Cellogram: On-the-Fly Traction Force Microscopy.

Traction force microscopy (TFM) derives maps of cell-generated forces, typically in the nanonewton range, transmitted to the extracellular environment upon actuation of complex biological processes. In traditional approaches, force rendering requires a terminal, time-consuming step of cell deadhesion to obtain a reference image. A conceptually opposite approach is provided by reference-free methods, opening to the on-the-fly generation of force maps from an ongoing experiment. This requires an image processing algorithm keeping the pace of the biological phenomena under investigation. Here, we introduce an integrated software pipeline rendering force maps from single reference-free TFM images seconds to minutes after their acquisition. The algorithm tackles image processing, reference image estimation, and finite element analysis as a single problem, yielding a robust and fully automatic solution. The method's capabilities are demonstrated in two applications. First, the mechanical annihilation of cancer cells is monitored as a function of rising environmental temperature, setting a population threshold at 45 °C. Second, the fast temporal correlation of forces produced across individual cells is used to map physically connected adhesion points, yielding typical lengths that vary as a function of the cell cycle phase.

Superhydrophobicity enhancement through substrate flexibility.

Inspired by manifestations in nature, microengineering and nanoengineering of synthetic materials to achieve superhydrophobicity has been the focus of much work. Generally, hydrophobicity is enhanced through the combined effects of surface texturing and chemistry; being durable, rigid materials are the norm. However, many natural and technical surfaces are flexible, and the resulting effect on hydrophobicity has been largely ignored. Here, we show that the rational tuning of flexibility can work synergistically with the surface microtexture or nanotexture to enhance liquid repellency performance, characterized by impalement and breakup resistance, contact time reduction, and restitution coefficient increase. Reduction in substrate areal density and stiffness imparts immediate acceleration and intrinsic responsiveness to impacting droplets (∼350 × g), mitigating the collision and lowering the impalement probability by ∼60% without the need for active actuation. Furthermore, we exemplify the above discoveries with materials ranging from man-made (thin steel or polymer sheets) to nature-made (butterfly wings).

Potential of Bryophyllum pinnatum as a Detrusor Relaxant: An in Vitro Exploratory Study.

An earlier prospective, randomised, placebo-controlled clinical trial had suggested that Bryophyllum pinnatum might have potential in the treatment of overactive bladder. Here we investigated the effects of B. pinnatum leaf press juice, fractions enriched in flavonoids and bufadienolides, and a flavonoid aglycon mixture and individual aglycons on detrusor contractility as a major target in overactive bladder treatment. The strength of the detrusor contractions was investigated using porcine muscle strips stimulated with KCl. B. pinnatum leaf press juice increased the contraction force of muscle strips. Treatment with the flavonoid-enriched fraction had almost no effect on contractility, while the bufadienolide-enriched fraction and flavonoid aglycons led to a concentration-dependent lowering of the contraction force. The data indicate that several components of B. pinnatum leaf press juice may contribute to the inhibitory effect on detrusor contractility, which in turn provides support to overactive bladder treatment with B. pinnatum.

Quantification of Bufadienolides in Bryophyllum pinnatum Leaves and Manufactured Products by UHPLC-ESIMS/MS.

A quantitative assay for determination of the main bufadienolides bersaldegenin-1-acetate (1), bersaldegenin-3-acetate (2), bryophyllin A (3), and bersaldegenin-1,3,5-orthoacetate (4) in Bryophyllum pinnatum leaves and manufactured products was developed and validated. The assay involved extraction by pressurised liquid extraction, followed by quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy. The ultrahigh performance liquid chromatography-tandem mass spectroscopy method was applied to various batches of leaves harvested on several dates from plants grown at two locations (Brazil and Germany). In addition, press juices prepared from plants cultivated in Germany and Brazil were analysed. The total bufadienolide content ranged from 16.28 to 40.50 mg/100 g dry weight in leaves from plants grown in Brazil. The total content of these four bufadienolides was significantly lower in plants cultivated in Germany (3.78-12.49 mg/100 g dry weight, resp.). The total amounts of bufadienolides were 0.091-0.163 mg/100 mL and 0.89-1.16 mg/100 mL in press juices obtained from plants cultivated in Germany and Brazil, respectively. When analysing single leaves from individual plants, the content of bufadienolides was markedly higher in young leaves. For comparative purposes, the content of these bufadienolides was also determined in Bryophyllum daigremontianum and Bryophyllum tubiflorum. Bersaldegenin-1,3,5-orthoacetate (4) was predominant in the leaves of B. daigremontianum and in the stems of B. tubiflorum, while the leaves of B. tubiflorum contained very low amounts of 1-4.

Microscale investigation on interfacial slippage and detachment of ice from soft materials.

Surface icing is detrimental to applications ranging from transportation to biological systems. Soft elastomeric coatings can engender remarkably low ice adhesion strength, but mechanisms at the microscale and resulting ice extraction outcomes need to be understood. Here we investigate dynamic ice-elastomer interfacial events and show that the ice adhesion strength can actually vary by orders of magnitude due to the shear velocity. We study the detailed deformation fields of the elastomer using confocal traction force microscopy and elucidate the underlying mechanism. The elastomer initially undergoes elastic deformation having a shear velocity dependent threshold, followed by partial relaxation at the onset of slip, where velocity dependent "stick-slip" micropulsations are observed. The results of the work provide important information for the design of soft surfaces with respect to removal of ice, and utility to fields exemplified by adhesion, contact mechanics, and biofouling.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.

Wetting transitions in droplet drying on soft materials.

Droplet interactions with compliant materials are familiar, but surprisingly complex processes of importance to the manufacturing, chemical, and garment industries. Despite progress-previous research indicates that mesoscopic substrate deformations can enhance droplet drying or slow down spreading dynamics-our understanding of how the intertwined effects of transient wetting phenomena and substrate deformation affect drying remains incomplete. Here we show that above a critical receding contact line speed during drying, a previously not observed wetting transition occurs. We employ 4D confocal reference-free traction force microscopy (cTFM) to quantify the transient displacement and stress fields with the needed resolution, revealing high and asymmetric local substrate deformations leading to contact line pinning, illustrating a rate-dependent wettability on viscoelastic solids. Our study has significance for understanding the liquid removal mechanism on compliant substrates and for the associated surface design considerations. The developed methodology paves the way to study complex dynamic compliant substrate phenomena.

CBP-1/p300 acetyltransferase regulates SKN-1/Nrf cellular levels, nuclear localization, and activity in C. elegans.

SKN-1/Nrf transcription factors regulate diverse biological processes essentially stress defense, detoxification, and longevity. Studies in model organisms have identified a broad range of regulatory processes and mechanisms that profoundly influence SKN-1/Nrf functions. Defining the mechanisms how SKN-1 is regulated will provide insight how cells defend against diverse stressors contributing to aging and disease. In this study, we demonstrate a crucial role for the acetyltransferase CBP-1, the C. elegans homolog of mammalian CREB-binding protein CBP/p300 in the activation of SKN-1. cbp-1 is essential for tolerance of oxidative stress and normal lifespan. CBP-1 directly interacts with SKN-1 and increases SKN-1 protein abundance. In particular CBP-1 modulates SKN-1 nuclear translocation under basal conditions and in response to stress and promotes SKN-1-dependent transcription of protective genes. Moreover, CBP-1 is required for SKN-1 nuclear recruitment, transcriptional activity, and longevity due to reduced insulin/IGF-1-like signaling, mTOR-, and GSK-3 signaling. Our findings establish the acetyltransferase CBP-1 as a critical activator of SKN-1 that directly modulates SKN-1 protein stability, nuclear localization, and function to ascertain normal stress response and lifespan.

CGEF-1 regulates mTORC1 signaling during adult longevity and stress response in C. elegans.

The mechanistic target of rapamycin (mTOR) kinase is central to metabolism and growth, and has a conserved role in aging. mTOR functions in two complexes, mTORC1 and mTORC2. In diverse eukaryotes, inhibition of mTORC1 signaling increases lifespan. mTORC1 transduces anabolic signals to stimulate protein synthesis and inhibits autophagy. In this study, we demonstrate that CGEF-1, the C. elegans homolog of the human guanine nucleotide exchange factor Dbl, is a novel binding partner of RHEB-1 and activator of mTORC1 signaling in C. elegans. cgef-1 mutants display prolonged lifespan and enhanced stress resistance. The transcription factors DAF-16/FoxO and SKN-1/Nrf are required for increased longevity and stress tolerance, and induce protective gene expression in cgef-1 mutants. Genetic evidence indicates that cgef-1 functions in the same pathway with rheb-1, the mTOR kinase let-363, and daf-15/Raptor. When cgef-1 is inactivated, phosphorylation of 4E-BP, a central mTORC1 substrate for protein translation is reduced in C. elegans. Moreover, autophagy is increased upon cgef-1 and mTORC1 inhibition. In addition, we show that in human cells Dbl associates with Rheb and stimulates mTORC1 downstream targets for protein synthesis suggesting that the function of CGEF-1/Dbl in the mTORC1 signaling pathway is evolutionarily conserved. These findings have important implications for mTOR functions and signaling mechanisms in aging and age-related diseases.