Validation and implementation of telephone-administered version of the Multidimensional Prognostic Index (TELE-MPI) for remote monitoring of community-dwelling older adults.

BACKGROUND: During the recent lockdown measures adopted by national authorities to contain the COVID-19 pandemic, many vulnerable older patients with chronic conditions, normally followed in ambulatory setting, needed to be monitored and managed in alternative ways, including telemedicine. AIMS: In the framework of a telemedicine program, we aimed to validate and implement a telephone-administered version of the Multidimensional Prognostic Index (TELE-MPI) among community-dwelling older outpatients. METHOD: From March 9 to May 11, 2020, 131 older patients (82.1 years; 74% females) were interviewed using a telephone-based survey to calculate the TELE-MPI. The standard MPI was performed face-to-face three months apart. The Bland-Altman methodology measured the agreement between the two tools. Multivariate logistic regression models were built to ascertain the prognostic value of TELE-MPI and TELE-MPI classes (low, moderate, or severe risk) on negative outcomes occurring during the lockdown period. RESULTS: Mean MPI and TELE-MPI values were 0.523 and 0.522, respectively. Lower and upper 95% limits of agreement were - 0.122 and + 0.124, respectively, with only 4.6% of observations outside the limits. Each 0.1 increase of TELE-MPI score was significantly correlated with higher incidence of psychiatric disorders [odd ratio (OR): 1.57; 95% confidence interval (CI) 1.27, 1.95] and falls (OR: 1.41; 95% CI 1.08, 1.82) in community-dwelling-older adults. DISCUSSION: TELE-MPI showed a strong agreement with the standard MPI and was able to predict psychiatric disorders and falls during lockdown period. CONCLUSION: TELE-MPI may represent a useful way to follow by remote the health status of older adults.

Multidimensional prognostic index (MPI) predicts successful application for disability social benefits in older people.

AIM: The economic recognition of disability is of importance in daily practice, but the tools used in older people are still limited. Therefore, we aimed to investigate the effectiveness of the multidimensional prognostic index (MPI) to identify frail older subjects to be submitted to civil invalidity application for disability benefits including Attendance Allowance (AA) indemnity, Carer's Leave (Law 104) and/or Parking Card for people with disabilities. METHODS: From March 2018 to January 2019, 80 older people were included. The MPI was calculated from comprehensive geriatric assessment information including eight different domains. Civil benefits included attendance allowance (AA) indemnity by the Local Medico-Legal Committee (MLC-NHS) and by the National Institute of Social Security Committee (INPS), Carer's Leave (Law 104), and Parking Card for people with disabilities. RESULTS: MPI values were associated with an increased probability to obtain a 100% civil disability, AA indemnity, Carer's Leave and a parking card for people with disabilities. MPI score showed a very good accuracy in predicting the civil invalidity benefits with a area-under-curve (AUC) of 87.3 (95% CI 80.6-97.4) to predict the release of AA indemnity, 81.3 (95% CI 68.5-91.1) to predict Care's leave and 70.7 (95% CI 59.4-84.7) to predict the Parking Card release. Moreover, data showed that a cut-off score of MPI ≥ 0.75 could identify the 100% of older subjects who successfully obtained the indemnity release. CONCLUSION: MPI is an excellent predictor of social benefits' release by local and national agencies.

Effect of exposure to CeO2 nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours.

BACKGROUND: Cerium oxide nanoparticles (CeO2 NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO2 NPs for 96 h on the main structural and kinematic parameters of spermatozoa. METHODS: The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO2 NPs (0, 44 and 220 µg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H2DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni's as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. RESULTS: Exposure of ram spermatozoa to increasing doses of CeO2 NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO2 NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. CONCLUSIONS: We reported beneficial effects of CeO2 NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO2 NPs. These effects could not be related to an antioxidant activity of CeO2 NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

Hard X-rays for processing hybrid organic-inorganic thick films.

Hard X-rays, deriving from a synchrotron light source, have been used as an effective tool for processing hybrid organic-inorganic films and thick coatings up to several micrometres. These coatings could be directly modified, in terms of composition and properties, by controlled exposure to X-rays. The physico-chemical properties of the coatings, such as hardness, refractive index and fluorescence, can be properly tuned using the interaction of hard X-rays with the sol-gel hybrid films. The changes in the microstructure have been correlated especially with the modification of the optical and the mechanical properties. A relationship between the degradation rate of the organic groups and the rise of fluorescence from the hybrid material has been observed; nanoindentation analysis of the coatings as a function of the X-ray doses has shown a not linear dependence between thickness and film hardness.

Tuning the phase transition of ZnO thin films through lithography: an integrated bottom-up and top-down processing.

An innovative approach towards the physico-chemical tailoring of zinc oxide thin films is reported. The films have been deposited by liquid phase using the sol-gel method and then exposed to hard X-rays, provided by a synchrotron storage ring, for lithography. The use of surfactant and chelating agents in the sol allows easy-to-pattern films made by an organic-inorganic matrix to be deposited. The exposure to hard X-rays strongly affects the nucleation and growth of crystalline ZnO, triggering the formation of two intermediate phases before obtaining a wurtzite-like structure. At the same time, X-ray lithography allows for a fast patterning of the coatings enabling microfabrication for sensing and arrays technology.

Getting order in mesostructured thin films, from pore organization to crystalline walls, the case of 3-glycidoxypropyltrimethoxysilane.

A new type of mesostructured hybrid organic-inorganic film has been synthesised by evaporation-induced self-assembly using 3-glycidoxypropyltrimethoxysilane as the precursor and a tri-block copolymer, Pluronic F127, as the template. The chemistry has been tuned to form bridged polysilsesquioxanes that self-organise into ordered lamellar structures. Controlled aging under highly basic conditions, which has been monitored by Raman and infrared spectroscopy, has been used to obtain the layered ordered hybrid structures in the precursor sol. The pH of the sol has been adjusted to form the micelles that act as templates during solvent evaporation. The self-assembly of the system has been studied in situ by small and wide angle X-ray scattering using a synchrotron light source, which has confirmed both the formation of hybrid layered structures and the long-range organization of the mesophase in the hybrid films. The present approach allows ordering the hybrid film on two different length scales; at the end of film processing, hybrid crystals are incorporated into the pore walls and the micelles are arranged within the films with long range order.

Lithiated porous silicon nanowires stimulate periodontal regeneration.

Periodontal disease is a significant burden for oral health, causing progressive and irreversible damage to the support structure of the tooth. This complex structure, the periodontium, is composed of interconnected soft and mineralised tissues, posing a challenge for regenerative approaches. Materials combining silicon and lithium are widely studied in periodontal regeneration, as they stimulate bone repair via silicic acid release while providing regenerative stimuli through lithium activation of the Wnt/ß-catenin pathway. Yet, existing materials for combined lithium and silicon release have limited control over ion release amounts and kinetics. Porous silicon can provide controlled silicic acid release, inducing osteogenesis to support bone regeneration. Prelithiation, a strategy developed for battery technology, can introduce large, controllable amounts of lithium within porous silicon, but yields a highly reactive material, unsuitable for biomedicine. This work debuts a strategy to lithiate porous silicon nanowires (LipSiNs) which generates a biocompatible and bioresorbable material. LipSiNs incorporate lithium to between 1% and 40% of silicon content, releasing lithium and silicic acid in a tailorable fashion from days to weeks. LipSiNs combine osteogenic, cementogenic and Wnt/ß-catenin stimuli to regenerate bone, cementum and periodontal ligament fibres in a murine periodontal defect.

Nanocomposite Hydrogels with Polymer Grafted Silica Nanoparticles, Using Glucose Oxidase.

Nanocomposite hydrogels offer remarkable potential for applications in bone tissue engineering. They are synthesized through the chemical or physical crosslinking of polymers and nanomaterials, allowing for the enhancement of their behaviour by modifying the properties and compositions of the nanomaterials involved. However, their mechanical properties require further enhancement to meet the demands of bone tissue engineering. Here, we present an approach to improve the mechanical properties of nanocomposite hydrogels by incorporating polymer grafted silica nanoparticles into a double network inspired hydrogel (gSNP Gels). The gSNP Gels were synthesised via a graft polymerization process using a redox initiator. gSNP Gels were formed by grafting 2-acrylamido-2-methylpropanesulfonic acid (AMPS) as the first network gel followed by a sequential second network acrylamide (AAm) onto amine functionalized silica nanoparticles (ASNPs). We utilized glucose oxidase (GOx) to create an oxygen-free atmosphere during polymerization, resulting in higher polymer conversion compared to argon degassing. The gSNP Gels showed excellent compressive strengths of 13.9 ± 5.5 MPa, a strain of 69.6 ± 6.4%, and a water content of 63.4% ± 1.8. The synthesis technique demonstrates a promising approach to enhance the mechanical properties of hydrogels, which can have significant implications for bone tissue engineering and other soft tissue applications.

3D printed superparamagnetic stimuli-responsive starfish-shaped hydrogels.

Magnetic-stimuli responsive hydrogels are quickly becoming a promising class of materials across numerous fields, including biomedical devices, soft robotic actuators, and wearable electronics. Hydrogels are commonly fabricated by conventional methods that limit the potential for complex architectures normally required for rapidly changing custom configurations. Rapid prototyping using 3D printing provides a solution for this. Previous work has shown successful extrusion 3D printing of magnetic hydrogels; however, extrusion-based printing is limited by nozzle resolution and ink viscosity. VAT photopolymerization offers a higher control over resolution and build-architecture. Liquid photo-resins with magnetic nanocomposites normally suffer from nanoparticle agglomeration due to local magnetic fields. In this work, we develop an optimised method for homogenously infusing up to 2 wt % superparamagnetic iron oxide nanoparticles (SPIONs) with a 10 nm diameter into a photo-resin composed of water, acrylamide and PEGDA, with improved nanoparticle homogeneity and reduced agglomeration during printing. The 3D printed starfish hydrogels exhibited high mechanical stability and robust mechanical properties with a maximum Youngs modulus of 1.8 MPa and limited shape deformation of 10% when swollen. Each individual arm of the starfish could be magnetically actuated when a remote magnetic field is applied. The starfish could grab onto a magnet with all arms when a central magnetic field was applied. Ultimately, these hydrogels retained their shape post-printing and returned to their original formation once the magnetic field had been removed. These hydrogels can be used across a wide range of applications, including soft robotics and magnetically stimulated actuators.

Hybrid materials with an increased resistance to hard X-rays using fullerenes as radical sponges.

The protection of organic and hybrid organic-inorganic materials from X-ray damage is a fundamental technological issue for broadening the range of applications of these materials. In the present article it is shown that doping hybrid films with fullerenes C(60) gives a significant reduction of damage upon exposure to hard X-rays generated by a synchrotron source. At low X-ray dose the fullerene molecules act as `radical scavengers', considerably reducing the degradation of organic species triggered by radical formation. At higher doses the gradual hydroxylation of the fullerenes converts C(60) into fullerol and a bleaching of the radical sinking properties is observed.

Double-Network Hydrogels Reinforced with Covalently Bonded Silica Nanoparticles via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemistry.

Hydrogels have progressed from single-network materials with low mechanical integrity to double-network hydrogels (DNHGs) with tough, tunable properties. In this work, we introduce a nanocomposite structure into the first network of a DNHG. Amine-functionalized silica nanoparticles (ASNPs) were covalently cross-linked by forming amide bonds through the carboxylic groups of polyacrylic acid (PAAc) in the first network. DNHGs with varying sizes of ASNPs (50, 100, and 150 nm) and varying concentrations (2.5, 10, 20, and 40 wt %) were explored and compared to a control without a nanocomposite structure. Compressive strengths improved from 0.10 MPa for the control to a maximum of 1.28 MPa for the PAAc/PAAm DNHGs. All hydrogels experienced increased resistance to strain with a maximum of 74% compared to 45% for the control. SEM images of freeze-dried gels showed that ASNPs were integrated into the gel mesh. Nanoparticle retention was calculated using thermal gravimetric analysis (TGA) with improved retention values for larger ASNPs. New DNHG composites have been formed with improved mechanical properties and a potential use in tissue engineering and biomaterial applications.

Interaction of monodispersed strontium containing bioactive glass nanoparticles with macrophages.

The cellular response of murine primary macrophages to monodisperse strontium containing bioactive glass nanoparticles (SrBGNPs), with diameters of 90 ± 10 nm and a composition (mol%) of 88.8 SiO2-1.8CaO-9.4SrO (9.4% Sr-BGNPs) was investigated for the first time. Macrophage response is critical as applications of bioactive nanoparticles will involve the nanoparticles circulating in the blood stream and macrophages will be the first cells to encounter the particles, as part of inflammatory response mechanisms. Macrophage viability and total DNA measurements were not decreased by particle concentrations of up to 250 µg/mL. The Sr-BGNPs were actively internalised by the macrophages via formation of endosome/lysosome-like vesicles bordered by a membrane inside the cells. The Sr-BGNPs degraded inside the cells, with the Ca and Sr maintained inside the silica network. When RAW264.7 cells were incubated with Sr-BGNPs, the cells were polarised towards the pro-regenerative M2 population rather than the pro-inflammatory M1 population. Sr-BGNPs are potential biocompatible vehicles for therapeutic cation delivery for applications in bone regeneration.

Nanostars Carrying Multifunctional Neurotrophic Dendrimers Protect Neurons in Preclinical In Vitro Models of Neurodegenerative Disorders.

A challenge in neurology is the lack of efficient brain-penetrable neuroprotectants targeting multiple disease mechanisms. Plasmonic gold nanostars are promising candidates to deliver standard-of-care drugs inside the brain but have not been trialed as carriers for neuroprotectants. Here, we conjugated custom-made peptide dendrimers (termed H3/H6), encompassing motifs of the neurotrophic S100A4-protein, onto star-shaped and spherical gold nanostructures (H3/H6-AuNS/AuNP) and evaluated their potential as neuroprotectants and interaction with neurons. The H3/H6 nanostructures crossed a model blood-brain barrier, bound to plasma membranes, and induced neuritogenesis with the AuNS, showing higher potency/efficacy than the AuNP. The H3-AuNS/NP protected neurons against oxidative stress, the H3-AuNS being more potent, and against Parkinson's or Alzheimer's disease (PD/AD)-related cytotoxicity. Unconjugated S100A4 motifs also decreased amyloid beta-induced neurodegeneration, introducing S100A4 as a player in AD. Using custom-made dendrimers coupled to star-shaped nanoparticles is a promising route to activate multiple neuroprotective pathways and increase drug potency to treat neurodegenerative disorders.

Nanoceria provides antioxidant and osteogenic properties to mesoporous silica nanoparticles for osteoporosis treatment.

Osteoporosis, a chronic metabolic bone disease, is the most common cause of fractures. Drugs for treating osteoporosis generally inhibit osteoclast (OC) activity, but are rarely aimed at encouraging new bone growth and often cause severe systemic side effects. Reactive oxygen species (ROS) are one of the key triggers of osteoporosis, by inducing osteoblast (OB) and osteocyte apoptosis and promoting osteoclastogenesis. Here we tested the capability of the ROS-scavenger nanoceria encapsulated within mesoporous silica nanoparticles (Ce@MSNs) to treat osteoporosis using a pre-osteoblast MC3T3-E1 cell monoculture in stressed and normal conditions. Ce@MSNs (diameter of 80 ± 10 nm) were synthesised following a scalable two-step process involving sol-gel and wet impregnation methods. The Ce@MSNs at concentration of 100 µg mL-1 induced a significant reduction in oxidative stress produced by t-butyl hydroperoxide and did not alter cell viability significantly. Confocal microscopy showed that MSNs and Ce@MsNs were internalised into the cytoplasm of the pre-osteoblasts after 24 h but were not in the nucleus, avoiding any DNA and RNA modifications. Ce@MSNs provoked mineralisation of the pre-osteoablasts without osteogenic supplements, which did not occur when the cells were exposed to MSN without nanoceria. In a co-culture system of MC3T3-E1 and RAW264.7 macrophages, the Ce@MSNs exhibited antioxidant capability and stimulated cell proliferation and osteogenic responses without adding osteogenic supplements to the culture. The work brings forward an effective platform based for facile synthesis of Ce@MSNs to interact with both OBs and OCs for treatment of osteoporosis.

An overview of methods of fine and ultrafine particle collection for physicochemical characterisation and toxicity assessments.

Particulate matter (PM) is a crucial health risk factor for respiratory and cardiovascular diseases. The smaller size fractions, ≤2.5 µm (PM2.5; fine particles) and ≤0.1 µm (PM0.1; ultrafine particles), show the highest bioactivity but acquiring sufficient mass for in vitro and in vivo toxicological studies is challenging. We review the suitability of available instrumentation to collect the PM mass required for these assessments. Five different microenvironments representing the diverse exposure conditions in urban environments are considered in order to establish the typical PM concentrations present. The highest concentrations of PM2.5 and PM0.1 were found near traffic (i.e. roadsides and traffic intersections), followed by indoor environments, parks and behind roadside vegetation. We identify key factors to consider when selecting sampling instrumentation. These include PM concentration on-site (low concentrations increase sampling time), nature of sampling sites (e.g. indoors; noise and space will be an issue), equipment handling and power supply. Physicochemical characterisation requires micro- to milli-gram quantities of PM and it may increase according to the processing methods (e.g. digestion or sonication). Toxicological assessments of PM involve numerous mechanisms (e.g. inflammatory processes and oxidative stress) requiring significant amounts of PM to obtain accurate results. Optimising air sampling techniques are therefore important for the appropriate collection medium/filter which have innate physical properties and the potential to interact with samples. An evaluation of methods and instrumentation used for airborne virus collection concludes that samplers operating cyclone sampling techniques (using centrifugal forces) are effective in collecting airborne viruses. We highlight that predictive modelling can help to identify pollution hotspots in an urban environment for the efficient collection of PM mass. This review provides guidance to prepare and plan efficient sampling campaigns to collect sufficient PM mass for various purposes in a reasonable timeframe.

Fulleropyrrolidine-functionalized ceria nanoparticles as a tethered dual nanosystem with improved antioxidant properties.

Dual-tethered nanosystems which combine different properties at the nano scale represent a new fascinating frontier of research. In the present work, we present an example of a dual nanosystem designed to enhance the radical scavenging performances. Fulleropyrrolidine has been bonded to cerium oxide nanoparticles (nanoceria) to form a dual tethered system. Fulleropyrrolidine, bearing a silyl-alkoxide group, has been chemically bonded to the nanoceria surface, providing unprecedented antioxidant activity. This effect has been evaluated using an L929 mouse fibroblast cell line exposed to UV light. The fulleropyrrolidine molecules tethered to nanoceria enhance the radical scavenging properties of the oxide. At the same time, fulleropyrrolidine mitigates the potential toxicity of nanoceria at high doses. On the other hand, cerium oxide nanoparticles provide a strong hydrophilicity to the dual nanosystem, ensuring the administration in a cellular environment and preventing macroscopic aggregation of fulleropyrrolidine. The rational assembly of two different components in one nanosystem appears as a promising route for the development of "smarter" medical and cosmetic devices.

Auto-catalytic redox polymerisation using nanoceria and glucose oxidase for double network hydrogels.

A novel auto-catalytic reaction, a combination of naturally occurring enzyme glucose oxidase (GOx) and amine-functionalised cerium oxide nanoparticles (nanoceria), was employed for open vessel free radical polymerisation of double network hydrogels (DNHGs). The nanoceria also incorporated into the gels to enhance mechanical strength. GOx reduces atmospheric O2 to H2O2, causing a cyclic change of cerium ion states, resulting in propagating free radicals in the carbon group in the amino functionalised nanoceria surface. We synthesised novel nanocomposite DNHGs by grafting polymers onto amine-functionalised nanoceria (ANC), with poly(2-acrylamido-2-methylpropanesulfonic acid), PAMPS, and polyacrylamide (PAAm) in the first and second networks respectively. The graft polymerisation was initiated using the alternating cerium states on the ANC. GOx held two major roles within the reaction: to provide an oxygen free system, without any other form of degassing, and to provide cyclical cerium ion states between Ce4+ and Ce3+, creating new free radicals for polymerisation. Polymer conversion using ANC as the sole initiator in the presence of GOx resulted in 83% conversion for PAMPS and 64% PAAm. Polymers degassed only with argon resulted in less than 55% conversion for both PAAm and PAMPS, proving that the addition of GOx enhanced the reaction. The new gels (1.76 MPa) showed an order of magnitude improvement in mechanical properties compared to DNHG made without ANC/GOx (0.10 MPa).

The potential antimalarial efficacy of hemocompatible silver nanoparticles from Artemisia species against P. falciparum parasite.

Malaria represents one of the most common infectious diseases which becoming an impellent public health problem worldwide. Antimalarial classical medications include quinine-based drugs, like chloroquine, and artesunate, a derivative of artemisinin, a molecule found in the plant Artemisia annua. Such therapeutics are very effective but show heavy side effects like drug resistance. In this study, "green" silver nanoparticles (AgNPs) have been prepared from two Artemisia species (A. abrotanum and A. arborescens), traditionally used in folk medicine as a remedy for different conditions, and their potential antimalarial efficacy have been assessed. AgNPs have been characterized by UV-Vis, dynamic light scattering and zeta potential, FTIR, XRD, TEM and EDX. The structural characterization has demonstrated the spheroidal shape of nanoparticles and dimensions under 50 nm, useful for biomedical studies. Zeta potential analysis have shown the stability and dispersion of green AgNPs in aqueous medium without aggregation. AgNPs hemocompatibility and antimalarial activity have been studied in Plasmodium falciparum cultures in in vitro experiments. The antiplasmodial effect has been assessed using increasing doses of AgNPs (0.6 to 7.5 µg/mL) on parasitized red blood cells (pRBCs). Obtained data showed that the hemocompatibility of AgNPs is related to their synthetic route and depends on the administered dose. A. abrotanum-AgNPs (1) have shown the lowest percentage of hemolytic activity on pRBCs, underlining their hemocompatibility. These results are in accordance with the lower levels of parasitemia observed after A. abrotanum-AgNPs (1) treatment respect to A. arborescens-AgNPs (2), and AgNPs (3) derived from a classical chemical synthesis. Moreover, after 24 and 48 hours of A. abrotanum-AgNPs (1) treatment, the parasite growth was locked in the ring stage, evidencing the effect of these nanoparticles to hinder the maturation of P. falciparum. The anti-malarial activity of A. abrotanum-AgNPs (1) on pRBCs was demonstrated to be higher than that of A. arborescens-AgNPs (2).

A MOF-based carrier for in situ dopamine delivery.

MIL-88A (Fe) MOF crystals were nucleated and grown around a polymer core containing superparamagnetic nanoparticles to assemble a new class of biocompatible particles for magnetophoretic drug delivery of dopamine. The carrier enabled efficient targeted release, dopamine protection from oxidative damage, long-term delivery and improved drug delivery cost-efficiency. After loading, dopamine was stable within the carrier and did not undergo oxidation. Drug release monitoring via spectrofluorimetry revealed a shorter burst effect and higher release efficiency than silica based carriers. The in vitro cytotoxicity at different MOF concentrations and sizes was assessed using PC12 cells as the neuronal cell model. The drug was directly uptaken into the PC12 cells avoiding possible side effects due to oxidation occurring in the extracellular environment.