A single atom cobalt anchored MXene bifunctional platform for rapid, label-free and high-throughput biomarker analysis and tissue imaging.

Few of single-atom materials have been served as platform to analyze small molecules for surface assisted laser desorption/ionization mass spectrometry (SALDI-MS). Herein, a novel single Co atom-anchored MXene (Co-N-Ti3C2) is prepared to achieve enhanced SALDI-MS and mass spectrometry imaging (MSI) performance for the first time. The Co-N-Ti3C2 films were prepared by a simple in situ self-assembly strategy to generate an efficient SALDI-MS platform. Compared to typical inorganic/organic matrices, Co-N-Ti3C2 films exhibit superior performance in small molecules detection with ultra-high sensitivity (LOD at amol level), excellent repeatability (CV <4%), clean background and wide analyte coverage, enabling accurate quantitative analysis of various low-concentration metabolites from 1 µL biofluid in seconds. Its usage efficiently enhanced SALDI-MS detection of various small-molecule biomarkers such as amino acids, succinic acid, itaconic acid, arachidonic acid, citrulline, prostaglandin E2, creatinine, uric acid, glutamine, D-mannose, cholesterol and inositol in positive ion mode. The blood glucose level in humans was successfully determined from a linearity concentration range (0.25-10 mM). Notably, the Co-N-Ti3C2 assisted SALDI-MSI enables study the spatial distribution of small molecules covering the range central to metabolomics at a high resolution on a tissue section. Furthermore, Co-N-Ti3C2 platform revealed a specific peak profile that distinguishes osteoarthritis (OA) from rheumatoid arthritis (RA) tissue. Density functional theory theoretical investigation revealed that single Co atoms anchored on Ti3C2 could highly enhanced the ionization ability of metabolites, resulting in high-sensitivity and heterogeneous metabolome coverage.

SULFORAPHANE ADMINISTRATION AFTER HEMORRHAGIC SHOCK/RESUSCITATION IN MICE REDUCES THE SECRETION OF INFLAMMATORY CYTOKINES AND INCREASES THE IMMUNOCOMPETENCE OF SPLENIC MACROPHAGES.

ABSTRACT: Objective : The purpose of this study was to investigate the immunomodulatory effects of sulforaphane (SFN), a nuclear factor erythroid 2-related factor (Nrf2) pathway activator, on splenic macrophages' immunocompetence after hemorrhagic shock/resuscitation (HS/R). Methods : Male C57/BL6 wild-type mice (n = 6 per group) were subjected to either pressure-controlled HS (MAP, 35-45 mm Hg) or a sham procedure surgery (without HS). After 90 minutes of HS, fluid resuscitation with withdrawn blood and 0.9% NaCl was performed. Sulforaphane (50 mg/kg of body weight) was applied intraperitoneally immediately after the resuscitation phase as well as 24 and 48 h thereafter, depending on group allocation. The mice were killed at 6, 24, and 72 h after resuscitation. After killing, spleens were harvested to perform Nrf2 immunofluorescence histology. Splenic macrophages were isolated and cultured to measure cytokine secretion in the cell culture supernatant. Furthermore, macrophages isolated after 24-hour resuscitation were treated with 100 ng/mL of bacterial LPS to measure immunocompetence. Matrix-assisted laser desorption/ionization mass spectrometry imaging was performed to verify the distribution of SFN in the spleen after intraperitoneal injection. Results : We showed that administered SFN reached the spleen within the first hour after administration. Furthermore, we identified that SFN increased splenic Nrf2 activation and decreased cytokine expression in splenic macrophages after HS/R. In addition, we showed that SFN exhibited splenic anti-inflammatory properties of macrophages in vitro (IL-6/IL-10-ratio of the HS/R group: 51.79 ± 9.99 [at 6 h] and 15.70 ± 3.35 [at 24 h] vs. HS/R + SFN group: 20.54 ± 5.35 [at 6 h] and 8.60 ± 2.37 [at 24 h], P < 0.05). Furthermore, SFN improved in vitro splenic macrophage immunocompetence after HS/R, as evidenced by the increased secretion of inflammatory cytokines in response to LPS stimulation in vitro . Conclusions : Our study shows that SFN can reduce inflammatory cytokines secreted by splenic macrophages after HS/R and increase their immunocompetence toward a more anti-inflammatory profile.

Total knee arthroplasty exhibits satisfactory long-term clinical efficacy in the treatment of hemophilia patients with stiff knees.

Objective: This study aimed to (1) determine the long-term clinical efficacy of total knee arthroplasty (TKA) in the treatment of hemophilia patients with stiff knessknees, (2) assess the 5- and 10-year prosthesis survival in hemophilia, and (3) determine whether the severity of preoperative stiffness would affect postoperative clinical outcomes and complication rates. Methods: The clinical data of 71 patients (78 knees) with hemophilia and concomitant knee stiffness who had undergone TKA between September 2007 and June 2018 were retrospectively analyzed. All patients were male, their mean age at the time of surgery was 38.4. ± 7.9 years (range: 21-63 years), and the mean follow-up time was 8.7 years. To determine the effect of stiffness severity on clinical outcomes, the participants were categorized into two groups: severe [preoperative range of motion (ROM): <50°, 34 knees] and moderate (preoperative ROM: 50-90°, 44 knees). At preoperative and final follow-up, patients' post-TKA clinical and radiological outcomes, quality of life, complications, and long-term survival were assessed. Results: Flexion contracture improved from 23.2 ± 10.8° before surgery to 5.9 ± 7.5° upon final follow-up, the Knee Society Score (KSS) increased from 31.4 ± 12.4 to 74.9 ± 11.5, and the KSS functional score increased from 37.6 ± 9.3 to 81.4 ± 12.8. The mean ROM improved from 54.6 ± 32.6° preoperatively to 80.9 ± 34.5° postoperatively. The 36-Item Short Form Survey physical and mental scores also improved significantly. All these differences were statistically significant before and after surgery (P < 0.001). The following postoperative complications occurred in 10 knees (12.8%): hemarthrosis (n = 3), stiffness (n = 4), superficial infection (n = 1), skin necrosis (n = 1), and periprosthetic infection (n = 2), and revision TKA was performed on two knees. The 5- and 10-year survival rates of the prostheses were 98.5% and 93.7%, respectively. The mean ROM in the severe group increased from 30.7 ± 18.7° preoperatively to 70.5 ± 28.3° postoperatively (p < 0.001). The mean flexion contracture decreased from 27.3 ± 10.8° to 6.4 ± 12.0° (p < 0.001). The mean KSS improved from 27.0 ± 7.8 to 68.3 ± 9.6 (p < 0.001). The mean ROM in the moderate group improved from 84.3 ± 22.7 to 92.9 ± 28.8 (p < 0.001), while the mean flexion contracture decreased from 12.8 ± 11.0° to 4.8 ± 5.0° (p < 0.001) and the mean KSS improved from 41.3 ± 11.5 to 81.3 ± 12.2 (p < 0.001). The severe group had worse postoperative ROM and functional scores than the moderate group. Furthermore, the severe group used varus-valgus constrained or hinged prostheses more frequently (52.8% vs. 18.1%) and had more complications (18.9% vs. 9.0%) than the moderate group. Conclusion: TKA exhibits satisfactory long-term efficacy in patients with hemophilic knee joint disease involving preoperative stiffness, thus potentially providing a significant improvement in function and reducing pain. Furthermore, severely stiff knee joints have worse clinical outcomes and more complications than moderately stiff knee joints.

1,25-Dihydroxyvitamin D3 attenuates disease severity and induces synoviocyte apoptosis in a concentration-dependent manner in rats with adjuvant-induced arthritis by inactivating the NF-κB signaling pathway.

An aggressive proliferation of synoviocytes is the hallmark of rheumatoid arthritis (RA). Emerging evidence shows that inhibiting the NF-κB signaling pathway with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] may be a therapeutic approach for controlling inflammatory diseases. In this study, we demonstrated the protective effects of three different 1,25(OH)2D3 concentration on adjuvant-induced arthritis (AA) rats through the NF-κB signaling pathway and their pro-apoptotic roles in cultured adjuvant-induced arthritis synoviocytes (AIASs). AA rats were prepared by injecting complete Freund's adjuvant and independently given daily intraperitoneal injection of 1,25(OH)2D3 at concentrations of 50, 100, and 300 ng/day/kg. Subsequently, AIASs were isolated from the inflamed joints of AA rats to test the effects of 1,25(OH)2D3 on AIASs in vitro. Intraperitoneal injection of 1,25-(OH)2D3 was found to induce a concentration- and time-dependent improvement in relieving the symptoms of AA. We found an increased paw withdrawal thermal latency (PWTL) in the affected paw of AA rats as the concentration of 1,25-(OH)2D3 increased. 1,25-(OH)2D3 treatment reduced levels of inflammatory factors in synovial tissues of AA rats. In the case of cultured AIASs, 1,25-(OH)2D3 was shown to inhibit cell proliferation and induce cell apoptosis in a concentration-dependent manner. Additionally, 1,25-(OH)2D3 inhibited the activation of the NF-κB signaling pathway. In conclusion, our study provides evidence emphasizing that 1,25(OH)2D3 has the potential to attenuate disease severity in RA potentially due to its contributory role in synoviocyte proliferation and apoptosis. The protective role of 1,25(OH)2D3 against RA depends on the NF-κB signaling pathway.

Regulation of DMT1 on autophagy and apoptosis in osteoblast.

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 µmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Role of miR-155 in the regulation of MMP-16 expression in intervertebral disc degeneration.

The molecular mechanisms of intervertebral disc degeneration (IDD) remain elusive. We found that miR-155 is down-regulated in degenerative nucleus pulposus (NP), and more severe degeneration is correlated with higher matrix metallopeptidase 16 (MMP-16) expression. MMP-16 also degraded matrix aggrecan. Here, we addressed the in vivo miR-155-mediated pathological impact on IDD using a classic puncture mouse model. Lentiviral upregulated-miR-155 or downregulated-miR-155 was transduced into the discs of C57 mice, which was validated by real-time polymerase chain reaction (real-time PCR) and in situ hybridization. Immunohistochemistry and western blotting revealed that up-regulation of miR-155 resulted in down-regulation of MMP-16 and an increase in aggrecan and collagen type II in mouse NP; whereas, down-regulation of miR-155 resulted in up-regulation of MMP-16 and a decrease in aggrecan in mouse NP. Radiographic and histological analysis showed that the up-regulation of miR-155 attenuated IDD, while down-regulation of miR-155 resulted in the deterioration of IDD. These findings indicate that decreased miR-155 contributed to the up-regulation of MMP-16 in vivo, and MMP-16 further degraded aggrecan and collagen type II, leading to the dehydration and degeneration of discs. Our findings revealed a therapeutic role for miR-155 in IDD. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1323-1334, 2017.

Melatonin suppresses autophagy in type 2 diabetic osteoporosis.

Type 2 diabetes mellitus is often complicated by osteoporosis, a process which may involve osteoblast autophagy. As melatonin suppresses autophagy under certain conditions, we its investigated the effects on bone autophagy during diabetes. We first assessed different body parameters in a diabetic rat model treated with various concentrations of melatonin. Dynamic biomechanicalmeasurements, bone organization hard slice dyeing and micro-CT were used to observe the rat bone microstructure, and immunohistochemistry was used to determine levels of autophagy biomarkers. We also performed in vitro experiments on human fetal osteoblastic (hFOB1.19) cells cultured with high glucose, different concentrations of melatonin, and ERK pathway inhibitors. And we used Western blotting and immunofluorescence to measure the extent of osteogenesis and autophagy. We found that melatonin improved the bone microstructure in our rat diabetes model and reduced the level of autophagy(50 mg/kg was better than 100 mg/kg). Melatonin also enhanced osteogenesis and suppressed autophagy in osteoblasts cultured at high glucose levels (10 µM was better than 1 mM). This suggests melatonin may reduce the level of autophagy in osteoblasts and delay diabetes-induced osteoporosis by inhibiting the ERK signaling pathway.

Autophagy protects osteoblasts from advanced glycation end products-induced apoptosis through intracellular reactive oxygen species.

Patients with type II diabetes are susceptible to fracture; however, these patients typically have normal bone mineral density. Thus, such fractures cannot be entirely explained by advanced glycation end products (AGEs)-induced osteoblast apoptosis. Autophagy is a molecular process allowing cells to degrade unnecessary or dysfunctional cellular organelles, and closely interacts with apoptosis. The aim of this study was to determine whether autophagy participated in the pathology of AGEs-treated osteoblasts, and the possible mechanism of such an involvement. Osteoblastic MC3T3-E1 cells were used. Autophagy was evaluated by detecting the level of LC3 via western blotting and immunofluorescence. p62/SQSTM1 expression was also assessed by western blotting. The autophagy inducer rapamycin (RA) and the autophagy inhibitor 3-methyladenine were used to determine whether autophagy has effect on AGEs-induced apoptosis. N-Acetylcysteine (NAC), reactive oxygen species (ROS) inhibitor, was used to determine whether ROS and mitochondrial damage were involved in autophagy regulation. The results showed that the autophagy level was increased in MC3T3-E1 cells treated with AGEs, as represented by an increase in both the total LC3 level and the LC3II/LC3I ratio, as well as a decrease in p62/SQSTMI expression. Further inducing autophagy by RA attenuated AGEs-induced apoptosis. The antioxidant NAC suppresses AGEs-induced autophagy in osteoblastic MC3T3-E1 cells. These results demonstrate that autophagy participates in the pathology of AGEs-treated osteoblasts, and may play a protective role in AGEs-induced apoptosis in osteoblastic MC3T3-E1 cells. ROS and mitochondrial damage are essential in upregulating AGEs-induced autophagy.

Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

Regulation of DMT1 on Bone Microstructure in Type 2 Diabetes.

Diabetic osteoporosis is gradually attracted people's attention. However, the process of bone microstructure changes in diabetic patients, and the exact mechanism of osteoblast iron overload are unclear. Therefore, the present study aimed to explore the function of DMT1 in the pathological process of diabetic osteoporosis. We build the type two diabetes osteoporosis models with SD rats and Belgrade rats, respectively. Difference expression of DMT1 was detected by using the method of immunohistochemistry and western blotting. Detection of bone microstructure and biomechanics and iron content for each group of samples. We found that DMT1 expression in type 2 diabetic rats was higher than that in normal rats. The bone biomechanical indices and bone microstructure in the rat model deficient in DMT1 was significantly better than that in the normal diabetic model. The loss of DMT1 can reduce the content of iron in bone. These findings indicate that DMT1 expression was enhanced in the bone tissue of type 2 diabetic rats, and plays an important role in the pathological process of diabetic osteoporosis. Moreover, DMT1 may be a potential therapeutic target for diabetic osteoporosis.

Radiographic angles in hallux valgus: Comparison between protractor and iPhone measurements.

Radiographic angles are used to assess the severity of hallux valgus deformity, make preoperative plans, evaluate outcomes after surgery, and compare results between different methods. Traditionally, hallux valgus angle (HVA) has been measured by using a protractor and a marker pen with hardcopy radiographs. The main objective of this study is to compare HVA measurements performed using a smartphone and a traditional protractor. The secondary objective was to compare the time taken between those two methods. Six observers measured major HVA on 20 radiographs of hallux valgus deformity with both a standard protractor and an Apple iPhone. Four of the observers repeated the measurements at least a week after the original measurements. The mean absolute difference between pairs of protractor and smartphone measurements was 3.2°. The 95% confidence intervals for intra-observer variability were ±3.1° for the smartphone measurement and ±3.2° for the protractor method. The 95% confidence intervals for inter-observer variability were ±9.1° for the smartphone measurement and ±9.6° for the protractor measurement. We conclude that the smartphone is equivalent to the protractor for the accuracy of HVA measurement. But, the time taken in smartphone measurement was also reduced.