Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients.

In the early phase of the pandemic, we were among the first to postulate that neutrophil extracellular traps (NETs) play a key role in COVID-19 pathogenesis. This exploratory prospective study based on 279 individuals showed that plasma levels of neutrophil elastase, myeloperoxidase and circulating DNA of nuclear and mitochondrial origins in nonsevere (NS), severe (S) and postacute phase (PAP) COVID-19 patients were statistically different as compared to the levels in healthy individuals, and revealed the high diagnostic power of these NETs markers in respect to the disease severity. The diagnostic power of NE, MPO, and cir-nDNA as determined by the Area Under Receiver Operating Curves (AUROC) was 0.95, 097, and 0.64; 0.99, 1.0, and 0.82; and 0.94, 1.0, and 0.93, in NS, S, and PAP patient subgroups, respectively. In addition, a significant fraction of NS, S as well as of PAP patients exhibited aCL IgM/IgG and anti-B2GP IgM/IgG positivity. We first demonstrate persistence of these NETs markers in PAP patients and consequently of sustained innate immune response imbalance, and a prolonged low-level pro-thrombotic potential activity highlighting the need to monitor these markers in all COVID-19 PAP individuals, to investigate postacute COVID-19 pathogenesis following intensive care, and to better identify which medical resources will ensure complete patient recovery.

Room temperature deoxofluorination of aromatic aldehydes with XtalFluor-E under highly concentrated conditions.

The synthesis of difluoromethyl-containing compounds exploiting the deoxofluorination reaction of aromatic aldehydes using XtalFluor-E is described. This transformation occurs at room temperature under highly concentrated conditions, i.e., with no added solvent. A wide range of difluoromethyl-containing compounds was obtained in 21 to 87% isolated yields.

Fast and ergonomic extraction of adherent mammalian cells for NMR-based metabolomics studies.

Cellular metabolomics has become key to elucidate mechanistic aspects in various fields such as cancerology or pharmacology, and is rapidly becoming a standard phenotyping tool accessible to the broad biological community. Acquisition of reliable spectroscopic datasets, such as nuclear magnetic resonance (NMR) spectra, to characterize biological systems depends on the elaboration of robust methods for cellular metabolites extraction. Previous studies have addressed many issues raised by these protocols, however with little pondering on ergonomic and practical aspects of the methods that impact their scalability, reproducibility and hence their suitability to high-throughput studies or their use by non-metabolomics experts. Here, we optimize a fast and ergonomic protocol for extraction of metabolites from adherent mammalian cells for NMR metabolomics studies. The proposed extraction protocol, including cell washing, metabolism quenching and actual extraction of intracellular metabolites, was first optimized on HeLa cells. Efficiency of the protocol, in its globality and for the different individual steps, was assessed by NMR quantification of 27 metabolites from cellular extracts. We show that a single PBS wash provides a seemly compromise between contamination from growth medium and leakage of intracellular metabolites. In HeLa cells, extraction using pure methanol, without cell scraping, recovered a higher amount of intracellular metabolites than the reference methanol/water/chloroform method with cell scraping, with yields varying across metabolite classes. Optimized and reference protocols were further tested on eight cell lines of miscellaneous nature, and inter-operator reproducibility was demonstrated. Our results stress the need for tailored extraction protocols and show that fast protocols minimizing time-consuming steps, without compromising extraction yields, are suitable for high-throughput metabolomics studies. Graphical abstract.

WatchPose: A View-Aware Approach for Camera Pose Data Collection in Industrial Environments.

Collecting correlated scene images and camera poses is an essential step towards learning absolute camera pose regression models. While the acquisition of such data in living environments is relatively easy by following regular roads and paths, it is still a challenging task in constricted industrial environments. This is because industrial objects have varied sizes and inspections are usually carried out with non-constant motions. As a result, regression models are more sensitive to scene images with respect to viewpoints and distances. Motivated by this, we present a simple but efficient camera pose data collection method, WatchPose, to improve the generalization and robustness of camera pose regression models. Specifically, WatchPose tracks nested markers and visualizes viewpoints in an Augmented Reality- (AR) based manner to properly guide users to collect training data from broader camera-object distances and more diverse views around the objects. Experiments show that WatchPose can effectively improve the accuracy of existing camera pose regression models compared to the traditional data acquisition method. We also introduce a new dataset, Industrial10, to encourage the community to adapt camera pose regression methods for more complex environments.

Pivotal roles of environmental sensing and signaling mechanisms in plant responses to climate change.

Climate change reshapes the physiology and development of organisms through phenotypic plasticity, epigenetic modifications, and genetic adaptation. Under evolutionary pressures of the sessile lifestyle, plants possess efficient systems of phenotypic plasticity and acclimation to environmental conditions. Molecular analysis, especially through omics approaches, of these primary lines of environmental adjustment in the context of climate change has revealed the underlying biochemical and physiological mechanisms, thus characterizing the links between phenotypic plasticity and climate change responses. The efficiency of adaptive plasticity under climate change indeed depends on the realization of such biochemical and physiological mechanisms, but the importance of sensing and signaling mechanisms that can integrate perception of environmental cues and transduction into physiological responses is often overlooked. Recent progress opens the possibility of considering plant phenotypic plasticity and responses to climate change through the perspective of environmental sensing and signaling. This review aims to analyze present knowledge on plant sensing and signaling mechanisms and discuss how their structural and functional characteristics lead to resilience or hypersensitivity under conditions of climate change. Plant cells are endowed with arrays of environmental and stress sensors and with internal signals that act as molecular integrators of the multiple constraints of climate change, thus giving rise to potential mechanisms of climate change sensing. Moreover, mechanisms of stress-related information propagation lead to stress memory and acquired stress tolerance that could withstand different scenarios of modifications of stress frequency and intensity. However, optimal functioning of existing sensors, optimal integration of additive constraints and signals, or memory processes can be hampered by conflicting interferences between novel combinations and novel changes in intensity and duration of climate change-related factors. Analysis of these contrasted situations emphasizes the need for future research on the diversity and robustness of plant signaling mechanisms under climate change conditions.

Multimodal acquisition of articulatory data: Geometrical and temporal registration.

Acquisition of dynamic articulatory data is of major importance for studying speech production. It turns out that one technique alone often is not enough to get a correct coverage of the whole vocal tract at a sufficient sampling rate. Ultrasound (US) imaging has been proposed as a good acquisition technique for the tongue surface because it offers a good temporal sampling, does not alter speech production, is cheap, and is widely available. However, it cannot be used alone and this paper describes a multimodal acquisition system which uses electromagnetography sensors to locate the US probe. The paper particularly focuses on the calibration of the US modality which is the key point of the system. This approach enables US data to be merged with other data. The use of the system is illustrated via an experiment consisting of measuring the minimal tongue to palate distance in order to evaluate and design Magnetic Resonance Imaging protocols well suited for the acquisition of three-dimensional images of the vocal tract. Compared to manual registration of acquisition modalities which is often used in acquisition of articulatory data, the approach presented relies on automatic techniques well founded from geometrical and mathematical points of view.

Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition.

BACKGROUND: Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues. METHODS: We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel(®)). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential. RESULTS: In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes. CONCLUSIONS: The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.

Correlation of a self-report and direct measure of physical activity level in the electron-beam tomography and risk assessment among Japanese and US Men in the post World War II birth cohort (ERA JUMP) study.

BACKGROUND: Physical activity (PA) is complex and a difficult behavior to assess as there is no ideal assessment tool(s) that can capture all contexts of PA. Therefore, it is important to understand how different assessment tools rank individuals. We examined the extent to which self-report and direct assessment PA tools yielded the same ranking of PA levels. METHODS: PA levels were measured by the Modifiable Activity Questionnaire (MAQ) and pedometer at baseline among 855 white (W), African-American (AA), Japanese-American (JA), and Korean (K) men (mean age 45.3 years) in 3 geographic locations in the ERA JUMP study. RESULTS: Korean men were more active than W, AA, and JA men, according to both the MAQ and pedometer (MAQ total PA [mean ± SD]: 41.6 ± 17.8, 20.9 ± 9.9, 20.0 ± 9.1, and 29.4 ± 10.3 metabolic equivalent [MET] hours/week, respectively; pedometer: 9584.4 ± 449.4, 8363.8 ± 368.6, 8930.3 ± 285.6, 8335.7 ± 368.6 steps/day, respectively). Higher levels of total PA in Korean men, as shown by MAQ, were due to higher occupational PA. Spearman correlations between PA levels reported on the MAQ and pedometer indicated positive associations ranging from rho = 0.29 to 0.42 for total activity, rho = 0.13 to 0.35 for leisure activity, and rho = 0.10 to 0.26 for occupational activity. CONCLUSIONS: The 2 assessment methods correlated and were complementary rather than interchangeable. The MAQ revealed why Korean men were more active. In some subpopulations it may be necessary to assess PA domains other than leisure and to use more than 1 assessment tool to obtain a more representative picture of PA levels.

Deformation-based augmented reality for hepatic surgery.

In this paper we introduce a method for augmenting the laparoscopic view during hepatic tumor resection. Using augmented reality techniques, vessels, tumors and cutting planes computed from pre-operative data can be overlaid onto the laparoscopic video. Compared to current techniques, which are limited to a rigid registration of the pre-operative liver anatomy with the intra-operative image, we propose a real-time, physics-based, non-rigid registration. The main strength of our approach is that the deformable model can also be used to regularize the data extracted from the computer vision algorithms. We show preliminary results on a video sequence which clearly highlights the interest of using physics-based model for elastic registration.

Rhodium-Catalyzed Intramolecular Cyclopropanation of Trifluoromethyl- and Pentafluorosulfanyl-Substituted Allylic Cyanodiazoacetates.

The synthesis of trifluoromethyl (CF3)- and pentafluorosulfanyl (SF5)-substituted cyclopropane-fused γ-lactones was carried out through Rh2(esp)2-catalyzed intramolecular cyclopropanation in up to 99% yields. Twelve examples of this interesting scaffold are reported, as well as postfunctionalizations that provide access to highly functionalized CF3- and SF5-substituted cyclopropanes. These novel SF5-substituted analogues join the very short list of available pentafluorosulfanyl intermediates.

Warburg-associated acidification represses lactic fermentation independently of lactate, contribution from real-time NMR on cell-free systems.

Lactate accumulation and acidification in tumours are a cancer hallmark associated with the Warburg effect. Lactic acidosis correlates with cancer malignancy, and the benefit it offers to tumours has been the subject of numerous hypotheses. Strikingly, lactic acidosis enhances cancer cell survival to environmental glucose depletion by repressing high-rate glycolysis and lactic fermentation, and promoting an oxidative metabolism involving reactivated respiration. We used real-time NMR to evaluate how cytosolic lactate accumulation up to 40 mM and acidification up to pH 6.5 individually impact glucose consumption, lactate production and pyruvate evolution in isolated cytosols. We used a reductive cell-free system (CFS) to specifically study cytosolic metabolism independently of other Warburg-regulatory mechanisms found in the cell. We assessed the impact of lactate and acidification on the Warburg metabolism of cancer cytosols, and whether this effect extended to different cytosolic phenotypes of lactic fermentation and cancer. We observed that moderate acidification, independently of lactate concentration, drastically reduces the glucose consumption rate and halts lactate production in different lactic fermentation phenotypes. In parallel, for Warburg-type CFS lactate supplementation induces pyruvate accumulation at control pH, and can maintain a higher cytosolic pyruvate pool at low pH. Altogether, we demonstrate that intracellular acidification accounts for the direct repression of lactic fermentation by the Warburg-associated lactic acidosis.

Mid-Term Sequelae of Surviving Patients Hospitalized in Intensive Care Unit for COVID-19 Infection: The REHCOVER Study.

OBJECTIVES: The objective of this prospective, single-center study was to explore the mid-term outcomes 6 to 9 months after hospitalization in an Intensive Care Unit (ICU) for severe COVID-19 infection. METHODS: Patients systematically underwent biological tests, pulmonary function tests, chest computed tomography (CT) scan, and psychological tests. RESULTS: Among 86 patients, including 71 (82.6%) men, median age of 65.8 years (56.7; 72.4), 57 (71.3%) patients presented post-COVID-19 asthenia, 39 (48.1%) muscle weakness, and 30 (36.6%) arthralgia. Fifty-two (64.2%) patients had a decreased diffusion capacity for carbon monoxide (DLCO) <80% and 16 (19.8%) had DLCO <60%. Chest CT-scans showed ground glass opacities in 35 (40.7%) patients, and reticular changes in 28 patients (33.7%), including fibrosis-like changes in 18 (21.7%) patients. Reticular changes and DLCO <60% were associated with length of stay in ICU, and reticular changes with higher maximal CRP level. The psychological questionnaires found 37.7% suffered from depression, 23.5% from anxiety, 42.4% from insomnia, and 9.4% from post-traumatic stress. Being female was associated with a higher frequency of depression and anxiety, with depression scores being associated with obesity. CONCLUSIONS: Many patients hospitalized in ICU for severe COVID-19 infection have mid-term sequelae. Additional studies on the prognostic factors seem necessary.

Capturing contact in mitral valve dynamic closure with fluid-structure interaction simulation.

PURPOSE: Realistic fluid-structure interaction (FSI) simulation of the mitral valve opens the way toward planning for surgical repair. In the literature, blood leakage is identified by measuring the flow rate, but detailed information about closure efficiency is missing. We present in this paper an FSI model that improves the detection of blood leakage by building a map of contact. METHODS: Our model is based on the immersed boundary method that captures a map of contact and perfect closure of the mitral valve, without the presence of orifice holes, which often appear with existing methods. We also identified important factors influencing convergence issues. RESULTS: The method is demonstrated in three typical clinical situations: mitral valve with leakage, bulging, and healthy. In addition to the classical ways of evaluating MV closure, such as stress distribution and flow rate, the contact map provides easy detection of leakage with identification of the sources of leakage and a quality assessment of the closure. CONCLUSIONS: Our method significantly improves the quality of the simulation and allows the identification of regurgitation as well as a spatial evaluation of the quality of valve closure. Comparably fast simulation, ability to simulate large deformation, and capturing detailed contact are the main aspects of the study.

Occurrence of Candidemia in Patients with COVID-19 Admitted to Five ICUs in France.

Whether severe COVID-19 is by itself a significant risk factor for the development of candidemia currently remains an open question as conflicting results have been published. We aim to assess the occurrence of candidemia in patients with severe COVID-19 admitted to the intensive care unit (ICU). We conducted a retrospective study on patients with severe SARS-CoV-2-related pneumonia admitted to 5 ICUs in France who were specifically screened for fungal complications between March 2020 and January 2021. The study population included a total of 264 patients; the median age was 56 years old and most of them were male (n = 186; 70.5%) and immunocompetent (n = 225; 87.5%), and 62.7% (n = 153/244) were on extracorporeal membrane oxygenation support. Microbiological analysis included 4864 blood culture samples and beta-glucan test performed on 975 sera. Candidemia was diagnosed in 13 (4.9%) patients. The species involved were mainly C. albicans (n = 6) and C. parapsilosis (n = 5). Almost all patients (12/13; 92.3%) had a colonization by yeasts. ICU mortality was not significantly impacted by the occurrence of candidemia. Unrelated positive beta-glucan tests were observed in 49 patients (23.4%), including 6 with mold infections and 43 with false positive results. In our series, patients with severe SARS-CoV-2-related pneumonia seemed at low risk of developing invasive candidiasis.

Wet chemical synthesis and self-assembly of SnS2 nanoparticles on TiO2 for quantum dot-sensitized solar cells.

SnS2 nanoparticles were synthesized through a simple wet chemical process at room temperature. The SnS2 nanoparticles were approximately spherical in shape and had diameter about 3-4 nm. SnS2-sensitized TiO2 electrodes were fabricated by the immersion of chemically modified TiO2 to well-dispersed SnS2 solution for 72 h (i.e., self-assembly method.) SnS2-sensitized TiO2 electrodes were applied in quantum dot-sensitized solar cells (QDSSCs). Under AM1.5 irradiation with 100 mW/cm2 light intensity (at 1 sun), the short-circuit current density (J(sc)), the open-circuit voltage (V(oc)), the fill factor (FF), and the energy conversion efficiency (eta) were 0.47 mA/cm2, 0.29 V, 0.58 and 0.081%, respectively.

Synthesis of SnS nanoparticles by SILAR method for quantum dot-sensitized solar cells.

SnS-sensitized TiO2 electrodes were applied in quantum dot-sensitized solar cells (QDSSCs) which are environmentally more favorable than conventional Cd or Pb-chalcogenide-sensitized electrodes. SnS nanoparticles were well-distributed over the surface of TiO2 nanoparticles by the successive ionic layer adsorption and reaction (SILAR) method. Deposited SnS nanoparticles had diameter about 3 nm. Under AM1.5 irradiation with 100 mW/cm2 light intensity (at 1 sun), the energy conversion efficiency of obtained cells reached a value of 0.21% (0.25 cm2) at SILAR coating cycles of 5. In addition, the photovoltaic performance was improved by additional ZnS coating on the surface of SnS-sensitized TiO2 electrodes.

Arterial stiffness, the hidden face of cardiovascular risk in autoimmune and chronic inflammatory rheumatic diseases.

BACKGROUND AND OBJECTIVE: Cardiovascular diseases (CVD) are the leading causes of death in chronic inflammatory rheumatic diseases and are not solely explained by the increased prevalence of cardiovascular (CV) risk factors in this population. Arterial stiffness, assessed primarily by pulse wave velocity (PWV) and more indirectly by augmentation index (AIx), is a surrogate marker of CVD that should be considered. The objective of this review was to investigate the relationship between arterial stiffness and chronic inflammatory and/or autoimmune diseases. METHODS: We performed a systemic literature review of articles published in Medline from January 2012 to April 2020 restricted to English languages and to human adults. We selected relevant articles about the relationship between arterial stiffness and rheumatoid arthritis, systemic lupus erythematosus, psoriasis, Sjogren's syndrome and ankylosing spondylitis. For each selected article, data on PWV and AIx were extracted and factors that may have an impact on arterial stiffness were identified. RESULTS: A total of 214 references were identified through database searching and 82 of them were retained for analysis. Arterial stiffness is increased in chronic inflammatory and autoimmune diseases. Traditional CV risk factors such as hypertension and dyslipidemia accentuate this relationship. Current data are insufficient to determine whether disease activity significantly influences arterial stiffness, whereas disease duration seems rather critical. TNF-alpha inhibitors and cardiorespiratory fitness tend to decrease arterial stiffness. Finally, increased arterial stiffness leads to diastolic dysfunction, which is the main mechanism of heart failure in chronic inflammatory rheumatic diseases. CONCLUSION: CV risk assessment in chronic inflammatory and autoimmune diseases should also rely on PWV and AIx.

C-terminal lysine clipping of IgG1: impact on binding to human FcγRIIIa and neonatal Fc receptors.

Monoclonal antibodies (mAbs) display numerous structural attributes, some of them may impact their safety and/or efficacy profiles. C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants. If Fc-glycosylation has been by far the most documented critical quality attribute (CQA), the potential impacts of mAb C-terminal lysine content is far less reported, particularly on the ability of these basic variants to bind human Fc receptors. To address this question, three charge variant species having zero (K0), one (K1) and two (K2) C-terminal lysine(s) were isolated with high purity from an in-house human IgG1 by preparative strong-cation exchange (SCX) chromatography. A comprehensive biophysical characterization of these three fractions was undertaken, demonstrating their high similarity in terms of structural homogeneity, with a particular attention paid on their respective N-glycosylation profiles. The binding affinity of the fractions to human FcγRIIIa-Val176 was assessed both by affinity chromatography and surface plasmon resonance (SPR), and to human neonatal Fc receptor (FcRn) by affinity chromatography. Results demonstrate that the three charge variants did not show any significant binding difference for the two tested human Fc receptors, translating certainly to comparable biological properties. As a consequence, C-terminal lysine clipping of the present therapeutic IgG1 should not impact both FcRn-dependent pharmacokinetic profiles and FcγRIIIa-driven cytotoxic activities. The methods used in this study can be widely applied to other IgG1 to define criticality of the C-terminal lysine clipping as a CQA.

Darkfield and Fluorescence Macrovision of a Series of Large Images to Assess Anatomical and Chemical Tissue Variability in Whole Cross-Sections of Maize Stems.

The proportion and composition of plant tissues in maize stems vary with genotype and agroclimatic factors and may impact the final biomass use. In this manuscript, we propose a quantitative histology approach without any section labelling to estimate the proportion of different tissues in maize stem sections as well as their chemical characteristics. Macroscopic imaging was chosen to observe the entire section of a stem. Darkfield illumination was retained to visualise the whole stem cellular structure. Multispectral autofluorescence images were acquired to detect cell wall phenolic compounds after UV and visible excitations. Image analysis was implemented to extract morphological features and autofluorescence pseudospectra. By assimilating the internode to a cylinder, the relative proportions of tissues in the internode were estimated from their relative areas in the sections. The approach was applied to study a series of 14 maize inbred lines. Considerable variability was revealed among the 14 inbred lines for both anatomical and chemical traits. The most discriminant morphological descriptors were the relative amount of rind and parenchyma tissues together with the density and size of the individual bundles, the area of stem and the parenchyma cell diameter. The rind, as the most lignified tissue, showed strong visible-induced fluorescence which was line-dependant. The relative amount of para-coumaric acid was associated with the UV-induced fluorescence intensity in the rind and in the parenchyma near the rind, while ferulic acid amount was significantly correlated mainly with the parenchyma near the rind. The correlation between lignin and the tissue pseudospectra showed that a global higher amount of lignin resulted in a higher level of lignin fluorescence whatever the tissues. We demonstrated here the potential of darkfield and autofluorescence imaging coupled with image analysis to quantify histology of maize stem and highlight variability between different lines.

Automatic extraction of the mitral valve chordae geometry for biomechanical simulation.

PURPOSE: Mitral valve computational models are widely studied in the literature. They can be used for preoperative planning or anatomical understanding. Manual extraction of the valve geometry on medical images is tedious and requires special training, while automatic segmentation is still an open problem. METHODS: We propose here a fully automatic pipeline to extract the valve chordae architecture compatible with a computational model. First, an initial segmentation is obtained by sub-mesh topology analysis and RANSAC-like model-fitting procedure. Then, the chordal structure is optimized with respect to objective functions based on mechanical, anatomical, and image-based considerations. RESULTS: The approach has been validated on 5 micro-CT scans with a graph-based metric and has shown an [Formula: see text] accuracy rate. The method has also been tested within a structural simulation of the mitral valve closed state. CONCLUSION: Our results show that the chordae architecture resulting from our algorithm can give results similar to experienced users while providing an equivalent biomechanical simulation.