Mechanical Stress Via Muscle Contractile Exercise Suppresses Atrophic Alterations of Bone-microstructure in Immobilized Rat Femurs.

OBJECTIVES: This study aimed to determine whether mechanical stress via muscle contractile exercise with belt electrode-skeletal muscle electrical stimulation (B-SES) device effectively prevents immobilization-induced bone atrophy. METHODS: Wistar rats were randomly divided into the control (CON) group, immobilization (IM) group (immobilized treatment only), HES and LES groups (immobilized treatment and high or low-intensity electrical muscular stimulation through B-SES device). Bilateral femurs were used for X-ray micro-CT and biomechanical tests. RESULTS: The maximum load value was significantly lower in the IM and HES groups than in the CON group and significantly higher in the LES group than in the IM group. The maximum crushing load was significantly lower in the IM, HES, and LES groups than in the CON group, and significantly higher in the HES and LES groups than that in the IM group. In micro-CT, the mechanical stress by B-SES device did not affect degenerative microstructural changes in the cortical bone, but prevented those changes in the cancellous bone. CONCLUSIONS: Applying mechanical stress via B-SES device suppressed the loss of cancellous bone density and degenerative microstructural changes caused by immobilization, which in turn suppressed the reduction of bone strength. From these findings, muscle contractile exercise may be effective in preventing immobilization-induced bone atrophy.

Multiple trauma complicated by traumatic vertebral artery injury without cervical vertebral injury: A case report.

Background: Blunt traumatic vertebral artery injury is commonly associated with head and cervical spinal trauma. However, those associated with chest or upper extremity injuries without cervical spine-related trauma are rare. Case presentation: A 94-year-old woman was injured in a motor vehicle crash. She was diagnosed with traumatic subarachnoid hemorrhage, bilateral subdural hematomas, right vertebral artery injury, and right clavicle fracture. No cervical spine injuries were observed. It was possible that the fracture fragment of the right clavicle may have directly injured the right vertebral artery. Coil embolization was performed for the vertebral artery injury. The patient had a good postoperative course and was transferred to the hospital for rehabilitation on day 65. Conclusion: Regarding the high-risk injury mechanism, blunt traumatic vertebral artery injuries in the V1-2 segment may occur in cases with clavicle fractures.

Regular walking exercise prior to knee osteoarthritis reduces joint pain in an animal model.

We investigated the effect of regular walking exercise prior to knee osteoarthritis (OA) on pain and synovitis in a rat monoiodoacetic acid (MIA)-induced knee OA model. Seventy-one male Wistar rats were divided into three groups: (i) Sedentary + OA, (ii) Exercise + OA, and (iii) Sedentary + Sham groups. The Exercise + OA group underwent a regular treadmill walking exercise at 10 m/min (60 min/day, 5 days/week) for 6 weeks, followed by a 2-mg MIA injection in the right knee. The right knee joint was removed from rats in this group at the end of the 6-week exercise period and at 1 and 6 weeks after the MIA injection. After the 6 weeks of treadmill exercise but before MIA injection, there were no significant differences among the three groups in the pressure pain threshold, whereas at 1 week post-injection, the Exercise + OA group's pressure pain threshold was significantly higher than that in the Sedentary + OA group, and this difference persisted until the end of the experimental period. The histological changes in articular cartilage and subchondral bone revealed by toluidine blue staining showed no difference between the Sedentary + OA and EX + OA groups. The expression levels of interleukin (IL)-4 and IL-10 mRNA in the infrapatellar fat pad and synovium were significantly increased by the treadmill exercise. Significant reductions in the number of CD68-, CD11c-positive cells and IL-1ß mRNA expression and an increase in the number of CD206-positive cells were observed at 1 week after the MIA injection in the Exercise + OA group compared to the Sedentary + OA group. These results suggest that regular walking exercise prior to the development of OA could alleviate joint pain through increases in the expressions of anti-inflammatory cytokines in the rat infrapatellar fat pad and synovium.

The Relationship Between Physical Activity and Limited Range of Motion in the Older Bedridden Patients.

Background: The purpose of this study is to examine the association between physical activity and contracture in older patients confined to bed in long-term care (LTC) facilities. Methods: Patients wore ActiGraph GT3X+ for 8 hours on their wrists, and vector magnitude (VM) counts were obtained as the amount of activity. The passive range of motion (ROM) of joints was measured. The severity of ROM restriction classified, as the tertile value of the reference ROM of each joint, was scored 1-3 points. Spearman's rank correlation coefficients (Rs) were used to measure the association between the VM counts per day and ROM restrictions. Results: The sample comprised 128 patients with a mean (SD) age of 84.8 (8.8) years. The mean (SD) of VM was 84574.6 (115195.2) per day. ROM restriction was observed in most joints and movement directions. ROMs in all joints and movement directions, except wrist flexion and hip abduction, were significantly correlated with VM. Furthermore, the VM and ROM severity scores showed a significant negative correlation (Rs = -0.582, p < .0001). Conclusions: A significant correlation between the physical activity and ROM restrictions indicates that a decrease in the amount of physical activity could be one of the causes of contracture.

Effect of exercise and/or educational interventions on physical activity and pain in patients with hip/knee osteoarthritis: A systematic review with meta-analysis.

OBJECTIVE: To investigate the effectiveness of exercise and/or educational intervention on physical activity and pain in patients with hip/knee osteoarthritis (OA) using systematic review and meta-analysis. METHODS: We searched randomized controlled trials that investigated physical activity and pain and compared exercise and/or educational intervention with usual care in patients with hip/knee OA in MEDLINE (PubMed), ProQuest, Scopus, and the Physiotherapy Evidence Database (PEDro), including all those published by April 30, 2022 and written in English. Studies that newly applied analgesics after onset of the intervention were excluded. The revised Cochrane risk-of-bias tool for randomized trials was used to assess the methodological qualities. The random-effects model was used for meta-analysis with standard mean differences using RevMan version 5.4. The body of evidence for each study was synthesized using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach. RESULTS: Twenty studies including 2,350 patients were included (7 exercise studies, 8 educational intervention studies and 5 combination studies). The meta-analysis demonstrated that there is very low evidence that combination therapy of exercise and educational intervention improve the physical activity level at the endpoint (4 articles; SMD 0.33, 95% CI 0.04 to 0.51, P = 0.03). Low evidence was observed for combination therapy reducing pain (4 articles; SMD -0.15, 95% CI -0.29 to -0.02, P = 0.03). DISCUSSION: The current evidence indicated that combination therapy of exercise and educational intervention leads to improved physical activity and pain reduction in hip/knee OA patients, but the risk of bias in each study, especially in allocation concealment, downgraded the evidence level. These findings support the use of a combination therapy of exercise and educational intervention to promote physical activity levels in patients with hip/knee OA. TRAIL REGISTRATION: There was no financial support for this research. The protocol was registered at the International Prospective Register of Systematic Reviews (registration code: CRD42020205804).

Muscle contractile exercise through a belt electrode device prevents myofiber atrophy, muscle contracture, and muscular pain in immobilized rat gastrocnemius muscle.

PURPOSE: Immobilization of skeletal muscles causes muscle atrophy, muscle contracture, and muscle pain, the mechanisms of which are related to macrophage accumulation. However, muscle contractile exercise through a belt electrode device may mitigate macrophage accumulation. We hypothesized that such exercise would be effective in preventing myofiber atrophy, muscle contracture, and muscular pain. This study tested this hypothesis in immobilized rat gastrocnemius muscle. MATERIALS AND METHODS: A total of 32 rats were divided into the following control and experimental groups: immobilization (immobilized treatment only), low-frequency (LF; immobilized treatment and muscle contractile exercise with a 2 s (do) /6 s (rest) duty cycle), and high-frequency (HF; immobilized treatment and muscle contractile exercise with a 2 s (do)/2 s (rest) duty cycle). Electrical stimulation was performed at 50 Hz and 4.7 mA, and muscle contractile exercise was applied to the lower limb muscles for 15 or 20 min/session (once daily) for 2 weeks (6 times/week). After the behavioral tests, the bilateral gastrocnemius muscles were collected for analysis. RESULTS: The number of macrophages, the Atrogin-1 and MuRF-1 mRNA expression, and the hydroxyproline content in the HF group were lower than those in the immobilization and LF groups. The cross-sectional area (CSA) of type IIb myofibers in the superficial region, the PGC-1α mRNA expression, and the range of motion of dorsiflexion in the HF group were significantly higher than those in the immobilization and LF groups. The pressure pain thresholds in the LF and HF groups were significantly higher than that in the immobilization group, and the nerve growth factor (NGF) content in the LF and HF groups was significantly lower than that in the immobilization group. CONCLUSION: Muscle contractile exercise through the belt electrode device may be effective in preventing immobilization-induced myofiber atrophy, muscle contracture, and muscular pain in the immobilized rat gastrocnemius muscle.

Fecal Gram staining of phagocytosed bacteria to differentiate methicillin-resistant Staphylococcus aureus: A case report.

Antibiotic-associated diarrhea is a relatively common problem, and the main bacterial cause is Clostridioides difficile followed by Staphylococcus aureus and other pathogens. The diagnostic procedure for methicillin-resistant S. aureus enteritis is not well established. Phagocytosis is a key antimicrobial process involved in host defense. Phagocytosed bacteria identified by Gram staining are one marker to identify causative microorganisms and select subsequent treatment strategies. However, there are few reports on fecal Gram staining using phagocytosed bacteria as a target for diarrhea treatment. We report the successful use of fecal Gram staining to diagnose and treat methicillin-resistant S. aureus enteritis.

Effects of full-length human amelogenin on the differentiation of dental epithelial cells and osteoblastic cells.

BACKGROUND AND OBJECTIVE: Amelogenins are major components of extracellular matrix proteins in developing teeth, and regulate the growth of enamel crystals. They also function as signaling molecules in cell differentiation. This study aimed to determine the biological effects of amelogenins on the differentiation of HAT-7 dental epithelial cells and MC3T3-E1 pre-osteoblastic cells using full-length recombinant human amelogenin (rh-AMEL). DESIGN: rh-AMEL was expressed in a mammalian cell line (Expi293F™) and was purified by DDK agarose beads. Effects of rh-AMEL on differentiation were evaluated by Mineralization and Alkaline phosphatase (ALP) activity using Alizarin Red S staining and colorimetric substrate p-nitrophenol, respectively. RESULTS: Western blotting and silver staining confirmed the successful purification of rh-AMEL. Mineralization and ALP activity in HAT-7 cells were significantly higher after treatment with 4 µg/mL rh-AMEL, but not after treatment with Emdogain® (EMD). In MC3T3-E1 cells, on the other hand, rh-AMEL showed biphasic effects on differentiation. Treatment with low concentrations of rh-AMEL (0.001-0.1 µg/mL) and EMD (0.01-1 µg/mL) increased mineralization and ALP activity in MC3T3-E1 cells, whereas treatment with high concentrations of rh-AMEL (4 µg/mL) and EMD (100 µg/mL) had the opposite effect. CONCLUSION: High concentrations of rh-AMEL and EMD decreased the differentiation of MC3T3-E1 cells. By contrast, a high concentration of rh-AMEL, but not that of EMD, promoted the differentiation of HAT-7 cells. This study demonstrates that the effects of rh-AMEL on cell differentiation differ between HAT-7 and MC3T3-E1 cells, and suggests that different regions on AMEL may induce the differentiation of these cell types.

Antibacterial activity of lysozyme-binding proteins from chicken egg white.

The purpose of this study was to establish a method for determining the bacteriolytic activity after separation of lysozyme-binding proteins from egg white. Lysozyme-binding proteins such as ovotransferrin and ovalbumin were separated by non-denaturing two-dimensional electrophoresis (2DE) and transferred to a membrane. The lysozyme activity of the separated and immobilized egg white proteins was assessed directly to produce a non-denaturing 3D map of the egg white proteins by incorporating an axis that combined each spot's lysozyme-activity with the non-denaturing 2DE pattern. Lysozyme-ovotransferrin and lysozyme-ovalbumin complexes could be reconstructed in vitro after the cathode end fraction containing lysozyme was added to purified ovotransferrin and ovalbumin, respectively. These complexes retained lysozyme activity even after separation by non-denaturing 2DE. Furthermore, when the lysozyme-ovotransferrin complex from egg white was extracted after separation by isoelectric focusing by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, the complex possessed bacteriolytic activity against both Bacillus subtilis and Escherichia coli. These methods can be applied to investigate protein complexes possessing bacteriolytic activity against a wide range of both Gram-positive and Gram-negative bacteria.

Cutaneous squamous cell carcinoma, thyroid cancer and Langerhans cell histiocytosis in a patient with X-linked recessive Mendelian susceptibility to mycobacterial diseases with a nuclear factor-κB essential modifier mutation.

Nuclear factor (NF)-κB essential modifier (NEMO), also known as IκB kinase subunit-γ (IKKγ), is a pivotal molecule in the NF-κB signaling pathway. Mutations of NEMO cause incontinentia pigmenti and X-linked ectodermal dysplasia with immunodeficiency. Mendelian susceptibility to mycobacterial diseases (MSMD), which confers an almost selective predisposition to mycobacterial infection, is also caused by NEMO mutations. We herein report the first case of a patient with X-linked recessive (XR) MSMD who developed cutaneous squamous cell carcinoma, thyroid cancer and Langerhans cell histiocytosis. The relationship between NEMO mutation and oncogenesis is discussed.

Effect of 1,25-dihydroxyvitamin D3 on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging.

Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca2+ release from intracellular stores and subsequent Ca2+ entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 µM ATP. We applied long-term imaging (for ~10 h) to monitor Ca2+ responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca2+ concentration in the presence of 100 nM 1,25-dihydroxyvitamin D3. Many of these Ca2+ responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca2+ responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca2+ responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.

Analysis of CYP2R1 and CYP26A1 Expression Patterns in Regeneration in Mice with Liver Injury.

Cytochrome P450 enzymes (CYPs) are involved in the metabolism of various substances in the liver and small intestine and show markedly higher expression levels in the liver compared to other organs. The liver exhibits a remarkable capacity to regenerate. After excision of 70% of the liver, the organ can regenerate to its original size in approximately 1 week. Unlike the normal liver, in the injured liver, hepatic stem cells known as oval cells are considered to play an important role in regeneration. However, the role of CYPs in liver regeneration remains unclear. In the present study, we investigated the role of CYPs in the regeneration of injured liver. Liver injury was induced by 4-week repeated doses of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in the diet. Next, both DDC-fed mice and control diet (containing no DDC)-fed mice were subjected to 70% hepatectomy, and the hepatic gene expression patterns measured during regeneration were analyzed. Mice with DDC-induced liver injury expressed the oval cell markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM), and partial hepatectomy increased the expression levels of CYP2R1 and CYP26A1 as well as the hepatoblast marker alpha-fetoprotein (AFP) in these mice. The results of this study suggest that CYP2R1 and CYP26A1 are important in the differentiation of oval cells into hepatoblast-like cells in the injured liver.

Removal of bisphenol A and its derivatives from aqueous medium through laccase-catalyzed treatment enhanced by addition of polyethylene glycol.

In this study, enzymatic removal of bisphenol A (BPA) from the aqueous medium was investigated through the generation of water-insoluble oligomers, and this procedure was applied to removal of bisphenol derivatives. The experimental parameters, such as the temperature, pH value, enzyme concentration, and concentration and molecular weight of polyethylene glycol (PEG), were determined for the laccase-catalyzed treatment of BPA. The optimum conditions were determined to be pH 7.0 and 40°C in the absence of PEG. Water-insoluble oligomers generated under these conditions were readily removed by filtration or centrifugation. The optimum pH value was decreased to 5.0 in the presence of PEG and the laccase dose was reduced to one-fiftieth of that in the absence of PEG. This indicates that the addition of PEG protects the enzymatic activity and prevents capture of laccase molecules in the oligomers. The oligomers generated in the presence of PEG were removed from the aqueous medium by filtration with a membrane filter or by centrifugation. The oligomers were completely filtrated out with a filter paper by decreasing the pH value to 3.0. In addition, several bisphenol derivatives were also treated and subsequently removed by adjusting the laccase dose in the presence of PEG using the above procedure.

Prognostic significance of ß2-adrenergic receptor expression in malignant melanoma.

Recent studies cite ß2-adrenergic receptor (ß2AR) antagonists as novel therapeutic agents for melanoma, as they may reduce the disease progression. The ß2AR has shown to be expressed in malignant melanoma. However, it remains unclear whether the ß2AR expression has a clinical and pathological significance in patients with cutaneous malignant melanoma. We herein conducted a clinicopathological study to investigate the protein expression of ß2AR in malignant melanoma of the skin and its prognostic significance. One hundred thirty-three patients with surgically resected cutaneous malignant melanoma were evaluated. Tumor sections were stained by immunohistochemistry for ß2AR, Ki-67, the microvessel density (MVD) determined by CD34, and p53. ß2AR was highly expressed in 44.4 % (59 out of 133) of the patients. The expression of ß2AR was significantly associated with the tumor thickness, ulceration, T factor, N factor, disease stage, tumor size, cell proliferation (Ki-67), and MVD (CD34). Using Spearman's rank test, the ß2AR expression was correlated with Ki-67 (r = 0.278; 95 % CI, 0.108 to 0.432; P = 0.001), CD34 (r = 0.445; 95 %CI, 0.293 to 0.575; P < 0.001), and the tumor size (r = 0.226; 95 % CI, 0.053 to 0.386; P = 0.008). Using a univariate analysis, the tumor thickness, ulceration, disease stage, ß2AR, Ki-67, and CD34 had a significant relationship with the overall and progression-free survivals. A multivariable analysis confirmed that ß2AR was an independent prognostic factor for predicting a poor overall survival (HR 1.730; 95 % CI 1.221-2.515) and progression-free survival (HR 1.576; 95 % CI 1.176-2.143) of malignant melanoma of the skin. ß2AR can serve as a promising prognostic factor for predicting a worse outcome after surgical treatment and may play an important role in the development and aggressiveness of malignant melanoma.

Prognostic significance of L-type amino acid transporter 1 (LAT1) expression in cutaneous melanoma.

Amino acid transporters play a crucial role in the development and invasiveness of cancer cells. However, it remains unclear whether or not the expression of L-type amino acid transporter 1 (LAT1) has prognostic significance in patients with cutaneous melanoma. A total of 128 patients with cutaneous melanoma were evaluated. Tumor sections were stained by immunohistochemistry for LAT1, CD98, Ki-67, and microvessel density determined by CD34 and p53. We also analyzed 30 specimens of patients with melanocytic nevi as negative controls. LAT1 and CD98 were highly expressed in 58% (75/128) and 75% (97/128), respectively. The rates of positivity for LAT1 in the melanocytic nevi were 0% (0/30). The expression of LAT1 was associated significantly with tumor thickness, T factor, CD98 expression, cell proliferation (Ki-67), and microvessel density (CD34). By Spearman's rank test, LAT1 expression was correlated with CD98, Ki-67, and CD34. By univariate analysis, tumor thickness, ulceration, disease staging, LAT1, and CD34 showed a significant relationship with overall survival and disease-free survival. Furthermore, a multivariate analysis confirmed that LAT1 was an independent prognostic factor for predicting a poor prognosis. This study had a small sample size. LAT1 can serve as a significant prognostic factor to predict a poor outcome and it may therefore play an important role in the aggressiveness of cutaneous melanoma.

Effect of epithelial cells derived from periodontal ligament on osteoblast-like cells in a Transwell membrane coculture system.

BACKGROUND AND OBJECTIVE: The epithelial rests of Malassez (ERM) are developmental residues of Hertwig's epithelial root sheath, and they are present throughout life in the periodontal ligament. Previous studies have shown that ERM induce cementogenesis or inhibit cement-osteogenesis. This study investigated how ERM cells are involved in osteoblast mineralization in an in vitro coculture system. MATERIALS AND METHODS: ERM cells were isolated from porcine periodontal ligament by an outgrowth method. Osteoblast-like MC3T3-E1 cells were cocultured with ERM or gingival epithelium (GE) cells in six-well Transwell units. Mineralization of MC3T3-E1 cells was confirmed by Alizarin Red staining after 30 days of culture. Alkaline phosphatase (ALP) activity was measured in the culture media. To examine whether enamel matrix proteins are involved in the mineralization of MC3T3-E1 cells, an anti-amelogenin antibody was added to the culture media. RESULTS: The staining by Alizarin Red of MC3T3-E1 cells cocultured with ERM cells was clearly weaker than that in the GE cell coculture. The ALP activity in the ERM cell coculture was significantly lower than that in the GE cell coculture (p < 0.05). Alizarin Red staining of MC3T3-E1 cells in the ERM cell coculture with anti-amelogenin antibody was clearly stronger than in those cocultures without the antibody at 30 days. The ALP activity in the ERM cell coculture with anti-amelogenin antibody was significantly higher than in those cultures without the antibody (p < 0.05). CONCLUSION: This study demonstrated that ERM cells inhibit the calcification of osteoblast-like cells in a Transwell coculture system and that this inhibition is reversed with an anti-amelogenin antibody.

Removal of bisphenol derivatives through quinone oxidation by polyphenol oxidase and subsequent quinone adsorption on chitosan in the heterogeneous system.

In this study, the combined use of a biopolymer chitosan and an oxidoreductase polyphenol oxidase (PPO) was systematically investigated for the removal of bisphenol derivatives from aqueous medium. The process parameters, such as the pH value, temperature, and PPO concentration, were estimated to conduct the enzymatic quinone oxidation of bisphenol derivatives by as little enzyme as possible. Bisphenol derivatives effectively underwent PPO-catalysed quinone oxidation without H2O2 unlike other oxidoreductases, such as peroxidase and tyrosinase, and the optimum conditions were determined to be pH 7.0 and 40°C for bisphenol B, bisphenol E, bisphenol O, and bisphenol Z; pH 7.0 and 30°C for bisphenol C and bisphenol F; and pH 8.0 and 40°C for bisphenol T. They were completely removed through adsorption of enzymatically generated quinone derivatives on chitosan beads or chitosan powders. Quinone adsorption on chitosan beads or chitosan powders in the heterogeneous system was found to be a more effective procedure than generation of aggregates in the homogeneous system with chitosan solution. The removal time was shortened by increasing the amount of chitosan beads or decreasing the size of the chitosan powders.