4 research outputs found
Single cell imaging of nuclear architecture changes
No full textThis is the final version. Available from Frontiers Media via the DOI in this record.Data and materials availability: Data obtained in this work are available upon request.The dynamic architecture of chromatin, the macromolecular complex comprised primarily of
DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to
chromatin are important markers of functional and developmental processes in cells. However,
chromatin architecture regulation has not yet been fully elucidated. Therefore, novel approaches
to assessing chromatin changes at the single-cell level are required. Here we report the use of
FTIR imaging and microfluidic cell-stretcher chips to assess changes to chromatin architecture
and its effect on the mechanical properties of the nucleus in immune cells. FTIR imaging enables
label-free chemical imaging with subcellular resolution. By optimizing the FTIR methodology
and couple it with cell segmentation analysis approach, we have identified key spectral changes
corresponding to changes in DNA levels and chromatin conformation at the single cell level. By
further manipulating live single cells using pressure-driven microfluidics, we found that
chromatin decondensation – either during general transcriptional activation or during specific
immune cell maturation – can ultimately lead to nuclear auxeticity which is a new biological
phenomenon recently identified. Taken together our findings demonstrate the tight and,
potentially bilateral, link between extra-cellular mechanotransduction and intra-cellular nuclear
architecture.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology and Biological Sciences Research Council (BBSRC)Academy of Medical SciencesRoyal Societ
Mid-IR hyperspectral imaging for label-free histopathology and cytology
No full textThis is the author accepted manuscript. The final version is available from IOP Publishing via the DOI in this record.Mid-infrared (MIR) imaging has emerged as a valuable tool to investigate biological samples,
such as tissue histological sections and cell cultures, by providing non-destructive chemical
specificity without recourse to labels. While feasibility studies have shown the capabilities of
MIR imaging approaches to address key biological and clinical questions, these techniques are
still far from being deployable by non-expert users. In this review, we discuss the current state of
the art of MIR technologies and give an overview on technical innovations and developments
with the potential to make MIR imaging systems more readily available to a larger community.
The most promising developments over the last few years are discussed here. They include
improvements in MIR light sources with the availability of quantum cascade lasers and
supercontinuum IR sources as well as the recently developed upconversion scheme to improve
the detection of MIR radiation. These technical advances can substantially speed up data
acquisition of multispectral or hyperspectral datasets thus providing the end user with vast
amounts of data when imaging whole tissue areas of many mm2
. Therefore, effective data
analysis is of tremendous importance, and progress in method development is discussed with
respect to the specific biomedical context.Funding within the scope of Horizon 2020 by the European
Union is highly appreciated. This work was conducted as part
of the Mid-TECH Marie Curie innovative training network
[H2020-MSCA-ITN-2014-642661]
Epigenomic modifications mediating antibody maturation
No full textThis is the final version of the article. Available from the publisher via the DOI in this record.Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody-antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer.The authors wish to thank
Matthew Scharff, Shanzhi Wang, Sergio Roa, and Emma Knight
for their insight, and funding for RC from the Biotechnology
and Biological Research Council [BB/N017773/1], Royal Society
[IE150290], and Academy of Medical Sciences Springboard
Award. ECS and RBM are funded by Ph.D. studentships from
the Biotechnology and Biological Research Council-funded
South West Doctoral Training Partnership [BB/J014400/1] and
the Engineering and Physical Sciences Research Council-funded
Doctoral Training Partnership [EP/M506527/1], respectively
Single cell imaging of nuclear architecture changes
No full textThe dynamic architecture of chromatin, the macromolecular complex comprised primarily of
DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to
chromatin are important markers of functional and developmental processes in cells. However,
chromatin architecture regulation has not yet been fully elucidated. Therefore, novel approaches
to assessing chromatin changes at the single-cell level are required. Here we report the use of
FTIR imaging and microfluidic cell-stretcher chips to assess changes to chromatin architecture
and its effect on the mechanical properties of the nucleus in immune cells. FTIR imaging enables
label-free chemical imaging with subcellular resolution. By optimizing the FTIR methodology
and couple it with cell segmentation analysis approach, we have identified key spectral changes
corresponding to changes in DNA levels and chromatin conformation at the single cell level. By
further manipulating live single cells using pressure-driven microfluidics, we found that
chromatin decondensation – either during general transcriptional activation or during specific
immune cell maturation – can ultimately lead to nuclear auxeticity which is a new biological
phenomenon recently identified. Taken together our findings demonstrate the tight and,
potentially bilateral, link between extra-cellular mechanotransduction and intra-cellular nuclear
architecture
