calibration-free-vitro-quantification-protein-homo-oligomerization

Calibration-free <em>In Vitro</em> Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

University of Oxford

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State.

In spite of the utility of unit tests, most R package developers do not write them.  makes it easier to  writing unit tests by creating shells/skeletons of unit tests based on the examples in the user's package documentation. When completed, these unit tests test whether said examples run  . By combining the functionality of  with that of , having ensured that their examples adequately demonstrate the features of their package, the developer can have much of the work of constructing a comprehensive set of unit tests done for them.

-An easy start to unit testing R packages.

HIV-1 disseminates to diverse tissues through different cell types and establishes long-lived reservoirs. The exact cellular compartment where fusion occurs differs depending on the cell type and mode of viral transmission. This implies that HIV-1 may modulate a number of common host cell factors in different cell types. In this review, we evaluate recent advances on the host cell factors that play an important role in HIV-1 entry and fusion. New insights from restriction factors inhibiting virus-cell fusion in vitro may contribute to the development of future therapeutic interventions. Collectively, novel findings underline the need for potent, host-directed therapies that disrupt the earliest stages of the virus life cycle and preclude the emergence of resistant viral variants.

On the Whereabouts of HIV-1 Cellular Entry and Its Fusion Ports.

An R package for performing number and brightness image analysis, with the implementation of a novel automatic detrending algorithm.

nandb-number and brightness in R with a novel automatic detrending algorithm.

The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.

Rory Nolan

Cellular Imaging Group,
Wellcome Centre Human Genetics,
Cellular Imaging Group, Wellcome Centre Human Genetics

University of Oxford

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Rory Nolan

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University of Oxford

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Cellular Imaging Group,
Wellcome Centre Human Genetics,
Cellular Imaging Group, Wellcome Centre Human Genetics