Characterization of Patient-Derived GNAQ Mutated Endothelial Cells from Capillary Malformations.

Capillary malformations (CM) (port-wine stains) are congenital skin lesions that are characterized by dilated capillaries and postcapillary venules. CMs are caused by altered functioning of the vascular endothelium. Somatic genetic mutations have predominantly been identified in the endothelial cells of CMs, providing an opportunity for the development of targeted therapies. However, there is currently limited in-depth mechanistic insight into the pathophysiology and a lack of preclinical research approaches. In a monocenter exploratory study of 17 adult patients with CMs, we found somatic sequence variants in the GNAQ (p.R183Q, p.R183G, or p.Q209R) or GNA11 (p.R183C) genes. We applied an endothelial-selective cell isolation protocol to culture primary endothelial cells from skin biopsies from these patients. We successfully expanded patient-derived cells in culture in 3 of the 17 cases while maintaining endothelial specificity as demonstrated by vascular endothelial-cadherin immunostainings. In addition, we tested the angiogenic capacity of endothelial cells from a patient with a GNAQ (p.R183G) sequence substitution. These proof-of-principle results reveal that primary cells isolated from CMs may represent a functional research model to investigate the role of endothelial somatic mutations in the etiology of CMs, but improved isolation and culture methodologies are urgently needed to advance the field.

Early adipogenesis is repressed through the newly identified FHL2-NFAT5 signaling complex.

The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells towards osteoblast and myocyte phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation. For these studies pre-adipocytes isolated from mouse adipose tissue and the 3T3-L1 (pre)adipocyte cell line were applied. We performed FHL2 gain of function and knockdown experiments followed by extensive RNAseq analyses and phenotypic characterization of the cells by oil-red O (ORO) lipid staining. Through affinity-purification mass spectrometry (AP-MS) novel FHL2 interacting proteins were identified. Here we report that FHL2 is expressed in pre-adipocytes and for accurate adipocyte differentiation, this protein needs to be downregulated during the early stages of adipogenesis. More specifically, constitutive overexpression of FHL2 drastically inhibits adipocyte differentiation in 3T3-L1 cells, which was demonstrated by suppressed activation of the adipogenic gene expression program as shown by RNAseq analyses, and diminished lipid accumulation. Analysis of the protein-protein interactions mediating this repressive activity of FHL2 on adipogenesis revealed the interaction of FHL2 with the Nuclear factor of activated T-cells 5 (NFAT5). NFAT5 is an established inhibitor of adipocyte differentiation and its knockdown rescued the inhibitory effect of FHL2 overexpression on 3T3-L1 differentiation, indicating that these proteins act cooperatively. We present a new regulatory function of FHL2 in early adipocyte differentiation and revealed that FHL2-mediated inhibition of pre-adipocyte differentiation is dependent on its interaction with NFAT5. FHL2 expression increases with aging, which may affect mesenchymal stem cell differentiation, more specifically inhibit adipocyte differentiation.

Posterior Tibial Nerve Stimulation in Children with Lower Urinary Tract Dysfunction: A Mixed-Methods Analysis of Experiences, Quality of Life and Treatment Effect.

BACKGROUND: Posterior tibial nerve stimulation (PTNS) is one of the treatment modalities for children with therapy-refractory lower urinary tract dysfunction (LUTD). This study used a mixed-methods analysis to gain insight into the experiences of children treated with PTNS and their parents, the effect of treatment on quality of life (QOL) and the effect of PTNS on urinary symptoms. METHODS: Quantitative outcomes were assessed through a single-centre retrospective chart analysis of all children treated with PTNS in a group setting between 2016-2021. Voiding parameters and QOL scores before and after treatment were compared. Qualitative outcomes were assessed by an explorative study involving semi-structured interviews transcribed verbatim and inductively analysed using the constant-comparative method. RESULTS: The data of 101 children treated with PTNS were analysed. Overall improvement of LUTD was seen in 42% and complete resolution in 10%. Average and maximum voided volumes significantly increased. QOL improved in both parents and children independent of the actual effect on urinary symptoms. Interviews revealed PTNS to be well-tolerated. Facilitating PTNS in a group setting led to feelings of recognition in both children and parents. CONCLUSIONS: PTNS is a good treatment in children with therapy-refractory LUTD and provides valuable opportunities for peer support if given in a group setting.

A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis.

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.

Creation of Conductive Graphene Materials by Bacterial Reduction Using Shewanella Oneidensis.

Graphene's maximized surface-to-volume ratio, high conductance, mechanical strength, and flexibility make it a promising nanomaterial. However, large-scale graphene production is typically cost-intensive. This manuscript describes a microbial reduction approach for producing graphene that utilizes the bacterium Shewanella oneidensis in combination with modern nanotechnology to enable a low-cost, large-scale production method. The bacterial reduction approach presented in this paper increases the conductance of single graphene oxide flakes as well as bulk graphene oxide sheets by 2.1 to 2.7 orders of magnitude respectively while simultaneously retaining a high surface-area-to-thickness ratio. Shewanella-mediated reduction was employed in conjunction with electron-beam lithography to reduce one surface of individual graphene oxide flakes. This methodology yielded conducting flakes with differing functionalization on the top and bottom faces. Therefore, microbial reduction of graphene oxide enables the development and up-scaling of new types of graphene-based materials and devices with a variety of applications including nano-composites, conductive inks, and biosensors, while avoiding usage of hazardous, environmentally-unfriendly chemicals.

Mechanically controlled quantum interference in individual π-stacked dimers.

Recent observations of destructive quantum interference in single-molecule junctions confirm the role of quantum effects in the electronic conductance properties of molecular systems. These effects are central to a broad range of chemical and biological processes and may be beneficial for the design of single-molecule electronic components to exploit the intrinsic quantum effects that occur at the molecular scale. Here we show that destructive interference can be turned on or off within the same molecular system by mechanically controlling its conformation. Using a combination of ab initio calculations and single-molecule conductance measurements, we demonstrate the existence of a quasiperiodic destructive quantum-interference pattern along the breaking traces of π-stacked molecular dimers. The results demonstrate that it is possible to control the molecular conductance over more than one order of magnitude and with a sub-ångström resolution by exploiting the subtle structure-property relationship of π-stacked dimers.