Novel closed-loop control system of dual rotary blood pumps in total artificial heart based on the circulatory equilibrium framework: a proof-of-concept in vivo study.

OBJECTIVE: Total artificial heart (TAH) using dual rotary blood pumps (RBPs) is a potential treatment for end-stage heart failure. A well-noted challenge with RBPs is their low sensitivity to preload, which can lead to venous congestion and ventricular suction. To address this issue, we have developed an innovative closed-loop control system of dual RBPs in TAH. This system emulates the Frank-Starling law of the heart in controlling RBPs while monitoring stressed blood volume (V) based on the circulatory equilibrium framework. We validated the system in in-vivo experiments. METHODS: In 9 anesthetized dogs, we prepared a TAH circuit using 2 centrifugal-type RBPs. We first investigated whether the flow and inlet atrial pressure in each RBP adhered to a logarithmic Frank-Starling curve. We then examined whether the RBP flows and atrial pressures were maintained stably during aortic occlusion (AO) and pulmonary cannula stenosis (PS), whether averaged flow of dual RBPs and bilateral atrial pressures were controlled to their predefined target values for a specific V, and whether this system could maintain the atrial pressures within predefined control ranges under significant changes in V. RESULTS: This system effectively emulated the logarithmic Frank-Starling curve. It robustly stabilized the flow and atrial pressures during AO and PS without venous congestion or ventricular suction, accurately achieved target values in averaged flow and atrial pressures, and efficaciously maintained these pressures within the control ranges. CONCLUSION: This system controls dual RBPs in TAH accurately and stably. SIGNIFICANCE: This system may accelerate clinical application of TAH with dual RBPs.

Influence of angiotensin II and telmisartan on in vivo high-resolution renal arterial impedance in rats.

Angiotensin II (ANGII) is known to play an important role in regulating renal hemodynamics. We sought to quantify this effect in an in vivo rat model with high-resolution renal arterial (RA) impedance. This study examines the effects of ANGII and its type 1 receptor blocker, telmisartan (TELM), on RA impedance. In baroreflex deactivated rats, we measured RA pressure (Pr) and blood flow (Fr) during random ventricular pacing to induce pressure fluctuation at three different mean Pr (60, 80, and 100 mmHg). We then estimated RA impedance as the transfer function from Fr to Pr. The RA impedance was found to align with a three-element Windkessel model consisting of proximal (Rp) and distal (Rd) resistance and compliance (C). Our study showed Rd reflected the composite characteristics of afferent and efferent arterioles. Rd increased with increasing Pr under the baseline condition with a slope of 1.03 ± 0.21 (× 10-1) min·mL-1. ANGII significantly increased the slope by 0.72 ± 0.29 (× 10-1) min·mL-1 (P < 0.05) without affecting the intercept. TELM significantly reduced the intercept by 34.49 ± 4.86 (× 10-1) mmHg·min·mL-1 (P < 0.001) from the baseline value of 37.93 ± 13.36 (× 10-1) mmHg·min·mL-1, whereas it did not affect the slope. In contrast, Rp was less sensitive than Rd to ANGII or TELM, suggesting Rp may represent the characteristics of elastic large arteries. Our findings provide valuable insights into the influence of ANGII on the dynamics of the renal vasculature.

Inverse ESPVR Estimation with Singularity Avoidance via Constrained EDPVR Parameter Optimization.

Left ventricular end-systolic elastance Ees, as an index of cardiac contractility, can play a key role in continuous patient monitoring during cardiac treatment scenarios such as drug therapies. The clinical feasibility of Ees estimation remains challenging because most techniques have been built on left ventricular pressure and volume, which are difficult to measure or estimate in the regular ICU/CCU setting. The purpose of this paper is to propose and validate a novel approach to estimate Ees, which is independent of left ventricular pressure and volume. Our methods first derive an analytical representation of Ees as the inverse function of the gradient of the Frank-Starling Curve based on cardiac mechanics. Second, elucidating the mechanism of singularities in the inverse function, we derive multiple conditions in both end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relationship (EDPVR) parameters to avoid these singularities analytically. Third, we formulate a constrained nonlinear least squares problem to optimize both ESPVR and EDPVR parameters simultaneously to avoid singularities. The effectiveness of the proposed method in avoiding singularities was evaluated in an animal experiment. Compared to the conventional Ees estimation by linear regression, our proposed method reproduced in-vivo hemodynamics more accurately when simulating the estimated Ees variation during drug administration. Our method can be applied using the available data in the regular ICU/CCU setting. The improved clinical feasibility can support not only physicians' decision-making, including adjusting drug dosages in current clinical treatment, but also a closed-loop hemodynamic control system requiring accurate continuous Ees estimation.

Analytical Representation of Four-dimensional Hemodynamics for Drug Therapy Simulation in Acute Heart Failure Treatment.

Acute heart failure imperils multiple organs, including the heart. Elucidating the impact of drug therapies across this multidimensional hemodynamic system remains a challenge. This paper proposes a simulator that analyzes the impact of drug therapies on four dimensions of hemodynamics: left atrial pressure, cardiac output, mean arterial pressure, and myocardial oxygen consumption. To mathematically formulate hemodynamics, the analytical solutions of four-dimensional hemodynamics and the direction of its change are derived as functions of cardiovascular parameters: systemic vascular resistance, cardiac contractility, heart rate, and stressed blood volume. Furthermore, a drug library which represents the multi-dependency effect of drug therapies on cardiovascular parameters was identified in animal experiments. In evaluating the accuracy of our derived hemodynamic direction, the average angular error of predicted versus observed direction was 18.85[deg] after four different drug infusions for acute heart failure in animal experiments. Finally, the impact of drug therapies on four-dimensional hemodynamics was analyzed in three different simulation settings. One result showed that, even when drug therapies were simulated with simple rules according to the Forrester classification, the predicted direction of hemodynamic change matched the expected direction in more than 80% in 963 different AHF patient scenarios. Our developed simulator visualizes the impact of drug therapies on four-dimensional hemodynamics so intuitively that it can support clinicians' decision-making to protect multiple organs.

System Design for Optimizing Drug Infusions Using Cardiovascular Space Mapping for Acute Heart Failure.

Acute heart failure is caused by various factors and requires multiple drug therapies to remedy underlying causes. Due to the complexity of pharmacologic effects of cardiovascular agents, few studies have theoretically addressed the multidrug optimization problem. This paper proposes a drug infusion system for acute heart failure that controls cardiovascular performance metrics (cardiac output, left atrial pressure, and mean arterial pressure) within desired ranges as dictated by the cardiovascular parameters (systemic vascular resistance, cardiac contractility, heart rate, and stressed blood volume). The key to our system design is modeling and controlling cardiovascular parameters to yield the desired cardiovascular metrics. A 'tailored drug infusion' technique controls parameters by solving the optimization problem in order to conquer the complexity of multi-dependencies and the different dosage limits among multiple drugs. A 'cardiovascular space mapping' technique identifies the desired parameters from the desired metrics by deriving the analytical solutions of the metrics as functions of the parameters. To facilitate clinical discussions, parameters were set to realistic values in 5,600 simulated patients. Our results showed not only that the optimized drug combinations and dosages controlled the cardiovascular metrics to within the desired ranges, but also that they mostly corresponded to the recommended clinical use guidelines. An additional value of our system is that it proactively predicts the limitations of the tailored drug therapy, which supports the clinical decision of pivoting to alternative treatment strategies such as mechanical circulatory support.

A Reprieve from US wildlife mortality on roads during the COVID-19 pandemic.

High traffic volume is one of the main contributors to wildlife-vehicle collision (WVC) and wildlife mortality on roads. Government shelter-in-place (SIP) orders have been used to help mitigate the spread of COVID-19, resulting in unprecedented reductions in global traffic volumes. Using traffic and collision data from four US states (California, Idaho, Maine, and Washington), we investigated changes in total WVC, following the state and local SIP orders. From mid-March to mid-April 2020, these orders have resulted in up to 71%, 63%, 73%, and 72% reduction in driving, as measured by vehicle miles traveled (VMT), in CA, ID, ME, and WA respectively. The daily WVC rates from the 4 weeks prior to SIP orders going into effect, to the 4 weeks after, declined 34%, with 21, 36, 44, and 33% declines for CA, ID, ME, and WA, respectively. For mountain lions (Puma concolor) in CA, there was a 58% decline in mortality during the traffic reduction. The changes in WVC from 1 month pre-SIP orders to 1 month post-order only occurred in 2020 and not 2015, 2016, 2017, 2018, or 2019, suggesting that the reductions were associated with the reductions in traffic. The measured declines in WVC reversed in ME and WA during May, June and July 2020, paralleling reversals in traffic volumes. A 34% reduction in WVC would potentially equate to 10s of millions fewer vertebrates killed on US roadways during one month of traffic reduction, representing an unintentional conservation action unprecedented in modern times.

Calibrator material selection: a key criteria during biomarker assay method development.

Biomarker assay method development is a multistep rigorous process and calibrant material selection is integral to ensuring the quality of such assays. However, the impact of selection of calibrator material may often get overlooked. In this article, we highlight three case studies where biomarker calibrant material selection was deemed an essential criterion for consideration. Through these case studies we highlight challenges faced, steps taken and discuss the impact on assay-related decision-making. We also provide additional perspectives for selection and characterization of calibrant proteins in the setting of an evolving biomarker context of use.

Integration of Acoustic Liquid Handling into Quantitative Analysis of Biological Matrix Samples.

Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.

Considerations for Soluble Protein Biomarker Blood Sample Matrix Selection.

Blood-based soluble protein biomarkers provide invaluable clinical information about patients and are used as diagnostic, prognostic, and pharmacodynamic markers. The most commonly used blood sample matrices are serum and different types of plasma. In drug development research, the impact of sample matrix selection on successful protein biomarker quantification is sometimes overlooked. The sample matrix for a specific analyte is often chosen based on prior experience or literature searches, without good understanding of the possible effects on analyte quantification. Using a data set of 32 different soluble protein markers measured in matched serum and plasma samples, we examined the differences between serum and plasma and discussed how platelet or immune cell activation can change the quantified concentration of the analyte. We have also reviewed the effect of anticoagulant on analyte quantification. Finally, we provide specific recommendations for biomarker sample matrix selection and propose a systematic and data-driven approach for sample matrix selection. This review is intended to raise awareness of the impact and considerations of sample matrix selection on biomarker quantification.

ESOPHAGEAL MEASUREMENT OF CORE BODY TEMPERATURE IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS).

Cold-stress syndrome (CSS) is one of the leading natural threats to free-ranging Florida manatees (Trichechus manatus latirostris). Cold water exposure below the species' acceptable physiologic range is a frequent occurrence for manatees during cold weather months causing CSS-induced systemic illness and significant annual mortality. Although CSS is a commonly presented condition at manatee rehabilitation facilities, the core body temperatures in CSS manatees are currently unknown due to the lack of clinically applicable and accurate temperature measurement methodologies. Our objective was to establish a clinically applicable measurement methodology of core body temperature in manatees. A novel, minimally invasive temperature technique to obtain esophageal temperature by placing a temperature sensor through an oro-gastric tube was compared to current oral and nasal methods in 20 clinically healthy manatees. Results identified the esophageal measurement as the best performing and most precise temperature methodology. The superior performance of esophageal temperature measurements differed significantly from both nasal and oral measurements, while nasal and oral measurements did not differ when compared with each other. The esophageal measurements were consistent with manatee core body temperature, facilitating generation of a reference interval for core body temperature in healthy manatees (35.0-35.8 C). Four CSS medical cases were evaluated with the newly validated esophageal temperature method, facilitating diagnosis of hypothermia. The application of this temperature measurement technique to CSS manatees in field or rehabilitation settings will help in understanding CSS pathophysiology, improve medical assessments during rehabilitation, and contribute to conservation efforts.

Surface plasmon resonance as a tool for ligand-binding assay reagent characterization in bioanalysis of biotherapeutics.

Ligand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process. By evaluating surface plasmon resonance technology that offers high-throughput kinetic information, this article aims to provide perspectives on applying the surface plasmon resonance technology to strategic LBA critical reagent screening and characterization supported by a number of case studies from multiple biotherapeutic programs.

Highly selective and sensitive measurement of active forms of FGF21 using novel immunocapture enrichment with LC-MS/MS.

AIM: Recombinant FGF21 analogs are under wide ranging investigations as a potential therapeutic agent for Type 2 diabetes, as well as other metabolic disorders. The endogenous FGF21 is often used as a surrogate pharmacodynamic(PD) biomarker to assess drug efficacy and safety. Results & methodology: Immunocapture was performed using a monoclonal antibody which had been generated to bind to specific domain of native FGF21 as the capture reagent. After immunocapture, enzymatic digestion was performed and a native FGF21-specific tryptic peptide was monitored using LC-MS/MS by selective reaction monitoring. CONCLUSION: We have successfully developed and validated a bioanalytical assay which provides the specificity to differentiate the endogenous FGF21 from the recombinant therapeutic agent which has nearly identical sequence to the endogenous molecule.

Strategy for the Quantitation of a Protein Conjugate via Hybrid Immunocapture-Liquid Chromatography with Sequential HRMS and SRM-Based LC-MS/MS Analyses.

With the development of modern instrumentation and technologies, mass spectrometry based assays have played an important role in protein bioanalysis. We have developed a novel strategy by combining the "bottom-up" and "top-down" approaches using both high-resolution (HRMS) and selected reaction monitoring (SRM) based mass spectrometric detection to quantify a positron emission tomography (PET) detection tracer for an oncology marker. Monkey plasma samples were processed by immunocapture purification, followed by liquid chromatography (LC) with HRMS full scan analysis. Summed multiple charge states and multiple isotopes per charge state of the analyte were used during quantitation for optimized sensitivity. After the HRMS analysis, the remaining samples were digested by trypsin, followed by SRM detection. The HRMS approach provided the solution to a unique problem related to stability of the protein conjugate by quantifying the intact protein. The SRM method only measured a signature peptide generated from enzymatic digestion, but had a lower quantitation limit to meet the sensitivity requirement to assess the pharmacokinetics in a toxicology study. Both methods demonstrated good sensitivity, accuracy, precision and robustness, and the results revealed that there was no significant difference between the data sets obtained from both methods, indicating no in vivo or ex vivo degradation occurred in the incurred samples after dosing. This workflow not only provided the quantitative results for pharmacokinetic evaluation, but also revealed valuable in vivo stability information on the intact protein level.

Development and Validation of Electrochemiluminescence Assays to Measure Free and Total sSLAMF7 in Human Serum in the Absence and Presence of Elotuzumab.

Elotuzumab is a first in class humanized IgG1 monoclonal antibody for the treatment of multiple myeloma (MM). Elotuzumab targets the glycoprotein signaling lymphocyte activation molecule family 7 (SLAMF7, also described as CS1 or CRACC) which is expressed on the surface of myeloma cells and a subset of immune cells, including natural killer cells. A soluble version of SLAMF7 (sSLAMF7) has also been reported in MM patients but has not been evaluated as a potential biomarker following therapeutic intervention. In order to measure serum levels of sSLAMF7, two immunoassays were developed to monitor changes in circulating sSLAMF7 before and after elotuzumab treatment. Free (drug-unbound) and total (drug-bound and unbound) electrochemiluminescence (ECL) ELISA assays were developed and validated following a fit for purpose (FFP) methodology. Both assays met analytical acceptance criteria for precision, drug interference, dilution linearity, spike recovery, parallelism, and stability. Both exhibited the range and sensitivity necessary to measure clinical samples with an LLOQ of 51.2 pg/mL and ULOQs of 160 (free) and 800 ng/mL (total). Previously described assays were unable to detect sSLAMF7 in healthy individuals. However, due to the increased sensitivity of these new assays, low but measurable sSLAMF7 levels were detected in all normal healthy sera evaluated and were significantly elevated in MM patients. Cohort statistics revealed a significant increase of circulating sSLAMF7 in MM patients versus normal controls and both significant decreases in free and increases in total levels of protein post-elotuzumab treatment.

Measuring nerve growth factor in saliva by immunoassay: A cautionary note.

Nerve growth factor (NGF), a neurotrophin, modulates a diverse set of physiologic processes in the nervous, immune, and endocrine systems. Studies suggest that NGF can be measured in saliva (sNGF). Historically, the method for measuring sNGF involves the off-label use of an enzyme immunoassay designed for use with cell-culture supernatants/tissue extracts (Nam et al., 2007; Ruhl et al., 2004). In a series of experiments we reveal this measurement strategy is subject to non-specific interference by constituents present in oral fluids. We conclude that the measurement of sNGF by this assay is not optimal for use with oral fluid specimens.

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

U.S. dental students' attitudes toward research and science: impact of research experience.

This study aimed to provide a first nationwide assessment of dental students' attitudes toward the importance of research and its integration into the dental curriculum. For this purpose, the American Association for Dental Research National Student Research Group developed an online survey that was distributed to 89 percent of U.S. dental students in May 2012. The survey consisted of twenty-one Likert-type items divided into three groups: importance of research in dentistry, barriers to research involvement, and exposure to research in the dental curriculum. There were 733 responses (3.9 percent response rate), including students in all stages of education representing fifty-eight out of sixty-one dental schools. Age and race/ethnic distributions corresponded with U.S. dental school enrollees. Results showed that 63 percent of respondents had conducted research before matriculation, and of the 34 percent that participated in research during dental school, only 27 percent were newcomers. Respondents strongly agreed that scientific research enabled their progress in dentistry. Inadequate time in the curriculum was an obstacle they perceived to research involvement during dental school. Respondents agreed that dental curricula emphasize evidence-based practices but may be inadequately teaching biostatistics and research methodologies. Students with research experience tended to have stronger positive opinions about the importance of research in dental education. Efforts to foster research in schools have been well received by students, but several issues remain for enriching dental education through greater involvement of students in research.

Facilitating long-term changes in student approaches to learning science.

Undergraduates entering science curricula differ greatly in individual starting points and learning needs. The fast pace, high enrollment, and high stakes of introductory science courses, however, limit students' opportunities to self-assess and modify learning strategies. The University of Washington's Biology Fellows Program (BFP) intervenes through a 20-session, premajors course that introduces students to the rigor expected of bioscience majors and assists their development as science learners. This study uses quantitative and qualitative approaches to assess whether the 2007-2009 BFP achieved its desired short- and long-term impacts on student learning. Adjusting for differences in students' high school grade point average and Scholastic Aptitude Test scores, we found that participation in the BFP was associated with higher grades in two subsequent gateway biology courses, across multiple quarters and instructors. Two to 4 yr after participating in the program, students attributed changes in how they approached learning science to BFP participation. They reported having learned to "think like a scientist" and to value active-learning strategies and learning communities. In addition, they reported having developed a sense of belonging in bioscience communities. The achievement of long-term impacts for a short-term instructional investment suggests a practical means to prepare diverse students for the rigors of science curricula.

VEGF, PF4 and PDGF are elevated in platelets of colorectal cancer patients.

Platelets sequester angiogenesis regulatory proteins which suggests an avenue for developing biomarkers to monitor disease. We describe a comparison of angiogenesis regulatory proteins found in platelets of colorectal cancer patients and normal controls. Platelet and plasma content of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1) and endostatin in 35 patients with colon cancer were compared with 84 age-matched healthy controls using ELISAs. We standardized the platelet preparation procedure, introduced process controls and normalized the respective protein levels to platelet numbers using an actin ELISA. Statistically significant differences were found in the median levels of VEGF, PF4 and PDGF in platelets of patients with cancer compared to healthy individuals. Platelet concentrations in cancer patients versus controls were: VEGF 1.3 versus 0.6 pg/10(6), PF4 18.5 versus 9.4 ng/10(6), and PDGF 34.1 versus 21.0 pg/10(6). Multivariable logistic regression analysis indicated that PDGF, PF4 and VEGF were independent predictors of colorectal carcinoma and as a set provided statistically significant discrimination (area under the curve = 0.893, P < .0001). No significant differences were detected for bFGF, endostatin, or TSP-1. Reference Change Value analysis determined that the differences seen were not clinically significant. Plasma levels yielded no correlations.

Normal ranges of angiogenesis regulatory proteins in human platelets.

Platelets sequester angiogenesis regulatory proteins early in tumor growth, which suggests a new avenue for monitoring disease. To date, there are no clinically relevant reference ranges for markers of early angiogenesis. We introduce a new ELISA-based method for accurate and reproducible measurement of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1), fibroblast growth factor, basic (bFGF), and endostatin in platelets. To facilitate clinical applicability, the platelet levels in isolated samples were determined utilizing a new actin ELISA method. Platelets from healthy donors at single and repetitive time points were used for the assessment of normal ranges of these proteins. The physiological levels in platelets were: VEGF (0.74 +/- 0.37 pg/10(6) platelets); PDGF (23 +/- 6 pg/10(6)); PF4 (12 +/- 5 ng/10(6)); TSP-1 (31 +/- 12 ng/10(6)); bFGF (0.44 +/- 0.15 pg/10(6)); and endostatin (5.6 +/- 3.0 pg/10(6)). There was an excellent correlation (R(2) = 0.7) between the platelet levels calculated with the actin ELISA and complete blood count. The levels of the platelets were higher than those in platelet-poor plasma by factors of: VEGF (215-fold); PDGF (914-fold); PF-4 (516-fold); TSP-1 (813-fold); and bFGF (17-fold). The endostatin levels were nearly equivalent. The biovariability of the platelet proteins in eight healthy subjects over a 5-week period was found to be minimal. We describe accurate and direct measurements of the concentrations of VEGF, bFGF, PDGF, TSP-1, endostatin, and PF4 in platelets of healthy human subjects. In contrast to the highly variable levels in plasma and serum, the platelet-derived measurements were accurate and reproducible with minimal biovariability.