Construction of an instant structured illumination microscope

A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The “instant structured illumination microscope” (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 μm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x-y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation

Ultracool White Dwarfs and the Age of the Galactic Disc

We present parallax observations and a detailed model atmosphere analysis of 54 cool and ultracool (Teff \u3c 4000 K) white dwarfs (WDs) in the solar neighbourhood. For the first time, a large number of cool and ultracool WDs have distance and tangential velocities measurements available. Our targets have distances ranging from 21 pc to \u3e100 pc, and include five stars within 30 pc. Contrary to expectations, all but two of them have tangential velocities smaller than 150 km s−1 thus suggesting Galactic disc membership. The oldest WDs in this sample have WD cooling ages of 10 Gyr, providing a firm lower limit to the age of the thick disc population. Many of our targets have uncharacteristically large radii, indicating that they are low mass WDs. It appears that we have detected the brighter population of cool and ultracool WDs near the Sun. The fainter population of ultracool CO-core WDs remain to be discovered in large numbers. The Large Synoptic Survey Telescope should find these elusive, more massive ultracool WDs in the solar neighbourhood

Persistent Replication of a Chikungunya Virus Replicon in Human Cells is Associated with Presence of Stable Cytoplasmic Granules Containing Non-structural Protein 3

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2); (ii) were next to double-stranded RNA foci and nsP1-positive structures; and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at varying speeds. Granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the non-cytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).ImportanceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of non-cytotoxic replicons (Utt, Das, Varjak, Lulla, Lulla, Merits, J Virol 89:3145-62, 2015, doi:10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged non-structural protein 3. The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms

Visualisation and analysis of hepatitis C virus non-structural proteins using super-resolution microscopy

Hepatitis C virus (HCV) RNA replication occurs in the cytosol of infected cells within a specialised membranous compartment. How the viral non-structural (NS) proteins are associated and organised within these structures remains poorly defined. We employed a super-resolution microscopy approach to visualise NS3 and NS5A in HCV infected cells. Using single molecule localisation microscopy, both NS proteins were resolved as clusters of localisations smaller than the diffraction-limited volume observed by wide-field. Analysis of the protein clusters identified a significant difference in size between the NS proteins. We also observed a reduction in NS5A cluster size following inhibition of RNA replication using daclatasvir, a phenotype which was maintained in the presence of the Y93H resistance associated substitution and not observed for NS3 clusters. These results provide insight into the NS protein organisation within hepatitis C virus RNA replication complexes and the mode of action of NS5A inhibitors

Rapidly finding CAD features using database optimization

Automatic feature recognition aids downstream processes such as engineering analysis and manufacturing planning. Not all features can be defined in advance; a declarative approach allows engineers to specify new features without having to design algorithms to find them. Naive translation of declarations leads to executable algorithms with high time complexity. Database queries are also expressed declaratively; there is a large literature on optimizing query plans for efficient execution of database queries. Our earlier work investigated applying such technology to feature recognition, using a testbed interfacing a database system (SQLite) to a CAD modeler (CADfix). Feature declarations were translated into SQL queries which are then executed. The current paper extends this approach, using the PostgreSQL database, and provides several new insights: (i) query optimization works quite differently in these two databases, (ii) with care, an approach to query translation can be devised that works well for both databases, and (iii) when finding various simple common features, linear time performance can be achieved with respect to model size, with acceptable times for real industrial models. Further results also show how (i) lazy evaluation can be used to reduce the work performed by the CAD modeler, and (ii) estimating the time taken to compute various geometric operations can further improve the query plan. Experimental results are presented to validate our main conclusions

Antimalarial activity of cupredoxins: the interaction of Plasmodium Merozoite Surface Protein 119 (MSP119) and Rusticyanin

Background: The interaction of MSP119 with the cupredoxin azurin inhibits the growth of Plasmodium falciparum in red blood cells. Results: Rusticyanin forms a well-defined complex with MSP119 upon binding at the same surface area than inhibitory antibodies. Conclusion: Rusticyanin becomes an excellent therapeutic agent for malaria. Significance: Knowing the rusticyanin- MSP119 interface will allow the design of novel anti-malarial drugsJunta de Andalucía P08-CVI-3876, BIO198Ministerio de Economía y Competitividad SAF2011- 26611Fundación Séneca de la Región de Murcia 15354/PI/10Ministerio de Ciencia e Innovación BFU2010-19451Medical Research Council U117574558, U11753206