesis.

A prospective clinical study on the use of reverse transcription-polymerase chain reaction for the early diagnosis of Dengue fever.

Laboratory testing for dengue virus is used to confirm the diagnosis of dengue virus infection and to differentiate dengue from other febrile tropical illnesses. There are few data on the clinical use of reverse transcription-polymerase chain reaction (RT-PCR) for diagnosis of dengue virus infection. We prospectively evaluated 121 consecutive patients with possible dengue who had samples submitted for RT-PCR, IgM serology, and virus culture. Results were compared with the final discharge diagnosis. Semi-nested RT-PCR was performed using genus- and serotype-specific NS3 consensus primers. Results of 112 patients were available for the final analysis. The RT-PCR was positive in 40 of 62 patients with dengue. Patients who were RT-PCR-positive alone showed a mean of 4.4 days to RT-PCR positivity compared with 5.9 days in patients who were RT-PCR-negative and IgM serology-positive (P = 0.03, Mann-Whitney U-test). The sensitivity, specificity, negative predictive value, and positive predictive value were 70, 100, 84, and 100%, respectively, for samples analyzed within 5 days of illness onset. The RT-PCR also provided epidemiological data regarding the prevailing dengue virus serotypes: 25 with Den-2, eight with Den-3, and seven with Den-1 infection. We propose an algorithm of dengue testing that uses RT-PCR within 5 days of illness onset, whereas IgM capture enzyme-linked immunosorbent assay is preferred for those presenting later.