Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range

Background: The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes. Findings: Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d’Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 – 588 nt) of unknown function in the 5’ region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. Conclusions: In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide

Application of circulating cell-free tumor DNA profiles for therapeutic monitoring and outcome prediction in genetically heterogeneous metastatic melanoma

PURPOSE Circulating cell-free tumor DNA (ctDNA) reflects the heterogeneousspectrum of tumor-specific mutations, especially in systemic disease. We validated plasma-based assays that allow the dynamic quantitative detection of ctDNA as a prognostic biomarker for tumor load and prediction of therapy response in melanoma. MATERIALS and METHODS We analyzed plasma-derived ctDNA from a large training cohort (n = 96) of patients with advanced-stage melanoma, with assays for the BRAFV600E and NRASQ61 driver mutations as well as TERTC250T and TERTC228T promoter mutations. An independent patient cohort (n = 35) was used to validate the utility of ctDNA monitoring under mitogen-activated protein kinase–targeted or immune checkpoint therapies. RESULTS Elevated plasma ctDNA level at baseline was an independent prognostic factor of disease progression when compared with serum S100 and lactate dehydrogenase levels in multivariable analyses (hazard ratio [HR], 7.43; 95% CI, 1.01 to 55.19; P = .05). The change in ctDNA levels during therapy correlated with treatment response, where increasing ctDNA was predictive for shorter progression-free survival (eg, for BRAFV600EctDNA, HR, 3.70; 95% CI, 1.86 to 7.34; P < .001). Increasing ctDNA levels predicted disease progression significantly earlier than did routine radiologic scans (P < .05), with a mean lead time of 3.5 months. NRAS-mutant ctDNA was detected in a significant proportion of patients with BRAF-mutant tumors under therapy, but unexpectedly also at baseline. In vitro sensitivity studies suggested that this represents higher-than-expected intratumoral heterogeneity. The detection of NRASQ61 ctDNA in baseline samples of patients with BRAFV600E mutation who were treated with mitogen-activated protein kinase inhibitors significantly correlated with shorter progression-free survival (HR, 3.18; 95% CI, 1.31 to 7.68; P = .03) and shorter overall survival (HR, 4.08; 95% CI, 1.57 to 10.58; P = .01). CONCLUSION Our results show the potential role of ctDNA measurement as a sensitive monitoring and prediction tool for the early assessment of disease progression and therapeutic response in patients with metastaticmelanoma

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors

Printing, characterising, and assessing transparent 3D printed lenses for optical imaging

The production of high-quality lenses for optical instrumentation has involved subtractive manufacturing methods for centuries. These methods have demanded specialist equipment and expertise that often render custom high-grade bulk glass optics inaccessible. We aimed to develop a low-cost, accessible, and reproducible method using consumer-grade technology to manufacture high-quality three-dimensional (3D) printed lenses with comparable performance to glass lenses. Various 3D printed planoconvex lenses were produced using a consumer grade 3D printer and low-cost spin coating setup, and printed lenses were compared to their commercial glass counterparts. A range of mechanical and optical methods are introduced to determine the surface curvature of 3D printed lenses, providing a wide range of quality control methods. Amongst others, high-resolution interference reflection microscopy methods were used to reconstruct the convex surface of printed lenses and quantify their radius of curvature. Moreover, the optical throughput and focusing performance of 3D printed lenses were assessed using optical transmissivity measurements and classical beam characterisation methods. We determined that all 3D printed lens prescriptions tested had comparable surface curvature and optical performance to commercial glass lenses. Finally, we demonstrated the application of 3D printed lenses for brightfield transmission microscopy, resolving sub-cellular structures over a 2.3 mm field of view. The high reproducibility and comparable performance of 3D printed lenses present a great opportunity for additive manufacturing in the production of bespoke optical elements for low-cost rapid prototyping and improved accessibility to high-quality optics in low-resource settings

Printing, characterizing, and assessing transparent 3D printed lenses for optical imaging

High-quality lens production has involved subtractive manufacturing methods for centuries. These methods demand specialist equipment and expertise that often render custom high-grade glass optics inaccessible. A low-cost, accessible, and reproducible method is developed to manufacture high-quality three dimensional (3D) printed lenses using consumer-grade technology. Various planoconvex lenses are produced using a consumer-grade 3D printer and low-cost spin coating setup, and printed lenses are compared to commercial glass counterparts. A range of mechanical and optical methods are introduced to determine the surface quality and curvature of 3D printed lenses. Amongst others, high-resolution interference reflection microscopy methods are used to reconstruct the convex surface of printed lenses and quantify their radius of curvature. The optical throughput and performance of 3D printed lenses are assessed using optical transmissivity measurements and classical beam characterization methods. It is determined that 3D printed lenses have comparable curvature and performance to commercial glass lenses. Finally, the application of 3D printed lenses is demonstrated for brightfield transmission microscopy, resolving sub-cellular structures over a 2.3 mm field-of-view. The high reproducibility and comparable performance of 3D printed lenses present great opportunities for additive manufacturing of bespoke optics for low-cost rapid prototyping and improved accessibility to high-quality optics in low-resource settings

The tumour-suppressive function of CLU is explained by its localisation and interaction with HSP60

The product of the CLU gene promotes or inhibits tumourigenesis in a context-dependent manner. It has been hypothesised that different CLU isoforms have different and even opposing biological functions, but this theory has not been experimentally validated. Here we show that molecules involved in survival pathways are differentially modulated by the intracellular or secreted forms of CLU. Secreted CLU, which is selectively increased after transformation, activates the survival factor AKT, whereas intracellular CLU inhibits the activity of the oncogenic transcription factor nuclear factor kappa B. Furthermore, intracellular CLU is inactivated by the pro-proliferative and pro-survival activity of the chaperone protein HSP60 in neuroblastoma cells by forming a physical complex. Thus, localisation is key for CLU physiology, explaining the wide range of effects in cell survival and transformation

Emergence potential of sylvatic dengue virus type 4 in the urban transmission cycle is restrained by vaccination and homotypic immunity

Sylvatic dengue viruses (DENV) are both evolutionarily and ecologically distinct from human DENV and are maintained in an enzootic transmission cycle. Evidence of sylvatic human infections from West Africa and Southeast Asia suggests that sylvatic DENV come into regular contact with humans. Thus, this potential of emergence into the human transmission cycle could limit the potential for eradicating this cycle with vaccines currently in late stages of development. We assessed the likelihood of sylvatic DENV-4 emergence in the face of natural immunity to current human strains and vaccination with two DENV-4 vaccine candidates. Our data indicate homotypic neutralization of sylvatic and human DENV-4 strains by human primary convalescent and vaccinee sera but limited heterotypic immunity. These results suggest that emergence of sylvatic strains into the human cycle would be limited by homotypic immunity mediated by virus neutralizing antibodies produced by natural infection or vaccination

Emergence potential of sylvatic dengue virus type 4 in the urban transmission cycle is restrained by vaccination and homotypic immunity

Sylvatic dengue viruses (DENV) are both evolutionarily and ecologically distinct from human DENV and are maintained in an enzootic transmission cycle. Evidence of sylvatic human infections from West Africa and Southeast Asia suggests that sylvatic DENV come into regular contact with humans. Thus, this potential of emergence into the human transmission cycle could limit the potential for eradicating this cycle with vaccines currently in late stages of development. We assessed the likelihood of sylvatic DENV-4 emergence in the face of natural immunity to current human strains and vaccination with two DENV-4 vaccine candidates. Our data indicate homotypic neutralization of sylvatic and human DENV-4 strains by human primary convalescent and vaccinee sera but limited heterotypic immunity. These results suggest that emergence of sylvatic strains into the human cycle would be limited by homotypic immunity mediated by virus neutralizing antibodies produced by natural infection or vaccination

Gene expression profiling in sinonasal adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers.</p> <p>Methods</p> <p>To identify genes involved in this disease, we performed gene expression profiling using cancer-dedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors.</p> <p>Results</p> <p>Among the genes with significant differential expression we selected <it>LGALS4, ACS5, CLU, SRI and CCT5 </it>for further exploration. The overexpression of <it>LGALS4, ACS5, SRI</it>, <it>CCT5 </it>and the downregulation of <it>CLU </it>were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4), ACS5 (Acyl-CoA synthetase) and CLU (Clusterin) proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type.</p> <p>Conclusion</p> <p>Within our microarray study in sinonasal adenocarcinoma we identified two proteins, LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers.</p

Effects of clusterin over-expression on metastatic progression and therapy in breast cancer

<p>Abstract</p> <p>Background</p> <p>Clusterin is a secreted glycoprotein that is upregulated in a variety of cell lines in response to stress, and enhances cell survival. A second nuclear isoform of clusterin that is associated with cell death has also been identified. The aim of this study was to determine the role(s) of the secretory isoform in breast tumor progression and metastasis.</p> <p>Methods</p> <p>To investigate the role of secretory clusterin in the biology of breast cancer tumor growth and resistance to therapy we have engineered an MCF-7 cell line (MCF-7CLU) that over-expresses clusterin. We have measured the <it>in vitro </it>effects of clusterin over-expression on cell cycle, cell death, and sensitivity to TNFalpha and tamoxifen. Using an orthotopic model of breast cancer, we have also determined the effects of over-expression of clusterin on tumor growth and metastatic progression.</p> <p>Results</p> <p>In vitro, over-expression of secretory clusterin alters the cell cycle kinetics and decreases the rate of cell death, resulting in the enhancement of cell growth. Over-expression of secretory clusterin also blocks the TNFalpha-mediated induction of p21 and abrogates the cleavage of Bax to t-Bax, rendering the MCF-7CLU cells significantly more resistant to the cytokine than the parental cells. Orthotopic primary tumors derived from MCF-7CLU cells grow significantly more rapidly than tumors derived from parental MCF-7 cells and, unlike the parental cells, metastasize frequently to the lungs.</p> <p>Conclusions</p> <p>These data suggest that secretory clusterin, which is frequently up-regulated in breast cancers by common therapies, including anti-estrogens, may play a significant role in tumor growth, metastatic progression and subsequent drug resistance in surviving cells.</p