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1.  Contrast enhancement for early cancer imaging by Gd-nanoparticles and active feedback MRI 
Cancer Imaging  2015;15(Suppl 1):P2.
doi:10.1186/1470-7330-15-S1-P2
PMCID: PMC4601756
2.  Adolescents with Type 1 diabetes mellitus experience psychosensorial symptoms during hypoglycaemia 
Aim
To describe mood and psychosensorial symptoms of hypoglycaemia in adolescents with Type 1 diabetes mellitus in two countries with different cultures, Turkey and the USA.
Methods
We developed a 68-item questionnaire assessing physical, behavioural, mood and psychosensorial symptom frequency and ratings [`good', `bad', or `both' (sometimes good, sometimes bad)]. Adolescents with Type 1 diabetes were recruited from paediatric diabetes clinics at the University of North Carolina at Chapel Hill in the USA and Kocaeli University in Turkey. The percentages of participants at each clinic who endorsed individual symptoms, symptom categories and symptom ratings were calculated and compared.
Results
Cronbach's α values were > 0.7 for each real symptom category. No symptom items were excluded from the questionnaire analysis based on item-total correlation results which were all > 0.2. Data were collected from 132 participants (69 from University of North Carolina, 63 from Kocaeli University, 54% male). The mean (sd) age of the participants was 14.9 (1.9) years, HbA1c level was 8.7 (1.8) % and duration of Type 1 diabetes was 5.8 (3.7) years. On average, each physical symptom was experienced by 65.2% of participants, each behavioural symptom by 46.5%, each mood symptom by 42.8%, and each psychosensorial symptom by 48.9%. On average, each physical, behavioral, mood and psychosensorial symptom was rated as `good' or `both' by 23.0, 29.1, 36.9 and 37.2% of participants, respectively. There were no symptom differences between the groups in each country.
Conclusions
In addition to the classic physical symptoms experienced during hypoglycaemia, adolescents with Type 1 diabetes report psychosensorial, mood and behavioral symptoms, and some describe them as positive experiences. Symptom experiences were similar in these two countries with different cultures.
doi:10.1111/dme.12533
PMCID: PMC4167966  PMID: 24965522
3.  Survival, quality-adjusted survival, and other clinical end points in older advanced non-small-cell lung cancer patients treated with albumin-bound paclitaxel 
British Journal of Cancer  2015;113(1):20-29.
Background:
This analysis compared the quality-adjusted survival and clinical outcomes of albumin-bound paclitaxel+carboplatin (nab-PC) vs solvent-based paclitaxel+carboplatin (sb-PC) as first-line therapy in advanced non-small-cell lung cancer (NSCLC) in older patients.
Methods:
Using age-based subgroup data from a randomised Phase-3 clinical trial, nab-PC and sb-PC were compared with respect to overall response rate (ORR), overall survival (OS), progression-free survival (PFS), quality of life (QoL), safety/toxicity, and quality-adjusted time without symptoms or toxicity (Q-TWiST) with ages ⩾60 and ⩾70 years as cut points.
Results:
Among patients aged ⩾60 years (N=546), nab-PC (N=265) significantly increased ORR and prolonged OS, despite a non-significant improvement in PFS, vs sb-PC (N=281). Nab-PC improved QoL and was associated with less neuropathy, arthralgia, and myalgia but resulted in more anaemia and thrombocytopenia. Nab-PC yielded significant Q-TWiST benefits (11.1 vs 9.8 months; 95% CI of gain: 0.2–2.6), with a relative Q-TWiST gain of 10.8% (ranging from 6.4% to 15.1% in threshold analysis). In the ⩾70 years age group, nab-PC showed similar, but non-significant, ORR, PFS, and Q-TWiST benefits and significantly improved OS and QoL.
Conclusion:
Nab-PC as first-line therapy in older patients with advanced NSCLC increased ORR, OS, and QoL and resulted in quality-adjusted survival gains compared with standard sb-PC.
doi:10.1038/bjc.2015.181
PMCID: PMC4647528  PMID: 26035702
albumin-bound (nab) paclitaxel; solvent-based paclitaxel; non-small-cell lung cancer; quality-adjusted survival; Q-TWiST
4.  Association of Hearing Impairment with Brain Volume Changes in Older Adults 
NeuroImage  2014;90:84-92.
Hearing impairment in older adults is independently associated in longitudinal studies with accelerated cognitive decline and incident dementia, and in cross-sectional studies, with reduced volumes in the auditory cortex. Whether peripheral hearing impairment is associated with accelerated rates of brain atrophy is unclear. We analyzed brain volume measurements from magnetic resonance brain scans of individuals with normal hearing versus hearing impairment (speech-frequency pure tone average > 25 dB) followed in the neuroimaging substudy of the Baltimore Longitudinal Study of Aging for a mean of 6.4 years after the baseline scan (n = 126, age 56–86 years). Brain volume measurements were performed with semi-automated region-of-interest (ROI) algorithms, and brain volume trajectories were analyzed with mixed-effects regression models adjusted for demographic and cardiovascular factors. We found that individuals with hearing impairment (n = 51) compared to those with normal hearing (n = 75) had accelerated volume declines in whole brain and regional volumes in the right temporal lobe (superior, middle, and inferior temporal gyri, parahippocampus, p < .05). These results were robust to adjustment for multiple confounders and were consistent with voxel-based analyses, which also implicated right greater than left temporal regions. These findings demonstrate that peripheral hearing impairment is independently associated with accelerated brain atrophy in whole brain and regional volumes concentrated in the right temporal lobe. Further studies investigating the mechanistic basis of the observed associations are needed.
doi:10.1016/j.neuroimage.2013.12.059
PMCID: PMC3951583  PMID: 24412398
Hearing loss; hearing impairment; MRI; brain volume; aging
5.  Artesunate induces necrotic cell death in schwannoma cells 
Cell Death & Disease  2014;5(10):e1466-.
Established as a potent anti-malaria medicine, artemisinin-based drugs have been suggested to have anti-tumour activity in some cancers. Although the mechanism is poorly understood, it has been suggested that artemisinin induces apoptotic cell death. Here, we show that the artemisinin analogue artesunate (ART) effectively induces cell death in RT4 schwannoma cells and human primary schwannoma cells. Interestingly, our data indicate for first time that the cell death induced by ART is largely dependent on necroptosis. ART appears to inhibit autophagy, which may also contribute to the cell death. Our data in human schwannoma cells show that ART can be combined with the autophagy inhibitor chloroquine (CQ) to potentiate the cell death. Thus, this study suggests that artemisinin-based drugs may be used in certain tumours where cells are necroptosis competent, and the drugs may act in synergy with apoptosis inducers or autophagy inhibitors to enhance their anti-tumour activity.
doi:10.1038/cddis.2014.434
PMCID: PMC4649524  PMID: 25321473
6.  Survivin is essential for fertile egg production and female fertility in mice 
Cell Death & Disease  2014;5(3):e1154-.
Survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family and acts as a bifunctional protein involved in mitosis regulation and apoptosis inhibition. To identify the physiological role of Survivin in female reproduction, we selectively disrupted Survivin expression in oocytes and granulosa cells (GCs), two major cell types in the ovary, by two different Cre-Loxp conditional knockout systems, and found that both led to defective female fertility. Survivin deletion in oocytes did not affect oocyte growth, viability and ovulation, but caused tetraploid egg production and thus female infertility. Further exploration revealed that Survivin was essential for regulating proper meiotic spindle organization, spindle assembly checkpoint activity, timely metaphase-to-anaphase transition and cytokinesis. Mutant mice with Survivin depleted in GCs showed reduced ovulation and subfertility, caused by defective follicular growth, increased follicular atresia and impaired luteinization. These findings suggest that Survivin has an important role in regulating folliculogenesis and oogenesis in the adult mouse ovary.
doi:10.1038/cddis.2014.126
PMCID: PMC3973204  PMID: 24675472
folliculogenesis; apoptosis; granulosa cell; meiosis; oocyte; Survivin
7.  Protogenin prevents premature apoptosis of rostral cephalic neural crest cells by activating the α5β1-integrin 
Cell Death & Disease  2013;4(6):e651-.
The bones and connective tissues of the murine jaws and skull are partly derived from cephalic neural crest cells (CNCCs). Here, we report that mice deficient of protogenin (Prtg) protein, an immunoglobulin domain-containing receptor expressed in the developing nervous system, have impairments of the palatine and skull. Data from lineage tracing experiments, expression patterns of neural crest cell (NCC) marker genes and detection of apoptotic cells indicate that the malformation of bones in Prtg-deficient mice is due to increased apoptosis of rostral CNCCs (R-CNCCs). Using a yeast two-hybrid screening, we found that Prtg interacts with Radil, a protein previously shown to affect the migration and survival of NCCs in zebrafish with unknown mechanism. Overexpression of Prtg induces translocation of Radil from cytoplasm to cell membrane in cultured AD293 cells. In addition, overexpression of Prtg and Radil activates α5β1-integrins to high-affinity conformational forms, which is further enhanced by the addition of Prtg ligand ERdj3 into cultured cells. Blockage of Radil by RNA interference abolishes the effect of ERdj3 and Prtg on the α5β1-integrin, suggesting that Radil acts downstream of Prtg. Prtg-deficient R-CNCCs display fewer activated α5β1-integrins in embryos, and these cells show reduced migratory ability in in vitro transwell assay. These results suggest that the inside-out activation of the α5β1-integrin mediated by ERdj3/Prtg/Radil signaling is crucial for proper functions of R-CNCCs, and the deficiency of this pathway causes premature apoptosis of a subset of R-CNCCs and malformation of craniofacial structures.
doi:10.1038/cddis.2013.177
PMCID: PMC3698544  PMID: 23744351
Radil; branchial arches; bone formation; integrin
8.  Diuretic Use is Associated with Better Learning and Memory in Older Adults in the GEM Study 
Background
To investigate the association between diuretics, angiotensin-converting-enzyme inhibitors (ACE-I) and angiotensin 2 receptor blockers (AT2RB) and cognitive function.
Methods
This post-hoc analysis of the randomized controlled Ginkgo Evaluation of Memory Study trial focuses on 3069 non-demented community dwelling participants over the age of 75. At basline visit detailed information about medication use was collected and five cognitive domains were assessed. Multivariable linear regression analyses were used to assess cross-sectional associations between medication use and cognitive function.
Results
36% reported history of hypertension and 51% antihypertensive medication use, with 17% reporting diuretic, 11% ACE-I, and 2% AT2RB use. Potassium-sparing diuretic use (N=192) was associated with better verbal learning and memory measured by California Verbal Learning Test (CVLT), compared to no antihypertensive medication users (β=.068, P =.01; β =.094, P <.001) and other antihypertensive medication users (β=.080, P =.03; β=.153, P <.001). Use of ACE-I or AT2RB was not associated with better cognitive function.
Conclusion
Results warrant further investigation into possible protective effects of potassium-sparing diuretics and the role of potassium in mitigating cognitive decline.
doi:10.1016/j.jalz.2011.03.010
PMCID: PMC3341535  PMID: 22465175
Cognitive function; Diuretic; Angiotensin converting enzyme inhibitor; Angiotensin receptor blocker
10.  Rituximab-associated hepatitis B virus (HBV) reactivation in lymphoproliferative diseases: meta-analysis and examination of FDA safety reports 
Annals of Oncology  2010;22(5):1170-1180.
Background: Rituximab has been associated with hepatitis B virus reactivation (HBV-R). However, the characteristics and scope of this association remain largely undefined.
Methods: We completed a comprehensive literature search of all published rituximab-associated HBV-R cases and from the Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) MedWatch database. Literature and FDA cases were compared for completeness, and a meta-analysis was completed.
Results: One hundred and eighty-three unique cases of rituximab-associated HBV-R were identified from the literature (n = 27 case reports, n = 156 case series). The time from last rituximab to reactivation was 3 months (range 0–12), although 29% occurred >6 months after last rituximab. Within FDA data (n = 118 cases), there was a strong signal for rituximab-associated HBV-R [proportional reporting ratio = 28.5, 95% confidence interval (CI) 23.9–34.1; Empiric Bayes Geometric Mean = 26.4, 95% CI 21.4–31.1]. However, the completeness of data in FDA reports was significantly inferior compared with literature cases (P < 0.0001). Among HBV core antibody (HBcAb(+)) series, the pooled effect of rituximab-based therapy showed a significantly increased risk of HBV-R compared with nonrituximab-treated patients (odds ratio 5.73, 95% CI 2.01–16.33; Z = 3.33, P = 0.0009) without heterogeneity (χ2 = 2.12, P = 0.5473).
Conclusions: The FDA AERS provided strong HBV-R safety signals; however, literature-based cases provided a significantly more complete description. Furthermore, meta-analysis of HBcAb(+) series identified a more than fivefold increased rate of rituximab-associated HBV-R.
doi:10.1093/annonc/mdq583
PMCID: PMC3082161  PMID: 21115603
FDA; HBV reactivation; hepatitis B virus; non-Hodgkin's lymphoma; rituximab
12.  Mitochondrial redox signaling by p66Shc is involved in regulating androgenic growth stimulation of human prostate cancer cells 
Oncogene  2008;27(37):5057-5068.
p66Shc is shown to negatively regulate the life span in mice through reactive oxygen species (ROS) production. Recent reports, however, revealed that p66Shc protein level is significantly elevated in several human cancer tissues and growth-stimulated carcinoma cells, suggesting a mitogenic and carcinogenic role for p66Shc. In this communication, we demonstrate for the first time that p66Shc mediates androgenic growth signals in androgen-sensitive human prostate cancer cells through mitochondrial ROS production. Growth stimulation of prostate cancer cells with 5α-dihydrotestosterone (DHT) is accompanied by increased p66Shc level and ROS production, which is abolished by antioxidant treatments. However, antioxidant treatments do not affect the transcriptional activity of androgen receptor (AR) as observed by its inability to block DHT-induced prostate-specific antigen expression, an AR-dependent correlate of prostate cancer progression. Elevated expression of p66Shc by cDNA transfection increases the basal cell proliferation and, thus, reduces additional DHT-induced cell proliferation. Furthermore, DHT increases the translocation of p66Shc into mitochondria and its interaction with cytochrome c. Conversely, both redox-negative p66Shc mutant (W134F), which is deficient in cytochrome c interaction, and p66Shc small interfering RNA decrease DHT-induced cell proliferation. These results collectively reveal a novel role for p66Shc–ROS pathway in androgen-induced prostate cancer cell proliferation and, thus, may play a role in early prostate carcinogenesis.
doi:10.1038/onc.2008.143
PMCID: PMC2776635  PMID: 18504439
p66Shc; prostate cancer; DHT; reactive oxygen species
14.  Decision Making in Fuzzy Discrete Event Systems1 
Information sciences  2007;177(18):3749-3763.
The primary goal of the study presented in this paper is to develop a novel and comprehensive approach to decision making using fuzzy discrete event systems (FDES) and to apply such an approach to real-world problems. At the theoretical front, we develop a new control architecture of FDES as a way of decision making, which includes a FDES decision model, a fuzzy objective generator for generating optimal control objectives, and a control scheme using both disablement and enforcement. We develop an online approach to dealing with the optimal control problem efficiently. As an application, we apply the approach to HIV/AIDS treatment planning, a technical challenge since AIDS is one of the most complex diseases to treat. We build a FDES decision model for HIV/AIDS treatment based on expert’s knowledge, treatment guidelines, clinic trials, patient database statistics, and other available information. Our preliminary retrospective evaluation shows that the approach is capable of generating optimal control objectives for real patients in our AIDS clinic database and is able to apply our online approach to deciding an optimal treatment regimen for each patient. In the process, we have developed methods to resolve the following two new theoretical issues that have not been addressed in the literature: (1) the optimal control problem has state dependent performance index and hence it is not monotonic, (2) the state space of a FDES is infinite.
doi:10.1016/j.ins.2007.03.011
PMCID: PMC2701703  PMID: 19562097
Discrete event systems; fuzzy logic; decision making; HIV/AIDS treatment; optimal control
15.  Clinical significance of anti‐GM‐CSF antibodies in idiopathic pulmonary alveolar proteinosis 
Thorax  2006;61(6):528-534.
Background
The role of anti‐granulocyte‐macrophage colony stimulating factor (GM‐CSF) antibodies as a diagnostic marker in idiopathic pulmonary alveolar proteinosis (iPAP) remains unclear.
Methods
Anti‐GM‐CSF antibodies were detected in blood and bronchoalveolar lavage fluid (BAL) fluid in 13 patients with iPAP. Three patients with secondary PAP, 35 with other pulmonary disorders, and 10 subjects without lung lesions acted as controls. Blood samples only were obtained from 30 healthy medical personnel. Anti‐GM‐CSF antibodies were detected using immunoblotting and measured semi‐quantitatively by serial dilution or concentration methods. The relationship between antibodies and reported severity indicators for iPAP was analysed.
Results
Anti‐GM‐CSF antibodies could be detected in both blood and BAL fluid samples in 12 of 13 iPAP patients and were undetectable in blood and/or BAL fluid from the other subjects studied. BAL fluid levels of anti‐GM‐CSF antibodies were highly correlated with the severity indicators for iPAP, including serum lactate dehydrogenase (LDH) levels, arterial oxygen tension, alveolar‐arterial oxygen tension difference, (AaPo2), lung carbon monoxide transfer factor, and some lesion scores on chest radiographs and computed tomographic scans. In contrast, blood anti‐GM‐CSF antibodies were not significantly correlated with the severity indicators evaluated. In addition, patients with iPAP who required subsequent therapeutic lung lavage had significantly higher values of serum LDH, AaPO2, and BAL fluid anti‐GM‐CSF antibodies, and significantly lower values of Pao2.
Conclusions
In addition to serum LDH levels, Pao2 and AaPo2, BAL fluid levels of anti‐GM‐CSF antibodies might reflect disease severity in patients with iPAP and predict the need for subsequent therapeutic lung lavage. These findings may expand the role of anti‐GM‐CSF antibodies in iPAP.
doi:10.1136/thx.2005.054171
PMCID: PMC2111220  PMID: 16517574
anti‐granulocyte‐macrophage colony stimulating factor antibodies; granulocyte‐macrophage colony stimulating factor; lactate dehydrogenase; pulmonary alveolar proteinosis; pulmonary function
16.  Expression of secreted Wnt antagonists in gastrointestinal tissues: potential role in stem cell homeostasis 
Journal of Clinical Pathology  2005;58(5):515-519.
Background: Wnt signalling dysregulation has been implicated in cancer, including colon and gastric cancer. Initiation of Wnt signalling is modulated by soluble Wnt antagonists (sWAs), including soluble frizzled related proteins, dickkopf (Dkk) proteins, and Wnt inhibitory factor-1 (Wif1).
Aims: To evaluate the role of sWAs in upper (gastric) and lower (colon) gastrointestinal tract tumorigenesis.
Methods: Dkk1–3, Wif1, and FrzB expression was evaluated by in situ RNA hybridisation on normal and malignant human gastric and colon tissues. Expression was graded semiquantitatively.
Results: Wif1, Dkk1, and Dkk2 were not expressed in normal gastric tissue. Dkk3 was expressed in some samples, with stronger expression in deep gastric glands. FrzB was expressed in several normal gastric samples, but not in matched tumour specimens. In contrast, Dkk1 and FrzB were not expressed in normal colon. Wif1 was expressed in most colon samples, with stronger expression at crypt bases. Dkk3 and Dkk2 expression was also concentrated at crypt bases. There were no differences between sWA expression in malignant colon and matched normal tissue.
Conclusions: sWA expression differed between upper and lower gastrointestinal tract. The loss of FrzB in gastric cancer suggests that it acts as a tumour suppressor. The graded expression of Dkk3 in gastric tissue, and Dkk2, Dkk3, and Wif1 in colon tissue, with increased expression in the deep gastric glands/colonic crypt bases, where gastrointestinal stem cells reside, suggests that sWAs may be crucial Wnt signalling regulators in these tissues, and may contribute to stem cell pool maintenance. sWAs are important components of the gastrointestinal proliferative regulatory network.
doi:10.1136/jcp.2004.018598
PMCID: PMC1770654  PMID: 15858124
Wnt signalling; cancer; colon; stomach; frizzled related proteins
18.  Predominance of Th1 cytokine in peripheral blood and pathological tissues of patients with active untreated adult onset Still's disease 
Chen, D | Lan, J | Lin, F | Hsieh, T | Wen, M
Annals of the Rheumatic Diseases  2004;63(10):1300-1306.
Objective: To determine the type 1 T helper (Th1)/type 2 T helper (Th2) balance in the peripheral blood (PB) and pathological tissues of patients with active untreated adult onset Still's disease (AOSD).
Methods: The percentages of interferon γ (IFNγ)- and interleukin (IL)4-producing Th cells in the PB of 20 patients with active untreated AOSD, 20 patients with active rheumatoid arthritis (RA), and 20 healthy controls were determined by intracellular staining and flow cytometry. Serum levels of IL18 and soluble IL2 receptor were measured by enzyme linked immunosorbent assay. Levels of IFNγ and IL4 messenger (m) RNA expression were examined by real time quantitative polymerase chain reaction in biopsy specimens of evanescent rash and synovitis from 8 patients with AOSD.
Results: Significantly higher IFNγ-producing Th cells and Th1/Th2 ratio in PB were found in patients with AOSD than in healthy controls. Percentages of IFNγ-producing Th cells and Th1/Th2 ratio in PB correlated significantly with clinical activity score and serum IL18 levels in patients with AOSD. Increased ratio of Th1/Th2 cytokine transcripts was seen in the biopsy specimens of evanescent rash and synovitis from patients with AOSD compared with normal skin controls and patients with OA. Th cell cytokine pattern in PB and cytokine mRNA expression in synovium were similar for patients with AOSD and with RA. After 3 months' treatment, clinical remission was associated with a marked decrease in the percentages of cytokine-producing Th1 cells, but not of the Th2 cells.
Conclusion: A predominance of Th1 cytokine may precipitate the pathogenesis of AOSD.
doi:10.1136/ard.2003.013680
PMCID: PMC1754751  PMID: 15361391
19.  Dynamic changes of gene expression profiles during postnatal development of the heart in mice 
Heart  2004;90(8):927-934.
Objective: To study postnatal cardiac differentiation in the mouse.
Hypothesis: There might be mechanisms or factors in cardiac differentiation that could be identified by systematic gene expression analysis during postnatal cardiac development.
Methods: Expression of 6144 genes was examined in mouse heart, from the newborn period (day 0), through day 7 and day 14 day, to adulthood, using the cDNA microarray approach. Northern blotting and immunohistochemical techniques were used to confirm the microarray results.
Results: Various cardiac development related genes involving the cell cycle (cyclin B1, proliferating cell nuclear antigen (PCNA), and Ki67), growth factors (IGF-II, pleiotrophin (PTN), and midkine (MK)), and transcriptional regulation, cytoskeleton, and detoxification enzymes were identified by microarray analysis. Some of these genes were also confirmed by Northern blotting and immunohistochemistry of their RNA and protein content. In vivo treatment with PTN (20 ng/g) increased bromodeoxyuridine incorporation (by 2.24-fold) and PCNA expression (by 1.71-fold) during day 7 to day 14, indicating that PTN induces cell proliferation in mouse heart.
Conclusions: Global gene expression analysis in the whole heart may be useful in understanding the orchestrated process of postnatal development or terminal differentiation in the cardiac environment. These data are likely to be helpful in studying developmental anomalies of the heart in neonates.
doi:10.1136/hrt.2002.006734
PMCID: PMC1768375  PMID: 15253972
developmental biology; gene expression; growth factors; microarray; pleiotrophin
20.  High-Level Expression of AmpC β-Lactamase Due to Insertion of Nucleotides between −10 and −35 Promoter Sequences in Escherichia coli Clinical Isolates: Cases Not Responsive to Extended-Spectrum-Cephalosporin Treatment 
Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC β-lactamase with isoelectric points of >9.0. The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates. One isolate (isolate EC44) had a point mutation (G→A at position −28) and insertion of thymidine between positions −20 and −19 of the ampC promoter gene (GenBank accession no. AE000487). The single nucleotide insertion of T between positions −19 and −20 created an optimal distance (17 bp) in the Pribnow box for ampC hyperproduction. The other isolate (isolate EC38) had two point mutations (G→A at position −28 and C→T at position +58) and a 2-base (GT) insertion between positions −14 and −15. Although the insertion of GT between positions −14 and −15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original −35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion. Instead, multiple start sites for ampC transcription at −1, +1, +2, and +3 were observed in an S1 nuclease protection assay. These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription. In conclusion, nucleotide insertions between the −35 and −10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC β-lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyimino-cephalosporins highlights the important role of such a resistance mechanism in the clinical setting.
doi:10.1128/AAC.47.7.2138-2144.2003
PMCID: PMC161857  PMID: 12821459
21.  Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma 
Molecular Pathology  2002;55(4):220-226.
Aims: Signalling through the Wnt pathway is integrally associated with colon carcinogenesis. Although activating mutations in the genes for adenomatous polyposis coli (APC) and β-catenin are clearly associated with colon cancer, less is understood about the role of the upstream secreted ligands (Wnts) and their receptors (frizzled, Fz) in this process. In other systems, increased Wnt signalling has been shown to alter the expression of components of this pathway. This study was designed to test the hypothesis that colon cancer is characterised by aberrant expression of specific Wnt genes and Fz receptors.
Methods: The expression of Wnt genes was assessed by in situ, antisense RNA hybridisation in paraffin wax embedded samples of normal and malignant human colon tissues with probes specific for the individual Wnt genes. The expression of Fz1 and Fz2 was determined by immunoperoxidase based antibody staining on human tissues.
Results: Changes in the expression of some ligands and receptors were seen in colon cancer. For example, Wnt2 mRNA was detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt5a in normal mucosa was also noted, with increased expression at the base of the crypts compared with the luminal villi and slightly increased expression in colon cancer. Wnt7a exhibited minimal expression in both normal and malignant colon tissues, whereas other Wnt ligands including Wnts 1, 4, 5b, 6, 7b, and 10b were expressed equally and strongly in both normal and malignant colon tissues. In defining cellular responses and phenotype, the type and distribution of Fz receptors may be as important as the pattern of Wnt ligand expression. No expression of Fz receptor 1 and 2 was seen in normal colonic mucosa and in well differentiated tumours. However, poorly differentiated tumours exhibited a high degree of Fz receptor expression, especially at the margin of cellular invasion.
Conclusions: These data indicate that the expression of members of the Wnt signal transduction pathway, distinct from APC and β-catenin, is integrally associated with the process of colon carcinogenesis. Wnt2, and possibly Wnt5a, may be involved in the progression from normal mucosa to cancer and the expression of Fz1/2 receptors may be involved in processes associated with tumour invasion. Altered expression of these Wnts and Fz receptors may prove useful as prognostic or diagnostic markers for patients with colon cancer.
PMCID: PMC1187182  PMID: 12147710
colon carcinogenesis; signal transduction; in situ hybridisation
22.  Bacteremia Due to Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in a Pediatric Oncology Ward: Clinical Features and Identification of Different Plasmids Carrying both SHV-5 and TEM-1 Genes 
Journal of Clinical Microbiology  1999;37(12):4020-4027.
Thirteen patients who had 16 episodes of bacteremia were observed between 1993 and 1997 in a pediatric oncology ward with a high background isolation rate of cefotaxime- or aztreonam-resistant gram-negative bacteria. Four blood isolates were Escherichia coli and 12 were Klebsiella pneumoniae, and these isolates harbored extended-spectrum β-lactamases (ESBLs). All episodes of bacteremia were nosocomial, all except one of the episodes occurred in neutropenic patients, and all patients were treated with piperacillin or ceftazidime with amikacin and cefazolin prior to the onset of bacteremia. Nine of 13 patients were receiving extended-spectrum β-lactam treatment when the bacteremias caused by ESBL producers occurred. Molecular studies revealed that four K. pneumoniae SHV-2-producing isolates from 1994 were of the same clone. Other ESBL producers, including six that carried both TEM-1 and SHV-5, five that carried SHV-5, and one that carried SHV-2 alone, were unrelated. In conclusion, SHV-5 was present in 11 of the 16 isolates and coexisted with TEM-1 in 6 isolates. Acquisition of resistance genes probably occurred under antibiotic selection pressure. This study highlights the importance of routine checks for and detection of ESBL producers. Effective therapy against ESBL producers should be considered early for children who have malignancies and neutropenia and who are septic, despite treatment with a regimen that includes an extended-spectrum β-lactam, in a clinical setting of an increased incidence of ESBL-producing bacteria.
PMCID: PMC85870  PMID: 10565924
23.  Hip osteoarthritis and dysplasia in Chinese men. 
Annals of the Rheumatic Diseases  1995;54(12):965-969.
OBJECTIVE--To estimate the prevalence of hip osteoarthritis (OA hip) and hip dysplasia in a sample of Hong Kong men who were unselected with respect to hip symptoms. METHODS--The postmicturition films of 999 men aged 60-75 years, consecutive attenders for intravenous urography between 1987 and 1990 at a regional hospital, were reviewed. OA hip was diagnosed as the occurrence of two or more features of OA using a modified version of the Kellgren and Lawrence scale, or a minimal joint space of 1.5 mm or less. Hip dysplasia was defined as a centre-edge angle of less than 25 degrees, or an acetabular depth of less than 9 mm. The results were compared with British data obtained by similar methods. RESULTS--In the Hong Kong sample, the proportion of men with two or more features of osteoarthritis in at least one hip was about 50% that of the men in the British study (5.4% and 11.0%, respectively). Severe joint space narrowing (of 1.5 mm or less) occurred in 0.7% of the hips in Hong Kong men, compared with 2% in the British men. The proportion of hips with centre-edge angles less than 25 degrees was 4.5% in Hong Kong, compared with 3.6% in Britain, and the prevalence of shallow acetabular depth was greater in Chinese (14.5%) than in the British (2.1%). Radiographic measures of hip dysplasia were not associated with minimal joint space. CONCLUSIONS--Our results have confirmed the lower prevalence of radiographic hip osteoarthritis in Hong Kong men compared with British men. However, acetabular dysplasia was as prevalent among Chinese men as in the British sample. This is evidence against the hypothesis that variations in the frequency of hip osteoarthritis are caused by differences in the occurrence of hip dysplasia.
PMCID: PMC1010061  PMID: 8546528
24.  Sp1 binds to the precise locus of end processing within the terminal repeats of Epstein-Barr virus DNA. 
Journal of Virology  1997;71(8):6136-6143.
Interconversion between the linear genome of Epstein-Barr virus (EBV) present in virions and intracellular circular EBV DNA is a novel DNA recombination process. A previously characterized DNA binding activity called terminal repeat or tandem repeat binding protein (TRBP) was found to recognize several G-rich recombinogenic sequences in the EBV genome and in cellular DNA. TRBP was also found to be an autoantigen recognized by sera from certain patients with undifferentiated connective-tissue disorders. Here the transcription factor Sp1 has been identified as a component of TRBP and has been shown to be an autoantigen. Sp1 bound to recombination junctions of EBV DNA, such as those in the terminal repeats and in the large internal repeats, as well as to recombinogenic regions of cellular DNA, such as variable-number tandem repeats and switch regions of the immunoglobulin genes. We defined the ends of the linear EBV genome present in virions and showed that Sp1 binds to the sequence (GGGGTGGGGCATGGG) within EBV terminal repeats at the precise locus of interconversion of linear and circular viral DNA. Sp1 may be involved in DNA recombination.
PMCID: PMC191874  PMID: 9223508
25.  Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen. 
Journal of Virology  1997;71(4):3069-3076.
We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.
PMCID: PMC191437  PMID: 9060668

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