Tolerance, Adaptation, and Cell Response Elicited by Micromonospora sp. Facing Tellurite Toxicity: A Biological and Physical-Chemical Characterization

The intense use of tellurium (Te) in industrial applications, along with the improper disposal of Te-derivatives, is causing their accumulation in the environment, where oxyanion tellurite (TeO32−) is the most soluble, bioavailable, and toxic Te-species. On the other hand, tellurium is a rare metalloid element whose natural supply will end shortly with possible economic and technological effects. Thus, Te-containing waste represents the source from which Te should be recycled and recovered. Among the explored strategies, the microbial TeO32− biotransformation into less toxic Te-species is the most appropriate concerning the circular economy. Actinomycetes are ideal candidates in environmental biotechnology. However, their exploration in TeO32− biotransformation is scarce due to limited knowledge regarding oxyanion microbial processing. Here, this gap was filled by investigating the cell tolerance, adaptation, and response to TeO32− of a Micromonospora strain isolated from a metal(loid)-rich environment. To this aim, an integrated biological, physical-chemical, and statistical approach combining physiological and biochemical assays with confocal or scanning electron (SEM) microscopy and Fourier-transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR) was designed. Micromonospora cells exposed to TeO32− under different physiological states revealed a series of striking cell responses, such as cell morphology changes, extracellular polymeric substance production, cell membrane damages and modifications, oxidative stress burst, protein aggregation and phosphorylation, and superoxide dismutase induction. These results highlight this Micromonospora strain as an asset for biotechnological purposes

Refractory Status Epilepticus in Genetic Epilepsy-Is Vagus Nerve Stimulation an Option?

Refractory and super-refractory status epilepticus (RSE, SRSE) are severe conditions that can have long-term neurological consequences with high morbidity and mortality rates. The usefulness of vagus nerve-stimulation (VNS) implantation during RSE has been documented by anecdotal cases and in systematic reviews; however, the use of VNS in RSE has not been widely adopted. We successfully implanted VNS in two patients with genetic epilepsy admitted to hospital for SRSE; detailed descriptions of the clinical findings and VNS parameters are provided. Our patients were implanted 25 and 58 days after status epilepticus (SE) onset, and a stable remission of SE was observed from the seventh and tenth day after VNS implantation, respectively, without change in anti-seizure medication. We used a fast ramp-up of stimulation without evident side effects. Our results support the consideration of VNS implantation as a safe and effective adjunctive treatment for SRSE

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process

A composite PLLA scaffold for regeneration of complex tissues.

A composite biodegradable scaffold incorporating an integrated network of synthetic blood vessels was designed and prepared, in line with the requirements of a scaffold effectively supporting the regeneration of highly vascularized tissues. In other words, this composite scaffold should allow the regeneration of complex injured tissue (e.g. dermis) and, at the same time, favour the development of a vascular network on its inner, i.e. a 3D polymeric scaffolds embedding synthetic blood vessel-like structures for nutrient supply and metabolite removal. PLLA assures a high degree of biocompatibility and a low level of inflammation response upon implantation, while the embedded tubular vessel-like structures with a porous internal surface and a porous wall should give rise to a successful in-vitro growth of the endothelial cells, leading to the generation of new vessels. In order to realize the composite scaffold, a technique for integration of vessel-like scaffolds into porous PLLA scaffolds prepared via TIPS was assessed. The scaffolds obtained present a porous bulk with a high degree of interconnection. Moreover, the vessel-like scaffold(s) are completely embedded into the porous structure produced via TIPS and a continuous porous structure at the border of the vessel-like scaffold in communication with the macropores of the “main” scaffold was detected. A preliminary in-vitro coculture test showed that both the cell types seeded into the composite scaffold are able to grow and mature towards a ”primordial” tissue

Bidimensional zymography analyses of proteolytic enzymes present in membranes and shed membrane vesicles of endothelial cells.

<p>Partially purified plasma membranes (a–b) and shed membrane vesicles (c–d) obtained by endothelial cells cultures to confluence (a–c) or in migrating (b–d) conditions were analyzed in bi-dimensional zymography on gelatin substrate digestion. First dimensions were performed on commercial electrophoretical preconstituted gels in an anpholyte continuous gradient pH range of 4.6 to 7.8 (Bio-Rad). Second dimensions were performed on SDS-PAGE at 7.5%, containing gelatin substrate. Gels were incubated at 37°C for 48 hrs in the presence of 2mM calcium ions (+CaCl₂). Pro-forms of MMP-2 and MMP-9 from human blood plasma were used as markers.</p

Solution-Based Processing for Scaffold Fabrication in Tissue Engineering Applications: A Brief Review

The fabrication of 3D scaffolds is under wide investigation in tissue engineering (TE) because of its incessant development of new advanced technologies and the improvement of traditional processes. Currently, scientific and clinical research focuses on scaffold characterization to restore the function of missing or damaged tissues. A key for suitable scaffold production is the guarantee of an interconnected porous structure that allows the cells to grow as in native tissue. The fabrication techniques should meet the appropriate requirements, including feasible reproducibility and time- and cost-effective assets. This is necessary for easy processability, which is associated with the large range of biomaterials supporting the use of fabrication technologies. This paper presents a review of scaffold fabrication methods starting from polymer solutions that provide highly porous structures under controlled process parameters. In this review, general information of solution-based technologies, including freeze-drying, thermally or diffusion induced phase separation (TIPS or DIPS), and electrospinning, are presented, along with an overview of their technological strategies and applications. Furthermore, the differences in the fabricated constructs in terms of pore size and distribution, porosity, morphology, and mechanical and biological properties, are clarified and critically reviewed. Then, the combination of these techniques for obtaining scaffolds is described, offering the advantages of mimicking the unique architecture of tissues and organs that are intrinsically difficult to design

Degradative effects of shed membrane vesicles released by endothelial cells on extra-cellular matrix components.

<p>Shed membrane vesicles obtained from endothelial cells cultured to confluence (C) or in migrating (M) conditions were assayed for their capability to degrade different ECM components, in particular they were assayed on basal lamina components, Laminin (LM), Type-IV collagen (Coll-IV) and Fibronectin (FN) (a), and on type-I collagen fibrils (b and c). In (a), we see the degradative patterns obtained by treatment for 24 hrs of basal lamina components. Type-I and IV collagens and Laminin were biotinilated and degradation stained by streptavidin conjugated to HPR and developed with ECL staining (Amersham); while Fibronectin (FN) degradation was stained with mAbs against human Fibronectin. In (b) we see the kinetic degradative patterns obtained by treating jellified type-I collagen with vesicles obtained in different cell culture conditions at different times of incubation. In (c) there are two controls; collagen fibrils treated with MMP-2 originated by molecular engineering (MMP-2) and spontaneous degradation of type-I collagen fibrils (Control) at different times. In (a) markers are in kDa; in (b) and (c) the positions of α₁(I) and α₂(I) monomers of interstitial type-I collagen are marked.</p