Inverse method based on 3D nonlinear physically constrained minimisation in the framework of traction force microscopy

Traction force microscopy is a methodology that enables to estimate cellular forces from the measurement of the displacement field of an extracellular matrix (ECM)-mimicking hydrogel that a cell is mechanically interacting with. In this paper, a new inverse and physically-consistent methodology is developed and implemented in the context of 3D nonlinear elasticity. The proposed method searches for a displacement field that approximates the measured one, through the imposition of fulfillment of equilibrium with real and known forces acting in the hydrogel. The overall mathematical formulation leads to a constrained optimisation problem that is treated through a Lagrange operator and that is solved numerically by means of a nonlinear finite element framework. In order to illustrate the potential and enhanced accuracy of the proposed inverse method, it is applied to a total of 5 different real cases of cells cultured in a 3D hydrogel that is considered to behave as a nonlinear elastic material. Different error indicators are defined in order to compare ground truth simulated displacements and tractions to the ones recovered by the new inverse as well as by the forward method. Results indicate that the evaluation of displacement gradients leads to errors, in terms of recovered tractions, that are more than three times lower (on average) for the inverse method compared to the forward method. They highlight the enhanced accuracy of the developed methodology and the importance of appropriate inverse methods that impose physical constraints to traction and stress recovery in the context of traction force microscopyMinisterio de Economía y Competitividad (MINECO). PGC2018-097257-B-C31Consejo Europeo de Resucitación 308223Consejo Europeo de Resucitación G087018NMinisterio de Educación, Cultura y Deporte (MECD). España CAS17/00096Hércules G0H6316NFonds Wetenschappelijk Onderzoek (FWO) 12ZR120NFonds Wetenschappelijk Onderzoek (FWO) V413019

New Insights on Structures Forming the Lignin-Like Fractions of Ancestral Plants

In the present work, lignin-like fractions were isolated from several ancestral plants –including moss (Hypnum cupressiforme and Polytrichum commune), lycophyte (Selaginella kraussiana), horsetail (Equisetum palustre), fern (Nephrolepis cordifolia and Pteridium aquilinum), cycad (Cycas revoluta), and gnetophyte (Ephedra fragilis) species– and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme, P. commune and E. palustre “lignins, ” where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, β−O−4′ alkyl-aryl ethers were the most abundant substructures, followed by condensed β−5′ phenylcoumarans and β−β′ resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4–1.2%) and ferns (0–0.7%)

Soluble platelet-endothelial cell adhesion molecule-1, a biomarker of ventilator-induced lung injury

Introduction: Endothelial cell injury is an important component of acute lung injury. Platelet-endothelial cell adhesion molecule-1 (PECAM1) is a transmembrane protein that connects endothelial cells to one another and can be detected as a soluble, truncated protein (sPECAM1) in serum. We hypothesized that injurious mechanical ventilation (MV) leads to shedding of PECAM1 from lung endothelial cells resulting in increasing sPECAM1 levels in the systemic circulation. Methods: We studied 36 Sprague–Dawley rats in two prospective, randomized, controlled studies (healthy and septic) using established animal models of ventilator-induced lung injury. Animals (n = 6 in each group) were randomized to spontaneous breathing or two MV strategies: low tidal volume (VT) (6 ml/kg) and high-VT (20 ml/kg) on 2 cmH2O of positive end-expiratory pressure (PEEP). In low-VT septic animals, 10 cmH2O of PEEP was applied. We performed pulmonary histological and physiological evaluation and measured lung PECAM1 protein content and serum sPECAM1 levels after four hours ventilation period. Results: High-VT MV caused severe lung injury in healthy and septic animals, and decreased lung PECAM1 protein content (P < 0.001). Animals on high-VT had a four- to six-fold increase of mean sPECAM1 serum levels than the unventilated counterpart (35.4 ± 10.4 versus 5.6 ± 1.7 ng/ml in healthy rats; 156.8 ± 47.6 versus 35.6 ± 12.6 ng/ml in septic rats) (P < 0.0001). Low-VT MV prevented these changes. Levels of sPECAM1 in healthy animals on high-VT MV paralleled the sPECAM1 levels of non-ventilated septic animals. Conclusions: Our findings suggest that circulating sPECAM1 may represent a promising biomarker for the detection and monitoring of ventilator-induced lung injury

Ozone Eliminates SARS-CoV-2 from Difficult-to-Clean Office Supplies and Clinical Equipment.

Background: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to cause profound health, economic, and social problems worldwide. The management and disinfection of materials used daily in health centers and common working environments have prompted concerns about the control of coronavirus disease 2019 (COVID-19) infection risk. Ozone is a powerful oxidizing agent that has been widely used in disinfection processes for decades. The aim of this study was to assess the optimal conditions of ozone treatment for the elimination of heat-inactivated SARS-CoV-2 from office supplies (personal computer monitors, keyboards, and computer mice) and clinical equipment (continuous positive airway pressure tubes and personal protective equipment) that are difficult to clean. (2) Methods: The office supplies and clinical equipment were contaminated in an area of 1 cm2 with 1 × 104 viral units of a heat-inactivated SARS-CoV-2 strain, then treated with ozone using two different ozone devices: a specifically designed ozonation chamber (for low–medium ozone concentrations over large volumes) and a clinical ozone generator (for high ozone concentrations over small volumes). SARS-CoV-2 gene detection was carried out using quantitative real-time polymerase chain reaction (RT-qPCR). (3) Results: At high ozone concentrations over small surfaces, the ozone eliminated SARS-CoV-2 RNA in short time periods—i.e., 10 min (at 4000 ppm) or less. The optimum ozone concentration over large volumes was 90 ppm for 120 min in ambient conditions (24 °C and 60–75% relative humidity). (4) Conclusions: This study showed that the appropriate ozone concentration and exposure time eliminated heat-inactivated SARS-CoV-2 RNA from the surfaces of different widely used clinical and office supplies, decreasing their risk of transmission, and improving their reutilization. Ozone may provide an additional tool to control the spread of the COVID-19 pandemic.TRUEInstituto de Salud Carlos III, Madrid, Spain, and by the European Regional Development Funds (FEDER)Fundación Canaria del Instituto de Investigación Sanitaria de Canarias (FIISC), Las PalmasFundación Mapfre Guanarteme, Las PalmasGobierno de Canarias, Las Palmaspu

El análisis de 52 genomas fúngicos aclara la evolución de los estilos de vida de los Agaricales

1 p.Los Agaricomycetes han desarrollado complejas maquinarias enzimáticas que les permiten descomponer los diferentes polímeros vegetales, incluida la lignina. Entre ellos, los Agaricales saprótrofos se caracterizan por su diversidad de hábitats y estilos de vida. El análisis de 52 genomas de Agaricomycetes aquí realizado revela que los Agaricales poseen una gran diversidad de enzimas hidrolíticas y oxidativas para la descomposición de la lignocelulosa. En base a las familias de genes con mayor velocidad evolutiva (dominios de unión a celulosa, glicosil hidrolasa GH43, monooxigenasas líticas de polisacáridos, peroxidasas ligninolíticas, enzimas de la superfamilia de glucosa-metanol-colina oxidasas/deshidrogenasas, lacasas y peroxigenasas), reconstruimos los estilos de vida de los ancestros que dieron lugar a los actuales Agaricomycetes degradadores de lignocelulosa. Los cambios en el conjunto de herramientas enzimáticas de los Agaricales ancestrales se correlacionaron con la evolución de su capacidad para crecer no solo sobre madera, sino también sobre hojarasca de bosques y madera en descomposición, siendo los descomponedores de la hojarasca de praderas el grupo ecofisiológico más reciente. En este contexto, las anteriores familias de enzimas se analizaron en relación con la diversidad de estilos de vida. Las peroxidasas aparecen como un componente central del set enzimático de los Agaricomycetes saprotrófos, consistente con su papel esencial en la degradación de la lignina y sus altas tasas evolutivas. Esto incluye no solo expansiones/pérdidas de genes de peroxidasas, sino también la presencia generalizada en Agaricales de nuevos tipos de peroxidasas que no se encuentran en Polyporales degradadores de madera, y en otros órdenes de Agaricomycetes.Projectos/contratos BIO2017-86559-R, BIO2015-73697-JIN, AGL2014-55971-R, NSF-grant-1457721, CEFOX-031B0831B, PIE-201620E081, ANR-11-LABX-0002-01, US-DOE-DE-AC02-05CH11231Peer reviewe

Lifestyle Evolution And Peroxidase Diversity In Agaricales As Revealed By Comparative Genomics

Descripción de 1 páginas de la comunicación oral presentada en Oxizymes2022 10th edition of the international “Oxizymes” meeting. Siena, Italy, July 5-8, 2022Basidiomycetes of the class Agaricomycetes have developed complex enzymatic machineries that allow them to decompose plant polymers, including lignin. Within this group, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles in comparison with fungi from other orders. With the aim of shedding light on the evolution of lignocellulose-decaying lifestyles in Agaricales we conducted a comparative analysis of 52 Agaricomycetes genomes [1]. This study revealed that Agaricales possess a large diversity of hydrolytic and oxidative enzymes. Surprisingly, computer-assisted gene-family evolution analysis of these enzymes revealed that a few oxidoreductase families showed significantly higher evolutionary rates. Based on these gene families we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. According to this, we determined that changes in the oxidative enzymatic toolkit of ancestral Agaricales correlate with the evolution of their ability to grow not only on wood, but also on leaf and grass litter and decayed wood. In this context, the aboye families were analyzed and special attention was paid to peroxidases as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes responsible for lignin degradation. We identified a widespread presence of new ligninolytic peroxidase types in Agaricales, some of them not previously identified in this order, and others also not found in woodrottingPolyporales and other orders of Agaricomycetes. Peroxidase evolution was analyzed in Agaricomycetes by ancestral sequence reconstruction and several major evolutionary pathways were unveiled. The study of the newly identified peroxidases will provide insight into their role in the lignin degradation process. In fact, these studies have already been initiated with the expression and characterization of the first lignin peroxidase identified in Agaricales. [1] Ruiz-Dueñas FJ, Barrasa JM, Sánchez-García M, Camarero S, Miyauchi S, Serrano A, et al., 2021, Mol Biol Evol, 38, 1428-1446.Projects/contracts BI02017-86559-R, BI02015-7369-JIN, AGL2014-55971-R, NSFgrant-1457721 , CEFOX-031 B0831 S, PIE-201620E081 , ANR-11-LABX-0002-01 , US-DOE-DE-AC02-05CH11231N

Long-term variability of bulk milk somatic cell and bacterial counts associated with dairy farms moving from conventional to automatic milking systems

When a farm that was using a conventional milking system introduces an automatic milking system (AMS) possible risk factors can affect milk quality. The aim of the study was to investigate the influence of milking with automatic milking systems on milk quality variables over a long time-period post-installation. Bulk milk total bacterial count (BMTBC) and somatic cell count (BMSCC) were analysed and compared from 2 years before introduction of automatic milking until 4 years after. Differences regarding these quality parameters were contrasted using t-test and one-way analysis of variance (ANOVA) and post hoc comparisons were performed. A significant increase in BMTBC was observed during the first three months after introduction of AMS, counts then declined to equivalent levels pre-AMS installation, from 25,000 to 50,000 cfu mL−1. Although differences were significant for the first two years post-installation, they became non-significant during the following two years. The difference in BMSCC was not statistically significant between pre and post-AMS installation time periods, but by grouping data into annual periods, significantly higher values of BMSCC were found during the first year after introduction. Nevertheless, these values decreased over time and even showed a significant improvement in the third year with respect to pre-introduction. The data show that the installation of AMS had a marked impact on milk quality. However, as soon as farmers become accustomed to managing the new equipment and the adaption of cows is real, a level of milk quality which can be maintained over time is achievable

Traction force reconstruction assessment on real three-dimensional matrices and cellular morphologies

Traction force microscopy (TFM) allows to estimate tractions on the surface of cells when they mechanically interact with hydrogel substrates that mimic the extracellular matrix (ECM). The field of mechanobiology has a strong interest in using TFM in 3D in vitro models. However, there are a number of challenges that hamper the accuracy of 3D TFM and that are often bypassed. In this study, the computational efficiency and accuracy of TFM, referred to traction reconstruction from synthetically generated (control) ground truth solutions, are assessed from four different perspectives: magnitude of cellular pulling force (and hence strain level achieved in the hydrogel), effect of the complexity of the cellular morphology, accuracy and computational efficiency of forward vs inverse traction recovery methods, and the effect of incorrectly selecting a constitutive model that describes the behavior of the ECM (i.e. linear/nonlinear). The main results showed: (i) traction reconstruction is more challenging for complex cell morphologies, (ii) there is no significant impact of the magnitude of cellular pulling force on the overall reconstruction accuracy, and (iii) modeling a nonlinear hydrogel with a linear constitutive model leads to non-negligible errors (up to 80% and 30% for forward and inverse methodologies, respectively) in traction reconstruction. This study expands the characterization of the accuracy and efficiency of 3D TFM, highlighting important factors to be considered in future 3D TFM in vitro applications