Comprehensive workflow encompassing discovery, verification, and quantification of indicator peptide in snail mucin using LC-quadrupole Orbitrap high-resolution tandem mass spectrometry.

Peptidomics analysis was conducted using high-resolution tandem mass spectrometry (MS2) to determine the peptide profile of snail-derived mucin extract (SM). The study was also aimed to identify an indicator peptide and validate a quantification method for this peptide. The peptide profiling and identification were conducted using discovery-based peptidomics analysis employing data-dependent acquisition, whereas the selected peptides were verified and quantified using parallel reaction monitoring acquisition. Among the 16 identified peptides, the selected octapeptide (TEAPLNPK) was quantified via precursor ion ionization (m/z 435.2400), followed by quantification of the corresponding quantifier ion fragment (m/z 639.3824) using MS2. The quantification method was optimized and validated in terms of specificity, linearity, accuracy, precision, and limit of detection/quantification. The validated method accurately quantified the TEAPLNPK content in the SM as 7.5 ± 0.2 µg/g. Our study not only identifies an indicator peptide from SM but also introduces a novel validation method, involving precursor ion ionization and quantification of specific fragments. Our findings may serve as a comprehensive workflow for the monitoring, selection, and quantification of indicator peptides from diverse food resources.

Pharmacological effects of biologically synthesized ginsenoside CK-rich preparation (AceCK40) on the colitis symptoms in DSS-induced Caco-2 cells and C57BL mice.

BACKGROUND: Despite the notable pharmacological potential of natural ginsenosides, their industrial application is hindered by low oral bioavailability. Recent research centers on the production of less-glycosylated minor ginsenosides. PURPOSE: This study aimed to explore the effect of a biologically synthesized ginsenoside CK-rich minor ginsenoside complex (AceCK40), on ameliorating colitis using DSS-induced colitis models in vitro and in vivo. METHODS: The ginsenoside composition of AceCK40 was determined by HPLC-ELSD and UHPLC-MS/MS analyses. In vitro colitis model was established using dextran sodium sulfate (DSS)-induced Caco-2 intestinal epithelial model. For in vivo experiments, DSS-induced severe colitis mouse model was established. RESULTS: In DSS-stimulated Caco-2 cells, AceCK40 downregulated mitogen-activated protein kinase (MAPK) activation (p < 0.05), inhibited monocyte chemoattractant protein-1 (MCP-1) production (p < 0.05), and enhanced MUC2 expression (p < 0.05), mediated via signaling pathway regulation. Daily AceCK40 administration at doses of 10 and 30 mg/kg/day was well tolerated by DSS-induced severe colitis mice. These doses led to significant alleviation of disease activity index score (> 36.0% decrease, p < 0.05), increased luminal immunoglobulin (Ig)G (> 37.6% increase, p < 0.001) and IgA (> 33.8% increase, p < 0.001), lowered interleukin (IL)-6 (> 65.7% decrease, p < 0.01) and MCP-1 (> 116.2% decrease, p < 0.05), as well as elevated serum IgA (> 51.4% increase, p < 0.001) and lowered serum IL-6 (112.3% decrease at 30 mg/kg, p < 0.001). Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining revealed that DSS-mediated thickening of the muscular externa, extensive submucosal edema, crypt distortion, and decreased mucin droplets were significantly alleviated by AceCK40 administration. Additionally, daily administration of AceCK40 led to significant recovery of colonic tight junctions damaged by DSS through the elevation in the expression of adhesion molecules, including occludin, E-cadherin, and N-cadherin. CONCLUSION: This study presents the initial evidence elucidating the anti-colitis effects of AceCK40 and its underlying mechanism of action through sequential in vitro and in vivo systems employing DSS stimulation. Our findings provide valuable fundamental data for the utilization of AceCK40 in the development of novel anti-colitis candidates.

Modulation of gut microbiota ecosystem by a glucan-rich snail mucin heteropolysaccharide attenuates loperamide-induced constipation.

The present study aimed to investigate the effect of oral administration of snail-derived mucin extract (SM) on ameliorating constipation symptoms of loperamide-induced constipated rats (n = 6). The analytical results indicated that SM mainly contains a glucan-rich snail mucin heteropolysaccharide with high molecular weights (108.5-267.9 kDa), comprising primarily of glucose (64.9 %) and galactose (22.4 %) with some deoxyhexoses (5.0 %) and hexosamines (4.9 %). Daily SM administration at doses of 10-40 mg/kg/day to the loperamide-induced constipated rats significantly (p < 0.05) ameliorated the deterioration in fecal parameters, such as numbers and weight of feces, fecal water contents, and gastrointestinal transit ratio. The histomorphometric results showed that the loperamide-induced decreases in the thickness of mucosal and muscularis mucosae layers as well as the distribution of mucin and c-KIT-positive areas were significantly (p < 0.05) improved via SM consumption at all doses tested. SM administration at all doses significantly increased the expression of genes encoding tryptophan hydroxylases (TPH1 and TPH2; p < 0.05), tight junction molecules (OCLN, CLDN1, and TJP1; p < 0.05), and mucin (MUC2 and MUC4; p < 0.05), but significantly decreased the aquaporin-encoding genes (AQP3 and AQP8; p < 0.05). Gut microbial community analysis indicated that SM administration could modulate loperamide-induced dysbiosis by increasing the phyla Actinobacteria (11.72-12.64 % at 10-40 mg/kg doses; p < 0.05) and Firmicutes (79.33 % and 74.24 % at 20 and 40 mg/kg doses; p < 0.05) and decreasing the phyla Bacteroidetes (5.98-12.47 % at 10-40 mg/kg doses; p < 0.05) and Verrucomicrobia (2.21 % and 2.78 % at 20 and 40 mg/kg doses; p < 0.05), suggesting that SM administration is effective in ameliorating constipation by controlling gut microbial communities. These findings can be utilized as fundamental data for developing novel functional materials using SM to prevent or treat constipation.

Identification of galacturonic acid-rich polysaccharide with intestinal immune system modulating activity via Peyer's patch from postbiotics of Phellinus linteus mycelial submerged culture.

Interests in the development and exploration of industrial applications of medicinal mushrooms as postbiotics have lately increased. We recently reported the potential use of Phellinus linteus mycelial-containing whole culture extract (PLME) prepared by submerged cultivation as a postbiotic that promotes immune system activation. Here, we aimed to isolate and structurally elucidate the active ingredients in PLME by activity-guided fractionation. The intestinal immunostimulatory activity was evaluated by bone marrow (BM) cell proliferation activity and related cytokine production in C3H-HeN mouse-derived Peyer's patch (PP) cells treated with polysaccharide fractions. The initially crude polysaccharide (PLME-CP) of PLME prepared using ethanol precipitation was further fractionated into four fractions (PLME-CP-0 to -III) by anion-exchange column chromatography. BM cell proliferation and cytokine production of PLME-CP-III were significantly improved compared to those of PLME-CP. PLME-CP-III was then fractionated into PLME-CP-III-1 and PLME-CP-III-2 by gel filtration chromatography. Based on the molecular weight distribution, monosaccharide, and glycosyl linkage analyses, PLME-CP-III-1 was revealed as a novel galacturonic acid-rich acidic polysaccharide and further shown to play an important role in facilitating PP-mediated intestinal immunostimulatory activity. This is the first study demonstrating the structural characteristics of a novel intestinal immune system modulating acidic polysaccharide from P. linteus mycelium-containing whole culture broth postbiotics.

An Education-Counseling Program for Young Prehypertensive Adults.

In this non-equivalent control group, non-synchronized study, we assessed the effects of an education-counseling program for young prehypertensive adults. We included 40 and 47 prehypertensive individuals in the experimental and control groups, respectively. A structured questionnaire (pretest) was used to assess prehypertension-related knowledge, attitudes, health-promoting behavior, and self-efficacy. The experimental group underwent the 8-week program, while the control group received basic prehypertension and self-management education. Subsequently, blood pressure (BP) was measured, and prehypertension-related knowledge, attitudes, health-promoting behavior, and self-efficacy were evaluated using a questionnaire (posttest). There were significant intergroup differences in knowledge (t = 3.04, p = .003), attitudes (t = 6.41, p < .001), behavior (t = 11.60, p < .001), self-efficacy (t = 11.76, p < .001), and systolic BP (t = -5.49, p < .001); however, diastolic BP was not significantly different (t = -0.73, p = .473). Our findings demonstrated that the program is effective in improving knowledge, attitudes, behavior, self-efficacy, and systolic BP. Therefore, it can be used to prevent progression to hypertension.

Antioxidant and Anti-Inflammatory Activities of Sargassum macrocarpum Extracts.

Oxidative stress and the inflammatory response are known to be the most important pathological factors for aging skin cells. Therefore, substances that protect skin cells from oxidative stress and inflammatory reactions of the skin have potential as functional ingredients for skin care. In the present study, we investigated the potential of Sargassum macrocarpum as an anti-inflammatory candidate for inflammatory skin disease. Antioxidant and anti-inflammatory activities are desirable properties in such functional materials. The total polyphenol content as well as antioxidant and anti-inflammatory activities were evaluated in hot-water (HES) and ethanol (EES) extracts of S. macrocarpum. The polyphenol content was higher in the HES (HES: 115.9 ± 15.3 mg GA/g, EES: 3.9 ± 0.5 mg GA/g), and the HES also had ABTS (HES: IC50 1.0 ± 0.0 mg/mL, EES: IC50 16.09 ± 0.7 mg/mL) and DPPH (HES: IC50 6.50 ± 0.3 mg/mL, EES: IC50 35.3 ± 3.1 mg/mL) radical scavenging capacities as well as FRAP activity (HES: IC50 18.8 ± 0.4 mg/mL, EES: IC50 n.d.). Compared with the EES at the equivalent concentration range (1.25-20 µg/mL), the HES exerted a more potent inhibitory activity on LPS-stimulated nitric oxide (10.3-43.1%), IL-6 (15.7-45.0%), and TNF-α (14.1-20.8%) in RAW 264.7 macrophage cells in addition to TNF-α and IFN-γ-facilitated IL-6 (10.9-84.1%) and IL-8 (7.7-73.2%) in HaCaT keratinocytes. These results suggested that water-soluble materials might be deeply involved in the antioxidant and anti-inflammatory activity in S. macrocarpum. General composition analysis indicated that the HES contains more carbohydrates and polyphenols than the EES, and the monosaccharide composition analysis suggested that fucose-containing sulfated polysaccharide and ß-glucan might be potent anti-inflammatory candidates in the HES. The present study presents important preliminary results and a valuable strategy for developing novel anti-skin dermatitis candidates using a hot-water extract of S. macrocarpum.

Immunostimulatory Effect of Postbiotics Prepared from Phellinus linteus Mycelial Submerged Culture via Activation of Spleen and Peyer's Patch in C3H/HeN Mice.

Medicinal mushrooms are an important natural resource promoting health benefits. Herein, Phellinus linteus mycelia were prepared under submerged cultivation, the mycelium-containing culture broth was extracted as a whole to obtain the postbiotic materials (PLME), and its effect on the immune system was evaluated in normal C3H/HeN mice. Oral administration of PLME for 4 weeks was well tolerated and safe. In the PLME-administered groups, in addition to the production of immunostimulatory cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), the mitogenic activity was significantly increased. PLME administration also significantly increased the levels of serum immunoglobulin G (IgG) and IgA in the small intestinal fluid and Peyer's patches and enhanced Peyer's patch-mediated bone marrow cell proliferation activity and cytokine production (IL-2, IL-6, and IFN-γ). Histomorphometric analyses showed an increase in immune cells in the spleen and small intestinal tissues of mice administered PLME, supporting the rationale for its immune system activation. PLME mainly contained neutral sugar (969.1 mg/g), comprising primarily of glucose as a monosaccharide unit. The ß-glucan content was 88.5 mg/g. Data suggest that PLME effectively promote immune function by stimulating the systemic immune system through the spleen and intestinal immune tissues. PLME can thus be developed as a functional ingredient to enhance immune functions.

Effects of particle size and polymorph type of TiO2 on the properties of BaTiO3 nanopowder prepared by solid-state reaction.

Barium titanate (BaTiO3) has attracted considerable attention as a perovskite ferroelectric ceramic material for electronic multilayer ceramic capacitors (MLCCs). Fine BaTiO3 nanopowders with a considerably high tetragonality directly influence the typical properties of nanopowders; however, their synthesis has remained challenging. In this study, we analyzed the effect of two different TiO2 powders with anatase and rutile phases in a solid-state reaction with barium carbonate (BaCO3). The effect of the particle size ratio (TiO2/BaCO3) of the raw materials on the tetragonality and particle size of the as-synthesized BaTiO3 powders was also determined through extensive characterization of the powders by X-ray diffraction, field-emission scanning electron microscopy, and Raman spectroscopy. The present investigation reveals that the design BaTiO3 structure is expected to advance the development of efficient catalytic and sensor materials for sustainable environmental applications.

Magnetic polymer bowl for enhanced catalytic activity and recyclability.

This work introduces the fabrication of a magnetic polymer bowl for enhanced catalytic activity and recyclability, which involves the synthesis of silica-coated Fe3O4 magnetic clusters, seeded dispersion polymerization using the magnetic clusters, and transformation into a bowl-like structure via a phase separation route. The additional treatment with tannic acid (TA) on the bowls allows the in situ formation of silver nanoparticles (AgNPs) on their surfaces. The openness and larger surface area of the bowls, as compared with those of other structured particles, such as spheres and flowers, enable a considerably higher immobilization of AgNPs, thus leading to an excellent catalytic reduction for 4-nitrophenol (4-NP), methylene blue (MB), and rhodamine B. Furthermore, the strong magnetic response originating from the magnetic clusters inside the bowls endows a good magnetic recovery and an excellent reusability for the repeated reduction of the organic dyes without loss of catalytic activity.

[Knowledge of Diaper Dermatitis and Diaper Hygiene Practices among Mothers of Diaper-wearing Children]. / ì˜ìœ ì•„ 어머니의 ê¸°ì €ê·€ 발진에 대한 지식 및 ê¸°ì €ê·€ 관리 실태.

PURPOSE: The purpose of this study was to investigate the prevalence of diaper dermatitis (DD), knowledge of DD prevention and treatment, and diaper hygiene practices among mothers with diaper-wearing children. METHODS: The participants were 176 mothers who presented to an outpatient clinic at a children's hospital with diaper-wearing children. Data were collected using a structured self-administered questionnaire. RESULTS: The percent of correct answer for knowledge about DD was 59.7%. Almost half of the participants' children had experienced at least 1 episode of DD during the last 6 months. Inappropriate diaper hygiene practices, such as using talcum powder on DD and rubbing with a dry towel after cleansing, were reported. Moreover, only 37% of mothers used the recommended skin barrier to prevent DD. Although many children suffer from DD, levels of educational experience and perceived need for education on this topic were low. Almost 70% of mothers obtained DD-related information through internet sites. CONCLUSION: Educating parents about the etiology of DD and evidence-based diaper hygiene practices is an important aspect of effective DD prevention and treatment. Internet sites or smartphone apps may be effective methods for education on DD prevention and treatment considering parents' preferences for ways to obtain health information.

In vivo imaging of activated macrophages by 18F-FEDAC, a TSPO targeting PET ligand, in the use of biologic disease-modifying anti-rheumatic drugs (bDMARDs).

Rheumatoid arthritis (RA) is a chronic disease with systemic inflammation resulting in destruction of multiple articular cartilages and bones. Activated macrophage plays a pivotal role during the disease course and has been one of main targets to inhibit inflammatory reaction of RA by using biological disease-modifying anti-rheumatic drugs (bDMARDs). 18F-FEDAC is one of PET imaging agents targeting TSPO, which is overexpressed in activated macrophages. The aim of this study was to evaluate the roles of 18F-FEDAC PET as an in vivo imaging of activated macrophages on etanercept (ETN), a TNF-antagonist as one of bDMARDs in collagen induced arthritis mice. In RAW 264.7 cells, the expressions of TSPO as well as iNOS and infiltrated nucleus of NF-κB were induced by activation with lipopolysaccharide and interferon-gamma. TSPO expression was slightly attenuated by ETN treatment, not by methotrexate (MTX) as a cytotoxic agent. However, cell uptake of 18F-FEDAC did not show significant changes according to both of the treatments. Similarly in CIA mice, 18F-FEDAC uptake in inflamed paws on PET imaging did not show significant changes during both of the treatments, contrary to the uptake decrease of 18F-FDG, a glucose analog to reflect metabolic or active inflammatory activity. Interestingly, when we divided joints according to the degree of 18F-FEDAC uptake before ETN treatment, the joints of high 18F-FEDAC uptake showed better response to ETN than the joints with low 18F-FEDAC uptakes. In case of 18F-FDG, there was no such kinds of patterns. We can speculate that 18F-FEDAC PET imaging may identify activated macrophage-induced arthritis because that 18F-FEDAC can reflect activated macrophages, which is the therapeutic target of ETN by TNF antagonistic effect. Thus, in vivo imaging using 18F-FEDAC may be used as a predictor of therapeutic effects among those kinds of bDMARDs having anti-inflammatory actions to inhibit activated macrophage.

Cerebrospinal fluid dynamics at the lumbosacral level in patients with spinal stenosis: A pilot study.

Spinal stenosis is a common degenerative condition. However, how neurogenic claudication develops has not been clearly elucidated. Moreover, cerebrospinal fluid physiology at the lumbosacral level has not received adequate attention. This study was conducted to compare cerebrospinal fluid hydrodynamics at the lumbosacral spinal level between patients with spinal stenosis and healthy controls. Twelve subjects (four patients and eight healthy controls; 25-77 years old; seven males) underwent phase-contrast magnetic resonance imaging to quantify cerebrospinal fluid dynamics. The cerebrospinal fluid flow velocities were measured at the L2 and S1 levels. All subjects were evaluated at rest and after walking (to provoke neurogenic claudication in the patients). The caudal peak flow velocity in the sacral spine (-0.25 ± 0.28 cm/s) was attenuated compared to that in the lumbar spine (-0.93 ± 0.46 cm/s) in both patients and controls. The lumbar caudal peak flow velocity was slower in patients (-0.65 ± 0.22 cm/s) than controls (-1.07 ± 0.49 cm/s) and this difference became more pronounced after walking (-0.66 ± 0.37 cm/s in patients, -1.35 ± 0.52 cm/s in controls; p = 0.028). The sacral cerebrospinal fluid flow after walking was barely detectable in patients (caudal peak flow velocity: -0.09 ± 0.03 cm/s). Cerebrospinal fluid dynamics in the lumbosacral spine were more attenuated in patients with spinal stenosis than healthy controls. After walking, the patients experiencing claudication did not exhibit an increase in the cerebrospinal fluid flow rate as the controls did. Altered cerebrospinal fluid dynamics may partially explain the pathophysiology of spinal stenosis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:104-112, 2017.

Morphological Feature Extraction From a Continuous Intracranial Pressure Pulse via a Peak Clustering Algorithm.

OBJECTIVE: An increase in intracranial pressure (ICP) is frequently observed in patients with severe traumatic brain injury (TBI). The information derived from the observation of temporal changes in the mean ICP is insufficient for assessment of the compensatory reserve of the injured brain. This assessment can be achieved via continuous morphological analysis of the pulse waveform of the ICP. METHODS: Continuous arterial blood pressure (ABP) and ICP recordings from 292 TBI patients were analyzed. The algorithm extracted morphological landmarks (peaks, troughs, and flats) from the ICP. Among the extracted peaks, P1, P2, and P3 were assigned through peak clustering. The performance of the proposed method was validated through a comparison of the algorithm-defined peaks and those manually identified by experienced observers. RESULTS: The proposed algorithm successfully identified the three distinguishing peaks of the ICP with satisfactory accuracy (95.3%, 87.8%, and 87.5% for P1, P2, and P3, respectively), even from minimally filtered raw signals. CONCLUSION: The algorithm extracted the morphological features from both ABP and ICP recordings with high accuracy. SIGNIFICANCE: The ABP and ICP pulse waveforms can be simultaneously analyzed in real time using the proposed algorithm. The morphological features from these signals may aid the continuous care of patients with TBI.

Spectral analysis of intracranial pressure: Is it helpful in the assessment of shunt functioning in-vivo?

OBJECTIVE: Shunt failure is common in hydrocephalic patients. The cerebrospinal fluid (CSF) infusion test enables the assessment of CSF absorption capacity, which is represented by the resistance to CSF outflow (ROUT) However, shunt failure may not only affect the CSF absorption capacity but also the intracranial compliance or compensatory properties. Spectral analysis of the ICP signal obtained during the infusion test may enable the comprehensive assessment of the overall deterioration caused by shunt failure. MATERIAL AND METHODS: A total of 121 hydrocephalic shunted patients underwent the infusion test with continuous intracranial pressure (ICP) and arterial blood pressure (ABP) recording. The maximum amplitudes of three major frequency bandwidths (0.2-2.6, 2.6-4.0 and 4.0-15 Hz, respectively) were calculated from the ICP. Statistical analyses were conducted to identify factors significantly associated with shunt failure, to construct an index (i.e., the shunt response parameter, SRP) for detecting shunt failure, and to define thresholds for ROUT and SRP. RESULTS: The ROUT threshold for detecting shunt failure was 7.59 mmHg/ml/min, and this threshold showed an accuracy of 82.64%. All spectral parameters were found to be significantly associated with shunt patency (p<0.05). The SRP exhibited significantly better accuracy than ROUT in detecting shunt failure (91.74%). CONCLUSION: The hydrodynamic assessment of shunted patients enhanced by spectral analysis during the infusion test detected shunt failure with high accuracy. Although further validation is needed, the SRP exhibited promising results.

Finite element analysis of periventricular lucency in hydrocephalus: extravasation or transependymal CSF absorption?

OBJECTIVE: Periventricular lucency (PVL) is often observed in the hydrocephalic brain on CT or MRI. Earlier studies have proposed the extravasation of ventricular CSF into the periventricular white matter or transependymal CSF absorption as possible causes of PVL in hydrocephalus. However, there is insufficient evidence for either theory to be conclusive. METHODS: A finite element (FE) model of the hydrocephalic brain with detailed anatomical geometry was constructed to investigate the possible mechanism of PVL in hydrocephalus. The initiation of hydrocephalus was modeled by applying a transmantle pressure gradient (TPG). The model was exposed to varying TPGs to investigate the effects of different geometrical characteristics on the distribution of PVL. The edema map was derived based on the interstitial pore pressure. RESULTS: The model simulated the main radiological features of hydrocephalus, i.e., ventriculomegaly and PVL. The degree of PVL, assessed by the pore pressure, was prominent in mild to moderate ventriculomegaly. As the degree of ventriculomegaly exceeded certain values, the pore pressure across the cerebrum became positive, thus inducing the disappearance of PVL. CONCLUSIONS: The results are in accordance with common clinical findings of PVL. The degree of ventriculomegaly significantly influences the development of PVL, but two factors were not linearly correlated. The results are indicative of the transependymal CSF absorption as a possible cause of PVL, but the extravasation theory cannot be formally rejected.

Finite element analysis for normal pressure hydrocephalus: The effects of the integration of sulci.

Finite element analysis (FEA) is increasingly used to investigate the brain under various pathological changes. Although FEA has been used to study hydrocephalus for decades, previous studies have primarily focused on ventriculomegaly. The present study aimed to investigate the pathologic changes regarding sulcal deformation in normal pressure hydrocephalus (NPH). Two finite element (FE) models-an anatomical brain geometric (ABG) model and the conventional simplified brain geometric (SBG) model-of NPH were constructed. The models were constructed with identical boundary conditions but with different geometries. The ABG model contained details of the sulci geometry, whereas these details were omitted from the SBG model. The resulting pathologic changes were assessed via four biomechanical parameters: pore pressure, von Mises stress, pressure, and void ratio. NPH was induced by increasing the transmantle pressure gradient (TPG) from 0 to a maximum of 2.0 mmHg. Both models successfully simulated the major features of NPH (i.e., ventriculomegaly and periventricular lucency). The changes in the biomechanical parameters with increasing TPG were similar between the models. However, the SBG model underestimated the degree of stress across the cerebral mantle by 150% compared with the ABG model. The SBG model also overestimates the degree of ventriculomegaly (increases of 194.5% and 154.1% at TPG = 2.0 mmHg for the SBG and ABG models, respectively). Including the sulci geometry in a FEA for NPH clearly affects the overall results. The conventional SBG model is inferior to the ABG model, which accurately simulated sulcal deformation and the consequent effects on cortical or subcortical structures. The inclusion of sulci in future FEA for the brain is strongly advised, especially for models used to investigate space-occupying lesions.