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Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

Developmentally, the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced both LP derived prechondrocytes and preosteoblasts from human pluripotent stem cells (hPSC) through a common progenitor in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocyte- and preosteoblast-like cells can be generated. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to investigate further cartilage development and in the development of joint cartilage for potential treatments.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

The response of stratospheric climate and circulation to increasing amounts of greenhouse gases (GHGs) and ozone recovery in the twenty-first century is analyzed in simulations of 11 chemistry–climate models using near-identical forcings and experimental setup. In addition to an overall global cooling of the stratosphere in the simulations (0.59 ± 0.07 K decade−1at 10 hPa), ozone recovery causes a warming of the Southern Hemisphere polar lower stratosphere in summer with enhanced cooling above. The rate of warming correlates with the rate of ozone recovery projected by the models and, on average, changes from 0.8 to 0.48 K decade−1at 100 hPa as the rate of recovery declines from the first to the second half of the century. In the winter northern polar lower stratosphere the increased radiative cooling from the growing abundance of GHGs is, in most models, balanced by adiabatic warming from stronger polar downwelling. In the Antarctic lower stratosphere the models simulate an increase in low temperature extremes required for polar stratospheric cloud (PSC) formation, but the positive trend is decreasing over the twenty-first century in all models. In the Arctic, none of the models simulates a statistically significant increase in Arctic PSCs throughout the twenty-first century. The subtropical jets accelerate in response to climate change and the ozone recovery produces a westward acceleration of the lower-stratospheric wind over the Antarctic during summer, though this response is sensitive to the rate of recovery projected by the models. There is a strengthening of the Brewer–Dobson circulation throughout the depth of the stratosphere, which reduces the mean age of air nearly everywhere at a rate of about 0.05 yr decade−1in those models with this diagnostic. On average, the annual mean tropical upwelling in the lower stratosphere (∼70 hPa) increases by almost 2% decade−1, with 59% of this trend forced by the parameterized orographic gravity wave drag in the models. This is a consequence of the eastward acceleration of the subtropical jets, which increases the upward flux of (parameterized) momentum reaching the lower stratosphere in these latitudes.

Background Polyvinyl chloride (PVC) foams are widely used in crashworthiness and energy absorption applications due to their low density and the capability of crushing up to large deformations with limited loads. This property is due to their particular constitutive behavior: the stress-strain curve is characterized, after an initial yield or peak stress, by a relevant plateau region followed by a steep increase due to foam densification. Furthermore, the mechanical response of PVC foam is strongly strain rate dependent. Objective This work aims to characterize the mechanical behavior of PVC foams and to develop a complete constitutive model for impact and energy absorption applications. Methods Compressive tests are carried out at different speeds on PVC foam samples having different relative densities. Quasi-static and intermediate strain rate tests are performed by a pneumatic machine, while high strain rate tests are conducted by means of a Split Hopkinson Pressure Bar. The uniaxial stress-strain curves are used to calibrate the visco-elastic and visco-plastic constitutive model. In particular, the material behavior is divided into two parallel branches: the former describes the elasto-plastic behavior, while the latter accounts for the visco-elastic one; the plastic branch also includes a multiplicative term accounting for the strain rate sensitivity of the base material. Results The tests highlight a strong compressibility of the foam with negligible lateral expansion. The energy absorption efficiency, as well as the densification strain, is evaluated. The material model is also implemented in Finite Element (FE) simulations of puncture impact tests, validating the results of the calibration procedure. Conclusions The calibration of the visco-elasto-plastic material model offers a physically consistent identification of the constitutive response of the PVC foams, showing an effective characterization of the impact behavior of the material.

Numerical analysis is regularly used in engineering practice as useful mean to study component behavior in service, especially when an experimental campaign is too expensive or not practicable. Numerical software is becoming more and more powerful and integrated, leaving the correct use of a material model as a weak point. As some materials could manifest an unstable behavior if deformed at high strain rate, it is of primary importance to use an appropriate constitutive law to reproduce their behaviour. This paper shows the results of the inverse calibration of two material (Ti-4Al-6V and AA 7075-T6) constitutive laws in their classical definition, as present in the literature. Moreover, both of them have been modified by the authors in the attempt of improving the complex material behaviour. The calibrated models are Johnson-Cook (JC) and Nowak-Pecherski (NP) models. The improvement of these models showed that JC is not suitable for calibrate materials that show an unstable behavior also after its improvement; conversely NP provided a better matching with the experimental data.

Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but is still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development (referred to as the RAPID-E protocol). Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II, but additionally expressed Collagen X, indicative of hypertrophy. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

ABSTRACT Osmoderma italicum Sparacio (2000) is a rare and localized saproxylic beetle (sometimes considered a subspecies of the widespread western European Osmoderma eremita Scopoli, 1783), currently known from a limited number of sites in southern Italy. Classified as Endangered according to The International Union for Conservation of Nature (IUCN) criteria B2ab(i,ii,iii); D, and listed under Annexes II and IV of the EU Habitats Directive, it is primarily associated with mature and old-growth forests that provide essential microhabitats such as hollow trees and decaying wood. This study presents an updated assessment of the O. italicum geographical distribution, ecological preferences, and altitudinal range in southern Italy, with a particular focus on the region of Calabria. The results indicate a broad but fragmented altitudinal distribution and highlight key habitat associations with tree species such as Fagus sylvatica, Quercus cerris, Q. petraea subsp. austrotyrrhenica, Q. pubescens, Castanea sativa, Alnus cordata, and Pinus heldreichii subsp. leucodermis. Citizen science data, combined with field surveys and Natura 2000 records, have contributed significantly to refining the known range of this taxon. Despite recent advances in data availability, critical knowledge gaps remain, particularly regarding population dynamics and habitat connectivity. We discuss current threats to the taxon and propose a set of conservation actions, including the protection of old-growth forest remnants, the promotion of deadwood retention in managed forests, and the integration of citizen science into long-term monitoring frameworks.

This article describes the design, construction, and first experimental results of a 90 m-long Hopkinson bar which can perform high strain rate tests in a combined tension–torsion state. The system configuration is analogous to the classic Hopkinson bar technique, consisting of three bars: a pre-stressed bar, an input bar, and an output bar; the sample is placed between the input and output bar. The measurement is also based on the classical three-wave method, where the incident, transmitted and reflected waves are measured. Incident compression and torsional waves are simultaneously generated by the failure of a fragile element that connects the pre-stressed bar to electromechanical actuators; the indirect Hopkinson tension bar technique is exploited, where the compression wave reaches the end of the output bar without stressing the sample and is reflected as a tensile input wave. The length of the bars is designed so that the tensile wave reaches the sample from the output bar side at the same time as the torsion wave comes from the input bar. Void tests were carried out first, for preliminary analysis of the system behaviour. Then, successful tests have been conducted on samples made of AA7075 T6, both in combined and pure tension–torsion states; it has been possible to measure the tension–torsion stress–strain curves, from which the equivalent flow stress–strain curve has been evaluated and compared to the quasi-static ones.

ABSTRACT Data on 6 months of fouling colonization on a series of panels, immersed at two different depths (0.5 m and 3 m) in two marinas along the Apulian coasts (Ionian Sea-Mar Grande of Taranto, and South Adriatic Sea-Brindisi), are reported. A total of 79 taxa were found, 59 in Taranto and 57 in Brindisi. Among them, a total of 21 NIS (non-indigenous species) were found (14 in Taranto and 19 in Brindisi), together with seven cryptogenic species (all present in Taranto and six in Brindisi). Species composition between the two sites was quite different, with only 37 species in common. By contrast, the percentage of species of each taxonomic group was quite similar, excluding the complete absence of Cnidaria in Brindisi. The best represented group was Crustacea with 24 taxa, most of which were vagile forms, followed by Polychaeta with 20 taxa, and Mollusca with only 11 taxa. Among sessile invertebrates, Tunicata and Bryozoa were represented by 15 and six taxa, respectively. Porifera and Cnidaria were poorly represented in both sites. The multivariate analysis showed the structure of the macrofouling assemblages in Taranto and Brindisi were significantly influenced by location, time and depth. Comparing the colonization pattern of the two sites, a more homogeneous situation was highlighted in Brindisi. Here, serpulids were the dominant group covering all the available substrate at both times of immersion and depths, especially after 6 months. This situation was displayed also by the MultiDimensional Scaling (MDS) analysis performed on qualitative data. The comparison of the sessile component with data relative to a previously investigated northern area (La Spezia-Ligurian Sea) revealed a different fouling assemblage with a richer community and a higher presence of NIS.