[Identification of a Novel Calcium (Ca

Calcium (Ca^(2+))-activated chloride channel accessories (CLCAs) are putative anion channel-related proteins with diverse physiological functions. Exploring CLCA diversity is important for prediction of gene structure and function. In an effort to identify novel CLCA genes in Xenopus laevis, we successfully cloned and characterized a Xenopus laevis cDNA predicted to encode the xCLCA3 gene. Cloning of xCLCA3 was achieved by computational analysis, rapid amplification of cDNA ends (RACE), and a tissue distribution analysis by semi-quantitative reverse transcription (RT) PCR or real-time PCR. We obtained a 2958 bp xCLCA3 cDNA sequence with an open reading frame encoding 943 amino acids. According to the primary structure analysis, xCLCA3 contains a predicted signal sequence, multiple sites of N-linked (N-) glycosylation, N-myristoylation, PKA, PKC, and casein kinase II phosphorylation sites, five putative hydrophobic segments, and the HExxH metalloprotease motif. Additionally, the transmembrane prediction server yielded a preserved N-terminal CLCA domain and a von Willebrand factor type A domain with one transmembrane domain in the C-terminal region. Expression analysis showed that xCLCA3 is expressed in a number of tissues, with strong expression in the brain, colon, small intestine, lung, kidney, and spleen, and poor expression in the heart and liver. These results suggest that xCLCA3 may be a candidate CLCA family member as well as a metalloprotease, rather than just an ion channel accessory protein.

Collective cell migration over long time scales reveals distinct phenotypes.

INTRODUCTION: Migratory phenotypes of metastasizing tumor cells include single and collective cell migration. While migration of tumor cells is generally less cooperative than that of normal epithelial cells, our understanding of precisely how they differ in long time behavior is incomplete. OBJECTIVES: We measure in a model system how cancer progression affects collective migration on long time scales, and determine how perturbation of cell-cell adhesions, specifically reduced E-cadherin expression, affects the collective migration phenotype. METHODS: Time lapse imaging of cellular sheets and particle image velocimetry (PIV) are used to quantitatively study the dynamics of cell motion over ten hours. Long time dynamics are measured via finite time Lyapunov exponents (FTLE) and changes in FTLE with time. RESULTS: We find that non-malignant MCF10A cells are distinguished from malignant MCF10CA1a cells by both their short time (minutes) and long time (hours) dynamics. In addition, short time dynamics distinguish non-malignant E-cadherin knockdown cells from the control, but long time dynamics and increasing spatial correlations remain unchanged. DISCUSSION: Epithelial sheet collective behavior includes long time dynamics that cannot be captured by metrics that assess cooperativity based on short time dynamics, such as instantaneous speed or directionality. The use of metrics incorporating migration data over hours instead of minutes allows us to more precisely describe how E-cadherin, a clinically relevant adhesion molecule, affects collective migration. We predict that the long time scale metrics described here will be more robust and predictive of malignant behavior than analysis of instantaneous velocity fields alone.

Bioassay and biochemical studies of the status of pirimiphos-methyl and cypermethrin resistance in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Singapore.

Aedes (Stegomyia) aegypti (Linnaeus) and Ae. (Stegomyia) albopictus (Skuse) were sampled from five regions of Singapore (Central, North East, North West, South East and South West) and tested with diagnostic concentrations of the technical grade insecticides, pirimiphos-methyl and cypermethrin. Biochemical assays were performed on the same populations of Ae. aegypti and Ae. albopictus to determine activities of detoxifying enzymes, including non-specific esterase (EST), monooxygenase (MFO) and acetylcholinesterase (AChE). The diagnostic test showed that all Ae. aegypti populations were susceptible to pirimiphos-methyl (mortality = 99 to 100%), but resistant to cypermethrin (mortality = 11 to 76%). Resistance to pirimiphos-methyl was observed in all Ae. albopictus populations (mortality = 49 to 74%) while cypermethrin resistance was detected in most Ae. albopictus populations (mortality = 40 to 75%), except those from Central (mortality = 86%) and South East (mortality = 94%) showing incipient resistance. The biochemical assays showed that there was significant enhancement (P < 0.001) of MFO activity in pyrethroid-resistant Ae. albopictus populations and most Ae. aegypti populations. The biochemical assay results suggested that AChE could play a role in pirimiphos-methyl resistance of Ae. albopictus in South West, South East and North East regions. The small but significant increase in EST activities in Ae. aegypti from all regions suggest that it may play a role in the observed cypermethrin resistance.

Bioassay and biochemical studies of the status of pirimiphos-methyl and cypermethrin resistance in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Singapore Singapore 138667

Aedes (Stegomyia) aegypti (Linnaeus) and Ae. (Stegomyia) albopictus (Skuse) were sampled from five regions of Singapore (Central, North East, North West, South East and South West) and tested with diagnostic concentrations of the technical grade insecticides, pirimiphos-methyl and cypermethrin. Biochemical assays were performed on the same populations of Ae. aegypti and Ae. albopictus to determine activities of detoxifying enzymes, including non-specific esterase (EST), monooxygenase (MFO) and acetylcholinesterase (AChE). The diagnostic test showed that all Ae. aegypti populations were susceptible to pirimiphos-methyl (mortality = 99 to 100%), but resistant to cypermethrin (mortality = 11 to 76%). Resistance to pirimiphos-methyl was observed in all Ae. albopictus populations (mortality = 49 to 74%) while cypermethrin resistance was detected in most Ae. albopictus populations (mortality = 40 to 75%), except those from Central (mortality = 86%) and South East (mortality = 94%) showing incipient resistance. The biochemical assays showed that there was significant enhancement (P < 0.001) of MFO activity in pyrethroid-resistant Ae. albopictus populations and most Ae. aegypti populations. The biochemical assay results suggested that AChE could play a role in pirimiphos-methyl resistance of Ae. albopictus in South West, South East and North East regions. The small but significant increase in EST activities in Ae. aegypti from all regions suggest that it may play a role in the observed cypermethrin resistance.

Stilbene 5c, a microtubule poison with vascular disrupting properties that induces multiple modes of growth arrest and cell death.

The stilbene derivative, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding site in tubulin. The current studies were designed to investigate the effectiveness of stilbene 5c against the HCT-116 human colon cancer cell line and B16/F10 melanoma cells as well as human endothelial cell tube formation and tumor perfusion. Stilbene 5c produced a time-dependent decrease in cell viability in both cell lines and the capacity of the cells to proliferate was not restored upon removal of the drug. Treatment with stilbene 5c also promoted both senescence and autophagy in both cell lines. TUNEL and annexin 5 staining indicated that apoptosis also occurs in stilbene 5c-treated HCT-116 cells, but not in B16/F10 melanoma cells. DAPI staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells) indicative of mitotic catastrophe in HCT-116 cells but not in the B16/F10 melanoma cells. p53-null HCT-116 cells demonstrated a similar growth arrest/cell death response to stilbene as p53-wild type HCT-116 cells. Stilbene 5c also completely inhibited human endothelial cell tube formation on Matrigel, consistent with potential anti-angiogenic actions. Using a new method developed for monitoring the pharmacodynamic effects of stilbene 5c in vivo, we found that a single injection of stilbene 5c reduced tumor perfusion by 65% at 4h, returning to baseline by 24h, while subsequent daily injections of stilbene 5c produced progressively larger reductions and smaller rebounds. This work indicates that stilbene 5c could potentially be effective against melanoma and colon cancer through the promotion of multiple modes of growth arrest and cell death coupled with anti-angiogenic and antivascular actions.

Unilateral isolated extraocular muscle lymphoma.

The authors would like to present an unusual case of ocular adnexal, mucosa-associated lymphoid tissue lymphoma, isolated to a single extraocular muscle. A 59-year-old woman presented with a 3-month history of slowly progressive double vision, worse on elevation, for which her optometrist had given her prisms. A swollen left upper eyelid was present for 10 days. CT scan of the brain and orbits revealed a 3 cm × 1.5 cm mass arising from the region of the left superior rectus with no signs of bone erosion. Histology showed infiltration by small lymphoid cells. Stage 1AE low-grade marginal zone B cell lymphoma was diagnosed. Possible aetiologies included Chlamydia psittaci infection and the recently recognised IgG4-related sclerosing disease. After oral doxycycline 200 mg once a day failed to show improvement, localised radiotherapy 30 Gy resulted in excellent clinical and radiological resolution of this isolated lymphoma.

Modulation of thiopental-induced vascular relaxation and contraction by perivascular adipose tissue and endothelium.

BACKGROUND: Thiopental induces relaxation of vascular smooth muscle cells through its direct and/or indirect vasodilator effects. The perivascular adipose tissue (PVAT) and the endothelium are known to attenuate vascular contraction, and we have recently reported that PVAT potentiates the relaxation effect of propofol through endothelium-dependent and -independent mechanisms. Here, we studied the mechanisms of thiopental-induced vascular responses in relation to the involvement of PVAT and endothelium. METHODS: Thoracic aortic rings from male Wistar rats were prepared with or without PVAT (PVAT+ and PVAT-) and with an intact endothelium (E+) or with the endothelium removed (E-) for functional studies. The contraction and relaxation responses of these vessels to thiopental in the presence of agonists and various receptor antagonists and channel blockers were studied. RESULTS: In vessels pre-contracted with phenylephrine or KCl, thiopental-induced relaxation was highest in vessels denuded of both PVAT and the endothelium. PVAT attenuated the relaxation response to thiopental, and this attenuation effect was reduced by both angiotensin II (Ang II) type 1 receptor antagonists CV-11974 (2-n-butyl-4-choloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)biphenyl-methyl]-imidazole) or losartan and the angiotensin-converting enzyme inhibitor enalaprilat. Thiopental at high concentration (3 × 10(-3) M) caused a contraction through an endothelin-dependent mechanism. CONCLUSIONS: Thiopental induced relaxation in rat aorta through an endothelium-independent pathway and the presence of PVAT, endothelium, or both attenuated this relaxation response through Ang II-dependent and endothelin-dependent mechanisms, respectively.

Cyclic vomiting syndrome and functional vomiting in adults: association with cannabinoid use in males.

BACKGROUND: Cyclic vomiting syndrome (CVS) is characterized by stereotypical episodes of vomiting separated by symptom-free intervals. However, the difficulty encountered in the management of patients with CVS may be a reflection of a deficiency in our understanding of the disorder. We aimed to evaluate whether clinical or gastric emptying (GE) data discriminate patients labeled as having CVS from functional vomiting (FV) or irritable bowel syndrome (IBS). METHODS: The medical records of patients diagnosed with any vomiting (including CVS, FV) over a 13-year period (1993-2006) at our institution were carefully reviewed. Disease controls were age and gender matched subjects with IBS. Gastric emptying was performed by scintigraphy (99mTc-egg meal). The associations of clinical factors and GE data with patient status (CVS vs FV or IBS) were analyzed. KEY RESULTS: A total of 82 patients with CVS and 62 FV patients were identified. Younger age [per 10 years, OR = 0.7 (0.5, 0.9)], male gender [OR = 0.4 (0.2, 0.9)], and cannabinoid use [OR = 2.9 (1.2, 7.2)] were significantly associated with CVS compared with FV. However, there were no significant associations between patient status (CVS vs FV) and age, BMI, smoking, alcohol use, gastrointestinal symptoms, or GE. The proportion of cannabinoid users was significantly higher in patients with CVS compared with patients with IBS, whereas proportions for headaches and psychiatric disease were higher in subjects with IBS. CONCLUSIONS & INFERENCES: Cyclic vomiting syndrome (vs FV) was not associated with clinical factors, but was associated with younger age, male gender and cannabinoid use. A larger proportion of CVS (vs IBS) patients had used cannabinoids.

Strategies for improving the efficiency of quantum Monte Carlo calculations.

We describe a number of strategies for minimizing and calculating accurately the statistical uncertainty in quantum Monte Carlo calculations. We investigate the impact of the sampling algorithm on the efficiency of the variational Monte Carlo method. We then propose a technique to maximize the efficiency of the linear extrapolation of diffusion Monte Carlo results to zero time step, finding that a relative time-step ratio of 1:4 is optimal. Finally, we discuss the removal of serial correlation from data sets by reblocking, setting out criteria for the choice of block length and quantifying the effects of the uncertainty in the estimated correlation length.

Polypseudophakia for cataract surgery: 10-year follow-up on safety and stability of two poly-methyl-methacrylate (PMMA) intraocular lenses within the capsular bag.

PURPOSE: To report the long-term follow-up (more than 10 years) of three patients who have undergone polypseudophakia phacoemulsification cataract surgery. METHODS: A case series of three patients and four procedures. Two poly-methyl-methacrylate (PMMA) intraocular lenses (IOLs) were placed within the capsular bag (P359UV, Storz, Tuttlingen, Germany). No complications occurred peri-operatively. A full ophthalmological examination was performed at 10-year follow-up, looking for decentration, tilt, and complications of interlenticular opacification (ILO) between the IOLs. RESULTS: Inferior ILO with Elschnig pearls was observed in only one case, and was likely to be visually insignificant. No ILO was observed in the other three eyes. In one patient, the piggyback IOL had been displaced 1-2 mm nasally, but there was no tilt of the IOLs, with the haptics remaining well aligned. There was no displacement or tilt of the piggyback IOL in the other three cases. Corneal endothelial cell count (SP-2000P, Topcon, Tokyo, Japan) was above 1000 cell/mm(2) in all cases. CONCLUSIONS: With the introduction of foldable IOLs, the piggyback IOL is usually placed in the sulcus, but we have shown good long-term stability and minimal complications of dual PMMA IOLs placed within the bag.

Malignant peripheral nerve sheath tumor mimicking carotid body tumor--case report and review.

Malignant peripheral nerve sheath tumor is a rare neurogenic tumor that usually presents in geriatic patients. Typically, it is found in the trunk and extremities and rarely presents in the head and neck region. It may mimic a carotid body tumor when it presents in the neck. We report the first case of malignant peripheral nerve sheath tumor of the vagus nerve in an adolescent boy. He presented with an asymptomatic lateral neck lump that was thought to be a benign schwannoma on preoperative imaging. We describe the diagnostic dilemma and management difficulties in this patient and review the literature.

New mosquito species records (Diptera: Culicidae) from Singapore.

Nine species of mosquitoes in eight genera are recorded for the first time in Singapore. An additional two species were overlooked in a 1986 checklist for mosquitoes in Singapore, and one was described after 1986. Location and habitat data are provided for the nine new records. With the inclusion of these new records the number of species reported from Singapore is 137.

Modulation of vascular function by perivascular adipose tissue: the role of endothelium and hydrogen peroxide.

BACKGROUND AND PURPOSE: Perivascular adipose tissue (PVAT) attenuates vascular contraction, but the mechanisms remain largely unknown. The possible involvement of endothelium (E) and hydrogen peroxide (H2O2) was investigated. EXPERIMENTAL APPROACH: Aortic rings from Wistar rats were prepared with both PVAT and E intact (PVAT+ E+), with either PVAT or E removed (PVAT- E+, or PVAT+ E-), or with both removed (PVAT- E-) for functional studies. Nitric oxide (NO) production was measured. KEY RESULTS: Contraction to phenylephrine and 5-HT respectively was highest in PVAT- E-, lowest in PVAT+ E+, and intermediate in PVAT+ E- or PVAT- E+. In bioassay experiments, transferring bathing solution incubated with a PVAT+ ring (donor) to a PVAT- ring (recipient) induced relaxation in the recipient. This relaxation was abolished by E removal, NO synthase inhibition, scavenging of NO, high extracellular K+, or blockade of calcium-dependent K+ channels (K(Ca)). The solution stimulated NO production in isolated endothelial cells and in PVAT- E+ rings. In E- rings, the contraction to phenylephrine of PVAT+ rings but not PVAT- rings was enhanced by catalase or soluble guanylyl cyclase (sGC) inhibitor, but reduced by superoxide dismutase and tiron. In PVAT- E- rings, H2O2 attenuated phenylephrine-induced contraction. This effect was counteracted by sGC inhibition. NO donor and H2O2 exhibited additive inhibition of the contraction to phenylephrine in PVAT- E- rings. CONCLUSION: PVAT exerts its anti-contractile effects through two distinct mechanisms: (1) by releasing a transferable relaxing factor which induces endothelium-dependent relaxation through NO release and subsequent K(Ca) channel activation, and (2) by an endothelium-independent mechanism involving H2O2 and subsequent activation of sGC.

Induction of melanoma cell apoptosis and inhibition of tumor growth using a cell-permeable Survivin antagonist.

The inhibitor of apoptosis gene family member Survivin is highly expressed in most tumors, and appears to be a promising target for cancer therapy. Although a variety of Survivin antagonists have been shown to induce apoptosis in malignant cells, the potential utility of these agents is limited by inefficient delivery and cell impermeability. We generated recombinant fusion proteins containing the TAT protein transduction domain and either wild-type Survivin (TAT-Surv-WT) or a dominant-negative mutant (TAT-Surv-T34A). The TAT-Surv proteins were purified by sequential affinity and ion-exchange chromatography, and at 30 nM concentration demonstrated rapid entry into cells at 30 min. Whereas TAT-Surv-WT had minimal effect on YUSAC2 or WM793 melanoma cells, TAT-Surv-T34A induced cell detachment, DNA fragmentation, caspase-3 activation and mitochondrial release of apoptosis-inducing factor at low microM concentrations. Intraperitoneal (i.p.) injection of mice bearing subcutaneous YUSAC2 xenografts with TAT-Surv-T34A (10 mg/kg) was associated with rapid tumor accumulation at 1 h, and increased tumor cell apoptosis and aberrant nuclei formation in situ. Repeated i.p. injection of TAT-Surv-T34A resulted in a 40-50% reduction in growth and mass of established tumors, compared to those similarly injected with saline buffer or TAT-Surv-WT. These studies demonstrate the feasibility of systemic tumor treatment using a cell-permeable Survivin antagonist.

Influenza a viruses upregulate neutrophil toll-like receptor 2 expression and function.

Neutrophils are involved in the initial host response to influenza A virus (IAV) infection and exhibit both activation and depressed function after exposure to the virus. We demonstrate that IAV causes rapid upregulation of Toll-like receptor 2 (TLR2) expression on neutrophils. The neutrophil agonists, formyl-methylpleucyl-alanine (fMLP), C5a and lipopolysaccharide did not alter neutrophil TLR2 expression, whereas PMA and the microbial TLR2 ligands, peptidoglycan (PGN) and zymosan, reduced it. To determine the functional significance of IAV-induced increase in TLR2 expression, IAV-treated neutrophils were exposed to PGN, Staphylococcus aureus (S. aureus) and zymosan. Pretreatment with IAV resulted in significantly increased uptake of S. aureus and zymosan and accelerated neutrophil apoptosis when combined with S. aureus. IAV-treated cells generated significantly more H(2)O(2) in response to PGN. These results indicate that IAV increases neutrophil surface expression of TLR2 and modulates functional responses to ligands that bind TLR2. These findings may clarify IAV-induced perturbation of neutrophil functions in vivo.