Evaluation of vertebral bone strength with a finite element method using low dose computed tomography imaging.

BACKGROUND: Focusing on compression fractures of bone by finite elements, we evaluated bone strength based on the computed tomography-based finite element method. However, the exposure dose is an issue. We aimed to investigate the quantity of reduction of the radiation dose with respect to the reference dose by comparing the calculation results of compression fractures of the vertebral body using experimental data obtained from the spine of a pig. METHODS: Computed tomography images of a self-made phantom that enclosed the lower lumbar vertebra of edible wild pigs were obtained under baseline-dose conditions using various lower tube currents. Images obtained under reference-dose conditions were reconstructed using the filtered back-projection method, whereas images obtained under low-dose conditions were reconstructed using both the filtered back-projection method and the iterative reconstruction method. Computer simulations involving the creation of finite element models using all images were implemented for the compression load calculation for vertebral body parts. Based on the calculated results, images of the low-dose and reference-dose conditions were compared. RESULTS: Using pigs' lower lumbar vertebrae, finite element model analysis of low-dose X-ray computed tomography images showed that equivalent results can be obtained with a dose of approximately 40% of the standard radiographic reference doses. As for the compression stress intensity, the same results as those under reference-dose conditions were obtained using the iterative reconstruction method in combination with computed tomography-based finite element method. CONCLUSIONS: The combination of the iterative reconstruction method with the computed tomography-based finite element method is an effective image reconstruction method for achieving dose reduction.

A Simulation Case Study of Knee Joint Compressive Stress during the Stance Phase in Severe Knee Osteoarthritis Using Finite Element Method.

Background and Objectives: Medial knee osteoarthritis is known to increase the mechanical load on the medial compartment of the knee joint during walking; however, it is not visually understood how much the mechanical load increases nor where in the medial compartment of the knee joint that load is focused. Therefore, we conducted a simulation study to determine the location and amount of the mechanical load in the medial compartment of the knee joint during the stance phase. Materials and Methods: Subject was a patient with right medial knee osteoarthritis. Computed tomography imaging and gait analysis were performed on subject. The CT image of the right knee was calculated using finite element analysis software. Since this software can set the flexion angle arbitrarily while maintaining the nonuniform material properties of the bone region, the model is constructed by matching the knee joint extension image obtained by CT to the loading response phase of gait analysis. The data of muscle exertion tension and vertical ground reaction force were inserted into the knee joint model created from the computed tomography-based finite element method, and the knee joint compressive stress was calculated. Results: With regard to compressive stress, the tibia showed high stress at 4.10 to 5.36 N/mm2. The femur showed high stress at 4.00 to 6.48 N/mm2. The joint compressive stress on the medial compartment of the knee joint was found to concentrate on the edge of the medial tibial condyle in the medial knee osteoarthritis subject. Conclusions: The measurement method of knee joint compressive stress by computed tomography-based finite element method can visually be a reliable method of measuring joint compressive stress in the medial knee osteoarthritis. This reflects the clinical findings because concentration of stress on the medial knee joint was observed at the medial osteophyte.

Development of a Knee Joint CT-FEM Model in Load Response of the Stance Phase During Walking Using Muscle Exertion, Motion Analysis, and Ground Reaction Force Data.

Background and objectives: There are no reports on articular stress distribution during walking based on any computed tomography (CT)-finite element model (CT-FEM). This study aimed to develop a calculation model of the load response (LR) phase, the most burdensome phase on the knee, during walking using the finite element method of quantitative CT images. Materials and Methods: The right knee of a 43-year-old man who had no history of osteoarthritis or surgeries of the knee was examined. An image of the knee was obtained using CT and the extension position image was converted to the flexion angle image in the LR phase. The bone was composed of heterogeneous materials. The ligaments were made of truss elements; therefore, they do not generate strain during expansion or contraction and do not affect the reaction force or pressure. The construction of the knee joint included material properties of the ligament, cartilage, and meniscus. The extensor and flexor muscles were calculated and set as the muscle exercise tension around the knee joint. Ground reaction force was vertically applied to suppress the rotation of the knee, and the thigh was restrained. Results: An FEM was constructed using a motion analyzer, floor reaction force meter, and muscle tractive force calculation. In a normal knee, the equivalent stress and joint contact reaction force in the LR phase were distributed over a wide area on the inner upper surface of the femur and tibia. Conclusions: We developed a calculation model in the LR phase of the knee joint during walking using a CT-FEM. Methods to evaluate the heteromorphic risk, mechanisms of transformation, prevention of knee osteoarthritis, and treatment may be developed using this model.

[Revision of Pelvic Rotation Angle and One Idea for Improving Reproducibility of Hip Joint Radiographic Images (False Profile View)].

Radiographic images of the hip joint can include a false profile view when the foot of the affected side is positioned parallel to the detector plane and the pelvic rotation angle is 65°. However, to the best of our knowledge, pelvic rotation angle has yet to be adequately investigated. The present study aimed to improve radiographic imaging reproducibility by testing pelvic rotation angles using the pubic symphysis and greater trochanter as a guide. The pubic bone angles were 50°-60° independent of gender or age in approximately 70% of 210 hip joints examined by computed tomography. When the line connecting the centers of the femoral neck and the pubic symphysis was based on a detector plane during hip joint magnetic resonance imaging of 12 healthy volunteers, pubic bone angles were approximately 65° and 62° when rotated outwards at 20° and 30°, respectively. Based on the detector plane, the difference between the angle at the intersection of the line connecting the femoral neck center and the pubic symphysis center and the angle at the intersection of the line connecting the pubic symphysis superior margin and the greater trochanter was <4° at external rotation angles of 10°, 20°, and 30°. The foot of the affected side corresponding to the detector plane in front of the body at approximately 65° and the second metatarsal at a pelvic rotation angle when collimated was rotated 25° outwards. Radiation is incident on the pubic symphysis and the greater trochanter can be used as an ejection point.

[A Low-temperature Manganese Chloride Tetrahydrate Improves the Image Quality of Magnetic Resonance Cholangiopancreatography].

Manganese chloride tetrahydrate (MCT) is one of the oral negative contrast agents which is indispensable for imaging of magnetic resonance cholangiopancreatography (MRCP). In this study, improvement of the image quality of MRCP by using low-temperature MCT is verified. All MR imagings were performed using 1.5 T scanner. The T(1) and T(2) values of the different temperature MCTs were measured in the phantom study. Different concentrations of MCT-doped water (30%, 50%, 70%, and 90%) were measured at several temperature conditions (10°C, 15°C, 23°C, 35°C, and 40°C). As a result, the T(1) and T(2) values became larger with a temperature rise. It was more remarkable in low-concentration MCT. Then, 17 healthy subjects were scanned two times with different temperatures of MCT. The MCT of the normal temperature (23°C) and low temperature (10°C) were taken at consecutive 2 days. The contrast between the stomach and the spleen were significantly higher in 2D half Fourier acquisition single shot turbo spin echo (HASTE) images by use of the low-temperature MCT. The contrast between the common bile duct and the adjacent background were significantly higher in the source images of 3D MRCP by use of the low temperature MCT. In addition, 76% of subjects answered in the questionnaire that the low temperature MCT is easier to drink. The low temperature MCT improves the image quality of MRCP and contributes to performing noninvasive examination.

Antiferromagnetism and insulator-metal transition of alkali metal-loaded sodalite.

Alkali metal clusters with a single unpaired s-electron can be arranged three-dimensionally in a sodalite crystal by loading the guest alkali atoms. Na, K, and K-Rb alloy clusters are known to be Mott insulators and to exhibit antiferromagnetic ordering. The Néel temperature increases from about 50 K to about 100 K in this order. In this study, Li-Na alloy, Na-K alloy, and pure Rb samples were newly prepared and their magnetic, electrical conductivity, and optical properties were investigated, including those of previous samples. The Na-K alloy samples showed antiferromagnetic properties, which were intermediate between those of the Na and K samples. However, the Rb sample showed a non-magnetic metallic state. The shallower ionization potential in Rb is thought to cause an insulator-metal transition (Mott transition) due to weaker on-site Coulomb repulsion between electrons and larger electron transfer energy between neighboring clusters. On the other hand, the Li-Na alloy sample showed a non-magnetic insulating state. It is thought that the two electrons form a spin-singlet pair due to the strong electron-lattice interaction. In terms of electron correlations and polaron effects, the full picture of the element species dependence of the alkali metal-loaded sodalite is reviewed.

Simulating Knee-Stress Distribution Using a Computed Tomography-Based Finite Element Model: A Case Study.

This study aimed to evaluate the mechanism of progression involved in knee osteoarthritis (OA). We used the computed tomography-based finite element method (CT-FEM) of quantitative X-ray CT imaging to calculate and create a model of the load response phase, wherein the greatest burden is placed on the knee joint while walking. Weight gain was simulated by asking a male individual with a normal gait to carry sandbags on both shoulders. We developed a CT-FEM model that incorporated walking characteristics of individuals. Upon simulating changes owing to a weight gain of approximately 20%, the equivalent stress increased extensively in both medial and lower leg aspects of the femur and increased medio-posteriorly by approximately 230%. As the varus angle increased, stress on the surface of the femoral cartilage did not change significantly. However, the equivalent stress on the surface of the subchondral femur was distributed over a wider area, increasing by approximately 170% in the medio-posterior direction. The range of equivalent stress affecting the lower-leg end of the knee joint widened, and stress on the posterior medial side also increased significantly. It was reconfirmed that weight gain and varus enhancement increase knee-joint stress and cause the progression of OA.

TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts.

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca(2+) signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (-/-)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) were increased in MLP (-/-) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (-/-) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (-/-) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM.

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol.

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.

Microbial production of glyceric acid, an organic acid that can be mass produced from glycerol.

Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

Production of glyceric acid by Gluconobacter sp. NBRC3259 using raw glycerol.

Gluconobacter sp. NBRC3259 converted glycerol to glyceric acid (GA). The enantiomeric composition of the GA produced was a mixture of DL-forms with a 77% enantiomeric excess of D-GA. After culture conditions, such as initial glycerol concentration, types and amounts of nitrogen sources, and initial pH, were optimized, Gluconobacter sp. NBRC3259 produced 54.7 g/l of GA as well as 33.7 g/l of dihydroxyacetone (DHA) from 167 g/l of glycerol during 4 d of incubation in a jar fermentor with pH control. GA production from raw glycerol samples, the main by-product of the transesterification process in the biodiesel production and oleochemical industries, was also evaluated after proper pretreatment of the samples. Using a raw glycerol sample with activated charcoal pretreatment, 45.9 g/l of GA and 28.2 g/l of DHA were produced from 174 g/l of glycerol.

Feasibility of navigator setting on the left diaphragm for whole-heart coronary MRA: a study in healthy volunteers.

PURPOSE: We prospectively compared the quality of images obtained by navigator setting on the left and right diaphragm on whole-heart coronary magnetic resonance angiography (WHCMRA). METHODS: In 10 healthy volunteers, we performed free-breathing, 3-dimensional segmented true fast imaging with steady-state precession (trueFISP) WHCMRA by setting the navigator on the left and right diaphragm in random order. For the left diaphragm, we set the navigator outside the scope of the heart to avoid the influence on coronary arteries. We compared image acquisition time and visible length of coronary arteries using paired t-test and subjective image quality on a 4-point scale (1, poor; 4, excellent) using Wilcoxon signed-rank test. RESULTS: Mean overall subjective image quality was significantly better in the left diaphragm than the right (3.3+/-0.7 versus 2.9+/-0.9, P=0.02). Mean overall visible length of the coronary arteries was significantly better in the left diaphragm than the right (115.4+/-31.1 vs. 112.6+/-29.9 mm, P=0.02). Mean acquisition time between the left and right diaphragm was not significantly different (15.6+/-5.0 versus 16.0+/-5.7 min, P=0.79). CONCLUSION: In this small group of healthy volunteers, navigator setting for WHCMRA was superior on the left diaphragm than the right; however, feasibility of the technique requires additional consideration in a larger group of actual patients.

Shorter telomere length with age in the loggerhead turtle: a new hope for live sea turtle age estimation.

We verified whether telomere length shortens with age in the loggerhead sea turtle (Caretta caretta) by measuring telomere lengths (relative telomere to single copy gene [T/S] ratios) in whole blood and epidermis from 20 captive individuals with a real-time PCR method. There was no significant correlation between age and relative T/S ratios in blood. Although the correlation between age and relative T/S ratios in epidermis was not significant, older turtles had smaller relative T/S ratios in epidermis. It was thus demonstrated that telomere length in epidermis could be a useful age estimator for sea turtles. Relative age information obtained with this simple, rapid, non-invasive technique may help to advance our understanding of the ecology of endangered sea turtles. This is the first publication on age-related changes in telomere length among chelonians.

Frequency of common bile duct motion artifacts caused by inferior vena cava pulsation on magnetic resonance cholangiopancreatography.

PURPOSE: We assessed the frequency of common bile duct (CBD) motion artifacts caused by inferior vena cava (IVC) pulsation on magnetic resonance cholangiopancreatography (MRCP). METHODS: We retrospectively evaluated CBD motion artifacts in 4 MRCP sequences from each of 115 consecutive patients. RESULTS: We observed 37 (32.2%) ghost artifacts at the ventral and dorsal aspects of the CBD on transaxial, half-Fourier acquisition single-shot turbo spin-echo (HASTE-ax) images; no such artifacts were observed on transaxial T(2)-weighted turbo spin-echo images. In 10 patients, we observed 9 (7.8%) pseudo-defects of the CBD on 3-dimensional T(2)-weighted turbo spin-echo with navigator-triggered prospective acquisition correction technique MRCP and 6 (5.2%) pseudo-defects on single-shot rapid acquisition with relaxation enhancement MRCP. Pseudo-defects were significantly more frequent in patients with ghost artifacts than without (9 of 37 [24.3%] versus one of 78 [1.3%]; P<0.01, McNemar test). CONCLUSION: Although uncommon, pseudo-defects of the CBD caused by IVC pulsation are observed on MRCP. MRCP interpretation that includes comparison with HASTE-ax images can diminish the potential misinterpretation of such CBD motion artifact as bile duct tumor or biliary stone.

Evaluation of the oxygen reduction activities of rare-earth oxide-supported silver catalysts using a channel flow double electrode.

The channel flow double electrode (CFDE) was used for the evaluation of the oxygen reduction activities in alkaline solution of rare-earth oxide-supported silver catalysts. The CFDE cell was modified for the experiment using the powder catalyst as a working electrode. In the present experiment, the silver electrode was supported with CeO2 in order to improve the performance of the oxygen reduction. The detecting electrode current for HO2- emitted from the working electrode was recorded simultaneously with the measurement of the i-E curve of each working electrode. Moreover, the average number of charge transfers n was calculated from the working and detecting electrode currents. The value of n for the oxygen reduction was approximately 4 for silver electrode supported with rare-earth oxide, compared with the n value of pure silver that was smaller than 4. On the basis of these results, the mechanisms of oxygen reduction on these electrodes and role of the rare-earth oxide in alkaline solution were discussed.

SI-27, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity: detection with a variable-pressure scanning electron microscope.

OBJECTIVE: Degradation of basement membrane is one the of crucial steps in tumor angiogenesis and is performed by matrix metalloproteinases (MMPs). This study was designed to investigate the suppression of tumor angiogenesis by SI-27, an MMP inhibitor. METHODS: SI-27 was applied at noncytotoxic concentrations (1-100 micromol/L), and its effect on nonmitogenic vascular endothelial growth factor (VEGF)-enhanced cell motility and in vitro angiogenesis by human umbilical vein endothelial cells was determined. The activity of MMP-1, MMP-2, and tissue inhibitor of metalloproteinase 1 was determined by enzyme-linked immunosorbent assay. The effect of SI-27 on in vitro angiogenesis stimulated by supernatants of human glioma cell lines (U87MG, U251MG, and U373MG) also was examined. Angiogenesis was detected with variable vacuum scanning electron microscopy. RESULTS: Cell motility and in vitro angiogenesis by human umbilical vein endothelial cells were significantly increased by VEGF. The maximal effect on cell motility by VEGF was noted at 5 ng/ml (P < 0.001), and the maximal effect on the capillary network was observed at 10 ng/ml (P < 0.001), along with elevated MMP-1 and MMP-2 activity. Whereas SI-27 significantly suppressed VEGF-mediated in vitro angiogenesis (50 micromol/L; P < 0.001) and inactivated both MMP-1 and MMP-2, the expression of tissue inhibitor of metalloproteinase 1 and VEGF-mediated cell motility were not affected by SI-27. The angiogenesis promoted by glioma supernatants showed a significant reduction in the presence of SI-27 (10 micromol/L; U87MG, P < 0.01; U251MG, P < 0.01; U373MG, P < 0.01). CONCLUSION: SI-27 inhibited in vitro tumor angiogenesis by suppression of MMP. This agent may be anticipated to prevent tumor growth through an angiosuppressive effect.

Anti-invasive effect of an anti-matrix metalloproteinase agent in a murine brain slice model using the serial monitoring of green fluorescent protein-labeled glioma cells.

OBJECTIVE: We aimed to analyze the anti-invasive effect of the anti-matrix metalloproteinase (anti-MMP) agent SI-27 by quantitative tracking of enhanced green fluorescent protein (EGFP)-labeled human malignant glioma cell lines in a brain slice model. METHODS: Persistent expression of EGFP in human malignant glioma cell clones (U87MG, U251MG, and U373MG) was established with the use of the pEGFP-C1 vector. Tumor spheroid in 1 microl Matrigel was implanted into the caudate nucleus-putamen of a severe combined immunodeficient mouse brain slice. To allow the quantitative assessment of tumor cell invasion, the invasion area index was measured on Days 1, 3, 5, and 7 with a fluorescence stereomicroscope and an image analyzer in the presence of various concentrations of SI-27 (0, 1, 10, 50, or 100 microg/ml). RESULTS: In the control group (0 microg/ml), all glioma cell lines invaded in a fingerlike fashion and reached the contralateral hemisphere through the corpus callosum. SI-27 at concentrations of 10, 50, and 100 microg/ml significantly suppressed the invasion area index on Days 5 and 7 in a dose-dependent manner, whereas 1 microg/ml had no effect. Transmission electron microscopy and laser confocal microscopy indicated that the tumor cells had penetrated the brain slice and that the normal structural integrity of the brain was maintained until Day 7. CONCLUSION: This model enabled unequivocal periodic tracking of individual invading tumor cells in normal brain. The significant suppression of glioma cell invasion by noncytotoxic concentrations of SI-27 indicates that anti-MMP treatment may represent an important future therapeutic strategy for malignant cerebral neoplasms.

Apoptotic induction by BE16627B on human malignant glioma cell lines by an anti-matrix metalloproteinase agent.

We have elucidated the pharmacological action of the anti-matrix metalloproteinase inhibitor BE16627B on glioma cells. The study was limited to the noncytotoxic dose range. The aim of the study was to investigate whether the cytotoxicity of BE16627B, an anti-MMP agent, is related to apoptosis in the human malignant glioma cell lines U87MG, U251MG, and U373MG. MTT assay was performed to detect the cytotoxic dose range. Agarose gel electrophoresis was performed with purified genomic DNA following exposure to 20 to 500 microM BE16627B for 24 h, compared with 0 microM for the control group. Transmission electron microscopy (TEM) was employed to study nuclear fragmentation following exposure to 0, 20, and 500 microM of the agent for 24 h. An in situ endolabeling assay was performed to determine the index of apoptotic induction. MTT assay revealed that concentrations of 100 microM and above were cytotoxic. DNA laddering was demonstrated in agarose gel electrophoresis. TEM disclosed condensing and fragmentation of the chromatin. None of these changes were observed in the control group and the noncytotoxic dose group. The in situ endolabeling study disclosed that the apoptotic index was significantly elevated by cytotoxic doses of this agent (U373MG; control, 4.0%; 500 microM, 68.5%). These results indicated that cytotoxic concentrations of BE16627B induced apoptosis in human malignant glioma cell lines. In our previous report, this agent inhibited activity of MMP in noncytotoxic concentrations. Further study should be done to determine the pharmacological action of toxic BE16627B.

Tracking cell invasion of human glioma cells and suppression by anti-matrix metalloproteinase agent in rodent brain-slice model.

Persistent expression of green fluorescent protein (GFP) in human malignant glioma cell clones (U87MG, U251MG, and U373MG) was established using the pEGFP-Cl vector. Tumor spheroid was implanted into the caudate nucleus-putamen of a severely compromised immunodeficient (SCID) mouse brain slice. To allow quantitative assessment of tumor cell invasion, the invasion area index was measured on days 1, 3, 5, and 7 by a fluorescence stereomicroscope and an image analyzer in the presence of varying concentrations of SI-27. In the control group (0 microg/ml), all glioma cell lines invaded in a fingerlike fashion, reaching the contralateral hemisphere via the corpus callosum. SI-27 at concentrations of 10, 50, or 100 microg/ml significantly suppressed the index on days 5 and 7 in a dose-dependent manner, whereas 1 microg/ml had no effect. Laser confocal microscopy indicated that the tumor cells penetrated through the brain slice. This model enabled unequivocal periodic tracking of individual invading tumor cells in the normal brain. The significant suppression of glioma cell invasion by SI-27 indicates that anti-matrix metalloproteinase (MMP) treatment may represent an important future therapeutic strategy for malignant cerebral neoplasms.