Oncolytic reovirus therapy: Pilot study in dogs with spontaneously occurring tumours.

Oncolytic virotherapy is a novel treatment involving replication-competent virus in the elimination of cancer. We have previously reported the oncolytic effects of reovirus in various canine cancer cell lines. This study aims to establish the safety profile of reovirus in dogs with spontaneously occurring tumours and to determine a recommended dosing regimen. Nineteen dogs with various tumours, mostly of advanced stages, were treated with reovirus, ranging from 1.0 × 108 to 5.0 × 109 TCID50 given as intratumour injection (IT) or intravenous infusion (IV) daily for up to 5 consecutive days in 1 or multiple treatment cycles. Adverse events (AEs) were graded according to the Veterinary Cooperative Oncology Group- Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines. Viral shedding, neutralizing anti-reovirus antibody (NARA) production and immunohistochemical (IHC) detection of reovirus protein in the tumours were also assessed. AE was not observed in most dogs and events were limited to Grade I or II fever, vomiting, diarrhoea and inflammation of the injected tumour. No infectious virus was shed and all dogs had elevated NARA levels post-treatment. Although IHC results were only available in 6 dogs, 4 were detected positive for reovirus protein. In conclusion, reovirus is well-tolerated and can be given safely to tumour-bearing dogs according to the dosing regimen used in this study without significant concerns of viral shedding. Reovirus is also potentially effective in various types of canine tumours.

Signature of type-II Weyl semimetal phase in MoTe2.

Topological Weyl semimetal (TWS), a new state of quantum matter, has sparked enormous research interest recently. Possessing unique Weyl fermions in the bulk and Fermi arcs on the surface, TWSs offer a rare platform for realizing many exotic physical phenomena. TWSs can be classified into type-I that respect Lorentz symmetry and type-II that do not. Here, we directly visualize the electronic structure of MoTe2, a recently proposed type-II TWS. Using angle-resolved photoemission spectroscopy (ARPES), we unravel the unique surface Fermi arcs, in good agreement with our ab initio calculations that have nontrivial topological nature. Our work not only leads to new understandings of the unusual properties discovered in this family of compounds, but also allows for the further exploration of exotic properties and practical applications of type-II TWSs, as well as the interplay between superconductivity (MoTe2 was discovered to be superconducting recently) and their topological order.

Distinct Electronic Structure for the Extreme Magnetoresistance in YSb.

An extreme magnetoresistance (XMR) has recently been observed in several nonmagnetic semimetals. Increasing experimental and theoretical evidence indicates that the XMR can be driven by either topological protection or electron-hole compensation. Here, by investigating the electronic structure of a XMR material, YSb, we present spectroscopic evidence for a special case which lacks topological protection and perfect electron-hole compensation. Further investigations reveal that a cooperative action of a substantial difference between electron and hole mobility and a moderate carrier compensation might contribute to the XMR in YSb.

The effects of oncolytic reovirus in canine lymphoma cell lines.

Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus.

Brief report: enhancement of patient recruitment in rheumatoid arthritis clinical trials using a multi-biomarker disease activity score as an inclusion criterion.

OBJECTIVE: Rheumatoid arthritis (RA) clinical trials often exclude patients who have low C-reactive protein (CRP) levels, which slows enrollment into the trial. The purpose of this study was to determine whether high Multi-Biomarker Disease Activity (MBDA) scores (>44) in RA patients with low CRP levels (≤10 mg/liter) could be used as a complement to CRP levels >10 mg/liter to enhance patient recruitment without affecting clinical trial outcomes. METHODS: We evaluated patients from the Swedish Pharmacotherapy (SWEFOT) trial, which did not include any selection criteria for CRP levels. Clinical outcomes were assessed after 3 months of methotrexate (MTX) monotherapy in MTX-naive RA patients (n = 220) and after 3-10 months of add-on therapy in patients who were incomplete responders to MTX alone (MTX-IR) (n = 127). Radiographic outcomes were assessed at 1 year in all patients. Within each cohort, the outcomes were compared between patients with a CRP level of ≤10 mg/liter and an MBDA score of >44 at the start of the respective treatment interval versus those with a CRP level of >10 mg/liter. RESULTS: Patients with both a CRP level of ≤10 mg/liter and an MBDA score of >44 at baseline had clinical and radiographic outcomes that were comparable to those in patients with a CRP level of >10 mg/liter at baseline. This broadened definition of the inclusion criteria identified an additional 24% of patients in the MTX-naive cohort and 47% in the MTX-IR cohort. CONCLUSION: Patient recruitment into RA clinical trials may be substantially enhanced, without any decrease in clinical and radiographic outcomes, by using as an inclusion criterion "a CRP level of >10 mg/liter and/or an MBDA score of >44."

Spin-induced band modifications of graphene through intercalation of magnetic iron atoms.

Intercalation of magnetic iron atoms through graphene formed on the SiC(0001) surface is found to induce significant changes in the electronic properties of graphene due mainly to the Fe-induced asymmetries in charge as well as spin distribution. From our synchrotron-based photoelectron spectroscopy data together with ab initio calculations, we observe that the Fe-induced charge asymmetry results in the formation of a quasi-free-standing bilayer graphene while the spin asymmetry drives multiple spin-split bands. We find that Fe adatoms are best intercalated upon annealing at 600 °C, exhibiting split linear π-bands, characteristic of a bilayer graphene, but much diffused. Subsequent changes in the C 1s, Si 2p, and Fe 3p core levels are consistently described in terms of Fe-intercalation. Our calculations together with a spin-dependent tight binding model ascribe the diffuse nature of the π-bands to the multiple spin-split bands originated from the spin-injected carbon atoms residing only in the lower graphene layer.

In-situ synchrotron radiation photoemission spectroscopy study of the initial atomic layer deposition of Al2O3 film on Si(001) substrate.

In-situ synchrotron radiation photoemission spectroscopy and X-ray photoemission spectroscopy have been used to investigate the initial stages of Al2O3 growth on a Si(001) substrate by atomic layer deposition (ALD). The core level spectra of Si 2p, O 1s, and Al 2p as well as the valence band spectra were measured at every half reaction in the trimethylaluminum (TMA)-H2O ALD process. The line shape changes and binding energy shifts of the core level spectra reveal that Al2O3 is predominantly formed with a small amount of Si oxide in the initial stages without the formation of Al silicate. All core level spectra were alternately shifted toward higher and lower binding energies sides at every half ALD reaction. This can be explained by the band bending effect induced by different chemical species on the surface during the TMA-H2O ALD reaction. The valence band spectra showed that four cycles of ALD reactions were necessary to complete the electronic structure of the Al2O3 film with a valence band offset of 3.73 eV.

Structure-activity relationships of modified C-terminal endomorphin-2 analogues by molecular dynamics simulations.

Motivated by recent experimental works on the modifications of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2) to develop better painkiller, we performed structure-activity-relationship (SAR) studies to investigate modified C-terminal ligands by using molecular dynamics (MD) simulations. Specifically, instead of the CONH2 for the unmodified EM2's C-terminus, the analogue 2 with its C-terminus being CONHNH2 and analogue 3 with its C-terminus being COOMe are studied. First, a systematic conformer search was performed via the quantum chemical AM1 calculations. The cis/trans isomers of the lowest energy were hence selected as MD initial structures. We further showed that EM2s in water exhibited similar dihedral angles to those in DMSO, obtained from the NMR experiment. This similarity indicates the reliability of our MD simulations, and enables us to discuss related bioactivity. Our results showed that the interactions of the Tyr(1)-Phe(3) pair for cis-/trans-EM2s played a considerable role for structural stability. Furthermore, we utilized the chi(1) rotamers of individual aromatic side chains to examine the structural bioactivity. It is shown that this criterion to determine the conformational bioactivity toward mu-opioid receptor (MOR) is insufficient. Thus, we have further employed rotamer-combination approaches to examine the characteristics of SAR for cis-/trans-EM2s. Our results suggested that the bioactive chi(1) rotamers for Tyr(1)-Phe(3) pair remained to favor the [trans-trans] status for MOR selectivity. Therefore, based on the analysis of the chi(1) rotamers, it is suggested that the analogue 2 exhibit greater structural bioactivity for MOR than the analogue 3, and both of them be greater than unmodified EM2 for trans isomers.

Effect of flow regimes on the presence of Legionella within the biofilm of a model plumbing system.

AIMS: Stagnation is widely believed to predispose water systems to colonization by Legionella. A model plumbing system was constructed to determine the effect of flow regimes on the presence of Legionella within microbial biofilms. METHODS AND RESULTS: The plumbing model contained three parallel pipes where turbulent, laminar and stagnant flow regimes were established. Four sets of experiments were carried out with Reynolds number from 10,000 to 40,000 and from 355 to 2,000 in turbulent and laminar pipes, respectively. Legionella counts recovered from biofilm and planktonic water samples of the three sampling pipes were compared with to determine the effect of flow regime on the presence of Legionella. Significantly higher colony counts of Legionella were recovered from the biofilm of the pipe with turbulent flow compared with the pipe with laminar flow. The lowest counts were in the pipe with stagnant flow. CONCLUSIONS: We were unable to demonstrate that stagnant conditions promoted Legionella colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Plumbing modifications to remove areas of stagnation including deadlegs are widely recommended, but these modifications are tedious and expensive to perform. Controlled studies in large buildings are needed to validate this unproved hypothesis.

Determination of tetrodotoxin in human urine and blood using C18 cartridge column, ultrafiltration and LC-MS.

Six fishermen were victims (including one death) of food poisoning from unknown fish on their boat in central Taiwan Strait, in April 2001. The symptoms were like those of tetrodotoxin (TTX) poisoning. As there was no remaining fish, a new protocol was developed to determine TTX in the urine and blood of the victims. The urine and blood samples were cleansed using a C18 Sep-Pak cartridge column, and the toxin was extracted by methanol. The eluate was filtered through a microcentrifuge filter. The filtrate was freeze-dried, dissolved in distilled water, and determined by LC-MS. The recovery was more than 88.9%. The detection limit was 15.6 nM. A linear relationship between response and concentration was obtained between 93.75 and 9375 nM of TTX. It was shown that the urine and blood of the victims contained TTX. The range of TTX was 4.5-40.6 nM in blood and 47-344 nM in urine. Judging from the symptoms of the victims and the experimental data, the causative agent of the food poisoning was identified as TTX.

Low-energy electronic properties of the AB-stacked few-layer graphites.

In the presence of a perpendicular electric field, the low-energy electronic properties of the AB-stacked N-layer graphites with layer number N = 2, 3, and 4, respectively, are examined through the tight-binding model. The interlayer interactions, the number of layers, and the field strength are closely related to them. The interlayer interactions can significantly change the energy dispersions and produce new band-edge states. Bi-layer and four-layer graphites are two-dimensional semimetals due to a tiny overlap between the valence and conduction bands, while tri-layer graphite is a narrow-gap semiconductor. The electric field affects the low-energy electronic properties: the production of oscillating bands, the cause of subband (anti)crossing, the change in subband spacing, and the increase in band-edge states. Most importantly, the aforementioned effects are revealed completely in the density of states, e.g. the generation of special structures, the shift in peak position, the change in peak height, and the alteration of the band gap.

Differences in antimicrobial susceptibility among hospitals in an integrated health system.

We evaluated the differences in antimicrobial susceptibility among hospitals in three different integrated healthcare systems. Each system provided antibiogram-susceptibility reports from representative hospitals. Reports were analyzed for statistically significant differences between hospitals in a given system for nine important organisms. We found numerous significant interhospital differences in antimicrobial-susceptibility patterns within health systems. For this reason, the practice of combining antibiotic-susceptibility data into a systemwide antibiogram should be discouraged.

Chromatographic resolution of the enantiomers of phenylpropanolamine by using molecularly imprinted polymer as the stationary phase.

In this study molecular imprinting technology was employed to prepare a specific affinity sorbent for the resolution of phenylpropanolamine, a chiral drug. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting with either (-)- or (+)-phenylpropanolamine as the template. Methacrylic acid and ethylene glycol dimethacrylate were copolymerized in the presence of the template molecule. The bulk polymerization was carried out in chloroform with 2,2'-azobisisobutyronitrile as the initiator, at 4 degrees C and under UV radiation. The resulting MIP was ground into powders, which were slurry packed into analytical columns. After removal of template molecules, the MIP-packed columns were found to be effective for the resolution of (+/-)-phenylpropanolamine racemates. The separation factor for the enantiomers ranged between 1.8 and 3.8 when the column was packed with MIP prepared with (+)-phenylpropanolamine as the template. A separation factor ranging from 2.1 to 3.6 could be achieved from the column packed with MIP, prepared with (-)-phenylpropanolamine as the template. Although the separation factor was higher with that previously obtained from reversed-phase column chromatography following derivatization with a chiral agent, elution peaks were broader due to the heterogeneity of binding sites on MIP particles and the possible non-specific interaction.

Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase.

4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase.

Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis.

The NAD-malic enzyme cDNA has been subcloned into the pQE expression vector, expressed with a six-His tag, and purified. The His-tagged enzyme is purified by a combination of Ni-NTA and orange A agarose column chromatography with a yield of 45% and an estimated purity of >90%. The tag and linker have no effect on the kinetic parameters of the enzyme compared to the wild-type enzyme. Alanine-scanning site-directed mutagenesis has been carried out on all of the conserved neutral acid residues of the NAD-malic enzyme from Ascaris suum. Data obtained confirm the predicted role of D178 and D295 in metal ion binding, the likely role of D294, D361, and E440 in the NAD binding site, and the role of E58 and D272 in malate binding. Decreases in V/E(t) by 10(4)-fold and in V/K(malate)E(t) by 10(7)-fold, when D295 is changed to alanine, suggest that it is a likely candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme.

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].

Molecular dynamics of a liquid drop spreading in a corner formed by two planar substrates.

Molecular-dynamics simulations were used to investigate the spreading of nonvolatile liquid drops in a solid corner formed by two planar substrates. To understand the effect of the corner on the spreading, liquid drops in a corner with angles of 45 degrees, 90 degrees, and 135 degrees as well as on a flat substrate were examined. Both the solid substrate and the liquid drop were modeled using the Lennard-Jones interaction potential in the present study. Simulation results show that the mass center of the liquid molecules migrated towards the corner as time evolved and the spreading rate increased as the corner angle decreased. It is found that the variation of the mean spreading area with time can be described by a general relation of A(t) approximately t, which is in agreement with results obtained by other investigators. The distribution of liquid atoms per unit normalized corner degree shows a similar trend for different corner angles.

Purpura fulminans in children: report of two cases.

Purpura fulminans, usually seen in previously healthy children acquiring severe infections, especially meningococcal sepsis and meningitis, is a rare catastrophic disease with initial hemorrhagic skin lesions rapidly progressing to gangrene accompanied by shock and frequently resulting in death. We report 2 cases of purpura fulminans who were diagnosed in the past 2 years. Both blood cultures yielded Neisseria meningitidis. One of them expired within 48 hours after admission despite aggressive therapy. The second patient, who received the treatment of heparin, antibiotics, and blood product replacement, survived with minimal sequelae. We deem young age and severe coagulopathy are especially associated with a fatal outcome in children with purpura fulminans. Early recognition of this disease and prompt appropriate therapy may be lifesaving for these patients.

Multiple isotope effects as a probe of proton and hydride transfer in the 6-phosphogluconate dehydrogenase reaction.

Primary solvent deuterium, primary substrate deuterium, multiple solvent deuterium/substrate deuterium, and multiple solvent deuterium/13C isotope effects on V/K6PG have been measured for the Candida utilis and sheep liver 6-phosphogluconate dehydrogenases (6PGDH). Proton inventory data suggest the presence of a significant medium effect in a step preceding hydride transfer and the presence of a kinetic solvent deuterium isotope effect on hydride transfer. Multiple isotope effect data confirm the presence of multiple solvent deuterium sensitive steps, likely including a conformational change preceding hydride transfer, hydride transfer, and decarboxylation.

Mitochondrial myopathy with predominant respiratory dysfunction in a patient with A3243G mutation in the mitochondrial tRNA(Leu(UUR))gene.

We report a patient with the A3243G point mutation of mitochondrial DNA (mtDNA) who presented with severe impairment of respiratory function and only mild involvement of limb muscles. This 55-year-old woman had a history of repeated episodes of respiratory failure unexplained by lung disease or central nervous system lesions. Needle electromyography suggested myopathy and muscle biopsy showed many ragged-red fibers. Molecular analysis of mtDNA in blood and muscle cells showed an A3243G point mutation in the tRNA(Leu(UUR))gene; the percentages of mutant mtDNA in blood and muscle cells were 65% and 71%, respectively. These findings suggest that mitochondrial myopathy should be considered as a cause of respiratory failure due to neuromuscular disorders, and that pure myopathy with predominant respiratory dysfunction is one of the heterogeneous phenotypic features associated with the A3243G point mutation of mtDNA.