Effect of Cell Age and Membrane Rigidity on Red Blood Cell Shape in Capillary Flow

Blood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity

Effect of Cell Age and Membrane Rigidity on Red Blood Cell Shape in Capillary Flow

peer reviewedBlood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing. © 2020 The American Society of Gene and Cell TherapySuzuki et al. report correction of the CFTR defect in cystic fibrosis airway basal stem cells. They utilized gene-editing strategies either specific for the ΔF508 CFTR mutation or applicable to most CFTR mutations. Both approaches yielded highly efficient correction without selection, restoring CFTR function to therapeutically relevant levels

Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c.

Acknowledgements: We thank GlaxoSmithKline for gifting inhibitor compounds for use in this study. This work was supported by funding from the Medical Research Council (MC_U120027516, MC_UP_1605/12 and MC_UP_1605/11) as well as awards from the Wellcome Trust (099276/Z/12/Z to M.M. and ISSF PS3125_WCMA to M.V.P.). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing

Endogenous bioluminescent reporters reveal a sustained increase in utrophin gene expression upon EZH2 and ERK1/2 inhibition.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD

Occult Pathologic Findings in Reduction Mammaplasty in 5781 Patients—An International Multicenter Study

Breast cancer is among the most commonly diagnosed cancers in the world, affecting one in eight women in their lifetimes. The disease places a substantial burden on healthcare systems in developed countries and often requires surgical correction. In spite of this, much of the breast cancer pathophysiology remains unknown, allowing for the cancer to develop to later stages prior to detection. Many women undergo reduction mammaplasties (RM) to adjust breast size, with over 500,000 operations being performed annually. Tissue samples from such procedures have drawn interest recently, with studies attempting to garner a better understanding of breast cancer's development. A number of samples have revealed nascent cancer developments that were previously undetected and unexpected. Investigating these so-called "occult" findings of cancer in otherwise healthy patients may provide further insight regarding risk factors and countermeasures. Here, we detail occult findings of cancer in reduction mammaplasty samples provided from a cohort of over 5000 patients from 16 different institutions in Europe. Although the majority of our resected breast tissue specimens were benign, our findings indicate that there is a continued need for histopathological examination. As a result, our study suggests that preoperative imaging should be routinely performed in patients scheduled for RM, especially those with risk factors of breast cancer, to identify and enable a primary oncologic approach