Oleanolic acid exerts bone anabolic effects via activation of osteoblastic 25-hydroxyvitamin D 1-alpha hydroxylase.

Oleanolic acid (OA) is previously shown to exert bone protective effects in aged animals. However, its role in regulating osteoblastic vitamin D bioactivation, which is one of major causes of age-related bone loss, remains unclear. Our results revealed that treatment of OA significantly increased skeletal CYP27B1 expression and circulating 1,25(OH)2D3 in ovariectomized mice (p <0.01). Moreover, OA upregulated CYP27B1 protein expression and activity, as well as the vitamin D-responsive bone markers alkaline phosphatase (ALP) activity and osteopontin (OPN) protein expression, in human osteoblast-like MG-63 cells (p<0.05). CYP27B1 expression increased along with the osteoblastic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). CYP27B1 expression and cellular 1,25(OH)2D3 production were further potentiated by OA in cells at mature osteogenic stages. Notably, our study suggested that the osteogenic actions of OA were CYP27B1 dependent. In summary, the bone protective effects of OA were associated with the induction of CYP27B1 activity and expression in bone tissues and osteoblastic lineages. Hence, OA might be a potential approach for management of age-related bone loss.

Kinematic difference and asymmetries during level walking in adolescent patients with different types of mild scoliosis.

BACKGROUND: Adolescent idiopathic scoliosis (AIS), three-dimensional spine deformation, affects body motion. Previous research had indicated pathological gait patterns of AIS. However, the impact of the curve number on the walking mechanism has not been established. Therefore, this study aimed to compare the gait symmetry and kinematics in AIS patients with different curve numbers to healthy control. RESULTS: In the spinal region, double curves AIS patients demonstrated a smaller sagittal symmetry angle (SA) and larger sagittal convex ROM of the trunk and lower spine than the control group. In the lower extremities, the single curve patients showed a significantly reduced SA of the knee joint in the frontal plane, while the double curves patients showed a significantly reduced SA of the hip in the transverse plane. CONCLUSION: The curve number indeed affects gait symmetry and kinematics in AIS patients. The double curves patients seemed to adopt a more "careful walking" strategy to compensate for the effect of spinal deformation on sensory integration deficits. This compensation mainly occurred in the sagittal plane. Compared to double curves patients, single curve patients unitized a similar walking strategy with healthy subjects.

Comparison of plugin and redundant marker sets to analyze gait kinematics between different populations.

BACKGROUND: Gait model consists of a marker set and a segment pose estimation algorithm. Plugin marker set and inverse kinematic algorithm (IK.) are prevalent in gait analysis, especially musculoskeletal motion analysis. Adding extra markers for the plugin marker set could increase the robustness to marker misplacement, motion artifacts, and even markers occlusion. However, how the different marker sets affect the gait analysis's kinematic output is unclear. Therefore, this study aims to investigate the effect of marker sets on the kinematic output during level walking in different populations. RESULTS: In all three planes, there are significant differences (P < 0.05) between marker sets in some kinematic variables at the hip, knee, and ankle. In different populations, the kinematic variables that show significant differences varied. When comparing the kinematic differences between populations using the two marker sets separately, the range of motion (ROM) of hip flexion was only found to be a significant difference using the redundant marker set, while the peak internal rotation at the knee was only found a significant difference using plugin marker set. In addition, the redundant marker set shows less intra-subject variation than the plugin marker set. CONCLUSION: The findings in this study demonstrate the importance of marker set selection since it could change the result when comparing the kinematic differences between populations. Therefore, it is essential to increase the caution in explaining the result when using different marker sets. It is crucial to use the same marker set, and the redundant marker set might be a better choice for gait analysis.

Handgrip strength assessment at baseline in addition to bone parameters could potentially predict the risk of curve progression in adolescent idiopathic scoliosis.

Introduction: Adolescent idiopathic scoliosis (AIS) is characterized by deranged bone and muscle qualities, which are important prognostic factors for curve progression. This retrospective case-control study aims to investigate whether the baseline muscle parameters, in addition to the bone parameters, could predict curve progression in AIS. Methods: The study included a cohort of 126 female patients diagnosed with AIS who were between the ages of 12 and 14 years old at their initial clinical visit. These patients were longitudinally followed up every 6 months (average 4.08 years) until they reached skeletal maturity. The records of these patients were thoroughly reviewed as part of the study. The participants were categorized into two sub-groups: the progressive AIS group (increase in Cobb angle of ≥6°) and the stable AIS group (increase in Cobb angle <6°). Clinical and radiological assessments were conducted on each group. Results: Cobb angle increase of ≥6° was observed in 44 AIS patients (34.9%) prior to skeletal maturity. A progressive AIS was associated with decreased skeletal maturity and weight, lower trunk lean mass (5.7%, p = 0.027) and arm lean mass (8.9%, p < 0.050), weaker dominant handgrip strength (8.8%, p = 0.027), deranged cortical compartment [lower volumetric bone mineral density (vBMD) by 6.5%, p = 0.002], and lower bone mechanical properties [stiffness and estimated failure load lowered by 13.2% (p = 0.005) and 12.5% (p = 0.004)]. The best cut-off threshold of maximum dominant handgrip strength is 19.75 kg for distinguishing progressive AIS from stable AIS (75% sensitivity and 52.4% specificity, p = 0.011). Discussion: Patients with progressive AIS had poorer muscle and bone parameters than patients with stable AIS. The implementation of a cut-off threshold in the baseline dominant handgrip strength could potentially be used as an additional predictor, in addition to bone parameters, for identifying individuals with AIS who are at higher risk of experiencing curve progression.

Low-Dose Staphylococcal Enterotoxin C2 Mutant Maintains Bone Homeostasis via Regulating Crosstalk between Bone Formation and Host T-Cell Effector Immunity.

Studies in recent years have highlighted an elaborate crosstalk between T cells and bone cells, suggesting that T cells may be alternative therapeutic targets for the maintenance of bone homeostasis. Here, it is reported that systemic administration of low-dose staphylococcal enterotoxin C2 (SEC2) 2M-118, a form of mutant superantigen, dramatically alleviates ovariectomy (OVX)-induced bone loss via modulating T cells. Specially, SEC2 2M-118 treatment increases trabecular bone mass significantly via promoting bone formation in OVX mice. These beneficial effects are largely diminished in T-cell-deficient nude mice and can be rescued by T-cell reconstruction. Neutralizing assays determine interferon gamma (IFN-γ) as the key factor that mediates the beneficial effects of SEC2 2M-118 on bone. Mechanistic studies demonstrate that IFN-γ stimulates Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling, leading to enhanced production of nitric oxide, which further activates p38 mitogen-activated protein kinase (MAPK) and Runt-related transcription factor 2 (Runx2) signaling and promotes osteogenic differentiation. IFN-γ also directly inhibits osteoclast differentiation, but this effect is counteracted by proabsorptive factors tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) secreted from IFN-γ-stimulated macrophages. Taken together, this work provides clues for developing innovative approaches which target T cells for the prevention and treatment of osteoporosis.

Mesenchymal stem cells alleviate dexamethasone-induced muscle atrophy in mice and the involvement of ERK1/2 signalling pathway.

BACKGROUND: High dosage of dexamethasone (Dex) is an effective treatment for multiple diseases; however, it is often associated with severe side effects including muscle atrophy, resulting in higher risk of falls and poorer life quality of patients. Cell therapy with mesenchymal stem cells (MSCs) holds promise for regenerative medicine. In this study, we aimed to investigate the therapeutic efficacy of systemic administration of adipose-derived mesenchymal stem cells (ADSCs) in mitigating the loss of muscle mass and strength in mouse model of DEX-induced muscle atrophy. METHODS: 3-month-old female C57BL/6 mice were treated with Dex (20 mg/kg body weight, i.p.) for 10 days to induce muscle atrophy, then subjected to intravenous injection of a single dose of ADSCs ([Formula: see text] cells/kg body weight) or vehicle control. The mice were killed 7 days after ADSCs treatment. Body compositions were measured by animal DXA, gastrocnemius muscle was isolated for ex vivo muscle functional test, histological assessment and Western blot, while tibialis anterior muscles were isolated for RNA-sequencing and qPCR. For in vitro study, C2C12 myoblast cells were cultured under myogenic differentiation medium for 5 days following 100 [Formula: see text]M Dex treatment with or without ADSC-conditioned medium for another 4 days. Samples were collected for qPCR analysis and Western blot analysis. Myotube morphology was measured by myosin heavy chain immunofluorescence staining. RESULTS: ADSC treatment significantly increased body lean mass (10-20%), muscle wet weight (15-30%) and cross-sectional area (CSA) (~ 33%) in DEX-induced muscle atrophy mice model and down-regulated muscle atrophy-associated genes expression (45-65%). Hindlimb grip strength (~ 37%) and forelimb ex vivo muscle contraction property were significantly improved (~ 57%) in the treatment group. Significant increase in type I fibres (~ 77%) was found after ADSC injection. RNA-sequencing results suggested that ERK1/2 signalling pathway might be playing important role underlying the beneficial effect of ADSC treatment, which was confirmed by ERK1/2 inhibitor both in vitro and in vivo. CONCLUSIONS: ADSCs restore the pathogenesis of Dex-induced muscle atrophy with an increased number of type I fibres, stronger muscle strength, faster recovery rate and more anti-fatigue ability via ERK1/2 signalling pathway. The inhibition of muscle atrophy-associated genes by ADSCs offered this treatment as an intervention option for muscle-associated diseases. Taken together, our findings suggested that adipose-derived mesenchymal stem cell therapy could be a new treatment option for patient with Dex-induced muscle atrophy.

Genetic insight into the putative causal proteins and druggable targets of osteoporosis: a large-scale proteome-wide mendelian randomization study.

Background: Osteoporosis is a major causative factor of the global burden of disease and disability, characterized by low bone mineral density (BMD) and high risks of fracture. We aimed to identify putative causal proteins and druggable targets of osteoporosis. Methods: This study utilized the largest GWAS summary statistics on plasma proteins and estimated heel BMD (eBMD) to identify causal proteins of osteoporosis by mendelian randomization (MR) analysis. Different GWAS datasets were used to validate the results. Multiple sensitivity analyses were conducted to evaluate the robustness of primary MR findings. We have also performed an enrichment analysis for the identified causal proteins and evaluated their druggability. Results: After Bonferroni correction, 67 proteins were identified to be causally associated with estimated BMD (eBMD) (p < 4 × 10-5). We further replicated 38 of the 67 proteins to be associated with total body BMD, lumbar spine BMD, femoral neck BMD as well as fractures, such as RSPO3, IDUA, SMOC2, and LRP4. The findings were supported by sensitivity analyses. Enrichment analysis identified multiple Gene Ontology items, including collagen-containing extracellular matrix (GO:0062023, p = 1.6 × 10-10), collagen binding (GO:0005518, p = 8.6 × 10-5), and extracellular matrix structural constituent (GO:0005201, p = 2.7 × 10-5). Conclusion: The study identified novel putative causal proteins for osteoporosis which may serve as potential early screening biomarkers and druggable targets. Furthermore, the role of plasma proteins involved in collagen binding and extracellular matrix in the development of osteoporosis was highlighted. Further studies are warranted to validate our findings and investigate the underlying mechanism.

Is dexamethasone-induced muscle atrophy an alternative model for naturally aged sarcopenia model?

Background: Primary sarcopenia is usually known as age-related skeletal muscle loss; however, other factors like endocrine, lifestyle and inflammation can also cause muscle loss, known as secondary sarcopenia. Although many studies have used different sarcopenia animal models for exploring the underlying mechanism and therapeutic approaches for sarcopenia, limited study has provided evidence of the relevance of these animal models. This study aims to investigate the similarity and difference in muscle qualities between primary and secondary sarcopenia mice models, using naturally aged mice and dexamethasone-induced mice. Methods: 21-month-old mice were used as naturally aged primary sarcopenia mice and 3-month-old mice received daily intraperitoneal injection of dexamethasone (20 mg/ kg body weight) for 10 days were used as secondary sarcopenia model. This study provided measurements for muscle mass and functions, including Dual-energy X-ray absorptiometry (DXA) scanning, handgrip strength test and treadmill running to exhaustion test. Besides, muscle contraction, muscle fibre type measurements and gene expression were also performed to provide additional information on muscle qualities. Results: The results suggest two sarcopenia animal models shared a comparable decrease in forelimb lean mass, muscle fibre size, grip strength and muscle contraction ability. Besides, the upregulation of protein degradation genes was also observed in two sarcopenia animal models. However, only primary sarcopenia mice were identified with an early stage of mtDNA deletion. Conclusion: Collectively, this study evaluated that the dexamethasone-induced mouse model could be served as an alternative model for primary sarcopenia, according to the comparable muscle mass and functional changes. However, whether dexamethasone-induced mice can be used as an animal model when studying the molecular mechanisms of sarcopenia needs to be carefully evaluated. The translational potential of this article: The purpose of sarcopenia research is to investigate appropriate treatments for reversing the loss of skeletal muscle mass and functions. Using animal models for the preclinical study could predict the safety and efficacy of the treatments. This study compared the typical age-related sarcopenia mice model and dexamethasone-induced secondary sarcopenia mice to provide evidence of the pathological and functional changes in the mice models.

Recent Advances in "Functional Engineering of Articular Cartilage Zones by Polymeric Biomaterials Mediated with Physical, Mechanical, and Biological/Chemical Cues".

Articular cartilage (AC) plays an unquestionable role in joint movements but unfortunately the healing capacity is restricted due to its avascular and acellular nature. While cartilage tissue engineering has been lifesaving, it is very challenging to remodel the complex cartilage composition and architecture with gradient physio-mechanical properties vital to proper tissue functions. To address these issues, a better understanding of the intrinsic AC properties and how cells respond to stimuli from the external microenvironment must be better understood. This is essential in order to take one step closer to producing functional cartilaginous constructs for clinical use. Recently, biopolymers have aroused much attention due to their versatility, processability, and flexibility because the properties can be tailored to match the requirements of AC. This review highlights polymeric scaffolds developed in the past decade for reconstruction of zonal AC layers including the superficial zone, middle zone, and deep zone by means of exogenous stimuli such as physical, mechanical, and biological/chemical signals. The mimicked properties are reviewed in terms of the biochemical composition and organization, cell fate (morphology, orientation, and differentiation), as well as mechanical properties and finally, the challenges and potential ways to tackle them are discussed.

Abnormal morphological features of osteocyte lacunae in adolescent idiopathic scoliosis: A large-scale assessment by ultra-high-resolution micro-computed tomography.

AIM: Abnormal osteocyte lacunar morphology in adolescent idiopathic scoliosis (AIS) has been reported while the results were limited by the number of osteocyte lacunae being quantified. The present study aimed to validate previous findings through (a) comparing morphological features of osteocyte lacunae between AIS patients and controls in spine and ilium using a large-scale assessment, and (b) investigating whether there is an association between the acquired morphological features of osteocyte lacunae and disease severity in AIS. METHOD: Trabecular bone tissue of the facet joint of human vertebrae on both concave and convex sides at the apex of the scoliotic curve were collected from 4 AIS and 5 congenital scoliosis (CS) patients, and also at the same anatomic site from 3 non-scoliosis (NS) subjects intraoperatively. Trabecular bone tissue from ilium was obtained from 12 AIS vs 9 NS subjects during surgery. Osteocyte lacunae were assessed using ultra-high-resolution micro-computed tomography. Clinical information such as age, body mass index (BMI) and radiological Cobb angle of the major curve were collected. RESULTS: There was no significant difference between density of osteocyte lacuna and bone volume fraction (BV/TV) between groups. A total of 230,076 and 78,758 osteocyte lacunae from facet joints of apical vertebra of scoliotic curve and iliac bone were included in the analysis, respectively. In facet joint bone biopsies, lacunar stretch (Lc.St) was higher, and lacunar equancy (Lc.Eq), lacunar oblateness (Lc.Ob), and lacunar sphericity (Lc.Sr) were lower in AIS and CS groups when compared with NS group. CA side was associated with higher Lc.St when compared with CX side. In iliac bone biopsies, Lc.Ob was higher and lacunar surface area (Lc.S) was lower in AIS group than NS group. Median values of Lc.St, Lc.Eq and Lc.Sr were significantly associated with radiological Cobb angle with adjustment for age and BMI (R-squared: 0.576, 0.558 and 0.543, respectively). CONCLUSIONS: This large-scale assessment of osteocyte lacunae confirms that AIS osteocyte lacunae are more oblate in iliac bone that is less influenced by asymmetric loading of the deformed spine than the vertebrae. Shape of osteocyte lacunae in iliac bone is associated with radiological Cobb angle of the major curve in AIS patients, suggesting the likelihood of systemic abnormal osteocyte morphology in AIS. Osteocyte lacunae from concave side of scoliotic curves were more stretched in both AIS and CS groups, which is likely secondary to asymmetric mechanical loading.

Enhancing cartilage repair with optimized supramolecular hydrogel-based scaffold and pulsed electromagnetic field.

Functional tissue engineering strategies provide innovative approach for the repair and regeneration of damaged cartilage. Hydrogel is widely used because it could provide rapid defect filling and proper structure support, and is biocompatible for cell aggregation and matrix deposition. Efforts have been made to seek suitable scaffolds for cartilage tissue engineering. Here Alg-DA/Ac-ß-CD/gelatin hydrogel was designed with the features of physical and chemical multiple crosslinking and self-healing properties. Gelation time, swelling ratio, biodegradability and biocompatibility of the hydrogels were systematically characterized, and the injectable self-healing adhesive hydrogel were demonstrated to exhibit ideal properties for cartilage repair. Furthermore, the new hydrogel design introduces a pre-gel state before photo-crosslinking, where increased viscosity and decreased fluidity allow the gel to remain in a semi-solid condition. This granted multiple administration routes to the hydrogels, which brings hydrogels the ability to adapt to complex clinical situations. Pulsed electromagnetic fields (PEMF) have been recognized as a promising solution to various health problems owing to their noninvasive properties and therapeutic potentials. PEMF treatment offers a better clinical outcome with fewer, if any, side effects, and wildly used in musculoskeletal tissue repair. Thereby we propose PEMF as an effective biophysical stimulation to be 4th key element in cartilage tissue engineering. In this study, the as-prepared Alg-DA/Ac-ß-CD/gelatin hydrogels were utilized in the rat osteochondral defect model, and the potential application of PEMF in cartilage tissue engineering were investigated. PEMF treatment were proven to enhance the quality of engineered chondrogenic constructs in vitro, and facilitate chondrogenesis and cartilage repair in vivo. All of the results suggested that with the injectable self-healing adhesive hydrogel and PEMF treatment, this newly proposed tissue engineering strategy revealed superior clinical potential for cartilage defect treatment.

Sirt3 mediates the benefits of exercise on bone in aged mice.

Exercise in later life is important for bone health and delays the progression of osteoporotic bone loss. Osteocytes are the major bone cells responsible for transforming mechanical stimuli into cellular signals through their highly specialized lacunocanalicular networks (LCN). Osteocyte activity and LCN degenerate with aging, thus might impair the effectiveness of exercise on bone health; however, the underlying mechanism and clinical implications remain elusive. Herein, we showed that deletion of Sirt3 in osteocytes could impair the formation of osteocyte dendritic processes and inhibit bone gain in response to exercise in vivo. Mechanistic studies revealed that Sirt3 regulates E11/gp38 through the protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway. Additionally, the Sirt3 activator honokiol enhanced the sensitivity of osteocytes to fluid shear stress in vitro, and intraperitoneal injection of honokiol reduced bone loss in aged mice in a dose-dependent manner. Collectively, Sirt3 in osteocytes regulates bone mass and mechanical responses through the regulation of E11/gp38. Therefore, targeting Sirt3 could be a novel therapeutic strategy to prevent age-related bone loss and augment the benefits of exercise on the senescent skeleton.

Efficient hepatic differentiation of hydrogel microsphere-encapsulated human pluripotent stem cells for engineering prevascularized liver tissue.

Liver tissue engineering is promising as an alternative strategy to treat liver failure. However, generating functional hepatocytes from stem cells is conventionally restricted by the immature status of differentiated cells. Besides, embedding hepatocytes in bulk scaffold is limited by a lack of vascularity and low cell-packing density. Here, we fabricate collagen type I (COL1) microspheres for efficient hepatic differentiation of pluripotent stem cells and subsequent assembly of prevascularized liver tissue (PLT). Using a microfluidic platform, we demonstrate that hydrogel COL1 microspheres (mCOL1) encapsulating human embryonic stem cells (hESCs) can be reproducibly generated and efficiently differentiated into hepatocyte-like cells (HLCs) microspheres for the first time. Compared with other culture configurations such as encapsulation of hESC in a bulk COL1 hydrogel and 2D monolayer culture, mCOL1 with high uniformity produce HLC microspheres of improved maturity based on comprehensive analyses of cell morphology, transcriptome profile, hepatic marker expression and hepatic functions. In addition, these HLC microspheres can be applied as building blocks to self-assemble with endothelial cells to construct a dense PLT. The PLT resembles native liver tissue with high cell-packing density, shows successful engraftment in mice liver following implantation, and exhibits improved hepatic functionin vivo. Overall, it is believed that this multiscale technology will advance the fabrication of stem cell-based liver tissue for regenerative medicine, drug screening, andin vitroliver modeling.

Randomized control trial of mesenchymal stem cells versus hyaluronic acid in patients with knee osteoarthritis - A Hong Kong pilot study.

Objective: This pilot study evaluated the efficacy of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) versus hyaluronic acid (HA) in surgically naïve patients with knee osteoarthritis (OA). Methods: Single-centre, single-blind randomized study of patients with knee OA. Twenty patients were randomized into groups of 10 each for intra-articular injection of cultured BM-MSCs (6 â€‹ml of BM-MSCs at 1 â€‹× â€‹106 â€‹cells/mL) or HA (6 â€‹ml). Clinical assessments of pain, quality of life, radiographic imaging, and magnetic resonance imaging (MRI) compositional change were performed at baseline and 12 months follow-up. Results: Compared with HA, BM-MSCs injection resulted in significant improvement in qualify of life and reduction in pain as reflected by visual analogue scale (VAS) pain score, Western Ontario and McMaster Universities Arthritis Index (WOMAC) score, and 36-Item Short Form Survey (SF-36) score collectively. T2-relaxation time tended to decrease more in the BM-MSCs group with a 38 â€‹± â€‹24.0% reduction in 6 out of 10 BM-MSC participants; while there was only a 12 â€‹± â€‹7.9% reduction in 4 out of 10 HA participants at the end of follow-up. The remaining participants showed either no response or had relaxation time increased on MRI assessment. Conclusions: This pilot study found that autologous BM-MSCs significantly reduced pain, improved functional assessment score, and improved quality of life parameters comparing with HA at one year follow-up. Further clinical trial with larger sample size and longer follow up duration is warranted. The Translational Potential of this Article: This pilot RCT demonstrated the feasibility and potential effectiveness of BM-MSCs advanced therapy for patients with knee OA compared to HA injection. Further multi-center clinical trial with a larger sample size and longer follow up duration in accordance with latest regulatory guidelines is warranted to ascertain the long term safety and effectiveness of MSCs therapy for cartilage regeneration in OA. Registration: The study was registered in the Centre for Clinical Research Biostatistics - Clinical Trials Registry (CUHK_CCT00469).

Emerging role of lncRNAs in osteoarthritis: An updated review.

Osteoarthritis (OA) is a prevalent joint disease, which is associated with progressive articular cartilage loss, synovial inflammation, subchondral sclerosis and meniscus injury. The molecular mechanism underlying OA pathogenesis is multifactorial. Long non-coding RNAs (lncRNAs) are non-protein coding RNAs with length more than 200 nucleotides. They have various functions such as modulating transcription and protein activity, as well as forming endogenous small interfering RNAs (siRNAs) and microRNA (miRNA) sponges. Emerging evidence suggests that lncRNAs might be involved in the pathogenesis of OA which opens up a new avenue for the development of new biomarkers and therapeutic strategies. The purpose of this review is to summarize the current clinical and basic experiments related to lncRNAs and OA with a focus on the extensively studied H19, GAS5, MALAT1, XIST and HOTAIR. The potential translational value of these lncRNAs as therapeutic targets for OA is also discussed.

Fetal muscle extract improves muscle function and performance in aged mice.

Background: Loss of skeletal muscle mass and function is one of the major musculoskeletal health problems in the aging population. Recent studies have demonstrated differential proteomic profiles at different fetal stages, which might be associated with muscle growth and development. We hypothesized that extract derived from fetal muscle tissues at the stage of hypertrophy could ameliorate the loss of muscle mass and strength in aged mice. Methods: To allow sufficient raw materials for investigation, skeletal muscle extract from fetal sheep at week 16 of gestation and maternal tissue were used in the present study. iTRAQ (isobaric tags for relative and absolute quantitation) and KEGG pathway analyses identified differentially expressed proteins in fetal sheep muscle extract vs. adult sheep muscle extract. Effects of FSME and ASME on human myoblast proliferation were studied. To examine the effect of FSME in vivo, C57BL/6 male mice at 20 months of age were subjected to intramuscular administration of FSME or vehicle control for 8 weeks. A grip strength test and ex vivo muscle force frequency test were conducted. Finally, serum samples were collected for multiplex analysis to determine potential changes in immunological cytokines upon FSME injection. Results: Compared with ASME, 697 and 412 peptides were upregulated and downregulated, respectively, in FSME, as indicated by iTRAQ analysis. These peptides were highly related to muscle development, function, and differentiation from GO enrichment analysis. FSME promoted cell proliferation of myoblast cells (+300%, p < 0.01) without causing significant cytotoxicity at the tested concentration range compared with ASME. After 8 weeks of FSME treatment, the percentage of lean mass (+10%, p < 0.05), grip strength (+50%, p < 0.01), and ability in fatigue resistance were significantly higher than those of the control group. Isometric forces stimulated by different frequencies were higher in the control group. Histologically, the control group showed a larger cross-sectional area (+20%, p < 0.01) than the FSME group. The multiplex assay indicated that FSME treatment did not lead to an elevated circulatory level of inflammatory cytokines. Of note, after FSME treatment, we observed a significant drop in the circulating level of IL-12 (p40) from 90.8 ± 48.3 pg/ml to 82.65 ± 4.4 pg/ml, G-CSF from 23476 ± 8341.9 pg/ml to 28.35 ± 24.2 pg/ml, KC from 97.09 ± 21.2 pg/ml to 29.2 ± 7.2 pg/ml, and RANTES from 325.4 ± 17.3 pg/ml to 49.96 ± 32.1 pg/ml. Conclusion: This is the first study demonstrating the beneficial effect of fetal muscle extract on muscle health in aged mice. Further analysis of the active ingredients of the extract will shed light on the development of a novel treatment for sarcopenia.

Pharmacogenomic Profiling of Pediatric Acute Myeloid Leukemia to Identify Therapeutic Vulnerabilities and Inform Functional Precision Medicine.

Despite the expanding portfolio of targeted therapies for adults with acute myeloid leukemia (AML), direct implementation in children is challenging due to inherent differences in underlying genetics. Here we established the pharmacologic profile of pediatric AML by screening myeloblast sensitivity to approved and investigational agents, revealing candidates of immediate clinical relevance. Drug responses ex vivo correlated with patient characteristics, exhibited age-specific alterations, and concorded with activities in xenograft models. Integration with genomic data uncovered new gene-drug associations, suggesting actionable therapeutic vulnerabilities. Transcriptome profiling further identified gene-expression signatures associated with on- and off-target drug responses. We also demonstrated the feasibility of drug screening-guided treatment for children with high-risk AML, with two evaluable cases achieving remission. Collectively, this study offers a high-dimensional gene-drug clinical data set that could be leveraged to research the unique biology of pediatric AML and sets the stage for realizing functional precision medicine for the clinical management of the disease. SIGNIFICANCE: We conducted integrated drug and genomic profiling of patient biopsies to build the functional genomic landscape of pediatric AML. Age-specific differences in drug response and new gene-drug interactions were identified. The feasibility of functional precision medicine-guided management of children with high-risk AML was successfully demonstrated in two evaluable clinical cases. This article is highlighted in the In This Issue feature, p. 476.

Upregulation of microRNA-96-5p is associated with adolescent idiopathic scoliosis and low bone mass phenotype.

Bone densitometry revealed low bone mass in patients with adolescent idiopathic scoliosis (AIS) and its prognostic potential to predict curve progression. Recent studies showed differential circulating miRNAs in AIS but their diagnostic potential and links to low bone mass have not been well-documented. The present study aimed to compare miRNA profiles in bone tissues collected from AIS and non-scoliotic subjects, and to explore if the selected miRNA candidates could be useful diagnostic biomarkers for AIS. Microarray analysis identified miR-96-5p being the most upregulated among the candidates. miR-96-5p level was measured in plasma samples from 100 AIS and 52 healthy girls. Our results showed significantly higher plasma levels of miR-96-5p in AIS girls with an area under the curve (AUC) of 0.671 for diagnostic accuracy. A model that was composed of plasma miR-96-5p and patient-specific parameters (age, body weight and years since menarche) gave rise to an improved AUC of 0.752. Ingenuity Pathway Analysis (IPA) indicated functional links between bone metabolic pathways and miR-96-5p. In conclusion, differentially expressed miRNAs in AIS bone and plasma samples represented a new source of disease biomarkers and players in AIS etiopathogenesis, which required further validation study involving AIS patients of both genders with long-term follow-up.

Biological effect of dysregulated LBX1 on adolescent idiopathic scoliosis through modulating muscle carbohydrate metabolism.

BACKGROUND CONTEXT: Abnormal energy metabolism such as lower body weight and body mass index (BMI) and less fat mass is widely reported in patients with adolescent idiopathic scoliosis (AIS) and has been implicated in deformity development. However, the underlying mechanism is largely unclear. LBX1 is one of the promising AIS predisposing genes validated by multicenter studies. PURPOSE: This study aimed to identify differentially expressed proteins (DEPs) relating to energy metabolism in AIS by using proteomic and metabolic analysis and to explore if the expression of these DEPs is associated with clinical parameters and modulated by LBX1. STUDY DESIGN: This is a cross-sectional study using clinical data and biological samples followed by basic study using a cellular model. PATIENT SAMPLE: Plasma samples were collected from Chinese girls with nonprogressive and progressive AIS (N=7 and 8, respectively) and age-matched healthy girls (N=50). Paraspinal muscle tissues were collected intraoperatively from concave and convex side of the apex of the major spinal curve in AIS (N=24) and either side from nonscoliosis patients (N=14). OUTCOME MEASURES: Radiological Cobb angle and basic anthropometric data of recruited subjects were measured. The DEPs and metabolites were compared in plasma using proteomics and metabolomics technique. The relative expression of selected genes was measured in muscles. METHODS: Plasma samples from AIS were collected at first clinical visit and were further divided into nonprogressive or progressive groups according to Cobb angle changes in 6-year follow-up. Age-matched healthy girls were recruited as control. High-performance liquid chromatography-mass spectrometry based proteomic analysis was carried out in three groups to identify DEPs and their annotated metabolic pathways. An independent cohort was used for validation by gas chromatography-mass spectrometry based metabolomic analysis. Paraspinal muscles were subjected to quantitative polymerase chain reaction (qPCR) followed by correlation analysis. Human skeletal muscle myoblast (HSMM) was used as the cellular model. RESULTS: The likelihood of aberrant galactose metabolism and glycolysis was found to be associated with AIS curve progression as evidenced by the thirteen DEPs and seven related metabolites according to proteomic and metabolomic analysis. Some of the DEPs showed significantly altered expression in AIS concave and convex sides paraspinal muscles compared with those in nonscoliosis control. Four DEPs were found significantly and negatively correlated with LBX1 in AIS convex side paraspinal muscles. Overexpressing LBX1 in HSMM cells led to increased expression of three DEPs and decreased expression of three DEPs, respectively. CONCLUSIONS: This is the first integrated proteomic and metabolomic analysis on AIS. Our findings show dysregulated galactose metabolism and glycolysis pathways in progressive group of AIS, suggesting the presence of abnormal energy metabolism at early stage of this disease, and their association with higher risk of progressing into more severe curvature. Evidence from ex vivo study with human muscle biopsies and in vitro study with human myoblast cells propose the possible effect of LBX1 on these two pathways in skeletal muscles. The present study provides new evidence of LBX1 function in AIS via modulating effect on the expression of energy metabolism related genes. This study might provide new insights into etiopathogenesis and development of novel treatment strategy targeting on abnormal body weight and BMI in patients with AIS. Additionally, the plasma proteomic and metabolomic studies suggested new candidates as biomarkers for establishing predictive model for AIS onset/progression.

Cranial Bone Transport Promotes Angiogenesis, Neurogenesis, and Modulates Meningeal Lymphatic Function in Middle Cerebral Artery Occlusion Rats.

BACKGROUND: Ischemic stroke is a leading cause of death and disability worldwide. However, the time window for quickly dissolving clots and restoring cerebral blood flow, using tissue-type plasminogen activator treatment is rather limited, resulting in many patients experiencing long-term functional impairments if not death. This study aims to determine the roles of cranial bone transport (CBT), a novel, effective, and simple surgical technique, in the recovery of ischemic stroke using middle cerebral artery occlusion (MCAO) rat model. METHODS: CBT was performed by slowly sliding a bone segment in skull with a special frame and a speed of 0.25 mm/12 hours for 10 days following MCAO. Morris water maze, rotarod test, and catwalk gait analysis were used to study the neurological behaviors, and infarct area and cerebral flow were evaluated during CBT process. Immunofluorescence staining of CD31 and Nestin/Sox2 (sex determining region Y box 2) was performed to study the angiogenesis and neurogenesis. OVA-A647 (ovalbumin-Alexa Fluor 647) was intracisterna magna injected to evaluate the meningeal lymphatic drainage function. RESULTS: CBT treatment has significantly reduced the ischemic lesions areas and improved the neurological deficits in MCAO rats compared with the rats in the control groups. CBT treatment significantly promoted angiogenesis and neurogenesis in the brain of MCAO rats. The drainage function of meningeal lymphatic vessels in MCAO rats was significantly impaired compared with normal rats. Ablation of meningeal lymphatic drainage led to increased neuroinflammation and aggravated neurological deficits and ischemic injury in MCAO rats. CBT treatment significantly improved the meningeal lymphatic drainage function and alleviated T-cell infiltration in MCAO rats. CONCLUSIONS: This study provided evidence for the possible mechanisms on how CBT attenuates ischemic stroke injury and facilitates rapid neuronal function recovery, suggesting that CBT may be an alternative treatment strategy for managing ischemic stroke.