Quantifying a spectrum of clinical response in atrial tachyarrhythmias using spatiotemporal synchronization of electrograms.

AIMS: There is a clinical spectrum for atrial tachyarrhythmias wherein most patients with atrial tachycardia (AT) and some with atrial fibrillation (AF) respond to ablation, while others do not. It is undefined if this clinical spectrum has pathophysiological signatures. This study aims to test the hypothesis that the size of spatial regions showing repetitive synchronized electrogram (EGM) shapes over time reveals a spectrum from AT, to AF patients who respond acutely to ablation, to AF patients without acute response. METHODS AND RESULTS: We studied n = 160 patients (35% women, 65.0 ± 10.4 years) of whom (i) n = 75 had AF terminated by ablation propensity matched to (ii) n = 75 without AF termination and (iii) n = 10 with AT. All patients had mapping by 64-pole baskets to identify areas of repetitive activity (REACT) to correlate unipolar EGMs in shape over time. Synchronized regions (REACT) were largest in AT, smaller in AF termination, and smallest in non-termination cohorts (0.63 ± 0.15, 0.37 ± 0.22, and 0.22 ± 0.18, P < 0.001). Area under the curve for predicting AF termination in hold-out cohorts was 0.72 ± 0.03. Simulations showed that lower REACT represented greater variability in clinical EGM timing and shape. Unsupervised machine learning of REACT and extensive (50) clinical variables yielded four clusters of increasing risk for AF termination (P < 0.01, χ2), which were more predictive than clinical profiles alone (P < 0.001). CONCLUSION: The area of synchronized EGMs within the atrium reveals a spectrum of clinical response in atrial tachyarrhythmias. These fundamental EGM properties, which do not reflect any predetermined mechanism or mapping technology, predict outcome and offer a platform to compare mapping tools and mechanisms between AF patient groups.

Intrinsically stretchable electrode array enabled in vivo electrophysiological mapping of atrial fibrillation at cellular resolution.

Electrophysiological mapping of chronic atrial fibrillation (AF) at high throughput and high resolution is critical for understanding its underlying mechanism and guiding definitive treatment such as cardiac ablation, but current electrophysiological tools are limited by either low spatial resolution or electromechanical uncoupling of the beating heart. To overcome this limitation, we herein introduce a scalable method for fabricating a tissue-like, high-density, fully elastic electrode (elastrode) array capable of achieving real-time, stable, cellular level-resolution electrophysiological mapping in vivo. Testing with acute rabbit and porcine models, the device is proven to have robust and intimate tissue coupling while maintaining its chemical, mechanical, and electrical properties during the cardiac cycle. The elastrode array records epicardial atrial signals with comparable efficacy to currently available endocardial-mapping techniques but with 2 times higher atrial-to-ventricular signal ratio and >100 times higher spatial resolution and can reliably identify electrical local heterogeneity within an area of simultaneously identified rotor-like electrical patterns in a porcine model of chronic AF.

Water reuse in the post-tanning process: minimizing environmental impact of leather production.

Post-tanning wastewater is very diversified, as the post-tanning stage should meet the desirable properties of the leather for the final product, with low standardization of the process (compared to beamhouse and tanning). This makes post-tanning effluent reuse less feasible, and reuse in the post-tanning stage still needs to be explored. This work aims to evaluate the reuse of liquid effluents in the post-tanning process. The work methodology consisted of (i) characterization of water streams (groundwater, liquid effluent after primary treatment, and liquid effluent after secondary treatment); (ii) pilot-scale post-tanning tests using groundwater, primary effluent, and secondary effluent; (iii) characterization of the residual baths from pilot-scale tests (pH, conductivity, total solids, chemical oxygen demand, biochemical oxygen demand, chloride, hardness and oil and grease); and (iv) testing the leather obtained for total sulfated ash and organoleptic properties. Results showed that the primary effluent and the secondary effluent could be reused in pilot-scale post-tanning tests. There was an increase in the conductivity of the residual baths when liquid effluents were reused, which confirms the accumulation of salts in the effluents after their reuse.

Metallothionein isoforms as double agents - Their roles in carcinogenesis, cancer progression and chemoresistance.

Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.

Novel three-dimensional imaging approach for cryoballoon navigation and confirmation of pulmonary vein occlusion.

BACKGROUND: Cryoballoon apposition is crucial for durable pulmonary vein isolation (PVI) in atrial fibrillation, yet the balloon is difficult to visualize by conventional mapping systems, and pulmonary venography may miss small or out-of-plane leaks. We report a novel imaging system that offers real-time 3D navigation of the cryoballoon within atrial anatomy that may circumvent these issues. METHODS AND RESULTS: A novel overlay guidance system (OGS) registers already-acquired segmented atrial cardiac tomography (CT) with fluoroscopy, enabling real-time visualization of the cryoballoon within tomographic left atrial imaging during PVI. Phantom experiments in a patient-specific 3D printed left atrium showed feasibility for confirming PV apposition and leaks. We applied OGS prospectively to 68 PVs during PVI in 17 patients. The cryoballoon was successfully reconstructed in all cases, and its apposition was compared to concurrent PV venography. The OGS uncovered leaks undetected by venography in nine veins (eight cases), which enabled repositioning, confirming apposition in remaining 68 veins. Concordance of OGS to venography was 83.8% (χ2 , P < .01) CONCLUSIONS: We report a new system for real-time imaging of cryoballoon catheters to ensure PV apposition within the tomography of the left atrium. While providing high concordance with other imaging modalities for confirming balloon apposition or leak, the system also identified leaks missed by venography. Future studies should determine if this tool can provide a new reference for cryoballoon positioning.

Soil protein as a potential antimicrobial agent against methicillin -resistant Staphylococcus aureus.

Recently, the interest is increasing to find alternatives to replace the usage of antibiotics since their massive and improper usage enhance the antibiotic resistance in human pathogens. In this study, for the first time we showed that the soil proteins have very high antibacterial activity (98% of growth inhibition) against methicillin resistant Staphylococcus aureus (MRSA), one of the most threatening human pathogens. We found that the protein extract (C3) from the forest with past intensive management showed higher antibacterial activity than that of unmanaged forest. The MIC and IC50 were found to be 30 and 15.0 µg protein g-1 dry soil respectively. C3 was found to kill the bacteria by cell wall disruption and genotoxicity which was confirmed by optical and fluorescent microscopy and comet assay. According to qPCR study, the mecA (the antibiotic resistant gene) expression in MRSA was found to be down-regulated after C3 treatment. In contrast, C3 showed no hemolytic toxicity on human red blood cells which was confirmed by hemolytic assay. According to ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), 144 proteins were identified in C3 among which the majority belonged to Gram negative bacteria (45.8%). Altogether, our results will help to develop novel, cost-effective, non-toxic and highly efficient antibacterial medicines from natural sources against antibiotic resistant infections.

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells.

BACKGROUND: Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS: The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% ß-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION: This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

Drug Sequestration in Lysosomes as One of the Mechanisms of Chemoresistance of Cancer Cells and the Possibilities of Its Inhibition.

Resistance to chemotherapeutics and targeted drugs is one of the main problems in successful cancer therapy. Various mechanisms have been identified to contribute to drug resistance. One of those mechanisms is lysosome-mediated drug resistance. Lysosomes have been shown to trap certain hydrophobic weak base chemotherapeutics, as well as some tyrosine kinase inhibitors, thereby being sequestered away from their intracellular target site. Lysosomal sequestration is in most cases followed by the release of their content from the cell by exocytosis. Lysosomal accumulation of anticancer drugs is caused mainly by ion-trapping, but active transport of certain drugs into lysosomes was also described. Lysosomal low pH, which is necessary for ion-trapping is achieved by the activity of the V-ATPase. This sequestration can be successfully inhibited by lysosomotropic agents and V-ATPase inhibitors in experimental conditions. Clinical trials have been performed only with lysosomotropic drug chloroquine and their results were less successful. The aim of this review is to give an overview of lysosomal sequestration and expression of acidifying enzymes as yet not well known mechanism of cancer cell chemoresistance and about possibilities how to overcome this form of resistance.

Proteomic Signature of Neuroblastoma Cells UKF-NB-4 Reveals Key Role of Lysosomal Sequestration and the Proteasome Complex in Acquiring Chemoresistance to Cisplatin.

Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analyzed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4CDDP) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analyzed using nano liquid chromatography with tandem mass spectrometry. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ATP-binding cassette superfamily proteins (ATP = adenosine triphosphate), solute carrier-mediated trans-membrane transporters, proteasome complex subunits, and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of the key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP.

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes.

BACKGROUND: Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. METHODS: To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. RESULTS: We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. CONCLUSIONS: Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets.

The Application of Curve Fitting on the Voltammograms of Various Isoforms of Metallothioneins-Metal Complexes.

The translation of metallothioneins (MTs) is one of the defense strategies by which organisms protect themselves from metal-induced toxicity. MTs belong to a family of proteins comprising MT-1, MT-2, MT-3, and MT-4 classes, with multiple isoforms within each class. The main aim of this study was to determine the behavior of MT in dependence on various externally modelled environments, using electrochemistry. In our study, the mass distribution of MTs was characterized using MALDI-TOF. After that, adsorptive transfer stripping technique with differential pulse voltammetry was selected for optimization of electrochemical detection of MTs with regard to accumulation time and pH effects. Our results show that utilization of 0.5 M NaCl, pH 6.4, as the supporting electrolyte provides a highly complicated fingerprint, showing a number of non-resolved voltammograms. Hence, we further resolved the voltammograms exhibiting the broad and overlapping signals using curve fitting. The separated signals were assigned to the electrochemical responses of several MT complexes with zinc(II), cadmium(II), and copper(II), respectively. Our results show that electrochemistry could serve as a great tool for metalloproteomic applications to determine the ratio of metal ion bonds within the target protein structure, however, it provides highly complicated signals, which require further resolution using a proper statistical method, such as curve fitting.

Effects of a Resistance and Stretching Training Program on Forward Head and Protracted Shoulder Posture in Adolescents.

OBJECTIVE: The purpose of this study was to evaluate the effects of a 16-week resistance and stretching training program applied in physical education (PE) classes on forward head posture and protracted shoulder posture in Portuguese adolescents. METHODS: This prospective, randomized, controlled study was conducted in 2 secondary schools. One hundred and thirty adolescents (aged 15-17 years) with forward head and protracted shoulder posture were randomly assigned to a control or experimental group. Sagittal head, cervical, and shoulder angles were measured with photogrammetry and Postural Assessment Software. The American Shoulder and Elbow Surgeons Shoulder Assessment was used to assess shoulder pain, and neck pain during the last month was self-reported with a single question. These variables were assessed before and after a 16-week intervention period. The control group (n = 46) attended the PE classes, whereas the exercise group (n = 84) received a posture corrective exercise program in addition to PE classes. RESULTS: A significant increase in cervical and shoulder angles was observed in the intervention group from pretest to posttest (P < .05). For the shoulder pain scores in both groups, there were no significant changes after the 16 weeks. CONCLUSIONS: A 16-week resistance and stretching training program decreased forward head and protracted shoulder postures in adolescents.

Noninvasive Estimation of Epicardial Dominant High-Frequency Regions During Atrial Fibrillation.

INTRODUCTION: Ablation of high dominant frequency (DF) sources in patients with atrial fibrillation (AF) is an effective treatment option for paroxysmal AF. The aim of this study was to evaluate the accuracy of noninvasive estimation of DF and electrical patterns determination by solving the inverse problem of the electrocardiography. METHODS: Four representative AF patients with left-to-right and right-to-left atrial DF patterns were included in the study. For each patient, intracardiac electrograms from both atria were recorded simultaneously together with 67-lead body surface recordings. In addition to clinical recordings, realistic mathematical models of atria and torso anatomy with different DF patterns of AF were used. For both mathematical models and clinical recordings, inverse-computed electrograms were compared to intracardiac electrograms in terms of voltage, phase, and frequency spectrum relative errors. RESULTS: Comparison between intracardiac and inverse computed electrograms for AF patients showed 8.8 ± 4.4% errors for DF, 32 ± 4% for voltage, and 65 ± 4% for phase determination. These results were corroborated by mathematical simulations showing that the inverse problem solution was able to reconstruct the frequency spectrum and the DF maps with relative errors of 5.5 ± 4.1%, whereas the reconstruction of the electrograms or the instantaneous phase presented larger relative errors (i.e., 38 ± 15% and 48 ± 14 % respectively, P < 0.01). CONCLUSIONS: Noninvasive reconstruction of atrial frequency maps can be achieved by solving the inverse problem of electrocardiography with a higher accuracy than temporal distribution patterns.

Treatment with Parkinsonia aculeata combats insulin resistance-induced oxidative stress through the increase in PPARγ/CuZn-SOD axis expression in diet-induced obesity mice.

Parkinsonia aculeata L. (Caesalpiniaceae) is a traditional ethnomedicine and has been used for the empiric treatment of hyperglycemia, without scientific background. Mechanistic analyses at molecular level from the antioxidant mechanism observed by P. aculeata are required. Herein the effects of the treatment by hydroethanolic extract partitioned with ethyl acetate of P. aculeata aerial parts (HEPa/EtOAc) in mice fed a high-fat diet that share many obesity phenotypes with humans were evaluated. The animals were treated orally with HEPa/EtOAc (125 and 250 mg/kg/day) and pioglitazone (5 mg/kg/day), for 16 days. After the treatment, HEPa/EtOAc reduced fasting serum glucose and insulin levels, as well as homeostasis model assessment for insulin resistance. In addition, an improvement in glucose intolerance was also observed. Indeed, a reduction in the circulating levels of TNF-α and IL-6 was also observed. Furthermore, at molecular level, it was demonstrated that the HEPa/EtOAc treatment was able to improve these physiological parameters, through the activation of peroxisome proliferator-activated receptor γ (PPARγ) per si, as well as the enhancement of antioxidant mechanism by an increase in PPARγ/Cu(2+), Zn(2+)-superoxide dismutase (CuZn-SOD) axis expression in liver and adipose tissue. In sum, P. aculeata is effective to improve insulin resistance in a mouse model of obesity and this effect seems to involve the antioxidant and anti-inflammatory mechanisms through the increase in PPARγ/CuZn-SOD axis expression.

Transport phenomena of nanoparticles in plants and animals/humans.

The interaction of a plethora nanoparticles with major biota such as plants and animals/humans has been the subject of various multidisciplinary studies with special emphasis on toxicity aspects. However, reports are meager on the transport phenomena of nanoparticles in the plant-animal/human system. Since plants and animals/humans are closely linked via food chain, discussion is imperative on the main processes and mechanisms underlying the transport phenomena of nanoparticles in the plant-animal/human system, which is the main objective of this paper. Based on the literature appraised herein, it is recommended to perform an exhaustive exploration of so far least explored aspects such as reproducibility, predictability, and compliance risks of nanoparticles, and insights into underlying mechanisms in context with their transport phenomenon in the plant-animal/human system. The outcomes of the suggested studies can provide important clues for fetching significant benefits of rapidly expanding nanotechnology to the plant-animal/human health-improvements and protection as well.

3D-printed chip for detection of methicillin-resistant Staphylococcus aureus labeled with gold nanoparticles.

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen occurring not only in hospitals but also in foodstuff. Currently, discussions on the issue of the increasing resistance, and timely and rapid diagnostic of resistance strains have become more frequent and sought. Therefore, the aim of this study was to design an effective platform for DNA isolation from different species of microorganisms as well as the amplification of mecA gene that encodes the resistance to ß-lactam antibiotic formation and is contained in MRSA. For this purpose, we fabricated 3D-printed chip that was suitable for bacterial cultivation, DNA isolation, PCR, and detection of amplified gene using gold nanoparticle (AuNP) probes as an indicator of MRSA. Confirmation of the MRSA presence in the samples was based on a specific interaction between mecA gene with the AuNP probes and a colorimetric detection, which utilized the noncross-linking aggregation phenomenon of DNA-functionalized AuNPs. To test the whole system, we analyzed several real refractive indexes, in which two of them were positively scanned to find the presence of mecA gene. The aggregation of AuNP probes were reflected by 75% decrease of absorbance (λ = 530 nm) and change in AuNPs size from 3 ± 0.05 to 4 ± 0.05 nm (n = 5). We provide the one-step identification of mecA gene using the unique platform that employs the rapid, low-cost, and easy-to-use colorimetric method for MRSA detection in various samples.

Intrarater and interrater reliability of photographic measurement of upper-body standing posture of adolescents.

OBJECTIVE: The purposes of this study were to determine the intrarater and interrater reliability of a photographic measurement of the sagittal postures of the cervical spine and shoulder, quantitatively characterize the postural alignment of the head and shoulders in the sagittal plane of Portuguese adolescents 15 to 17 years old in natural erect standing, and analyze differences in postural angles between sexes. METHODS: This cross-sectional study was conducted in 2 secondary schools in Portugal where 275 adolescent students (146 females and 129 males) aged 15 to 17 years were evaluated. Sagittal head, cervical, and shoulder angles were measured with photogrammetry and the Postural Assessment Software. RESULTS: For interrater reliability, all of the intraclass correlation coefficient (ICC) values for the 3 angles were higher than 0.85. For intrarater reliability, the ICC values for the sagittal head angle, shoulder angle, and cervical angle were 0.83, 0.78, and 0.66, respectively. Mean values of sagittal head, cervical, and shoulder angles were 17.2° ± 5.7°, 47.4° ± 5.2°, and 51.4° ± 8.5°, respectively. Anterior head carriage was demonstrated by 68% of the adolescents, whereas 58% had protraction of the shoulder(s). Males had significantly higher mean cervical and sagittal head angles. CONCLUSIONS: Forward head posture and protracted shoulders were common postural disorders in adolescents 15 to 17 years old, with females revealing a lower mean cervical angle. The intrarater and interrater evaluation of standing sagittal posture of the cervical spine and shoulders by photogrammetry was reliable.

Liposomal nanotransporter for targeted binding based on nucleic acid anchor system.

Microfluidic techniques have been developed intensively in recent years due to lower reagent consumption, faster analysis, and possibility of the integration of several analytical detectors into one chip. Electrochemical detectors are preferred in microfluidic systems, whereas liposomes can be used for amplification of the electrochemical signals. The aim of this study was to design a nanodevice for targeted anchoring of liposome as transport device. In this study, liposome with encapsulated Zn(II) was prepared. Further, gold nanoparticles were anchored onto the liposome surface allowing binding of thiol moiety-modified molecules (DNA). For targeted capturing of the transport device, DNA loops were used. DNA loops were represented by paramagnetic microparticles with oligo(DT)25 chain, on which a connecting DNA was bound. Capturing of transport device was subsequently done by hybridization to the loop. The individual steps were analyzed by electrochemistry and UV/Vis spectrometry. For detection of Zn(II) encapsulated in liposome, a microfluidic system was used. The study succeeded in demonstrating that liposome is suitable for the transport of Zn(II) and nucleic acids. Such transporter may be used for targeted binding using DNA anchor system.