Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.Massachusetts Institute of Technology. Presidential FellowshipNational Institutes of Health (U.S.). Biotechnology Training FellowshipSingapore-MIT Alliance for Research and TechnologyMassachusetts Institute of Biotechnology Training GrantMassachusetts Institute of Technology (Open-source Funding

Comparative phylogenetic methods and the cultural evolution of medicinal plant use

Human life depends on plant biodiversity and the ways in which plants are used are culturally determined. Whilst anthropologists have used phylogenetic comparative methods (PCMs) to gain an increasingly sophisticated understanding of the evolution of political, religious, social, and material culture, plant use has been almost entirely neglected. Medicinal plants are of special interest because of their role in maintaining people’s health across the world. PCMs in particular, and cultural evolutionary theory in general, provide a framework in which to study the diversity of medicinal plant applications cross-culturally, and to infer changes in plant use through time. These methods can be applied to single medicinal plants as well as the entire set of plants used by a culture for medicine, and they account for the non-independence of data when testing for floristic, cultural or other drivers of plant use. With cultural, biological, and linguistic diversity under threat, gaining a deeper and broader understanding of the variation of medicinal plant use through time and space is pressing

Neuronal circuitry for pain processing in the dorsal horn

Neurons in the spinal dorsal horn process sensory information, which is then transmitted to several brain regions, including those responsible for pain perception. The dorsal horn provides numerous potential targets for the development of novel analgesics and is thought to undergo changes that contribute to the exaggerated pain felt after nerve injury and inflammation. Despite its obvious importance, we still know little about the neuronal circuits that process sensory information, mainly because of the heterogeneity of the various neuronal components that make up these circuits. Recent studies have begun to shed light on the neuronal organization and circuitry of this complex region

Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Tactual perception: a review of experimental variables and procedures

This paper reviews literature on tactual perception. Throughout this review we will highlight some of the most relevant variables in touch literature: interaction between touch and other senses; type of stimuli, from abstract stimuli such as vibrations, to two- and three-dimensional stimuli, also considering concrete stimuli such as the relation between familiar and unfamiliar stimuli or the haptic perception of faces; type of participants, separating studies with blind participants, studies with children and adults, and an analysis of sex differences in performance; and finally, type of tactile exploration, considering conditions of active and passive touch, the relevance of movement in touch and the relation between exploration and time. This review intends to present an organised overview of the main variables in touch experiments, attending to the main findings described in literature, to guide the design of future works on tactual perception and memory.This work was funded by the Portuguese “Foundation for Science and Technology” through PhD scholarship SFRH/BD/35918/2007

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

BACKGROUND: In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. METHODS: To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm(2 )for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (μPIV) experiments. RESULTS: Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within ±10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. CONCLUSION: The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable

Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

Background: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery.Methodology/Principal Findings: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. the prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells.Conclusions/Significance: the extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institutes of Health (NIH)Universidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USAUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 06/05095-6FAPESP: 07/04757-8FAPESP: 07/59768-4CNPq: 301666/2010-5National Institutes of Health (NIH): 5G12RR008124-16A1National Institutes of Health (NIH): 5G12RR008124-16A1S1National Institutes of Health (NIH): G12MD007592Web of Scienc

Microvascular engineering in perfusion culture: immunohistochemistry and CLSM findings

BACKGROUND: One of the most challenging problems in tissue engineering is the establishment of vascular supply. A possible approach might be the engineering of microvasculature in vitro and the supply by engineered feeder vessels. METHODS: An in vitro model for a small-diameter vessel was developed and made from adipose tissue stromal cells and human umbilical vein endothelial cells in a tube-like gelatine scaffold. The number of "branches" emerging from the central lumen and the morphology of the central lumen of the vessel equivalent were assessed after 16 days of either pulsatile perfusion culture or culture in rotating containers by evaluation of immunohistochemically stained sections (n = 6 pairs of cultures). Intramural capillary network formation was demonstrated in five experiments with confocal laser scanning microscopy. RESULTS: Perfused specimens showed a round or oval lumen lined by a single layer of endothelial cells, whereas following rotation culture the lumen tended to collapse. Confocal laser scanning microscopy showed more extended network formation in perfused specimens as compared to specimens after rotation culture. Partially highly interconected capillary-like networks were imaged which showed orientation around the central lumen. Perfused specimens exhibited significantly more branches emerging from the central lumen. There were, however, hardly any capillary branches crossing the whole vessel wall. CONCLUSION: Pulsatile perfusion supports the development of vascular networks with physiological appearance. Advances in reactor development, acquisition of functional data and imaging procedures are however necessary in order to attain the ultimate goal of a fully functional engineered supplying vessel