Risk equations for prosthetic joint infections (PJIs) in UK: a retrospective study using the Clinical Practice Research Datalink (CPRD) AURUM and GOLD databases.

BACKGROUND: Prosthetic joint infections (PJIs) are a serious negative outcome of arthroplasty with incidence of about 1%. Risk of PJI could depend on local treatment policies and guidelines; no UK-specific risk scoring is currently available. OBJECTIVE: To determine a risk quantification model for the development of PJI using electronic health records. DESIGN: Records in Clinical Practice Research Datalink (CPRD) GOLD and AURUM of patients undergoing hip or knee arthroplasty between January 2007 and December 2014, with linkage to Hospital Episode Statistics and Office of National Statistics, were obtained. Cohorts' characteristics and risk equations through parametric models were developed and compared between the two databases. Pooled cohort risk equations were determined for the UK population and simplified through stepwise selection. RESULTS: After applying the inclusion/exclusion criteria, 174 905 joints (1021 developed PJI) were identified in CPRD AURUM and 48 419 joints (228 developed PJI) in CPRD GOLD. Patients undergoing hip or knee arthroplasty in both databases exhibited different sociodemographic characteristics and medical/drug history. However, the quantification of the impact of such covariates (coefficients of parametric models fitted to the survival curves) on the risk of PJI between the two cohorts was not statistically significant. The log-normal model fitted to the pooled cohorts after stepwise selection had a C-statistic >0.7. CONCLUSIONS: The risk prediction tool developed here could help prevent PJI through identifying modifiable risk factors pre-surgery and identifying the patients most likely to benefit from close monitoring/preventive actions. As derived from the UK population, such tool will help the National Health Service reduce the impact of PJI on its resources and patient lives.

Systematic reviews of machine learning in healthcare: a literature review.

INTRODUCTION: The increasing availability of data and computing power has made machine learning (ML) a viable approach to faster, more efficient healthcare delivery. METHODS: A systematic literature review (SLR) of published SLRs evaluating ML applications in healthcare settings published between1 January 2010 and 27 March 2023 was conducted. RESULTS: In total 220 SLRs covering 10,462 ML algorithms were reviewed. The main application of AI in medicine related to the clinical prediction and disease prognosis in oncology and neurology with the use of imaging data. Accuracy, specificity, and sensitivity were provided in 56%, 28%, and 25% SLRs respectively. Internal and external validation was reported in 53% and less than 1% of the cases respectively. The most common modeling approach was neural networks (2,454 ML algorithms), followed by support vector machine and random forest/decision trees (1,578 and 1,522 ML algorithms, respectively). EXPERT OPINION: The review indicated considerable reporting gaps in terms of the ML's performance, both internal and external validation. Greater accessibility to healthcare data for developers can ensure the faster adoption of ML algorithms into clinical practice.

A retrospective study of risk factors, causative micro-organisms and healthcare resources consumption associated with prosthetic joint infections (PJI) using the Clinical Practice Research Datalink (CPRD) Aurum database.

BACKGROUND: Prosthetic joint infection (PJI) is a serious complication after joint replacement surgery and it is associated with risk of mortality and morbidity along with high direct costs. METHODS: The Clinical Practice Research Datalink (CPRD) data were utilized to quantify PJI incidence after hip or knee replacement up to 5 years after implant and a variety of risk factors related to patient characteristics, medical and treatment history along with characteristics of the original surgery were analyzed through Cox proportional hazard. RESULTS: 221,826 patients (individual joints 283,789) met all the inclusion and exclusion criteria of the study; during the study follow-up period (5 years), 707 and 695 PJIs were diagnosed in hip and knee, respectively. Patients undergoing joint replacement surgery during an unscheduled hospitalization had greater risk of PJI than patients whose surgery was elective; similarly, the risk of developing PJI after a secondary hip or knee replacement was about 4 times greater than after primary arthroplasty when adjusted for all other variables considered. A previous diagnosis of PJI, even in a different joint, increased the risk of a further PJI. Distribution of average LoS per each hospitalization caused by PJI exhibited a right skewed profile with median duration [IQR] duration of 16 days [8-32] and 13 days [7.25-32] for hip and knee, respectively. PJIs causative micro-organisms were dependent on the time between initial surgery and infection offset; early PJI were more likely to be multispecies than later (years after surgery); the identification of Gram- pathogens decreased with increasing post-surgery follow-up. CONCLUSIONS: This study offers a contemporary assessment of the budgetary and capacity (number and duration of hospitalizations along with the number of Accident and Emergency (A&E) visits) posed by PJIs in UK for the national healthcare system (NHS). The results to provide risk management and planning tools to health providers and policy makers in order to fully assess technologies aimed at controlling and preventing PJI. The findings add to the existing evidence-based knowledge surrounding the epidemiology and burden of PJI by quantifying patterns of PJI in patients with a relatively broad set of prevalent comorbidities.

Feasibility and application of machine learning enabled fast screening of poly-beta-amino-esters for cartilage therapies.

Despite the large prevalence of diseases affecting cartilage (e.g. knee osteoarthritis affecting 16% of population globally), no curative treatments are available because of the limited capacity of drugs to localise in such tissue caused by low vascularisation and electrostatic repulsion. While an effective delivery system is sought, the only option is using high drug doses that can lead to systemic side effects. We introduced poly-beta-amino-esters (PBAEs) to effectively deliver drugs into cartilage tissues. PBAEs are copolymer of amines and di-acrylates further end-capped with other amine; therefore encompassing a very large research space for the identification of optimal candidates. In order to accelerate the screening of all possible PBAEs, the results of a small pool of polymers (n = 90) were used to train a variety of machine learning (ML) methods using only polymers properties available in public libraries or estimated from the chemical structure. Bagged multivariate adaptive regression splines (MARS) returned the best predictive performance and was used on the remaining (n = 3915) possible PBAEs resulting in the recognition of pivotal features; a further round of screening was carried out on PBAEs (n = 150) with small variations of structure of the main candidates from the first round. The refinements of such characteristics enabled the identification of a leading candidate predicted to improve drug uptake > 20 folds over conventional clinical treatment; this uptake improvement was also experimentally confirmed. This work highlights the potential of ML to accelerate biomaterials development by efficiently extracting information from a limited experimental dataset thus allowing patients to benefit earlier from a new technology and at a lower price. Such roadmap could also be applied for other drug/materials development where optimisation would normally be approached through combinatorial chemistry.

The distal C terminus of the dihydropyridine receptor ß1a subunit is essential for tetrad formation in skeletal muscle.

The skeletal muscle dihydropyridine receptor (DHPR) ß1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation­contraction (EC) coupling. Previously, we showed that all four vertebrate ß isoforms (ß1­ß4) facilitate α1S triad targeting and, except for ß3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. Consequently, ß3 failed to restore EC coupling despite the fact that both ß3 and ß1a restore tetrads. Thus, all ß-subunits are able to restore triad targeting, but only ß1a restores both tetrads and proper DHPR­RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. To investigate the molecular region(s) of ß1a responsible for the tetradic arrangement of DHPRs and thus DHPR­RyR1 coupling, we expressed loss- and gain-of-function chimeras between ß1a and ß4, with systematically swapped domains in zebrafish strain relaxed (ß1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. ß1a/ß4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from ß4 showed complete restoration of SR Ca2+ release. However, chimera ß1a/ß4(C) with ß4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera ß4/ß1a(C) with ß1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of ß1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR­RyR1 interaction, essential for skeletal muscle EC coupling.

The Builders of the Junction: Roles of Junctophilin1 and Junctophilin2 in the Assembly of the Sarcoplasmic Reticulum-Plasma Membrane Junctions in Striated Muscle.

Contraction of striated muscle is triggered by a massive release of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. This intracellular calcium release is initiated by membrane depolarization, which is sensed by voltage-gated calcium channels CaV1.1 (in skeletal muscle) and CaV1.2 (in cardiac muscle) in the plasma membrane (PM), which in turn activate the calcium-releasing channel ryanodine receptor (RyR) embedded in the SR membrane. This cross-communication between channels in the PM and in the SR happens at specialized regions, the SR-PM junctions, where these two compartments come in close proximity. Junctophilin1 and Junctophilin2 are responsible for the formation and stabilization of SR-PM junctions in striated muscle and actively participate in the recruitment of the two essential players in intracellular calcium release, CaV and RyR. This short review focuses on the roles of junctophilins1 and 2 in the formation and organization of SR-PM junctions in skeletal and cardiac muscle and on the functional consequences of the absence or malfunction of these proteins in striated muscle in light of recently published data and recent advancements in protein structure prediction.

Junctophilins 1, 2, and 3 all support voltage-induced Ca2+ release despite considerable divergence.

In skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1 that then activate RYR1 to release calcium from the SR. Being independent of extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPHs) 1 and 2 form the SR-PM junctions at which voltage-induced calcium release occurs. Previous work demonstrated that JPH2 is able to recapitulate voltage-induced calcium release when expressed in HEK293 cells together with CaV1.1, ß1a, Stac3, and RYR1. However, it is unknown whether JPH1 and the more distantly related neuronal JPH3 and JPH4 might also function in this manner, a question of interest because different JPH isoforms diverge in their interactions with RYR1. Here, we show that, like JPH2, JPH1 and JPH3, coexpressed with CaV1.1, ß1a, Stac3, and RYR1 in HEK293 cells, cause colocalization of CaV1.1 and RYR1 at ER-PM junctions. Furthermore, potassium depolarization elicited cytoplasmic calcium transients in cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D), indicating that JPH1, JPH2, and JPH3 can all support voltage-induced calcium release, despite sequence divergence and differences in interaction with RYR1. Conversely, JPH4-induced ER-PM junctions contain CaV1.1 but not RYR1, and cells expressing JPH4 are unable to produce depolarization-induced calcium transients. Thus, JPHs seem to act primarily to form ER-PM junctions and to recruit the necessary signaling proteins to these junctions but appear not to be directly involved in the functional interactions between these proteins.

Amplify antimicrobial photo dynamic therapy efficacy with poly-beta-amino esters (PBAEs).

Light-activated antimicrobial agents (photosensitisers) are promising alternatives to antibiotics for the treatment of skin infections and wounds through antimicrobial photo dynamic therapy (aPDT); utilisation of this technique is still restricted by general low efficacy requiring long exposure time (in the order of tens of minutes) that make the treatment very resource intensive. We report for the first time the possibility of harvesting the cell penetrating properties of poly-beta-amino esters (PBAEs) in combination with toluidine blue O (TBO) to shorten aPDT exposure time. Candidates capable of inactivation rates 30 times quicker than pure TBO were discovered and further improvements through PBAE backbone optimisation could be foreseen. Efficacy of the complexes was PBAE-dependent on a combination of TBO uptake and a newly discovered and unexpected role of PBAEs on reactive species production. Chemometric approach of partial least square regression was employed to assess the critical PBAE properties involved in this newly observed phenomenon in order to elicit a possible mechanism. The superior antimicrobial performance of this new approach benefits from the use of well established, low-cost and safe dye (TBO) coupled with inexpensive, widely tested and biodegradable polymers also known to be safe. Moreover, no adverse cytotoxic effects of the PBAEs adjuvated TBO delivery have been observed on a skin cells in vitro model demonstrating the safety profile of this new technology.

Neuronal junctophilins recruit specific CaV and RyR isoforms to ER-PM junctions and functionally alter CaV2.1 and CaV2.2.

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated CaV isoforms, but not a low-voltage-activated CaV. Also, JPH3 and JPH4 noticeably modify CaV2.1 and CaV2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2.

Rheometer enabled study of cartilage frequency-dependent properties.

Despite the well-established dependence of cartilage mechanical properties on the frequency of the applied load, most research in the field is carried out in either load-free or constant load conditions because of the complexity of the equipment required for the determination of time-dependent properties. These simpler analyses provide a limited representation of cartilage properties thus greatly reducing the impact of the information gathered hindering the understanding of the mechanisms involved in this tissue replacement, development and pathology. More complex techniques could represent better investigative methods, but their uptake in cartilage research is limited by the highly specialised training required and cost of the equipment. There is, therefore, a clear need for alternative experimental approaches to cartilage testing to be deployed in research and clinical settings using more user-friendly and financial accessible devices. Frequency dependent material properties can be determined through rheometry that is an easy to use requiring a relatively inexpensive device; we present how a commercial rheometer can be adapted to determine the viscoelastic properties of articular cartilage. Frequency-sweep tests were run at various applied normal loads on immature, mature and trypsinased (as model of osteoarthritis) cartilage samples to determine the dynamic shear moduli (G*, G' G″) of the tissues. Moduli increased with increasing frequency and applied load; mature cartilage had generally the highest moduli and GAG depleted samples the lowest. Hydraulic permeability (KH) was estimated from the rheological data and decreased with applied load; GAG depleted cartilage exhibited higher hydraulic permeability than either immature or mature tissues. The rheometer-based methodology developed was validated by the close comparison of the rheometer-obtained cartilage characteristics (G*, G', G″, KH) with results obtained with more complex testing techniques available in literature. Rheometry is relatively simpler and does not require highly capital intensive machinery and staff training is more accessible; thus the use of a rheometer would represent a cost-effective approach for the determination of frequency-dependent properties of cartilage for more comprehensive and impactful results for both healthcare professional and R&D.

Long acting anti-infection constructs on titanium.

Peri-prosthetic joint infections (PJI) are a serious adverse event following joint replacement surgeries; antibiotics are usually added to bone cement to prevent infection offset. For uncemented prosthesis, alternative antimicrobial approaches are necessary in order to prevent PJI; however, despite elution of drug from the surface of the device being shown one of the most promising approach, no effective antimicrobial eluting uncemented device is currently available on the market. Consequently, there is a clinical need for non-antibiotic antimicrobial uncemented prosthesis as these devices present numerous benefits, particularly for young patients, over cemented artificial joints. Moreover, non-antibiotic approaches are driven by the need to address the growing threat posed by antibiotic resistance. We developed a multilayers functional coating on titanium surfaces releasing chlorhexidine, a well-known antimicrobial agent used in mouthwash products and antiseptic creams, embedding the drug between alginate and poly-beta-amino-esters. Chlorhexidine release was sustained for almost 2 months and the material efficacy and safety was proven both in vitro and in vivo. The coatings did not negatively impact osteoblast and fibroblast cells growth and were capable of reducing bacterial load and accelerating wound healing in an excisional wound model. As PJI can develop weeks and months after the initial surgery, these materials could provide a viable solution to prevent infections after arthroplasty in uncemented prosthetic devices and, simultaneously, help the fight against antibiotic resistance.

Optimisation and feature selection of poly-beta-amino-ester as a drug delivery system for cartilage.

Drug localisation is still one of the main challenges in treating pathologies affecting cartilage; poly-beta-amino-esters (PBAEs) drug conjugates are a possible solution; however, their efficacy highly depends on the polymer structure hence the full potential of this delivery system is still unknown. For the purpose of optimising the delivery system design, a large library of PBAEs was synthesised and dexamethasone (DEX) uptake in cartilage was determined. All three components of PBAE (amine, acrylate and end-capping) impacted the outcome. The most effective PBAE identified enhanced DEX uptake by 8 folds compared to an equivalent dose of the commercial formulation and also prevented, through delivery of DEX, the cartilage degradation caused by IL-1α (interleukine1α). A chemometrics based predictive model was constructed and PBAEs properties most affecting the performance of the drug delivery systems were identified. This model will allow further computer based PBAEs optimisation and fast track the bench to market process for this delivery system.

Systematic Review and Meta-Analysis of Tobacco Use as a Risk Factor for Prosthetic Joint Infection After Total Hip Replacement.

BACKGROUND: A prosthetic joint infection (PJI) is one of the possible complications after total hip arthroplasty (THA). Several studies, but not all, have reported smoking as a risk factor of PJIs in orthopaedic surgery. This study summarizes the most recent evidence using a systematic review of whether tobacco use (not only tobacco smoking) is a risk factor in developing PJIs, specifically after THA. METHODS: Ovid Medline, EMBASE, Scopus, Web of Science, and Cochrane databases were searched from inception to July 2019 to identify case-control studies that examined the PJI risk in tobacco users and tobacco nonusers undergoing THA. Publication bias was also assessed through funnel plots. RESULTS: Searches identified 2689 articles, and 10 of these, involving a total of 20,640 patients, met the inclusion criteria. The overall odds ratio (pooled odds ratio) to develop either a superficial infection, a deep infection, or an infection requiring revision surgery for tobacco users vs nonusers was 1.54 (95% confidence interval: 1.25-1.91) when a fixed-effect model was used and 1.56 (95% confidence interval: 1.10-2.21) when a random-effect model was used. No publication bias was observed among the identified studies. CONCLUSIONS: The findings of the study indicated that tobacco use is associated with a higher risk of PJIs in patients undergoing THA.

Nanoparticle-based model of anti-inflammatory drug releasing LbL coatings for uncemented prosthesis aseptic loosening prevention.

INTRODUCTION: The only treatment for aseptic loosening is the replacement of the prosthesis through revision surgery. A preventive approach, achieved through anti-inflammatory drugs released from the device, has shown to be a viable strategy; however, the performance of these devices is not yet satisfactory thus further improvements are necessary. METHODS: We used titanium nanoparticles as a model for implant surfaces and developed a coating containing dexamethasone (DEX) using layer-by-layer deposition. RESULTS: The amount of deposited drug depended on the number of layers and the release was sustained for months. The efficiency of the released DEX in reducing inflammation markers (tumor necrosis factor alpha and IL-6) produced by human monocytes and macrophages was similar to the pure drug at the same concentration without negative impacts on the viability and morphology of these cells. CONCLUSION: These coatings were not inferior to medical grade titanium (the standard material used in uncemented devices) regarding their ability to sustain osteoblasts and fibroblasts growth.

Anti-inflammatory drug-eluting implant model system to prevent wear particle-induced periprosthetic osteolysis.

BACKGROUND: Aseptic loosening, as a consequence of an extended inflammatory reaction induced by wear particles, has been classified as one of the most common complications of total joint replacement (TJR). Despite its high incidence, no therapeutical approach has yet been found to prevent aseptic loosening, leaving revision as only effective treatment. The local delivery of anti-inflammatory drugs to modulate wear-induced inflammation has been regarded as a potential therapeutical approach to prevent aseptic-loosening. METHODS: In this context, we developed and characterized anti-inflammatory drug-eluting TiO2 surfaces, using nanoparticles as a model for larger surfaces. The eluting surfaces were obtained by conjugating dexamethasone to carboxyl-functionalized TiO2 particles, obtained by using either silane agents with amino or mercapto moieties. RESULTS: Zeta potential measurements, thermogravimetric analysis (TGA) and drug release results suggest that dexamethasone was successfully loaded onto the TiO2 particles. Release was pH dependent and greater amounts of drug were observed from amino route functionalized surfaces. The model-system was then tested for its cytotoxic and anti-inflammatory properties in LPS-stimulated macrophages. Dexamethasone released from amino route functionalized surfaces TiO2 particles was able to decrease LPS-induced nitric oxide (NO) and TNF-a production similarly to pure DEX at the same concentration; DEX released from mercapto route functionalized surfaces was at a too low concentration to be effective. CONCLUSION: Dexamethasone released from amino functionalized titanium can offer the possibility of preventing asepting loosening of joint replacement devices.

Prolonged Antimicrobial Activity of PMMA Bone Cement with Embedded Gentamicin-Releasing Silica Nanocarriers.

Antibiotic laden bone cements are regularly employed to prevent infections after joint replacement surgeries. We have developed silica nanocarriers loaded with gentamicin as a drug delivery system to be dispersed in poly methyl-methacrylate (PMMA) bone cement for controlling and extending the release of the antibiotic from bone cements, thus proving a prolonged antimicrobial activity. Layer-by-layer self-assembly was used to deposit gentamicin between alginate layers and two different poly ß-amino esters on the silica nanoparticles. The release of gentamicin from PMMA bone cement containing silica nanocarriers continued for about 30 days compared to 6 days when the same amount of antibiotic was added as a pure powder (as in commercial formulations); moreover, the medium containing the released antimicrobial drug was capable of preventing the growth of numerous bacteria species responsible for prosthetic joint infections (both catalogue strains and clinical isolates) for longer periods of time than in the case of commercial formulations, thus confirming the extended antimicrobial properties of the drug once released from the carrier. No detrimental effects toward human osteoblasts were also observed; moreover, bone cement material characteristics such as curing time, water uptake, and mechanical properties were unaffected when the silica nanocarriers were added.

LbL-assembled gentamicin delivery system for PMMA bone cements to prolong antimicrobial activity.

INTRODUCTION: Antibiotic-loaded poly(methyl methacrylate) bone cements (ALBCs) are widely used in total joint replacement (TJR), for local delivery of antibiotics to provide prophylaxis against prosthetic joint infections (PJI). One of the shortcomings of the current generation of ALBCs is that the antibiotic release profile is characterized by a burst over the first few hours followed by a sharp decrease in rate for the following several days (often below minimum inhibitory concentration (MIC)), and, finally, exhaustion (after, typically, ~ 20 d). This profile means that the ALBCs provide only short-term antimicrobial action against bacterial strains involved PJI. RATIONALE: The purpose of the present study was to develop an improved antibiotic delivery system for an ALBC. This system involved using a layer-by-layer technique to load the antibiotic (gentamicin sulphate) (GEN) on silica nanoparticles, which are then blended with the powder of the cement. Then, the powder was mixed with the liquid of the cement (NP-GEN cement). For controls, two GEN-loaded brands were used (Cemex Genta and Palacos R+G). Gentamicin release and a host of other relevant properties were determined for all the cements studied. RESULTS: Compared to control cement specimens, improved GEN release, longer antimicrobial activity (against clinically-relevant bacterial strains), and comparable setting time, cytocompatibility, compressive strength (both prior to and after aging in PBS at 37 oC for 30 d), 4-point bend strength and modulus, fracture toughness, and PBS uptake. CONCLUSIONS: NP-GEN cement may have a role in preventing or treating PJI.

Stac Proteins Suppress Ca2+-Dependent Inactivation of Neuronal l-type Ca2+ Channels.

Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with ß and α2-δ1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal l-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal l-type channels.SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.

Cobalt and Titanium nanoparticles influence on human osteoblast mitochondrial activity and biophysical properties of their cytoskeleton.

We investigated the biophysical effects (cell elasticity and spring constant) caused on Saos-2 human osteoblast-like cells by nanosized metal (Co and Ti) wear debris, as well as the adhesive characteristics of cells after exposure to the metal nanoparticles. Cell mitochondrial activity was investigated using the MTT assays; along with LDH assay, metal uptake, cell apoptosis and mineralisation output (alizarin red assay) of the cells. Osteoblasts mitochondrial activity was not affected by Ti nanoparticles at concentrations up to 1 mg/ml and by Cobalt nanoparticles at concentrations < 0.5 mg/ml; however elasticity and spring constant were significantly modified by the exposure to nanoparticles of these metals in agreement with the alteration of cell conformation (shape), as result of the exposure to simulated wear debris, demonstrated by fluorescence images after actin staining.