EV71:TLLcho virus murine model of enterovirus A71 neurological disease does not exhibit neurogenic pulmonary oedema.

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema.

Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Xylan utilization regulon in Xanthomonas citri pv. citri Strain 306: gene expression and utilization of oligoxylosides.

Xanthomonas citri pv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α-l-arabinofuranosidase, and the xyn43F gene, encoding a putative ß-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGXn) to oligoxylosides methylglucuronoxylotriose (MeGX3), xylotriose (X3), and xylobiose (X2). Xcc306 completely utilizes MeGXn predigested with Xyn10A or Xyn10C but shows little utilization of MeGXn. Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX3 for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGXn. Preferential growth on oligoxylosides compared to growth on polymeric MeGXn indicates that GH10 xylanases, either secreted by Xcc306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10, agu67, cirA, the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc306 GH10 xylanases on MeGXn. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc306 and, ultimately, to plant disease.

Phase 1 dose-escalation study of CP-690 550 in stable renal allograft recipients: preliminary findings of safety, tolerability, effects on lymphocyte subsets and pharmacokinetics.

CP-690 550 inhibits Janus kinase 3 with nanomolar potency. In this dose-escalation study, we assessed the safety, tolerability, effects on lymphocyte subsets, and pharmacokinetics of CP-690 550 when coadministered with mycophenolate mofetil in stable renal allograft recipients for 28 days. Twenty-eight patients were enrolled. Six patients received CP-690 550 5 mg twice daily (BID), 6 patients received 15 mg BID, 10 patients received 30 mg BID, and 6 patients received placebo. The most frequent adverse events were infections and gastrointestinal (abdominal pain, diarrhea, dyspepsia, and vomiting). CP-690 550 15 mg BID and 30 mg BID were associated with a mean decrease in hemoglobin from baseline of 11% and a mean decrease in absolute natural killer cell counts of 50%. CP-690 550 30 mg BID was also associated with a mean increase in absolute CD19(+) B-lymphocytes of 130%. There were no changes in the number of neutrophils, total lymphocytes, platelets, or CD4(+) or CD8(+) T cells; clinical chemistry; vital signs; or electrocardiograms from the pretreatment baseline. Administration of CP-690 550 without a concomitant calcineurin inhibitor resulted in CP-690 550 exposures consistent with previous studies in nontransplant subjects. Additional dose-ranging studies are warranted to evaluate the safety and efficacy of CP-690 550 in renal transplant recipients over longer treatment duration.

Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6.

Prevalence of hospital infection and antibiotic use at a university medical center in Hong Kong.

Hospital infection prevalence surveys were performed in our 1400-bed University medical centre in Hong Kong from 1985 to 1988. We investigated the rates of four major hospital-acquired infections (HAIs) (pneumonia, symptomatic urinary tract infection, surgical site infection and laboratory-confirmed bloodstream infection) in order to identify current distribution and any changes after 15 years. A one-day point prevalence study was performed on 7 September 2005. All inpatients were surveyed for HAIs, community-acquired infections (CAIs), risk factors, pathogenic isolates and antibiotics prescribed. Infections were diagnosed according to Centers for Disease Control and Prevention (CDC) criteria. In total, 1021 patients were surveyed; of these, 41 had 42 HAIs (4% prevalence) and 389 (38%) were receiving antibiotics. The commonest HAI was pneumonia (1.4%) followed by bloodstream infection (0.9%) and symptomatic urinary tract infection (0.8%). The prevalence of postoperative surgical site infection was 5.6%. The nosocomial prevalence rate was highest in the Intensive Care Unit, followed by the Pediatric and Neonatal Intensive Care Units, Children's Cancer Centre/Bone Marrow Transplant Unit and Orthopaedics with Traumatology. Meticillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were the commonest pathogens. The rates are significantly lower than previously and reflect the increased resources for infection control made available following the outbreak of severe acute respiratory syndrome (SARS).

Nanostructural characteristics, mechanical properties, and osteoblast response of spark plasma sintered hydroxyapatite.

This study aimed to fabricate bulk nanostructured hydroxyapatite (HA) pellets with improved properties using spark plasma sintering (SPS) for orthopedic applications. Spray-dried nanostructured HA (nSD-HA) powders were consolidated using the rapid SPS processing. The SPS processed nSD-HA was characterized using Raman spectroscopy and field emission scanning electron microscopy (FESEM). Mechanical properties of the consolidates were also evaluated through indentation approach. The nanostructures ( approximately 80 nm in grain size) of the starting powders were successfully retained after the SPS processing operated at 950 degrees C with <15 min holding time. The SPS consolidated nSD-HA showed promising mechanical properties, approximately 118 GPa for Young's modulus, and up to 2.22 MPa m(0.5) for fracture toughness. SPS holding time showed minor influence on the phases of the pellets. Furthermore, the spheroidized nanostructured HA retained the HA structure after the SPS consolidation. Preliminary cytotoxicity and cell attachment studies were also carried out using a human osteoblast cell line hFOB 1.19. Enhanced cell attachment and proliferation on the nanostructured pellets were revealed. The presence of the nanostructures accounts mainly for the enhanced mechanical properties and promoted proliferation of the osteoblast cells. This study suggests that the SPS technique is an appropriate process for fabrication of bulk nSD-HA from nanostructured powder.

PCR detection and typing of human papilloma virus DNA in squamous carcinoma of the cervix in a cohort of Sri Lankan women.

OBJECTIVE: To determine the prevalence of human papilloma virus (HPV) types 16 and 18 in squamous carcinomas of the cervix in Sri Lanka. DESIGN: Case control study. SETTING: One gynaecological unit at the Cancer Institute, Maharagama, Sri Lanka. PATIENTS: 15 patients with squamous carcinoma of the cervix, and 15 age matched controls with histologically normal cervices. MEASUREMENTS: DNA was extracted from paraffin embedded cervical biopsies. Polymerase chain reaction was performed on extracted DNA employing primers specific for HPV types 16 and 18. RESULTS: HPV 16 DNA was detected in 11 out of 15 cervical cancer biopsies (73.3%), in comparison with 3 out of 15 normal controls (20%). HPV 18 was detected in 3 out of 15 cervical cancer biopsies, but not in a single control biopsy. CONCLUSION: Despite the limited number of cases in this cohort, this study supports the strong association between HPV 16 and squamous cancer of the cervix.

Evaluation of DNA methylation at imprinted DMRs in the spermatozoa of oligozoospermic men in association with MTHFR C677T genotype.

Altered DNA methylation has been previously identified in the spermatozoa of infertile men; however, the origins of these errors are poorly understood. DNA methylation is an epigenetic modification which is thought to play a fundamental role in male germline development. DNA methylation reactions rely on the cellular availability of methyl donors, which are primarily products of folate metabolism, where a key enzyme is methylenetetrahydrofolate reductase (MTHFR). The MTHFR C677T single nucleotide polymorphism (SNP) reduces enzyme activity and may potentially alter DNA methylation processes during germline development. The objective of this study was to determine whether altered DNA methylation in spermatozoa is associated with the MTHFR C677T SNP. DNA methylation was evaluated at the H19, IG-GTL2, and MEST imprinted differentially methylated regions in the spermatozoa of 53 men - 44 oligozoospermic men and nine fertile men with normal sperm parameters via bisulfite sequencing of sperm clones. The 44 infertile men were stratified by severity of oligozoospermia - three normal (>15 million spermatozoa/mL), eight moderate (5-15 million spermatozoa/mL), 23 severe (1-5 million spermatozoa/mL), and 10 very severe (<1 million spermatozoa/mL). MTHFR C677T SNP genotyping was conducted in a subset of 44 peripheral blood samples via restriction fragment length polymorphism. A total of three men - severe oligozoospermic and CT genotype - were found to be altered, which is defined as having ≥50% of their clones altered, where an altered clone was in turn defined as ≥50% of CpGs with incorrect DNA methylation patterns. The incidence of three altered men within the CT subgroup, however, was not significantly higher than the incidence in the CC subgroup. Taken together, altered DNA methylation in spermatozoa was not significantly associated with the MTHFR C677T SNP; however, there was a trend for higher incidence of alterations among severe oligozoospermic infertile men with CT genotypes.

Inactivation of the p53 oncogene by internal deletion or retroviral integration in erythroleukemic cell lines induced by Friend leukemia virus.

The p53 gene is rearranged in a high proportion of erythroleukemic cell lines derived from the spleens of mice infected with Friend leukemia virus. These rearrangements result in either the synthesis of a truncated protein or the inactivation of the p53 gene. Here we have molecularly characterized the rearrangements in two murine erythroleukemic cell lines induced by Friend leukemia virus, DP20-1 and CB3, that contain a rearranged p53 gene and fail to express p53 protein. The rearrangement in the DP20-1 cell line is due to the insertion of Friend spleen focus-forming provirus (SFFV) in the 3' end of the p53 gene in intron sequences between exons 9 and 10. Transfection of molecular clones of this SFFV provirus into NIH3T3 cells results in the generation of infectious virus as determined by its ability, in the presence of helper virus, to induce rapid splenomegaly and polycythemia when injected into adult DBA/2J mice. Insertion of SFFV in DP20-1 cells resulted in the expression of an aberrant 2.9 kb RNA species. Analysis of a molecular clone of the rearranged p53 gene in a second cell line, CB3, revealed that the p53 gene in this clone has sustained a large deletion within the p53 gene resulting in the loss of coding sequences between exons 4 and 8. The 5' end of the deletion originates within exon 4 and extends 3' to within the eighth intron. The significance of these findings with regard to the multi-stage nature of Friend virus induced erythroleukemia is discussed.

The evolution of teaching and learning medical microbiology and infectious diseases at NUS.

Infectious diseases were rife during the early years of the Singapore Medical College, which was established in 1905. The current Department of Microbiology in the National University of Singapore (NUS) has its historical roots in the Departments of Bacteriology and Parasitology, which were established in 1925 and 1950 respectively. With the achievements since its inception, and with its present research focus on Infectious Diseases, Immunology, Applied and Environmental Microbiology, it is poised to face the microbiological challenges of the 21st century. Over the decades, the structure of the medical microbiology course in NUS has modernised, culminating in the current emphasis on its practical utility in clinical practice. Coordinated by the Department of Microbiology, the Microbiology and Infectious Diseases module and the Immunology module both adopt integrated multidisciplinary approaches that aim to introduce students to the language and fundamental concepts in microbiology, infectious diseases and immunology.

The NUS MBBS-PhD programme: nurturing clinician-scientists for tomorrow.

The MBBS-PhD programme is a significant milestone in medical education in Singapore. In July 2000, the Faculty of Medicine, National University of Singapore launched this programme in collaboration with the Institute of Molecular and Cell Biology, with support from the Economic Development Board, and the Agency for Science, Technology and Research, Singapore. The objectives of the programme are to nurture and develop the talents of the brightest medical students by integrating clinical and basic biomedical research training, as well as to stimulate advanced basic and applied research in areas of growing importance to clinical medicine. The programme also aims to train clinician-scientists who will interface basic biology and clinical practice to solve biomedical problems and spearhead biomedical research initiatives in Singapore. Successful MBBS-PhD graduates can pursue career tracks in clinical research, basic biomedical research or in the biotechnology industry.

The contribution of cardiovascular mortality to long term outcomes in a relatively young demographic following acute pulmonary embolism: a validation study.

BACKGROUND: Long-term studies following acute pulmonary embolism (PE) remain limited in the current era. A recent study from our collaborative group, in a contemporary adult population, showed substantially increased cardiovascular mortality following PE. We sought to evaluate the contribution of cardiovascular mortality to long-term outcomes in a different demographic that comprised of a significantly younger PE cohort. METHODS AND RESULTS: Demographic and clinical characteristics were retrospectively collected for this cohort, and similar methods and outcome measures were applied as detailed in the original study. We compared a population from a different metropolitan area (LH: Liverpool Hospital) to that from the original study (CRGH: Concord Hospital) over a similar time period. A total of 815 patients comprised this cohort with mean 5.3±3.8year follow-up. There were similar demographics between the two cohorts, though the mean age was significantly younger in LH group (60 vs 68years, p<0.001). Prior history of cardiovascular disease in the LH group was half of that present in the CRGH cohort. The overall mortality was 7.4% per patient-year. Patients with underlying cardiovascular disease when presenting with an acute PE had a 2.3-fold increased risk of death during follow-up compared to those without. Multivariate analysis showed that older age, male gender, malignancy, diabetes, cardiovascular disease and chronic pulmonary disease were independent predictors of post-discharge mortality. CONCLUSIONS: Despite our cohort being significantly younger with a lower incidence of pre-existing cardiovascular disease, cardiovascular disease was still a significant contributor to long-term outcomes and an important predictor of mortality following acute PE.

Diagnostic investigation of parotid neoplasms: a 16-year experience of freehand fine needle aspiration cytology and ultrasound-guided core needle biopsy.

This study aimed to examine the diagnostic yield of fine needle aspiration cytology (FNAC) and ultrasound-guided core needle biopsy (USCB) in the diagnosis of parotid neoplasia. A 16-year retrospective analysis was performed of patients entered into our pathology database with a final diagnosis of parotid neoplasia. FNAC and USCB data were compared to surgical excision where available. One hundred and twenty FNAC, 313 USCB, and 259 surgical specimens were analyzed from 397 patients. Fifty-six percent of FNAC and 4% of USCB were non-diagnostic. One hundred and thirty-two (33%) patients had a final diagnosis made by USCB and did not undergo surgery. Surgery was performed in 257 (65%) patients, 226 (88%) of whom had a preoperative biopsy. Most lesions were benign, but there were 62 parotid and 13 haematological malignancies diagnosed; false-negative results were obtained in three FNAC and two USCB samples. The sensitivity and specificity of FNAC were 70% and 89%, respectively, and for USCB were 93% and 100%, respectively. This study represents the largest series of patients with a parotid neoplasm undergoing USCB for diagnosis. USCB is highly accurate with a low non-diagnostic rate and should be considered an integral part of parotid assessment.

Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

HEP-COP, a novel human gene whose product is highly homologous to the alpha-subunit of the yeast coatomer protein complex.

A 4333-bp novel human cDNA sequence designated HEP-COP was isolated from the Hep3B hepatocellular carcinoma cell line by the RACE technique. Within HEP-COP was identified an ORF of 3672 bp encoding a deduced 1224-amino-acid (aa) sequence which exhibited striking homology with the 1201-aa sequence of RET1P, the alpha-subunit of the coatomer complex (alpha-COP) in Saccharomyces cerevisiae which participates in membrane transport between the endoplasmic reticulum and Golgi apparatus. The aa homology was highest in their N-terminal regions which each contained six WD-40 repeat motifs [Van der Voorn and Ploegh, FEBS Lett. 307 (1992) 131-134], and both proteins were predicted to be hydrophilic with similar estimated molecular masses of 138 324 and 135 599 Da, respectively. Northern blot hybridization demonstrated that HEP-COP was expressed in a wide range of human adult and fetal tissues. RT-PCR analysis revealed no differential expression of HEP-COP in 14 human cancer cell lines, as compared with normal control cells. Considering the close similarities between HEP-COP and yeast alpha-COP, and the ubiquitous expression of HEP-COP implying an essential cellular role, it is likely that HEP-COP is the human homologue of alpha-COP.

Biosynthesis of rabbit haptoglobin: chemical evidence for a single chain precursor.

The primary translation product of the mRNA for rabbit haptoglobin was obtained from a rabbit reticulocyte lysate cell-free system by immunoprecipitation with an antiserum that was directed to the beta chain of haptoglobin. Analysis of the translation product by gel electrophoresis and by protein sequencing analysis identified a single polypeptide of Mr 41 000. Sequence analysis established a signal region of 18 residues that was immediately followed by the alpha chain sequence. These results give strong evidence that haptoglobin is initially synthesized as a single chain composed of a signal peptide followed by alpha and beta chain regions, respectively.