ALD-R491 regulates vimentin filament stability and solubility, cell contractile force, cell migration speed and directionality.

Metastasizing cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumors in the clinic. However, the role of vimentin in cell motility, and if the assembly of non-filamentous variants of vimentin into filaments regulates cell migration remains unclear. We observed that the vimentin-targeting drug ALD-R491 increased the stability of vimentin filaments, by reducing filament assembly and/or disassembly. ALD-R491-treatment also resulted in more bundled and disorganized filaments and an increased pool of non-filamentous vimentin. This was accompanied by a reduction in size of cell-matrix adhesions and increased cellular contractile forces. Moreover, during cell migration, cells showed erratic formation of lamellipodia at the cell periphery, loss of coordinated cell movement, reduced cell migration speed, directionality and an elongated cell shape with long thin extensions at the rear that often detached. Taken together, these results indicate that the stability of vimentin filaments and the soluble pool of vimentin regulate the speed and directionality of cell migration and the capacity of cells to migrate in a mechanically cohesive manner. These observations suggest that the stability of vimentin filaments governs the adhesive, physical and migratory properties of cells, and expands our understanding of vimentin functions in health and disease, including cancer metastasis.

Procedural Fundamentals for Medical Students: Institutional Outcomes of a Novel Multimodal Course.

RATIONALE AND OBJECTIVES: There is a gap in current medical student education pertaining to procedural skills' exposure and acquisition. The aim of this study is to evaluate the institutional experience of a novel medical student procedural course and its impact on procedural confidence. MATERIALS AND METHODS: This is a single-center prospective study performed at a public medical school and its associated tertiary care medical center between June 2020 and January 2021. This study was deemed exempt by our Institutional Review Board and was performed with participant consent. The multimodal course developed by the radiology department consisted of four didactic lectures, four simulation sessions, and a minimum of 16 clinical rotation hours with the department's vascular access team. Primary outcomes were assessed by comparing participant pre and post course surveys including twenty-five 5-point Likert scaled questions. RESULTS: Twenty-five self-selected students completed the course in its entirety. The curriculum and the corresponding survey analysis were stratified into sections by procedure modality. An increase in participant confidence to a moderate or greater level was observed when comparing pre and post course survey data for each procedure: vascular access (4% vs 52%, p < 0.01), thoracentesis (8% vs 48%, p < 0.01), paracentesis (8% vs 72%, p < 0.01), lumbar puncture (4% vs 44%, p < 0.01), and bone marrow biopsy (0% vs 48%, p < 0.01). CONCLUSIONS: The creation of a medical-student-centric procedural course is feasible and fills a potential gap in undergraduate medical education. This study demonstrated that a comprehensive multimodal course, designed to include didactic, simulation and clinical experiences, increases participant exposure to, participation with, and confidence in bedside procedural performance abilities.

Rewetting does not return drained fen peatlands to their old selves.

Peatlands have been drained for land use for a long time and on a large scale, turning them from carbon and nutrient sinks into respective sources, diminishing water regulation capacity, causing surface height loss and destroying biodiversity. Over the last decades, drained peatlands have been rewetted for biodiversity restoration and, as it strongly decreases greenhouse gas emissions, also for climate protection. We quantify restoration success by comparing 320 rewetted fen peatland sites to 243 near-natural peatland sites of similar origin across temperate Europe, all set into perspective by 10k additional European fen vegetation plots. Results imply that rewetting of drained fen peatlands induces the establishment of tall, graminoid wetland plants (helophytisation) and long-lasting differences to pre-drainage biodiversity (vegetation), ecosystem functioning (geochemistry, hydrology), and land cover characteristics (spectral temporal metrics). The Paris Agreement entails the rewetting of 500,000 km2 of drained peatlands worldwide until 2050-2070. A better understanding of the resulting locally novel ecosystems is required to improve planning and implementation of peatland rewetting and subsequent management.

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice.

This corrects the article DOI: 10.1038/mi.2017.68.

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice.

We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ-/- mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ-/- mice. Mechanistically, increased bacterial growth in macrophages from PKCδ-/- mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.

Comment on "Mycorrhizal association as a primary control of the CO2 fertilization effect".

Terrer et al (Reports, 1 July 2016, p. 72) used meta-analysis of carbon dioxide (CO2) enrichment experiments as evidence of an interaction between mycorrhizal symbiosis and soil nitrogen availability. We challenge their database and biomass as the response metric and, hence, their recommendation that incorporation of mycorrhizae in models will improve predictions of terrestrial ecosystem responses to increasing atmospheric CO2.

Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of 15N tracer field studies.

Effects of anthropogenic nitrogen (N) deposition and the ability of terrestrial ecosystems to store carbon (C) depend in part on the amount of N retained in the system and its partitioning among plant and soil pools. We conducted a meta-analysis of studies at 48 sites across four continents that used enriched 15N isotope tracers in order to synthesize information about total ecosystem N retention (i.e., total ecosystem 15N recovery in plant and soil pools) across natural systems and N partitioning among ecosystem pools. The greatest recoveries of ecosystem 15N tracer occurred in shrublands (mean, 89.5%) and wetlands (84.8%) followed by forests (74.9%) and grasslands (51.8%). In the short term (< 1 week after 15N tracer application), total ecosystem 15N recovery was negatively correlated with fine-root and soil 15N natural abundance, and organic soil C and N concentration but was positively correlated with mean annual temperature and mineral soil C:N. In the longer term (3-18 months after 15N tracer application), total ecosystem 15N retention was negatively correlated with foliar natural-abundance 15N but was positively correlated with mineral soil C and N concentration and C:N, showing that plant and soil natural-abundance 15N and soil C:N are good indicators of total ecosystem N retention. Foliar N concentration was not significantly related to ecosystem 15N tracer recovery, suggesting that plant N status is not a good predictor of total ecosystem N retention. Because the largest ecosystem sinks for 15N tracer were below ground in forests, shrublands, and grasslands, we conclude that growth enhancement and potential for increased C storage in aboveground biomass from atmospheric N deposition is likely to be modest in these ecosystems. Total ecosystem 15N recovery decreased with N fertilization, with an apparent threshold fertilization rate of 46 kg N x ha(-1) x yr(-1) above which most ecosystems showed net losses of applied 15N tracer in response to N fertilizer addition.

Quantifying gene network connectivity in silico: scalability and accuracy of a modular approach.

Large, complex data sets that are generated from microarray experiments, create a need for systematic analysis techniques to unravel the underlying connectivity of gene regulatory networks. A modular approach, previously proposed by Kholodenko and co-workers, helps to scale down the network complexity into more computationally manageable entities called modules. A functional module includes a gene's mRNA, promoter and resulting products, thus encompassing a large set of interacting states. The essential elements of this approach are described in detail for a three-gene model network and later extended to a ten-gene model network, demonstrating scalability. The network architecture is identified by analysing in silico steady-state changes in the activities of only the module outputs, communicating intermediates, that result from specific perturbations applied to the network modules one at a time. These steady-state changes form the system response matrix, which is used to compute the network connectivity or network interaction map. By employing a known biochemical network, the accuracy of the modular approach and its sensitivity to key assumptions are evaluated.

Fine root chemistry and decomposition in model communities of north-temperate tree species show little response to elevated atmospheric CO2 and varying soil resource availability.

Rising atmospheric [CO2] has the potential to alter soil carbon (C) cycling by increasing the content of recalcitrant constituents in plant litter, thereby decreasing rates of decomposition. Because fine root turnover constitutes a large fraction of annual NPP, changes in fine root decomposition are especially important. These responses will likely be affected by soil resource availability and the life history characteristics of the dominant tree species. We evaluated the effects of elevated atmospheric [CO2] and soil resource availability on the production and chemistry, mycorrhizal colonization, and decomposition of fine roots in an early- and late-successional tree species that are economically and ecologically important in north temperate forests. Open-top chambers were used to expose young trembling aspen (Populus tremuloides) and sugar maple (Acer saccharum) trees to ambient (36 Pa) and elevated (56 Pa) atmospheric CO2. Soil resource availability was composed of two treatments that bracketed the range found in the Upper Lake States, USA. After 2.5 years of growth, sugar maple had greater fine root standing crop due to relatively greater allocation to fine roots (30% of total root biomass) relative to aspen (7% total root biomass). Relative to the low soil resources treatment, aspen fine root biomass increased 76% with increased soil resource availability, but only under elevated [CO2]. Sugar maple fine root biomass increased 26% with increased soil resource availability (relative to the low soil resources treatment), and showed little response to elevated [CO2]. Concentrations of N and soluble phenolics, and C/N ratio in roots were similar for the two species, but aspen had slightly higher lignin and lower condensed tannins contents compared to sugar maple. As predicted by source-sink models of carbon allocation, pooled constituents (C/N ratio, soluble phenolics) increased in response to increased relative carbon availability (elevated [CO2]/low soil resource availability), however, biosynthetically distinct compounds (lignin, starch, condensed tannins) did not always respond as predicted. We found that mycorrhizal colonization of fine roots was not strongly affected by atmospheric [CO2] or soil resource availability, as indicated by root ergosterol contents. Overall, absolute changes in root chemical composition in response to increases in C and soil resource availability were small and had no effect on soil fungal biomass or specific rates of fine root decomposition. We conclude that root contributions to soil carbon cycling will mainly be influenced by fine root production and turnover responses to rising atmospheric [CO2], rather than changes in substrate chemistry.

Microbial community structure and oxidative enzyme activity in nitrogen-amended north temperate forest soils.

Large regions of temperate forest are subject to elevated atmospheric nitrogen (N) deposition which can affect soil organic matter dynamics by altering mass loss rates, soil respiration, and dissolved organic matter production. At present there is no general model that links these responses to changes in the organization and operation of microbial decomposer communities. Toward that end, we studied the response of litter and soil microbial communities to high levels of N amendment (30 and 80 kg ha(-1) yr(-1)) in three types of northern temperate forest: sugar maple/basswood (SMBW), sugar maple/red oak (SMRO), and white oak/black oak (WOBO). We measured the activity of extracellular enzymes (EEA) involved directly in the oxidation of lignin and humus (phenol oxidase, peroxidase), and indirectly, through the production of hydrogen peroxide (glucose oxidase, glyoxal oxidase). Community composition was analyzed by extracting and quantifying phospholipid fatty acids (PLFA) from soils. Litter EEA responses at SMBW sites diverged from those at oak-bearing sites (SMRO, BOWO), but the changes were not statistically significant. For soil, EEA responses were consistent across forests types: phenol oxidase and peroxidase activities declined as a function of N dose (33-73% and 5-41%, respectively, depending on forest type); glucose oxidase and glyoxal oxidase activities increased (200-400% and 150-300%, respectively, depending on forest type). Principal component analysis (PCA) ordinated forest types and treatment responses along two axes; factor 1 (44% of variance) was associated with phenol oxidase and peroxidase activities, factor 2 (31%) with glucose oxidase. Microbial biomass did not respond to N treatment, but nine of the 23 PLFA that formed >1 mol% of total biomass showed statistically significant treatment responses. PCA ordinated forest types and treatment responses along three axes (36%, 26%, 12% of variance). EEA factors 1 and 2 correlated negatively with PLFA factor 1 ( r = -0.20 and -0.35, respectively, n = 108) and positively with PLFA factor 3 ( r = +0.36 and +0.20, respectively, n = 108). In general, EEA responses were more strongly tied to changes in bacterial PLFA than to changes in fungal PLFA. Collectively, our data suggests that N inhibition of oxidative activity involves more than the repression of ligninase expression by white-rot basidiomycetes.

Fine-root biomass and fluxes of soil carbon in young stands of paper birch and trembling aspen as affected by elevated atmospheric CO2 and tropospheric O3.

Rising atmospheric CO2 may stimulate future forest productivity, possibly increasing carbon storage in terrestrial ecosystems, but how tropospheric ozone will modify this response is unknown. Because of the importance of fine roots to the belowground C cycle, we monitored fine-root biomass and associated C fluxes in regenerating stands of trembling aspen, and mixed stands of trembling aspen and paper birch at FACTS-II, the Aspen FACE project in Rhinelander, Wisconsin. Free-air CO2 enrichment (FACE) was used to elevate concentrations of CO2 (average enrichment concentration 535 µl l-1) and O3 (53 nl l-1) in developing forest stands in 1998 and 1999. Soil respiration, soil pCO2, and dissolved organic carbon in soil solution (DOC) were monitored biweekly. Soil respiration was measured with a portable infrared gas analyzer. Soil pCO2 and DOC samples were collected from soil gas wells and tension lysimeters, respectively, at depths of 15, 30, and 125 cm. Fine-root biomass averaged 263 g m-2 in control plots and increased 96% under elevated CO2. The increased root biomass was accompanied by a 39% increase in soil respiration and a 27% increase in soil pCO2. Both soil respiration and pCO2 exhibited a strong seasonal signal, which was positively correlated with soil temperature. DOC concentrations in soil solution averaged ~12 mg l-1 in surface horizons, declined with depth, and were little affected by the treatments. A simplified belowground C budget for the site indicated that native soil organic matter still dominated the system, and that soil respiration was by far the largest flux. Ozone decreased the above responses to elevated CO2, but effects were rarely statistically significant. We conclude that regenerating stands of northern hardwoods have the potential for substantially greater C input to soil due to greater fine-root production under elevated CO2. Greater fine-root biomass will be accompanied by greater soil C efflux as soil respiration, but leaching losses of C will probably be unaffected.

Genotypic variation in physiological and growth responses of Populus tremuloides to elevated atmospheric CO2 concentration.

Physiological and biomass responses of six genotypes of Populus tremuloides Michx., grown in ambient t (357 micromol mol(-1)) or twice ambient (707 micromol mol(-1)) CO2 concentration ([CO2]) and in low-N or high-N soils, were studied in 1995 and 1996 in northern Lower Michigan, USA. There was a significant CO2 x genotype interaction in photosynthetic responses. Net CO2 assimilation (A) was significantly enhanced by elevated [CO2] for five genotypes in high-N soil and for four genotypes in low-N soil. Enhancement of A by elevated [CO2] ranged from 14 to 68%. Genotypes also differed in their biomass responses to elevated [CO2], but biomass responses were poorly correlated with A responses. There was a correlation between magnitude of A enhancement by elevated [CO2] and stomatal sensitivity to CO2. Genotypes with low stomatal sensitivity to CO2 had a significantly higher A at elevated [CO2] than at ambient [CO2], but elevated [CO2] did not affect the ratio of intercellular [CO2] to leaf surface [CO2]. Stomatal conductance and A of different genotypes responded differentially to recovery from drought stress. Photosynthetic quantum yield and light compensation point were unaffected by elevated [CO2]. We conclude that P. tremuloides genotypes will respond differentially to rising atmospheric [CO2], with the degree of response dependent on other abiotic factors, such as soil N and water availability. The observed genotypic variation in growth could result in altered genotypic representation within natural populations and could affect the composition and structure of plant communities in a higher [CO2] environment in the future.

Combined effects of atmospheric CO2 and N availability on the belowground carbon and nitrogen dynamics of aspen mesocosms.

It is uncertain whether elevated atmospheric CO2 will increase C storage in terrestrial ecosystems without concomitant increases in plant access to N. Elevated CO2 may alter microbial activities that regulate soil N availability by changing the amount or composition of organic substrates produced by roots. Our objective was to determine the potential for elevated CO2 to change N availability in an experimental plant-soil system by affecting the acquisition of root-derived C by soil microbes. We grew Populus tremuloides (trembling aspen) cuttings for 2 years under two levels of atmospheric CO2 (36.7 and 71.5 Pa) and at two levels of soil N (210 and 970 µg N g-1). Ambient and twice-ambient CO2 concentrations were applied using open-top chambers, and soil N availability was manipulated by mixing soils differing in organic N content. From June to October of the second growing season, we measured midday rates of soil respiration. In August, we pulse-labeled plants with 14CO2 and measured soil 14CO2 respiration and the 14C contents of plants, soils, and microorganisms after a 6-day chase period. In conjunction with the August radio-labeling and again in October, we used 15N pool dilution techniques to measure in situ rates of gross N mineralization, N immobilization by microbes, and plant N uptake. At both levels of soil N availability, elevated CO2 significantly increased whole-plant and root biomass, and marginally increased whole-plant N capital. Significant increases in soil respiration were closely linked to increases in root biomass under elevated CO2. CO2 enrichment had no significant effect on the allometric distribution of biomass or 14C among plant components, total 14C allocation belowground, or cumulative (6-day) 14CO2 soil respiration. Elevated CO2 significantly increased microbial 14C contents, indicating greater availability of microbial substrates derived from roots. The near doubling of microbial 14C contents at elevated CO2 was a relatively small quantitative change in the belowground C cycle of our experimental system, but represents an ecologically significant effect on the dynamics of microbial growth. Rates of plant N uptake during both 6-day periods in August and October were significantly greater at elevated CO2, and were closely related to fine-root biomass. Gross N mineralization was not affected by elevated CO2. Despite significantly greater rates of N immobilization under elevated CO2, standing pools of microbial N were not affected by elevated CO2, suggesting that N was cycling through microbes more rapidly. Our results contained elements of both positive and negative feedback hypotheses, and may be most relevant to young, aggrading ecosystems, where soil resources are not yet fully exploited by plant roots. If the turnover of microbial N increases, higher rates of N immobilization may not decrease N availability to plants under elevated CO2.

Genotypic variation for condensed tannin production in trembling aspen (POPULUS TREMULOIDES, salicaceae) under elevated CO2 and in high- and low-fertility soil.

The carbon/nutrient balance hypothesis suggests that leaf carbon to nitrogen ratios influence the synthesis of secondary compounds such as condensed tannins. We studied the effects of rising atmospheric carbon dioxide on carbon to nitrogen ratios and tannin production. Six genotypes of Populus tremuloides were grown under elevated and ambient CO(2) partial pressure and high- and low-fertility soil in field open-top chambers in northern lower Michigan, USA. During the second year of exposure, leaves were harvested three times (June, August, and September) and analyzed for condensed tannin concentration. The carbon/nutrient balance hypothesis was supported overall, with significantly greater leaf tannin concentration at high CO(2) and low soil fertility compared to ambient CO(2) and high soil fertility. However, some genotypes increased tannin concentration at elevated compared to ambient CO(2), while others showed no CO(2) response. Performance of lepidopteran leaf miner (Phyllonorycter tremuloidiella) larvae feeding on these plants varied across genotypes, CO(2), and fertility treatments. These results suggest that with rising atmospheric CO(2), plant secondary compound production may vary within species. This could have consequences for plant-herbivore and plant-microbe interactions and for the evolutionary response of this species to global climate change.

Effect of measurement CO(2) concentration on sugar maple root respiration.

Accurate estimates of root respiration are crucial to predicting belowground C cycling in forest ecosystems. Inhibition of respiration has been reported as a short-term response of plant tissue to elevated measurement [CO(2)]. We sought to determine if measurement [CO(2)] affected root respiration in samples from mature sugar maple (Acer saccharum Marsh.) forests and to assess possible errors associated with root respiration measurements made at [CO(2)]s lower than that typical of the soil atmosphere. Root respiration was measured as both CO(2) production and O(2) consumption on excised fine roots ( 20,000 micro l l(-1). Root respiration was significantly affected by the [CO(2)] at which measurements were made for both CO(2) production and O(2) consumption. Root respiration was most sensitive to [CO(2)] near and below normal soil concentrations (< 1500 micro l l(-1)). Respiration rates changed little at [CO(2)]s above 3000 micro l l(-1) and were essentially constant above 6000 micro l l(-1) CO(2). These findings call into question estimates of root respiration made at or near atmospheric [CO(2)], suggesting that they overestimate actual rates in the soil. Our results indicate that sugar maple root respiration at atmospheric [CO(2)] (350 micro l l(-1)) is about 139% of that at soil [CO(2)]. Although the causal mechanism remains unknown, the increase in root respiration at low measurement [CO(2)] is significant and should be accounted for when estimating or modeling root respiration. Until the direct effect of [CO(2)] on root respiration is fully understood, we recommend making measurements at a [CO(2)] representative of, or higher than, soil [CO(2)]. In all cases, the [CO(2)] at which measurements are made and the [CO(2)] typical of the soil atmosphere should be reported.

Fine root respiration in northern hardwood forests in relation to temperature and nitrogen availability.

We examined fine-root (< 2.0 mm diameter) respiration throughout one growing season in four northern hardwood stands dominated by sugar maple (Acer saccharum Marsh.), located along soil temperature and nitrogen (N) availability gradients. In each stand, we fertilized three 50 x 50 m plots with 30 kg NO(3) (-)-N ha(-1) year(-1) and an additional three plots received no N and served as controls. We predicted that root respiration rates would increase with increasing soil temperature and N availability. We reasoned that respiration would be greater for trees using NO(3) (-) as an N source than for trees using NH(4) (+) as an N source because of the greater carbon (C) costs associated with NO(3) (-) versus NH(4) (+) uptake and assimilation. Within stands, seasonal patterns of fine-root respiration rates followed temporal changes in soil temperature, ranging from a low of 2.1 micro mol O(2) kg(-1) s(-1) at 6 degrees C to a high of 7.0 micro mol O(2) kg(-1) s(-1) at 18 degrees C. Differences in respiration rates among stands at a given soil temperature were related to variability in total net N mineralized (48-90 micro g N g(-1)) throughout the growing season and associated changes in mean root tissue N concentration (1.18-1.36 mol N kg(-1)). The hypothesized increases in respiration in response to NO(3) (-) fertilization were not observed. The best-fit model describing patterns within and among stands had root respiration rates increasing exponentially with soil temperature and increasing linearly with increasing tissue N concentration: R = 1.347Ne(0.072T) (r(2) = 0.63, P < 0.01), where R is root respiration rate ( micro mol O(2) kg(-1) s(-1)), N is root tissue N concentration (mol N kg(-1)), and T is soil temperature ( degrees C). We conclude that, in northern hardwood forests dominated by sugar maple, root respiration is responsive to changes in both soil temperature and N availability, and that both factors should be considered in models of forest C dynamics.

[Technical complications of peritoneal dialysis due to catheter dysfunction]. / Techniczne powiklania dializy otrzewnowej zwiazane z nieprawidlowym funkcjonowaniem cewnika otrzewnowego. Doswiadczenia wlasne.

The authors analysed causes of Tenckhoff peritoneal catheter dysfunction in 17 patients treated with standard peritoneal dialysis during 23.8 +/- 8.7 months. In 8 cases any catheter dysfunction were observed. Exit-site infection/tunnel infection were observed in 5 cases. Catheter obstruction or one-way obstruction of catheters caused by peritonitis was observed in 6 patients and 1/3 of implanted catheters. 3 of these patients were transferred to hemodialysis. Non-infective complications/caused mainly by catheter migration/was observed in 3 patients and 1/3 of implanted catheters. In all these cases peritoneal dialysis was stopped. Mean one-year catheter survival was 58.8%, and two-year survival was 17.6%. The authors described the technique of catheter implantation and catheter care after operation.

Non-invasive feedback of external pubococcegii muscle activity as a treatment for urinary incontinence.

Urinary incontinence may result from medical disease or surgical intervention. This article reports the treatment of 18 patients using biofeedback techniques to strengthen pubococcegii muscles and increase awareness and control of urinary function. The results indicate varying amounts of improvement, with all patients reporting benefit from the procedure.