Rapid Visualisation of Microarray Copy Number Data for the Detection of Structural Variations Linked to a Disease Phenotype

Whilst the majority of inherited diseases have been found to be caused by single base substitutions, small insertions or deletions (<1Kb), a significant proportion of genetic variability is due to copy number variation (CNV). The possible role of CNV in monogenic and complex diseases has recently attracted considerable interest. However, until the development of whole genome, oligonucleotide micro-arrays, designed specifically to detect the presence of copy number variation, it was not easy to screen an individual for the presence of unknown deletions or duplications with sizes below the level of sensitivity of optical microscopy (3–5 Mb). Now that currently available oligonucleotide micro-arrays have in excess of a million probes, the problem of copy number analysis has moved from one of data production to that of data analysis. We have developed CNViewer, to identify copy number variation that co-segregates with a disease phenotype in small nuclear families, from genome-wide oligonucleotide micro-array data. This freely available program should constitute a useful addition to the diagnostic armamentarium of clinical geneticists

Unsaturated phosphatidylcholines lining on the surface of cartilage and its possible physiological roles

Background Evidence has strongly indicated that surface-active phospholipid (SAPL), or surfactant, lines the surface of cartilage and serves as a lubricating agent. Previous clinical study showed that a saturated phosphatidylcholine (SPC), dipalmitoyl-phosphatidylcholine (DPPC), was effective in the treatment of osteoarthritis, however recent studies suggested that the dominant SAPL species at some sites outside the lung are not SPC, rather, are unsaturated phosphatidylcholine (USPC). Some of these USPC have been proven to be good boundary lubricants by our previous study, implicating their possible important physiological roles in joint if their existence can be confirmed. So far, no study has been conducted to identify the whole molecule species of different phosphatidylcholine (PC) classes on the surface of cartilage. In this study we identified the dominant PC molecule species on the surface of cartilage. We also confirmed that some of these PC species possess a property of semipermeability. Methods HPLC was used to analyse the PC profile of bovine cartilage samples and comparisons of DPPC and USPC were carried out through semipermeability tests. Results It was confirmed that USPC are the dominant SAPL species on the surface of cartilage. In particular, they are Dilinoleoyl-phosphatidylcholine (DLPC), Palmitoyl-linoleoyl-phosphatidylcholine, (PLPC), Palmitoyl-oleoyl-phosphatidylcholine (POPC) and Stearoyl-linoleoyl-phosphatidylcholine (SLPC). The relative content of DPPC (a SPC) was only 8%. Two USPC, PLPC and POPC, were capable of generating osmotic pressure that is equivalent to that by DPPC. Conclusion The results from the current study confirm vigorously that USPC is the endogenous species inside the joint as against DPPC thereby confirming once again that USPC, and not SPC, characterizes the PC species distribution at non-lung sites of the body. USPC not only has better anti-friction and lubrication properties than DPPC, they also possess a level of semipermeability that is equivalent to DPPC. We therefore hypothesize that USPC can constitute a possible addition or alternative to the current commercially available viscosupplementation products for the prevention and treatment of osteoarthritis in the future

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

Bridging the resolution gap: correlative super-resolution imaging

This month’s Under the Lens discusses progress towards bridging the resolution gap in correlative super-resolution light and electron microscopy, and highlights its application for visualizing bacterial infections

SPEKcheck — fluorescence microscopy spectral visualisation and optimisation: a web application, javascript library, and data resource

Advanced fluorescence imaging methods require careful matching of excitation sources, dichroics, emission filters, detectors, and dyes to operate at their best. This complex task is often left to guesswork, preventing optimal dye:filter combinations, particularly for multicolour applications. To overcome this challenge we developed SPEKcheck, a web application to visualise the efficiency of the light path in a fluorescence microscope. The software reports values for the excitation efficiency of a dye, the collection efficiency of the emitted fluorescence, and a "brightness" score, allowing easy comparison between different fluorescent labels. It also displays a spectral plot of various elements in the configuration, enabling users to readily spot potential problems such as low efficiency excitation, emission, or high bleedthrough. It serves as an aid to exploring the performance of different dyes and filter sets

Microscope-AOtools: A generalised adaptive optics implementation

Aberrations arising from sources such as sample heterogeneity and refractive index mismatches are constant problems in biological imaging. These aberrations reduce image quality and the achievable depth of imaging, particularly in super-resolution microscopy techniques. Adaptive optics (AO) technology has been proven to be effective in correcting for these aberrations, thereby improving the image quality. However, it has not been widely adopted by the biological imaging community due, in part, to difficulty in set-up and operation of AO. The methods for doing so are not novel or unknown, but new users often waste time and effort reimplementing existing methods for their specific set-ups, hardware, sample types, etc. Microscope-AOtools offers a robust, easy-to-use implementation of the essential methods for set-up and use of AO elements and techniques. These methods are constructed in a generalised manner that can utilise a range of adaptive optics elements, wavefront sensing techniques and sensorless AO correction methods. Furthermore, the methods are designed to be easily extensible as new techniques arise, leading to a streamlined pipeline for new AO technology and techniques to be adopted by the wider microscopy community

Assessing resolution in super-resolution imaging.

Resolution is a central concept in all imaging fields, and particularly in optical microscopy, but it can be easily misinterpreted. The mathematical definition of optical resolution was codified by Abbe, and practically defined by the Rayleigh Criterion in the late 19th century. The limit of conventional resolution was also achieved in this period, and it was thought that fundamental constraints of physics prevented further increases in resolution. With the recent development of a range of super-resolution techniques, it is necessary to revisit the concept of optical resolution. Fundamental differences in super-resolution modalities mean that resolution is not a directly transferrable metric between techniques. This article considers the issues in resolution raised by these new technologies, and presents approaches for comparing resolution between different super-resolution methods

Super-resolution imaging of remodeled synaptic actin reveals different synergies between NK cell receptors and integrins.

Natural killer (NK) cells secrete lytic granules to directly kill virus-infected or transformed cells and secrete cytokines to communicate with other cells. Three-dimensional super-resolved images of F-actin, lytic granules, and IFN-γ in primary human NK cells stimulated through different activating receptors reveal that both IFN-γ and lytic granules accumulated in domains where the periodicity of the cortical actin mesh at the synapse opened up to be penetrable. Ligation of some activating receptors alone (eg, CD16 or NKG2D) was sufficient to increase the periodicity of the actin mesh, but surprisingly, ligation of others (eg, NKp46 or CD2) was not sufficient to induce cortical actin remodeling unless LFA-1 was coligated. Importantly, influenza virus particles that can be recognized by NK cells similarly did not open the actin mesh but could if LFA-1 was coligated. This leads us to propose that immune cells using germline-encoded receptors to directly recognize foreign proteins can use integrin recognition to differentiate between free pathogens and pathogen-infected cells that will both be present in blood. This distinction would not be required for NK cell receptors, such as NKG2D, which recognize host cell-encoded proteins that can only be found on diseased cells and not pathogens