Long non-coding and circular RNAs in osteoporosis: Translation to clinical practice.

Non-coding RNAs (ncRNAs) belong to a class of untranslated nucleic acids involved in regulation of gene expression. ncRNAs are categorized as small (<200 ribonucleotides in length), i.e., microRNAs (miRNAs), and long ncRNAs (lncRNAs) (200 to thousands of ribonucleotides in length) and circular RNAs (circRNAs). In contrast to miRNAs, the roles of lncRNAs in general and circRNAs in bone metabolism specifically are not well understood. As such, a comprehensive understanding of these RNA species in bone turnover could be of great value in the development of new diagnostic tools and therapeutic targets. Unfortunately, measurement of these unique RNAs lacks standardization, a component critical to clinical translation. This review examines the potential role of lncRNA and circRNA as bone biomarkers, the need for validated and standardized measurement and challenges thereof.

Plasma microRNA signature associated with skeletal muscle wasting in post-menopausal osteoporotic women.

BACKGROUND: Skeletal muscle mass wasting almost invariably accompanies bone loss in elderly, and the coexistence of these two conditions depends on the tight endocrine crosstalk existing between the two organs, other than the biomechanical coupling. Since the current diagnostics limitation in this field, and given the progressive population aging, more effective tools are needed. The aim of this study was to identify circulating microRNAs (miRNAs) as potential biomarkers for muscle mass wasting in post-menopausal osteoporotic women. METHODS: One hundred seventy-nine miRNAs were assayed by quantitative real-time polymerase chain reaction in plasma samples from 28 otherwise healthy post-menopausal osteoporotic women (73.4 ± 6.6 years old). The cohort was divided in tertiles based on appendicular skeletal muscle mass index (ASMMI) to better highlight the differences on skeletal muscle mass (first tertile: n = 9, ASMMI = 4.88 ± 0.40 kg·m-2; second tertile: n = 10, ASMMI = 5.73 ± 0.23 kg·m-2; third tertile: n = 9, ASMMI = 6.40 ± 0.22 kg·m-2). Receiver operating characteristic (ROC) curves were calculated to estimate the diagnostic potential of miRNAs. miRNAs displaying a statistically significant fold change ≥ ±1.5 and area under the curve (AUC) > 0.800 (P < 0.05) between the first and third tertiles were considered. A linear regression model was applied to estimate the association between miRNA expression and ASMMI in the whole population, adjusting for body mass index, age, total fat (measured by total-body dual-energy X-ray absorptiometry [DXA]) and bone mineral density (measured by femur DXA). Circulating levels of adipo-myokines were evaluated by bead-based immunofluorescent assays and enzyme-linked immunosorbent assays. RESULTS: Five miRNAs (hsa-miR-221-3p, hsa-miR-374b-5p, hsa-miR-146a-5p, hsa-miR-126-5p and hsa-miR-425-5p) resulted down-regulated and two miRNAs (hsa-miR-145-5p and hsa-miR-25-3p) were up-regulated in the first tertile (relative-low ASMMI) compared with the third tertile (relative-high ASMMI) (fold change ≥ ±1.5; P-value < 0.05). All the corresponding ROC curves had AUC > 0.8 (P < 0.05). Two signatures hsa-miR-126-5p, hsa-miR-146a-5p and hsa-miR-425-5p; and hsa-miR-126-5p, hsa-miR-146a-5p, hsa-miR-145-5p and hsa-miR-25-3p showed the highest AUC, 0.914 (sensitivity = 77.78%; specificity = 100.00%) and 0.901 (sensitivity = 88.89%; specificity = 100.00%), respectively. CONCLUSIONS: In this study, we identified, for the first time, two miRNA signatures, hsa-miR-126-5p, hsa-miR-146a-5p and hsa-miR-425-5p; and hsa-miR-126-5p, hsa-miR-146a-5p, hsa-miR-145-5p and hsa-miR-25-3p, specifically associated with muscle mass wasting in post-menopausal osteoporotic women.

Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study.

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-ßgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system.

Downhill running increases markers of muscle damage and impairs the maximal voluntary force production as well as the late phase of the rate of voluntary force development.

PURPOSE: To examined the time-course of the early and late phase of the rate of voluntary force development (RVFD) and muscle damage markers after downhill running. METHODS: Ten recreational runners performed a 30-min downhill run at 10 km h-1 and -20% (-11.3°) on a motorized treadmill. At baseline and each day up to 4 days RVFD, knee extensors maximum voluntary isometric force (MVIC), serum creatine kinase (CK) concentration, quadriceps swelling, and soreness were assessed. The early (0-50 ms) and late (100-200 ms) phase of the RVFD, as well as the force developed at 50 and 200 ms, were also determined. RESULTS: MVIC showed moderate decrements (p < 0.05) and recovered after 4 days (p > 0.05). Force at 50 ms and the early phase were not impaired (p > 0.05). Conversely, force at 200 ms and the late phase showed moderate decrements (p < 0.05) and recovered after 3 and 4 days, respectively (p > 0.05). CK concentration, quadriceps swelling, and soreness increased (p < 0.05) were overall fully resolved after 4 days (p > 0.05). CONCLUSION: Downhill running affected the knee extensors RVFD late but not early phase. The RVFD late phase may be used as an additional marker of muscle damage in trail running.

Short and long-term effects of high-intensity interval training applied alone or with whole-body cryostimulation on glucose homeostasis and myokine levels in overweight to obese subjects.

Background: COVID-19 pandemic has exacerbated the problem of physical inactivity and weight gain. Consequently, new strategies to counteract weight gain are being sought. Because of their accessibility, interval training and cold therapy are the most popular such strategies. We here aimed to examine the effect of 6 units of high-intensity interval training (HIIT), applied alone or in combination with 10 sessions of whole-body cryotherapy (WBC; 3 min at -110 ∘C per session) on incretins, myokines, and adipokines levels. Materials and methods: The study involved 65 subjects (body mass index of approximately 30 kg•m-2). The subjects were randomly divided into training group (TR; n = 27) and training supported by WBC group (TR-WBC; n = 38). Blood samples were collected before, immediately following, and 4 weeks after the intervention. Results: Fibroblast growth factor 21 (FGF21) levels significantly increased (p = 0.03) and adiponectin levels increased in the TR group (p = 0.05) compared with those recorded in TR-WBC group 24 h after the end of experimental protocol. Beneficial changes in the lipid profile (p = 0.07), a significant drop in visfatin levels (p < 0.05), and the improvement in ß-cell function (HOMA-B; p = 0.02) were also observed in the TR group in the same time point of study. While TR-WBC did not induce similar changes, it ameliorated blood glucose levels (p = 0.03). Changes induced by both interventions were only sustained for 4 weeks after treatment. Conclusion: Collectively, HIIT, alone and in combination with WBC, positively affects metabolic indicators, albeit, most likely, different mechanisms drive the beneficial effects of different treatments.

Peri-Surgical Inflammatory Profile Associated with Mini-Invasive or Standard Open Lumbar Interbody Fusion Approaches.

BACKGROUND: Different surgical approaches are available for lumbar interbody fusion (LIF) to treat disc degeneration. However, a quantification of their invasiveness is lacking, and the definition of minimally invasive surgery (MIS) has not been biochemically detailed. We aimed at characterizing the inflammatory, hematological, and clinical peri-surgical responses to different LIF techniques. METHODS: 68 healthy subjects affected by single-level discopathy (L3 to S1) were addressed to MIS, anterior (ALIF, n = 21) or lateral (LLIF, n = 23), and conventional approaches, transforaminal (TLIF, n = 24), based on the preoperative clinical assessment. Venous blood samples were taken 24 h before the surgery and 24 and 72 h after surgery to assess a wide panel of inflammatory and hematological markers. RESULTS: martial (serum iron and transferrin) and pro-angiogenic profiles (MMP-2, TWEAK) were improved in ALIF and LLIF compared to TLIF, while the acute phase response (C-reactive protein, sCD163) was enhanced in LLIF. CONCLUSIONS: MIS procedures (ALIF and LLIF) associated with a reduced incidence of post-operative anemic status, faster recovery, and enhanced pro-angiogenic stimuli compared with TLIF. LLIF associated with an earlier activation of innate immune mechanisms than ALIF and TLIF. The trend of the inflammation markers confirms that the theoretically defined mini-invasive procedures behave as such.

Circulating Carboxylated Osteocalcin Correlates With Skeletal Muscle Mass and Risk of Fall in Postmenopausal Osteoporotic Women.

Background: Bone and skeletal muscle represent a single functional unit. We cross-sectionally investigated body composition, risk of fall and circulating osteocalcin (OC) isoforms in osteoporotic postmenopausal women to test the hypothesis of an involvement of OC in the bone-muscle crosstalk. Materials and Methods: Twenty-nine non-diabetic, non-obese, postmenopausal osteoporotic women (age 72.4 ± 6.8 years; BMI 23.0 ± 3.3 kg/m2) underwent to: 1) fasting blood sampling for biochemical and hormone assays, including carboxylated (cOC) and uncarboxylated (uOC) osteocalcin; 2) whole-body dual energy X-ray absorptiometry (DXA) to assess total and regional body composition; 3) magnetic resonance imaging to determine cross-sectional muscle area (CSA) and intermuscular adipose tissue (IMAT) of thigh muscles; 4) risk of fall assessment through the OAK system. Results: Appendicular skeletal muscle index (ASMMI) was low in 45% of patients. Forty percent got a low OAK score, consistent with moderate-severe risk of fall, which was predicted by low legs lean mass and increased total fat mass. Circulating cOC levels showed significantly correlated with ßCTx-I, lean mass parameters including IMAT, and OAK score. Fractured and unfractured women did not differ for any of the analyzed parameters, though cOC and uOC positively correlated with legs lean mass, OAK score and bone markers only in fractured women. Conclusions: Data supported the relationship between OC and skeletal muscle mass and function in postmenopausal osteoporotic women. Serum cOC, but not uOC, emerges as mediator in the bone-muscle crosstalk. Circulating cOC and uOC levels may be differentially regulated in fractured and unfractured osteoporotic women, suggesting underlying differences in bone metabolism.

A Physically Active Status Affects the Circulating Profile of Cancer-Associated miRNAs.

Circulating miRNAs are ideal diagnostics and prognostics biomarkers in cancer since altered levels of specific miRNAs have been associated to development/progression of several cancers. Physical activity is a recognized preventive strategy against several cancers, but it may also modify the baseline levels of cancer-associated miRNAs and, hence, may act as a confounding pre-analytical variable. This study aimed at understanding whether physical activity-dependent changes in cancer-associated circulating miRNAs profile could act as a confounding variable. A panel comprising 179 miRNAs was assayed in plasma from 20 highly trained and 10 sedentary men. RT-qPCR data were analyzed with the 2-2ΔΔCT methods and normalized on hsa-miR-320d, as determined by bioinformatics analysis. miRNAs associated with the diagnosis of the most prevalent cancers were considered. Only those miRNAs, relevantly associated with cancers, found ≥2-fold up- or downregulated in highly trained subjects compared to sedentary were disclosed. The results reveal that chronic physical activity determined modifications altering the baseline level of several cancer-associated miRNAs and, hence, their diagnostic and prognostic potential. In conclusion, based on our results, a physically active status emerges as an important pre-analytical variable able to alter the basal level of circulating miRNAs, and these alterations might be considered as potentially misleading the analytical output.

Engineering the early bone metastatic niche through human vascularized immuno bone minitissues.

Bone metastases occur in 65%-80% advanced breast cancer patients. Although significant progresses have been made in understanding the biological mechanisms driving the bone metastatic cascade, traditional 2Din vitromodels and animal studies are not effectively reproducing breast cancer cells (CCs) interactions with the bone microenvironment and suffer from species-specific differences, respectively. Moreover, simplifiedin vitromodels cannot realistically estimate drug anti-tumoral properties and side effects, hence leading to pre-clinical testing frequent failures. To solve this issue, a 3D metastatic bone minitissue (MBm) is designed with embedded human osteoblasts, osteoclasts, bone-resident macrophages, endothelial cells and breast CCs. This minitissue recapitulates key features of the bone metastatic niche, including the alteration of macrophage polarization and microvascular architecture, along with the induction of CC micrometastases and osteomimicry. The minitissue reflects breast CC organ-specific metastatization to bone compared to a muscle minitissue. Finally, two FDA approved drugs, doxorubicin and rapamycin, have been tested showing that the dose required to impair CC growth is significantly higher in the MBm compared to a simpler CC monoculture minitissue. The MBm allows the investigation of metastasis key biological features and represents a reliable tool to better predict drug effects on the metastatic bone microenvironment.

Another Weapon against Cancer and Metastasis: Physical-Activity-Dependent Effects on Adiposity and Adipokines.

Physically active behavior has been associated with a reduced risk of developing certain types of cancer and improved psychological conditions for patients by reducing anxiety and depression, in turn improving the quality of life of cancer patients. On the other hand, the correlations between inactivity, sedentary behavior, and overweight and obesity with the risk of development and progression of various cancers are well studied, mainly in middle-aged and elderly subjects. In this article, we have revised the evidence on the effects of physical activity on the expression and release of the adipose-tissue-derived mediators of low-grade chronic inflammation, i.e., adipokines, as well as the adipokine-mediated impacts of physical activity on tumor development, growth, and metastasis. Importantly, exercise training may be effective in mitigating the side effects related to anti-cancer treatment, thereby underlining the importance of encouraging cancer patients to engage in moderate-intensity activities. However, the strong need to customize and adapt exercises to a patient's abilities is apparent. Besides the preventive effects of physically active behavior against the adipokine-stimulated cancer risk, it remains poorly understood how physical activity, through its actions as an adipokine, can actually influence the onset and development of metastases.

Effect of collection matrix, platelet depletion, and storage conditions on plasma extracellular vesicles and extracellular vesicle-associated miRNAs measurements.

OBJECTIVES: The interest around circulating extracellular vesicles and their cargo in diagnostics has greatly increased; however, several pre-analytical variables affect their determination. In this study, we investigated the effects of sample matrix, processing, and plasma storage delay and temperature on extracellular vesicles and their miRNA content. METHODS: Blood was collected from 10 male volunteers in dipotassium ethylendiaminotetraacetate-coated tubes (K2EDTA), either with plasma-preparation tube (PPT) or without (K2E) gel separator. A stepwise centrifugation was applied to K2E aliquots to obtain platelet-poor plasma (PPP). K2E, PPP and PPT plasma, stored under different conditions, were assayed for extracellular vesicles concentration and size distribution, through dynamic laser light scattering, and microRNAs content, by qPCR. RESULTS: PPP samples were characterized by the lowest extracellular vesicles count and miRNA detectability. Although having no effects on extracellular vesicles total concentration, storage conditions influenced microRNAs detectability, mainly in PPP and PPT samples. Extracellular vesicles-associated miRNAs levels in K2E were, in general, higher than in PPP and to a very limited extent to PPT. Storage temperature and delay did not affect their profile in K2E samples. CONCLUSIONS: Extracellular vesicles count and extracellular vesicles miRNA profile changed under the analyzed pre-analytical variables, showing the greatest stability in K2E samples. Since pre-analytical variables differently affected extracellular vesicles and their miRNA content, they should be considered in each experimental setting and clinical routine.

Correction to: Interplay between low plasma RANKL and VDR-FokI polymorphism in lumbar disc herniation independently from age, body mass, and environmental factors: a case-control study in the Italian population.

Under the headline "Correlation of RANKL concentrations and VDR-FokI polymorphism on disc herniation" in the description text for Table 2, the term "allelic frequency" was used erroneously for "genotypic frequency".

Study of the preanalytical variables affecting the measurement of clinically relevant free-circulating microRNAs: focus on sample matrix, platelet depletion, and storage conditions.

INTRODUCTION: Circulating microRNAs (miRNAs) are emerging as potential biomarkers. However, the lack of preanalytical and analytical standardization limits their use. The aim of this study was to determine the expression of different miRNAs in plasma according to different collection and storage conditions. MATERIALS AND METHODS: Venous blood from 10 volunteers was collected in tubes spray-coated with dipotassium salt of ethylendiaminetetraacetic acid, either with (plasma-preparation tube, PPT) or without (K2EDTA) gel separator. Platelet-poor plasma (PPP) was also obtained from K2EDTA plasma. After storage under different conditions, miRNA-enriched total RNA was isolated from plasma and reverse transcribed. A panel of 179 miRNAs was assayed by quantitative polymerase chain reaction and the results were analysed by GenEx software. Detectability and stability of miRNAs were determined. RESULTS: The number of undetected miRNAs was: 18, 24, and 22 in PPT; 83, 43, and 20 in K2EDTA; and 76, 106, and 104 in PPP samples, for plasma immediately frozen at - 80°C and plasma stored for 24h at room temperature or 4°C, respectively. Circulating miRNA expression in PPT samples was not affected by storage delay or temperature, while the percentage of up- and down-regulated miRNA in K2EDTA and PPP samples ranged from 2%, and 1% to 7%, and 5%, respectively. CONCLUSIONS: Sample matrix, temperature and delay in storage strongly influence the expression level of plasma miRNAs. Our results indicate PPT tubes as the most suitable matrix to improve total miRNA detectability and stability, independently of temperature.

Differences in Osteoimmunological Biomarkers Predictive of Psoriatic Arthritis among a Large Italian Cohort of Psoriatic Patients.

(1) Background: In literature it is reported that 20-30% of psoriatic patients evolve to psoriatic arthritis over time. Currently, no specific biochemical markers can either predict progression to psoriatic arthritis or response to therapies. This study aimed to identify osteoimmunological markers applicable to clinical practice, giving a quantitative tool for evaluating pathological status and, eventually, to provide prognostic support in diagnosis. (2) Methods: Soluble (serum) bone and cartilage markers were quantified in 50 patients with only psoriasis, 50 psoriatic patients with psoriatic arthritis, and 20 healthy controls by means of multiplex and enzyme-linked immunoassays. (3) Results: Differences in the concentrations of matrix metalloproteases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), receptor activator of nuclear factor kappa-B- ligand (RANK-L), procollagen type I N propeptide (PINP), C-terminal telopeptide of type I collagen (CTx-I), dickkopf-related protein 1 (DKK1), and sclerostin (SOST) distinguished healthy controls from psoriasis and psoriatic arthritis patients. We found that MMP2, MMP12, MMP13, TIMP2, and TIMP4 distinguished psoriasis from psoriatic arthritis patients undergoing a systemic treatment, with a good diagnostic accuracy (Area under the ROC Curve (AUC) > 0.7). Then, chitinase-3-like protein 1 (CHI3L1) and MMP10 distinguished psoriasis from psoriatic arthritis not undergoing systemic therapy and, in the presence of onychopathy, MMP8 levels were higher in psoriasis than in psoriatic arthritis. However, in these latter cases, the diagnostic accuracy of the identified biomarkers was low (0.5 < AUC < 0.7). (4) Conclusions. By highlighting never exploited differences, the wide osteoimmunological biomarkers panel provides a novel clue to the development of diagnostic paths in psoriasis and psoriasis-associated arthropathic disease.

Effects of 12-months treatment with zoledronate or teriparatide on intima-media thickness of carotid artery in women with postmenopausal osteoporosis: A pilot study.

Atherosclerosis and osteoporosis are interrelated entities and share similar pathogenic mechanisms. Recent studies showed that key proteins of bone metabolism, such as osteoprotegerin (OPG) and osteopontin (OPN), are also involved in vascular atherosclerosis and calcifications. The carotid intima-media thickness (CA-IMT) is an early quantitative marker of generalized atherosclerosis. Aim of study was to investigate whether 12-months treatment with zoledronate (ZLN) or teriparatide (TPT) affects CA-IMT and circulating OPG and OPN levels. In this study, 11 postmenopausal osteoporotic women (aged 73, 70.5-74.5 years; median, range interquartile) treated with 5 mg/year iv ZLN; 9 postmenopausal osteoporotic women (aged 70, 62.5-73.5 years) treated with 20 µg/day sc TPT; and 10 aged-, body mass index (BMI)-, glycemic, and lipid profiles-matched, free from anti-osteoporotic and hypocholesterolemic drugs, controls were prospectively investigated at baseline and after 12 months. At baseline, median CA-IMT was similar in the three groups and increased after 12 months. CA-IMT increased significantly in TPT-treated patients (1.0, 0.8-1.2 vs 1.1, 0.9-15 mm, P = 0.04), though the change was minimal. After 12 months of treatment, CA-IMT positively correlated with alkaline phosphatase (ALP) levels (r = 0.767, P = 0.008) and negatively with high-density lipoprotein (HDL) cholesterol levels (r = -0.65, P = 0.03), suggesting interplay between active bone remodeling and lipid profile. At baseline and after 12 months, median serum OPG and OPN levels did not differ among the groups and did not correlate with changes in CA-IMT. In conclusion, ZLN and TPT treatments are safe on carotid walls in osteoporotic women with subclinical atherosclerosis; circulating OPG and OPN are not affected by long-term anti-osteoporotic treatments and do not correlate with CA-IMT.

Normalization strategies differently affect circulating miRNA profile associated with the training status.

MicroRNAs are fine regulators of the whole-body adaptive response but their use as biomarkers is limited by the lack of standardized pre- and post-analytical procedures. This work aimed to compare different normalization approaches for RT-qPCR data analyses, in order to identify the most reliable and reproducible method to analyze circulating miRNA expression profiles in sedentary and highly-trained subjects. As the physically active status is known to affect miRNA expression, they could be effective biomarkers of the homeostatic response. Following RNA extraction from plasma, a panel of 179 miRNAs was assayed by RT-qPCR and quantified by applying different normalization strategies based on endogenous miRNAs and exogenous oligonucleotides. hsa-miR-320d was found as the most appropriate reference miRNA in reducing the technical variability among the experimental replicates and, hence, in highlighting the inter-cohorts differences. Our data showed an association between the physically active status and specific skeletal muscle- and bone-associated circulating miRNAs profiles, revealing that established epigenetic modifications affect the baseline physiological status of these tissues. Since different normalization strategies led to different outputs, in order to avoid misleading interpretation of data, we remark the importance of the accurate choice of the most reliable normalization method in every experimental setting.

Engineering an Environment for the Study of Fibrosis: A 3D Human Muscle Model with Endothelium Specificity and Endomysium.

The integration of vascular structures into in vitro cultured tissues provides realistic models of complex tissue-vascular interactions. Despite the incidence and impact of muscle-wasting disorders, advanced in vitro systems are still far from recapitulating the environmental complexity of skeletal muscle. Our model comprises differentiated human muscle fibers enveloped by a sheath of human muscle-derived fibroblasts and supported by a vascular network with mural-like cells. Here, we demonstrate the induction of muscle-specific endothelium and the self-organization of endomysial muscle fibroblasts mediated by endothelial cells. We use this model to mimic the fibrotic environment characterizing muscular dystrophies and to highlight key signatures of fibrosis that are neglected or underestimated in traditional 2D monocultures. Overall, this vascularized meso-scale cellular construct finely recapitulates the human skeletal muscle environment and provides an advanced solution for in vitro studies of muscle physiology and pathology.

Effects of repeated sprints training on fracture risk-associated miRNA.

Repeated-sprint training (RS, short-duration sprints at supramaximal intensities interspersed with brief recoveries) is a time-saving metabolically effective strategy whose effects on bone are unknown. Bone metabolism is a finely regulated process profoundly affected by exercise as assayable by studying specific systemic (e.g., hormones, cytokines) and bone-derived molecules (e.g., bone markers, miRNAs). Aim of this study was to determine the effect of a 8-week repeated-sprint on circulating levels of fracture risk-associated miRNA. Blood was collected from 9 subjects performing RS 3 times/week (EXP) and 9 age-matched inactive controls (CTRL) before the start of the protocol (T0) and after 4 (T1) and 8 weeks (T2). The relative expression of miR-21-5p, miR-23a-3p, miR-24-3p, miR93-5p, miR-100-5p, miR-122-5p, miR-124-3p, miR-125b-5p, miR-148a-3p, miR-637 was assayed by real-time PCR by the 2 -ΔΔCT method (housekeeping: miR-425-5p, miR-484). Serum concentrations of bone markers (DKK1, sclerostin, osteoprotegerin, osteocalcin, osteopontin), cytokines (IL-1ß, TNFα), and metabolic hormones (leptin, insulin, PTH) were assayed by multiplex assay. miR-637 and miR-124-3p were undetectable. In CTRL miRNA levels remained unchanged. In EXP miR-21-5p remained unchanged. Compared to T0 miR-23a-3p and miR-24-3p were significantly decreased at T1 and T2, also compared to CTRL, miR-100 was significantly decreased at T2, miR-122-5p, miR-125-5p, and miR148a-3p were significantly decreased at T1, while miR-93-5p was significantly increased at T1. None of the metabolic hormones was affected by the intervention while, among the bone markers, DKK1, osteocalcin and sclerostin were slightly but significantly decreased. In conclusion, an 8-week repeated-sprint training downregulates the expression of circulating miRNA associated with fracture risk.

Sodium butyrate has anti-proliferative, pro-differentiating, and immunomodulatory effects in osteosarcoma cells and counteracts the TNFα-induced low-grade inflammation.

Butyrate, an essential factor for colonocytes and regulator in the development of colon cancer, is partially absorbed by the gut. It influences the proliferation and differentiation of several cell types including osteoblasts. We evaluated the effects of different doses of butyrate on differentiation and functionality of osteosarcoma cells in vitro and the expression of a pro-inflammatory phenotype in a normal or inflammatory environment. SaOS-2 osteosarcoma cells were induced to differentiate and contemporarily treated for 24 h, 48 h, or 7 days with sodium butyrate 10-4, 5 × 10-4, or 10-3 M in the presence or absence of tumor necrosis factor alpha (TNFα) 1 ng/mL, a pro-inflammatory stimulus. Despite the mild effects on proliferation and alkaline phosphatase activity, butyrate dose- and time-dependently induced the expression of a differentiated phenotype (RUNX2, COL1A1 gene expression, and osteopontin gene and protein expression). This was associated with a partial inhibition of nuclear factor kappa B (NF-κB) activation and the induction of histone deacetylase 1 expression. The net effect was the expression of an anti-inflammatory phenotype and the increase in the osteoprotegerin-to-receptor activator of nuclear factor kappa-B ligand (RANKL) ratio. Moreover, butyrate, especially at the highest dose, counteracted the effects of the pro-inflammatory stimulus of TNFα 1 ng/mL. Butyrate affects osteosarcoma cell metabolism by anticipating the expression of a differentiated phenotype and by inducing the expression of anti-inflammatory mediators.

Measuring myokines with cardiovascular functions: pre-analytical variables affecting the analytical output.

In the last few years, a growing number of molecules have been associated to an endocrine function of the skeletal muscle. Circulating myokine levels, in turn, have been associated with several pathophysiological conditions including the cardiovascular ones. However, data from different studies are often not completely comparable or even discordant. This would be due, at least in part, to the whole set of situations related to the preparation of the patient prior to blood sampling, blood sampling procedure, processing and/or store. This entire process constitutes the pre-analytical phase. The importance of the pre-analytical phase is often not considered. However, in routine diagnostics, the 70% of the errors are in this phase. Moreover, errors during the pre-analytical phase are carried over in the analytical phase and affects the final output. In research, for example, when samples are collected over a long time and by different laboratories, a standardized procedure for sample collecting and the correct procedure for sample storage are acknowledged. In this review, we discuss the pre-analytical variables potentially affecting the measurement of myokines with cardiovascular functions.