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1.  Effect of a YAG laser iridotomy on intraocular pressure in pigmentary glaucoma 
The British Journal of Ophthalmology  2002;86(12):1443-1444.
PMCID: PMC1771416  PMID: 12446385
YAG laser iridotomy; intraocular pressure; pigmentary glaucoma
European cells & materials  2014;28:236-245.
In 2007 and 2008, we published two articles reporting a tamoxifen (TM)-inducible, chondrocyte-specific gene-targeting mouse model in which the expression of CreERT2 is driven by the type II collagen promoter (Col2CreERT2). The fusion protein is specifically expressed and translocated into the nucleus upon TM administration, which in turn triggers gene recombination. Since then, this animal model has become a powerful tool to study the molecular mechanism of skeletal development and degenerative cartilage diseases, including knee joint osteoarthritis (OA), temporomandibular joint (TMJ) OA, and intervertebral disc (IVD) degeneration. In this review article, we summarise the application of Col2CreERT2 mice and discuss the potential usage of this animal model in a broad spectrum of cartilage development and molecular pathology studies.
PMCID: PMC4288584  PMID: 25340803
Animals; cartilage development; cartilage physiology; chondrocytes; gene expression
3.  Clinical-dosimetric relationship between lacrimal gland dose and ocular toxicity after intensity-modulated radiotherapy for sinonasal tumours 
The British Journal of Radiology  2013;86(1032):20130459.
To characterise the relationship between lacrimal gland dose and ocular toxicity among patients treated by intensity-modulated radiotherapy (IMRT) for sinonasal tumours.
40 patients with cancers involving the nasal cavity and paranasal sinuses were treated with IMRT to a median dose of 66.0 Gy. Toxicity was scored using the Radiation Therapy Oncology Group morbidity criteria based on conjunctivitis, corneal ulceration and keratitis. The paired lacrimal glands were contoured as organs at risk, and the mean dose, maximum dose, V10, V20 and V30 were determined. Statistical analysis was performed using logistic regression and the Akaike information criterion (AIC).
The maximum and mean dose to the ipsilateral lacrimal gland were 19.2 Gy (range, 1.4–75.4 Gy) and 14.5 Gy (range, 11.1–67.8 Gy), respectively. The mean V10, V20 and V30 values were 50%, 25% and 17%, respectively. The incidence of acute and late Grade 3+ toxicities was 23% and 19%, respectively. Based on logistic regression and AIC, the maximum dose to the ipsilateral lacrimal gland was identified as a more significant predictor of acute toxicity (AIC, 53.89) and late toxicity (AIC, 32.94) than the mean dose (AIC, 56.13 and 33.83, respectively). The V20 was identified as the most significant predictor of late toxicity (AIC, 26.81).
A dose–response relationship between maximum dose to the lacrimal gland and ocular toxicity was established. Our data suggesting a threshold relationship may be useful in establishing dosimetric guidelines for IMRT planning that may decrease the risk of acute and late lacrimal toxicities in the future.
Advances in knowledge:
A threshold relationship between radiation dose to the lacrimal gland and ocular toxicity was demonstrated, which may aid in treatment planning and reducing the morbidity of radiotherapy for sinonasal tumours.
PMCID: PMC3856547  PMID: 24167183
4.  Therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia using Fe2O3 nanoparticles 
This study aimed to investigate the therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia (MFH) using Fe2O3 nanoparticles. Hepatocarcinoma SMMC-7721 cells cultured in vitro were treated with ferrofluid containing Fe2O3 nanoparticles and irradiated with an alternating radio frequency magnetic field. The influence of the treatment on the cells was examined by inverted microscopy, MTT and flow cytometry. To study the therapeutic mechanism of the Fe2O3 MFH, Hsp70, Bax, Bcl-2 and p53 were detected by immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). It was shown that Fe2O3 MFH could cause cellular necrosis, induce cellular apoptosis, and significantly inhibit cellular growth, all of which appeared to be dependent on the concentration of the Fe2O3 nanoparticles. Immunocytochemistry results showed that MFH could induce high expression of Hsp70 and Bax, decrease the expression of mutant p53, and had little effect on Bcl-2. RT-PCR indicated that Hsp70 expression was high in the early stage of MFH (<24 h) and became low or absent after 24 h of MFH treatment. It can be concluded that Fe2O3 MFH significantly inhibited the proliferation of in vitro cultured liver cancer cells (SMMC-7721), induced cell apoptosis and arrested the cell cycle at the G2/M phase. Fe2O3 MFH can induce high Hsp70 expression at an early stage, enhance the expression of Bax, and decrease the expression of mutant p53, which promotes the apoptosis of tumor cells.
PMCID: PMC4230284  PMID: 25296356
Fe2O3; Magnetic nanoparticles; Magnetic fluid hyperthermia; Hepatocarcinoma cells; Liver cancer
5.  The prognostic value of CDKN2A hypermethylation in colorectal cancer: a meta-analysis 
Xing, X | Cai, W | Shi, H | Wang, Y | Li, M | Jiao, J | Chen, M
British Journal of Cancer  2013;108(12):2542-2548.
The prognostic value of CDKN2A promoter hypermethylation in colorectal cancer remains controversial. We systematically reviewed the evidence for assessment of CDKN2A methylation in colorectal cancer to elucidate this issue.
Pubmed, Embase and ISI web of knowledge were searched to identify eligible studies to evaluate the association of CDKN2A hypermethylation and overall survival and clinicopathological features of colorectal cancer patients. Combined hazard ratios (HRs) or odds ratios (ORs) with 95% confidence interval (95% CI) were pooled using a random-effects model.
A total of 11 studies encompassing 3440 patients were included in the meta-analysis. CDKN2A hypermethylation had an unfavourable impact on OS of patients with colorectal cancer (HR 1.65, 95% CI 1.29–2.11). Subgroup analysis indicated that CDKN2A hypermethylation was significantly correlated with OS in Europe (HR 1.49; 95% CI 1.28–1.74) and Asia (HR 3.30; 95% CI 1.68–6.46). Furthermore, there was a significant association between CDKN2A hypermethylation and lymphovascular invasion (OR 1.68, 95% CI 1.15–2.47), lymph node metastasis (OR 1.68, 95% CI 1.09–2.59) and proximal tumour location (OR 2.09, 95% CI 1.34–3.26) of colorectal cancer.
This meta-analysis indicated that CDKN2A hypermethylation might be a predictive factor for unfavourable prognosis of colorectal cancer patients.
PMCID: PMC3694241  PMID: 23703248
CDKN2A; methylation; colorectal cancer; prognosis
6.  Therapeutic ROS targeting of GADD45γ in the induction of G2/M arrest in primary human colorectal cancer cell lines by cucurbitacin E 
Cell Death & Disease  2014;5(4):e1198-.
Cucurbitacin E (CuE) or α-elaterin is a natural compound previously shown to be an antifeedant as well as a potent chemopreventive agent against several types of cancer. The present study investigated the anticancer effects of CuE on colorectal cancer (CRC) using primary cell lines isolated from five CRC patients in Taiwan, Specifically, we explored the anti-proliferation and cell cycle G2/M arrest induced by CuE in CRC cells. MPM-2 flow cytometry tests show that CuE-treated cells accumulated in metaphase (CuE 2.5–7.5 μM). Results further indicate that CuE produced G2/M arrest as well as the downregulation of CDC2 and cyclin B1 expression and dissociation. Both effects increased proportionally with the dose of CuE; however, the inhibition of proliferation, arrest of mitosis, production of reactive oxygen species (ROS), and loss of mitochondrial membrane potential (ΔΨm) were found to be dependent on the quantity of CuE used to treat the cancer cells. In addition, cell cycle arrest in treated cells coincided with the activation of the gene GADD45(α, β, γ). Incubation with CuE resulted in the binding of GADD45γ to CDC2, which suggests that the delay in CuE-induced mitosis is regulated by the overexpression of GADD45γ. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activities in established CRC.
PMCID: PMC4001305  PMID: 24763055
Cucurbitacin E; Colorectal cancer (CRC); G2/M arrest; Growth arrest and DNA damage 45 (GADD45)
7.  Quantitative analysis of contrast-enhanced ultrasonography: differentiating focal nodular hyperplasia from hepatocellular carcinoma 
The British Journal of Radiology  2013;86(1023):20120536.
To explore the potential of quantitative analysis of contrast-enhanced ultrasonography (CEUS) in differentiating focal nodular hyperplasia (FNH) from hepatocellular carcinoma (HCC).
34 cases of FNH and 66 cases of HCC (all lesions <5 cm) were studied using CEUS to evaluate enhancement patterns and using analytic software Sonoliver® (Image-Arena™ v.4.0, TomTec Imaging Systems, Munich, Germany) to obtain quantitative features of CEUS in the region of interest. The quantitative features of maximum of intensity (IMAX), rise slope (RS), rise time (RT) and time to peak (TTP) were compared between the two groups and applied to further characterise both FNH and HCC with hypoenhancing patterns in the late phase on CEUS.
The sensitivity and specificity of CEUS for diagnosis of FNH were 67.6% and 93.9%, respectively. For quantitative analysis, IMAX and RS in FNHs were significantly higher than those in HCCs (p<0.05), while RT and TTP in FNHs were significantly shorter (p<0.05). Both the 11 FNHs and 62 HCCs with hypo-enhancing patterns in the late phase were further characterised with their quantitative features, and the sensitivity and specificity of IMAX for diagnosis of FNH were 90.9% and 43.5%, RS 81.8% and 80.6%, RT 90.9% and 71.0%, and TTP 90.9% and 71.0%, respectively.
The quantitative features of CEUS in FNH and HCC were significantly different, and they could further differentiate FNH from HCC following conventional CEUS.
Advances in knowledge:
Our findings suggest that quantitative analysis of CEUS can improve the accuracy of differentiating FNH from HCC.
PMCID: PMC3608056  PMID: 23392189
8.  Detection of acute cerebral hemorrhage in rabbits by magnetic induction 
Acute cerebral hemorrhage (ACH) is an important clinical problem that is often monitored and studied with expensive devices such as computed tomography, magnetic resonance imaging, and positron emission tomography. These devices are not readily available in economically underdeveloped regions of the world, emergency departments, and emergency zones. We have developed a less expensive tool for non-contact monitoring of ACH. The system measures the magnetic induction phase shift (MIPS) between the electromagnetic signals on two coils. ACH was induced in 6 experimental rabbits and edema was induced in 4 control rabbits by stereotactic methods, and their intracranial pressure and heart rate were monitored for 1 h. Signals were continuously monitored for up to 1 h at an exciting frequency of 10.7 MHz. Autologous blood was administered to the experimental group, and saline to the control group (1 to 3 mL) by injection of 1-mL every 5 min. The results showed a significant increase in MIPS as a function of the injection volume, but the heart rate was stable. In the experimental (ACH) group, there was a statistically significant positive correlation of the intracranial pressure and MIPS. The change of MIPS was greater in the ACH group than in the control group. This high-sensitivity system could detect a 1-mL change in blood volume. The MIPS was significantly related to the intracranial pressure. This observation suggests that the method could be valuable for detecting early warning signs in emergency medicine and critical care units.
PMCID: PMC4051184  PMID: 24519130
Acute cerebral hemorrhage; Magnetic induction phase shift; Electromagnetic properties of biological tissues; Intracranial pressure
9.  Inducement of mitosis delay by cucurbitacin E, a novel tetracyclic triterpene from climbing stem of Cucumis melo L., through GADD45γ in human brain malignant glioma (GBM) 8401 cells 
Cell Death & Disease  2014;5(2):e1087-.
Cucurbitacin E (CuE) is a natural compound previously shown to have anti-feedant, antioxidant and antitumor activities as well as a potent chemo-preventive action against cancer. The present study investigates its anti-proliferative property using MTT assay; CuE demonstrated cytotoxic activity against malignant glioma GBM 8401 cells and induced cell cycle G2/M arrest in these cells. CuE-treated cells accumulated in metaphase (CuE 2.5–10 μM) as determined using MPM-2 by flow cytometry. We attempted to characterize the molecular pathways responsible for cytotoxic effects of CuE in GBM 8401 cells. We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. The CuE reduced the expression of 558 genes and elevated the levels of 1354 genes, suggesting an existence of the common pathways involved in induction of G2/M arrest. We identified the RB (GADD45β and GADD45γ) and the p53 (GADD45α) signaling pathways as the common pathways, serving as key molecules that regulate cell cycle. Results indicate that CuE produced G2/M arrest as well as the upregulation of GADD45 γ and binding with CDC2. Both effects increased proportionally with the dose of CuE, suggesting that the CuE-induced mitosis delay is regulated by GADD45γ overexpression. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activity in glioma therapy.
PMCID: PMC3944240  PMID: 24577085
cucurbitacin E (CuE); malignant glioma; G2/M arrest; growth arrest and DNA damage 45 (GADD45)
10.  Corrigendum 
Li, J | Sima, W | Ouyang, B | Wang, T | Ziaf, K | Luo, Z | Liu, L | Li, H | Chen, M | Huang, Y | Feng, Y | Hao, Y | Ye, Z
PMCID: PMC3873127
11.  Association of p21 with NF-YA suppresses the expression of Polo-like kinase 1 and prevents mitotic death in response to DNA damage 
Cell Death & Disease  2014;5(1):e987-.
Polo-like kinase 1 (PLK1) is an important mitotic kinase and its expression is tightly regulated in the cell cycle and in the DNA damage response. PLK1 expression is previously shown to be suppressed by p53 and/or p21. Here, we demonstrate that the CCAAT box in the PLK1 promoter is pivotal for p53/p21-mediated PLK1 repression. Chromatin immunoprecipitation showed that cyclin-dependent kinase 2 (CDK2) associated with the CCAAT box-containing region of PLK1 promoter in unstressed cells, whereas adriamycin (ADR) induced the recruitment of p21 with a concomitant reduction in the occupancy of CDK2 in this region. Expression of p21 inhibited the interaction between CDK2 and the nuclear factor YA (NF-YA) subunit of the CCAAT box-binding transcription factor NF-Y. A mutant p21 that is defective in CDK2 binding was unable to disrupt the CDK2–NF-YA interaction or suppress PLK1 transcription. Co-immunoprecipitation experiments demonstrated the interaction between NF-YA and p21, and in vitro assays showed that p21 could directly bind to NF-YA. Knockdown of NF-YA decreased the amount of PLK1 promoter-associated p21 and abolished p21-mediated PLK1 repression in cells treated with ADR. Depletion of NF-YA diminished the p53-regulated transcriptional activation and suppressed the p53-mediated protection from mitotic death after DNA damage, and these effects of NF-YA deletion were alleviated by PLK1 depletion. Our findings have uncovered a novel p21/NF-YA/PLK1 axis critical for maintaining the checkpoint function of p53 to prevent mitotic death in the DNA damage-induced response.
PMCID: PMC4040702  PMID: 24407240
PLK1; NF-YA; p21; p53; mitotic death
12.  No increased mortality from donor or recipient hepatitis B- and/or hepatitis C-positive serostatus after related-donor allogeneic hematopoietic cell transplantation 
Limited data exist on allogeneic transplant outcomes in recipients receiving hematopoietic cells from donors with prior or current hepatitis B (HBV) or C (HCV) infection (seropositive donors) or for recipients with prior or current HBV or HCV infection (seropositive recipients). Transplant outcomes are reported for 416 recipients from 121 centers who received a human leukocyte antigen-identical related-donor allogeneic transplant for hematologic malignancies between 1995 and 2003. Of these, 33 seronegative recipients received grafts from seropositive donors and 128 recipients were seropositive. The remaining 256 patients served as controls. With comparable median follow-up (cases, 5.9 years; controls, 6.7 years), the incidence of treatment-related mortality, survival, graft-versus-host disease, and hepatic toxicity appears similar in all cohorts. The frequencies of hepatic toxicities as well as causes of death between cases and controls were similar. Prior exposure to HBV or HCV in either the donor or the recipient should not be considered an absolute contraindication to transplant.
PMCID: PMC3481161  PMID: 22548788
allogeneic transplantation; hepatitis B; hepatitis C
13.  WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma 
Cell Death & Disease  2013;4(9):e792-.
Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.
PMCID: PMC3789168  PMID: 24008736
tumor suppressor; methotrexate; chemotherapy; autophagy; apoptosis
14.  The HDAC inhibitor, MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells 
Cell Death & Disease  2013;4(9):e810-.
Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.
PMCID: PMC3789188  PMID: 24052078
lung cancer; HDAC; synergistic; EGFR; apoptosis; erlotinib
15.  Defective jejunal and colonic salt absorption and altered Na+/H+ exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice 
We investigated the role of the Na+/H+ exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na+ absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.
PMCID: PMC3746809  PMID: 18758809
PDZ domain proteins; Intestinal electrolyte transport; PDZK1; NHERF1; EPAC; Knockout mice
16.  Intensity-modulated radiotherapy for nasopharyngeal carcinoma: improvement of the therapeutic ratio with helical tomotherapy vs segmental multileaf collimator-based techniques 
The British Journal of Radiology  2012;85(1016):e537-e543.
The aim of the study was to compare differences in dosimetric, clinical and quality-of-life end points among patients treated with helical tomotherapy (HT) and segmental multileaf collimator (SMLC)-based intensity-modulated radiotherapy (IMRT) for nasopharyngeal carcinoma.
From June 2005 to August 2009, 30 consecutive patients were treated with IMRT for nasopharyngeal carcinoma to a dose of 70 Gy. 14 patients (47%) were treated using HT and 16 (53%) were treated using SMLC-based IMRT. 28 patients (93%) received concurrent chemotherapy. The patients were evenly balanced between the two radiotherapy groups with respect to clinical and pathological characteristics. Median follow-up was 30 months (range, 6–62 months).
The 2-year estimates of overall survival, local–regional control and progression-free survival were 81%, 87% and 82%, respectively. There were no significant differences in any of these end points with respect to IMRT technique (p>0.05 for all). Dosimetric analysis revealed that patients treated by HT had significantly improved salivary sparing with respect to mean dose (27.3 vs 34.1 Gy, p=0.03) and volume receiving greater than or equal to 30 Gy (31.7% vs 47.3%, p=0.01) to the contralateral (spared) parotid gland. The incidence of Grade 3+ late xerostomia was 13 and 7% among patients treated with SMLC-based IMRT and HT, respectively (p=0.62). The corresponding proportion of patients who subjectively reported “too little” or “no” saliva at final follow-up was 38% and 7%, respectively (p=0.04).
The superior dosimetric outcome observed with HT appeared to translate into moderately improved clinical outcomes with respect to salivary sparing. Prospective trials are needed to validate this gain in the therapeutic ratio.
PMCID: PMC3587075  PMID: 22253350
17.  Controllable Disorder in a Hybrid Nanoelectronic System: Realization of a Superconducting Diode 
Scientific Reports  2013;3:2274.
We have studied a hybrid nanoelectronic system which consists of an AlGaAs/GaAs two-dimensional electron gas (2DEG) in close proximity (~70 nm) to an Al superconducting nanofilm. By tuning the current through the Al film, we can change the conductance of the 2DEG and furthermore vary the effective disorder in the Al superconducting film in a controllable way. When a high current is injected into the film, screening which couples the Al film and the 2DEG results in a collapse of anti-symmetric behavior in the current-voltage characteristics, V(I) ~ -V(-I), which holds true in a conventional superconductor. Our results may open a new avenue of experimentally realizing a superconducting diode.
PMCID: PMC3721077  PMID: 23881449
18.  Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate 
The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina.
Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina.
Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections.
This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.
PMCID: PMC3673018  PMID: 22081232
Bruch’s membrane; RPE; Advanced glycation endproducts; Ubiquitin carboxyterminal hydrolase-1
19.  64-slice CT angiography for the detection of functionally significant coronary stenoses: comparison with stress myocardial perfusion imaging 
The British Journal of Radiology  2012;85(1012):368-376.
To evaluate the accuracy of 64-slice CT angiography (CTA) compared with single photon emission CT (SPECT) myocardial perfusion imaging (MPI), which served as the reference standard, for the detection of functionally significant coronary artery disease (CAD).
141 consecutive patients (60±10 years, 101 men) were investigated with 64-slice CTA and SPECT MPI; a subset of 35 patients had additional invasive coronary angiography (ICA). The data from CTA and ICA were compared with those from MPI for both cut-offs of ≥50% and ≥70% stenosis, respectively.
The sensitivity, specificity, positive and negative predictive values, and accuracy of CTA, using a cut-off of ≥50% for significant stenosis, in detecting inducible perfusion defects on MPI were 96% [95% confidence interval (CI) 88–100%], 61% (95% CI 52–70%), 37% (95% CI 23–49%), 99% (95% CI 97–100%) and 68%, respectively, in patient-based analysis and 97% (95% CI 91–100%), 86% (95% CI 83–89%), 33% (95% CI 24–42%), 100% (95% CI 99–100%) and 87%, respectively, in vessel-based analysis. Applying a cut-off of ≥70% for significant stenosis, CTA yielded the following sensitivity, specificity, positive and negative predictive values, and accuracy for the detection of inducible MPI defects: by patient, 65% (95% CI 46–84%), 95% (95% CI 91–99%), 74% (95% CI 50–92%), 92% (95% CI 87–97%) and 89%, respectively; by vessel, 58% (95% CI 42–74%), 97% (95% CI 95–99%), 62% (95% CI 45–79%), 97% (95% CI 95–99%) and 95%, respectively.
64-slice CTA is a reliable tool to exclude functionally significant CAD when using a cut-off of ≥50% diameter stenosis. By contrast, a cut-off of ≥70% diameter narrowing is a strong predictor of ischaemia.
PMCID: PMC3486657  PMID: 21224298
20.  Minimizing shoulder syndrome with intra-operative spinal accessory nerve monitoring for neck dissection 
The objective of this study was to analyze the safety and results of intra-operative SAN (spinal accessary nerve) monitoring during selective neck dissection, with emphasis on shoulder syndrome. Twenty-five consecutive patients with head and neck cancer were studied. Selective neck dissection was performed by a single clinical fellow under the supervision of the department chief using an intra-operative SAN monitor. Electrophysiological data were recorded after initial identification of the SAN and continued until just before closure. Electromyographic evaluation was carried out to assess SAN function one month postoperatively. Shoulder disability was also evaluated at this time using a questionnaire for shoulder syndrome (shrug, flexion, abduction, winging, and pain). No patients had postoperative shoulder syndrome involving shrug, flexion, abduction, or winging. Twenty-two of the 25 (88%) patients had shoulder pain, but the average pain score was low (2.3 ± 1.3). No patients had neck recurrence during at least 1 year of follow up. By using nerve monitoring during selective neck dissection, no patient developed significant "shoulder syndrome", with the exception of slight pain.
PMCID: PMC3665377  PMID: 23853398
Head and neck; Selective neck dissection; Axonal injury; Spinal accessory nerve; Electrophysiology
21.  Retinopathy in a patient with Fanconi anemia and vitamin B12 deficiency 
Eye  2011;26(2):331.
PMCID: PMC3272190  PMID: 22056860
22.  Cage-Forming Compounds in the Ba–Rh–Ge System: From Thermoelectrics to Superconductivity 
Inorganic Chemistry  2013;52(2):931-943.
Phase relations and solidification behavior in the Ge-rich part of the phase diagram have been determined in two isothermal sections at 700 and 750 °C and in a liquidus projection. A reaction scheme has been derived in the form of a Schulz–Scheil diagram. Phase equilibria are characterized by three ternary compounds: τ1-BaRhGe3 (BaNiSn3-type) and two novel phases, τ2-Ba3Rh4Ge16 and τ3-Ba5Rh15Ge36-x, both forming in peritectic reactions. The crystal structures of τ2 and τ3 have been elucidated from single-crystal X-ray intensity data and were found to crystallize in unique structure types: Ba3Rh4Ge16 is tetragonal (I4/mmm, a = 0.65643(2) nm, c = 2.20367(8) nm, and RF = 0.0273), whereas atoms in Ba5Rh15Ge36–x (x = 0.25) arrange in a large orthorhombic unit cell (Fddd, a = 0.84570(2) nm, b = 1.4725(2) nm, c = 6.644(3) nm, and RF = 0.034). The body-centered-cubic superstructure of binary Ba8Ge43□3 was observed to extend at 800 °C to Ba8Rh0.6Ge43□2.4, while the clathrate type I phase, κI-Ba8RhxGe46–x–y□y, reveals a maximum solubility of x = 1.2 Rh atoms in the structure at a vacancy level of y = 2.0. The cubic lattice parameter increases with increasing Rh content. Clathrate I decomposes eutectoidally at 740 °C: κI ⇔ (Ge) + κIX + τ2. A very small solubility range is observed at 750 °C for the clathrate IX, κIX-Ba6RhxGe25–x (x ∼ 0.16). Density functional theory calculations have been performed to derive the enthalpies of formation and densities of states for various compositions Ba8RhxGe46–x (x = 0–6). The physical properties have been investigated for the phases κI, τ1, τ2, and τ3, documenting a change from thermoelectric (κI) to superconducting behavior (τ2). The electrical resistivity of κI-Ba8Rh1.2Ge42.8□2.0 increases almost linearly with the temperature from room temperature to 730 K, and the Seebeck coefficient is negative throughout the same temperature range. τ1-BaRhGe3 has a typical metallic electrical resistivity. A superconducting transition at TC = 6.5 K was observed for τ2-Ba3Rh4Ge16, whereas τ3-Ba5Rh15Ge35.75 showed metallic-like behavior down to 4 K.
Phase equilibria in the Ba−Rh−Ge system are characterized by three ternary cage compounds: τ 1-BaRhGe3 (BaNiSn3-type) and two novel phases with unique structures, τ 2-Ba3Rh4Ge16 and τ 3-Ba5Rh15Ge36−x, besides κI-Ba8RhxGe46−x−y□y (x ≤ 1.2 and y ≥ 2.0). Density functional theory calculations for the enthalpies of formation and density of states for various compositions Ba8RhxGe46−x (x = 0−6) demonstrate a strong stabilizing influence of Ge/Rh substitution. The physical properties have been investigated for κI, τ 1, τ 2, and τ 3, documenting a change from n-type thermoelectric (κI) to superconducting behavior (τ 2; TC = 6.5 K).
PMCID: PMC3557931  PMID: 23286379
23.  Identification and Quantification of Dimethylamylamine in Geranium by Liquid Chromatography Tandem Mass Spectrometry 
A sensitive and reliable method of liquid chromatography–electrospray ionization/tandem mass spectrometry (LC-ESI/MS/ MS) was developed and validated for determining 1,3-dimethylamylamine (1,3-DMAA) and 1,4-dimethylamylamine (1,4-DMAA) in geranium plants (Pelargonium graveolens). The sample was extracted with 0.5 M HCl and purified by liquid-liquid partition with hexane. The parameters for reverse-phase (C18) LC and positive ESI/MS/MS were optimized. The matrix effect, specificity, linearity, precision, accuracy and reproducibility of the method were determined and evaluated. The method was linear over a range of 0.10–10.00 ng/mL examined, with R2 of 0.99 for both 1,3-DMAA and 1,4-DMAA. The recoveries from spiked concentrations between 5.00–40.00 ng/g were 85.1%–104.9% for 1,3-DMAA, with relative standard deviation (RSD) of 2.9%–11.0%, and 82.9%–101.8% for 1,4-DMAA, with RSD of 3.2%–11.7%. The instrument detection limit was 1–2 pg for both DMAAs. The quantification limit was estimated to be 1–2 ng/g for the plant sample. This method was successfully applied to the quantitative determination of 1,3- and 1,4-DMAA in both geranium plant and geranium oil.
PMCID: PMC3422085  PMID: 22915838
1,3-dimethylamylamine; 1,4-dimethylamylamine; geranium (Pelargonium graveolens); liquid chromatography-tandem mass spectrometry (LC/MS/MS)
25.  Differing Neisseria gonorrhoeae Bacterial Loads in the Pharynx and Rectum in Men Who Have Sex with Men: Implications for Gonococcal Detection, Transmission, and Control ▿  
Journal of Clinical Microbiology  2011;49(12):4304-4306.
The bacterial loads for gonococcal infections of the pharynx and rectum were determined among men with male sexual partners. The median bacterial load for rectal infections (18,960 copies/swab) was significantly higher than the load for pharyngeal infections (2,100 copies/swab; P = 0.001). Bacterial loads among men with symptomatic proctitis were strikingly high (median, 278,000 copies/swab).
PMCID: PMC3232985  PMID: 21956992

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