HuR-positive stress granules: Potential targets for age-related osteoporosis.

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target ß-catenin by binding and recruiting ß-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of ß-catenin into HuR-positive SGs, facilitated ß-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.

Integrated metabolomics and proteomics analysis to understand muscle atrophy resistance in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.

A novel recombinant ORF7-siRNA delivered by flexible nano-liposomes inhibits varicella zoster virus infection.

BACKGROUND: Varicella zoster virus (VZV), which is a human restricted alpha-herpesvirus, causes varicella (chickenpox) and zoster (shingles). The subsequent post-herpetic neuralgia (PHN) due to VZV infection is excruciating for most patients. Thus, developing specific therapeutics against VZV infection is imperative. RNA interference (RNAi) represents an effective approach for alternative antiviral therapy. This study aimed to develop a novel anti-VZV therapeutics based on RNAi. RESULTS: In this study, we screened and found the open reading frame 7 (ORF7) of the VZV genome was an ideal antiviral target based on RNAi. Therefore, a novel siRNA targeting ORF7 (si-ORF7) was designed to explore the potential of RNAi antiviral treatment strategy toward VZV. We used a bio-engineering approach to manufacture recombinant siRNA agents with high yield in E. coli. Then, the efficacy of recombinant ORF7-siRNA (r/si-ORF7) in inhibiting VZV infection both in cellular level and 3D human epidermal skin model was evaluated. The r/si-ORF7 was proved to inhibit the VZV replication and reduce the virus copy numbers significantly in vitro. Furthermore, flexible nano-liposomes were established to deliver r/si-ORF7 to 3D human epidermal skin model and found r/si-ORF7 also could inhibit the VZV infection, thus maintaining normal skin morphology. CONCLUSIONS: Taken together, our results highlighted that transdermal administration of antiviral r/si-ORF7 was a promising therapeutic strategy for functional cure of VZV infection.

U-MLP: MLP-based ultralight refinement network for medical image segmentation.

The convolutional neural network (CNN) and Transformer play an important role in computer-aided diagnosis and intelligent medicine. However, CNN cannot obtain long-range dependence, and Transformer has shortcomings in computational complexity and a large number of parameters. Recently, compared with CNN and Transformer, the Multi-Layer Perceptron (MLP)-based medical image processing network can achieve higher accuracy with smaller computational and parametric quantities. Hence, in this work, we propose an encoder-decoder network, U-MLP, based on the ReMLP block. The ReMLP block contains an overlapping sliding window mechanism and a Multi-head Gate Self-Attention (MGSA) module, where the overlapping sliding window can extract local features of the image like convolution, then combines MGSA to fuse the information extracted from multiple dimensions to obtain more contextual semantic information. Meanwhile, to increase the generalization ability of the model, we design the Vague Region Refinement (VRRE) module, which uses the primary features generated by network inference to create local reference features, thus determining the pixel class by inferring the proximity between local features and labeled features. Extensive experimental evaluation shows U-MLP boosts the performance of segmentation. In the skin lesions, spleen, and left atrium segmentation on three benchmark datasets, our U-MLP method achieved a dice similarity coefficient of 88.27%, 97.61%, and 95.91% on the test set, respectively, outperforming 7 state-of-the-art methods.

A potential therapeutic drug for osteoporosis: prospect for osteogenic LncRNAs.

Long non-coding RNAs (LncRNAs) play essential roles in multiple physiological processes including bone formation. Investigators have revealed that LncRNAs regulated bone formation through various signaling pathways and micro RNAs (miRNAs). However, several problems exist in current research studies on osteogenic LncRNAs, including sophisticated techniques, high cost for in vivo experiment, as well as low homology of LncRNAs between animal model and human, which hindered translational medicine research. Moreover, compared with gene editing, LncRNAs would only lead to inhibition of target genes rather than completely knocking them out. As the studies on osteogenic LncRNA gradually proceed, some of these problems have turned osteogenic LncRNA research studies into slump. This review described some new techniques and innovative ideas to address these problems. Although investigations on osteogenic LncRNAs still have obtacles to overcome, LncRNA will work as a promising therapeutic drug for osteoporosis in the near future.

MiR-12200-5p Targets Multiple Members of Wnt Signaling Pathway to Inhibit Osteoblast Differentiation and Bone Formation.

BACKGROUND: Osteoporosis is widespread and has become an emerging problem in the elderly. MicroRNAs could affect osteoblast differentiation and further regulate the occurrence of osteoporosis by targeting osteogenic differentiation signaling pathways. Our screening study found that miR-12200-5p simultaneously targeted six important factors within the Wnt signaling pathway (Apc, Tcf4, Tcf7, Wnt3a, Wnt5a, and Lrp6), indicating that miR-12200-5p might function as a strong regulator of this pathway. Since the Wnt pathway exists as one of the most essential pathways for osteogenic differentiation, miR-12200-5p may have an important role in the development of osteoporosis. OBJECTIVE: This study intended to explore the regulatory role and corresponding mechanism of miR-12200-5p in osteoblast differentiation. METHODS: We investigated the differentiation of osteoblast after the treatments of miR-12200-5p mimic and inhibitor. The interactions between miR-12200-5p and its target genes were also detected. Furthermore, the rescue effect of miR-12200-5p inhibitor on osteoporosis was evaluated using an ovariectomized osteoporosis mouse model. RESULTS: MiR-12200-5p significantly inhibited osteoblast differentiation, and bound with the 3'-UTR sequences of its target genes (Apc, Tcf4, Tcf7, Wnt3a, Wnt5a, and Lrp6) to reduce the expressions of these genes. The inhibition of miR-12200-5p would almost fully alleviate postmenopausal osteoporosis. CONCLUSION: MiR-12200-5p could strongly repress osteoblast differentiation and bone formation by targeting multiple members of the Wnt signaling pathway simultaneously. The study supplemented the theoretical and experimental basis for researching the mechanism of osteogenic differentiation and inspired the development of novel therapeutic strategies for osteoporosis.

Ellagic acid inhibits CDK12 to increase osteoblast differentiation and alleviate osteoporosis in hindlimb-unloaded and ovariectomized mice.

BACKGROUND: Osteoporosis is a highly prevalent bone disease occurred commonly in astronauts and postmenopausal women due to mechanical unloading and estrogen deficiency, respectively. At present, there are some traditional Chinese medicine compounds for preventing and treating osteoporosis induced by simulated microgravity, but the detailed components of the traditional Chinese medicines still need to be confirmed and osteoporosis is still untreatable due to a lack of effective small-molecule natural medicine. PURPOSE: To explore the role of cyclin-dependent kinase 12 (CDK12) in osteoporosis induced by simulated microgravity and the therapeutic effect of CDK12-targeted Ellagic Acid (EA) on osteoporosis. METHODS: Our previous study has suggested that CDK12 as a potential target for treating and preventing osteoporosis. In this study, the role of CDK12 in osteoblasts and mice bone tissues was further studied under simulated microgravity. And by targeting CDK12, natural small-molecule product EA was screened out based on a large scale through the weighted set similarity (WES) method and the therapeutic effects of EA on osteoporosis was investigated in hindlimb-unloaded (HU) mouse model and ovariectomized (OVX) model. RESULTS: The results demonstrated that simulated microgravity inhibited bone formation and up-regulated the expression of CDK12. Furthermore, CDK12-siRNA or THZ531 (an inhibitor of CDK 12) promoted osteoblast differentiation, while the overexpression of CDK12 inhibited osteoblasts differentiation. And we further proved that CDK12-targeted EA showed a rescue effect on osteoblast differentiation inhibition caused by simulated microgravity. EA (50 mg·kg-1·day-1) daily intragastric administration alleviated the symptoms of osteoporosis and accompanied with the improvement of trabecular bone and cortical bone parameters with significantly overexpression of CDK12. CONCLUSION: EA efficiently improves osteoporosis by targeting CDK12, which is a suppresser of osteoblast differentiation and a novel therapeutic target for treating osteoporosis.

MACF1 overexpression in BMSCs alleviates senile osteoporosis in mice through TCF4/miR-335-5p signaling pathway.

Background: The decreased osteogenic differentiation ability of mesenchymal stem cells (MSCs) is one of the important reasons for SOP. Inhibition of Wnt signaling in MSCs is closely related to SOP. Microtubule actin crosslinking factor 1 (MACF1) is an important regulator in Wnt/ß-catenin signal transduction. However, whether the specific expression of MACF1 in MSC regulates SOP and its mechanism remains unclear. Methods: We established MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-KI) mice, naturally aged male mice, and ovariectomized female mice models. Micro-CT, H&E staining, double calcein labeling, and the three-point bending test were used to explore the effects of MACF1 on bone formation and bone microstructure in the SOP mice model. Bioinformatics analysis, ChIP-PCR, qPCR, and ALP staining were used to explore the effects and mechanisms of MACF1 on MSCs' osteogenic differentiation. Results: Microarray analysis revealed that the expression of MACF1 and positive regulators of the Wnt pathway (such as TCF4, ß-catenin, Dvl) was decreased in human MSCs (hMSCs) isolated from aged osteoporotic than non-osteoporotic patients. The ALP activity and osteogenesis marker genes (Alp, Runx2, and Bglap) expression in mouse MSCs was downregulated during aging. Furthermore, Micro-CT analysis of the femur from 2-month-old MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-cKI) mice showed no significant trabecular bone changes compared to wild-type littermate controls, whereas 18- and 21-month-old MACF1 c-KI animals displayed increased bone mineral densities (BMD), improved bone microstructure, and increased maximum compression stress. In addition, the ovariectomy (OVX)-induced osteoporosis model of MACF1 c-KI mice had significantly higher trabecular volume and number, and increased bone formation rate than that in control mice. Mechanistically, ChIP-PCR showed that TCF4 could bind to the promoter region of the host gene miR-335-5p. Moreover, MACF1 could regulate the expression of miR-335-5p by TCF4 during the osteogenic differentiation of MSCs. Conclusion: These data indicate that MACF1 positively regulates MSCs osteogenesis and bone formation through the TCF4/miR-335-5p signaling pathway in SOP, suggesting that targeting MACF1 may be a novel therapeutic approach against SOP. The translational potential of this article: MACF1, an important switch in the Wnt signaling pathway, can alleviate SOP through the TCF4/miR-335-5p signaling pathway in mice model. It might act as a therapeutic target for the treatment of SOP to improve bone function.

Macrophage-Targeted Dextran Sulfate-Dexamethasone Conjugate Micelles for Effective Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic immune disease that causes joint affection and even disability. Activated macrophages play an important role in the pathogenesis and progression of RA by producing pro-inflammatory factors. The use of dexamethasone (DXM) is effective in relieving the intractable pain and inflammatory progression of RA. However, long-term use of DXM is strongly associated with increased rates of diabetes, osteoporosis, bone fractures, and mortality, which hinders its clinical use. In this study, the dextran sulfate-cisaconitic anhydride-dexamethasone (DXM@DS-cad-DXM) micelles were prepared to treat RA by selectively recognizing scavenger receptor (SR) on the activated macrophages. The potent targeting property of DXM@DS-cad-DXM micelles to SR was by fluorescence microscope. Additionally, the effective accumulation and powerful anti-inflammatory activity of DXM@DS-cad-DXM micelles were observed in the inflamed joints of adjuvant-induced arthritis (AIA) rats after intravenous administration. Overall, DXM@DS-cad-DXM micelles are a potentially effective nanomedicine for targeted therapy of RA.

Co-Polymer Carrier with Dual Advantages of Cartilage-Penetrating and Targeting Improves Delivery and Efficacy of MicroRNA Treatment of Osteoarthritis.

Osteoarthritis (OA) is a debilitating joint disease affecting nearly 400 million people with no efficient etiological therapies. OA is primarily identified by cartilage destruction, and gradual degeneration of the whole joint would happen when the OA progresses. Hence, cartilage has been identified as the primary therapeutic target of OA. Unfortunately, numerous barriers block the delivery of therapeutic agents into cartilage, including avascular traits and high hardness of the extracellular matrix. Herein, a cartilage-targeting peptide (CAP) modified polyvinylamine (PVAm)- poly (lactic-co-glycolic acid) (PLGA) copolymer (CAP-PVAm-PLGA) is designed, which can form spherical nanoparticles with the r-miR-140 (CPP-NPs). CPP-NPs possessed enhanced mechanical properties due to the introduction of PLGA to vehicles. Meanwhile, CAP endowed the cartilage targeting which facilitated CPP-NPs localization in cartilage. With such dual advantages, CPP-NPs exhibited outstanding penetrability and accumulation in cartilage even subchondral bone, and can penetrate to a depth of 1000 µm into human cartilage. The degeneration area of cartilage is reduced by 65% and synovial inflammation score by 80% in OA mice, and the microarchitecture of subchondral bone is also ameliorated. These studies established a promising platform for therapeutic RNA delivery in OA therapy that overcame the cartilage barriers.

Transcriptomic and proteomic time-course analyses based on Metascape reveal mechanisms against muscle atrophy in hibernating Spermophilus dauricus.

Hibernating Spermophilus dauricus is resistant to muscle atrophy. Comprehensive transcriptome and proteome time-course analyses based on Metascape can further reveal the underlying processes (pre-hibernation stage, PRE; torpor stage, TOR; interbout arousal stage, IBA; and post-hibernation stage, POST). Transcriptome analysis showed that the cellular responses to growth factor stimulus and discrete oxygen levels continuously changed during hibernation. Proteomic analysis showed that neutrophil degranulation, sulfur compound metabolic process, and generation of precursor metabolites and energy continuously changed during hibernation. Molecular complex detection (MCODE) analysis in both transcriptome and proteome indicated that smooth muscle contraction was involved in the POST versus IBA stage, and peroxisome proliferator-activated receptor delta (Ppard), Myc proto-oncogene (Myc), Sp1 transcription factor (Sp1), and nuclear factor Kappa B subunit 1 (NFκB1) are the common TFs during the hibernation process. Integrated transcriptome and proteome analyses found 18 molecules in the TOR versus PRE stage, 1 molecule in the IBA versus TOR stage, and 16 molecules in the POST versus IBA stage. Among these molecules, carnitine palmitoyltransferase 1A (Cpt1a), SET and MYND domain containing 2 (Smyd2), four and a half LIM domains 1(Fhl1), reactive oxygen species modulator 1 (Romo1), and translocase of the inner mitochondrial membrane 50 (Timm50) were testified by Western blot. In conclusion, novel muscle atrophy resistance mechanisms can be deciphered by time-course transcriptome and proteome analyses based on Metascape.

How do RNA binding proteins trigger liquid-liquid phase separation in human health and diseases?

RNA-binding proteins (RBPs) lie at the center of post-transcriptional regulation and protein synthesis, adding complexity to RNA life cycle. RBPs also participate in the formation of membrane-less organelles (MLOs) via undergoing liquid-liquid phase separation (LLPS), which underlies the formation of MLOs in eukaryotic cells. RBPs-triggered LLPS mainly relies on the interaction between their RNA recognition motifs (RRMs) and capped mRNA transcripts and the heterotypic multivalent interactions between their intrinsically disordered regions (IDRs) or prion-like domains (PLDs). In turn, the aggregations of RBPs are also dependent on the process of LLPS. RBPs-driven LLPS is involved in many intracellular processes (regulation of translation, mRNA storage and stabilization and cell signaling) and serves as the heart of cellular physiology and pathology. Thus, it is essential to comprehend the potential roles and investigate the internal mechanism of RPBs-triggered LLPS. In this review, we primarily expound on our current understanding of RBPs and they-triggered LLPS and summarize their physiological and pathological functions. Furthermore, we also summarize the potential roles of RBPs-triggered LLPS as novel therapeutic mechanism for human diseases. This review will help understand the mechanisms underlying LLPS and downstream regulation of RBPs and provide insights into the pathogenesis and therapy of complex diseases.

A lipid-polymer hybrid nanoparticle (LPN)-loaded dissolving microneedle patch for promoting hair regrowth by transdermal miR-218 delivery.

Alopecia is the most common multifactorial hair loss disorder, affecting almost 50% of the population and even having a serious psychological impact on the patients. miR-218 has therapeutic potential for alopecia since it can activate the Wnt/ß-catenin channel by down-regulating SFRP2, which is a key channel in hair follicle cycle transformation for hair regrowth. Although miR-218 has the potential to treat this disease, several barrier properties of the skin challenge miRNA's delivery to the target location, such as passing through the corneum and resistant enzymatic degradation. To address these challenges, we evaluated a device that combined the use of hyaluronic acid (HA)-based dissolving microneedle (MN) to enhance corneum permeability with the lipid polymer hybrid nanoparticles (LPNs) as a miRNA delivery carrier to protect miR-218 from degradation. The MN patches could promote LPNs/miR-218 diffusing in the dermis region, and significantly increase the bioavailability of miR-218. Furthermore, in the shaved mouse model, the MN patches showed higher efficacy in promoting hair growth than the topical smear treatment, while avoiding the safety concern. This work established a novel and effective combination device with MN and LPNs that can be used for localized transdermal miRNA delivery to promote hair regrowth.

Liquid-Liquid Phase Separation of Biomacromolecules and Its Roles in Metabolic Diseases.

Liquid-liquid phase separation (LLPS) compartmentalizes and concentrates biomacromolecules into liquid-like condensates, which underlies membraneless organelles (MLOs) formation in eukaryotic cells. With increasing evidence of the LLPS concept and methods, this phenomenon as a novel principle accounts for explaining the precise spatial and temporal regulation of cellular functions. Moreover, the phenomenon that LLPS tends to concentrate proteins is often accompanied by several abnormal signals for human diseases. It is reported that multiple metabolic diseases are strongly associated with the deposition of insoluble proteinaceous aggregating termed amyloids. At present, recent studies have observed the roles of LLPS in several metabolic diseases, including type 2 diabetes mellitus (T2DM), Alzheimer's disease (AD), and metabolic bone diseases (MBDs). This review aims to expound on the current concept and methods of LLPS and summarize its vital roles in T2DM, AD, and MBDs, uncover novel mechanisms of these metabolic diseases, and thus provide powerful potential therapeutic strategies and targets for ameliorating these metabolic diseases.

Comprehensive ceRNA network for MACF1 regulates osteoblast proliferation.

BACKGROUND: Previous studies have shown that microtubule actin crosslinking factor 1 (MACF1) can regulate osteoblast proliferation and differentiation through non-coding RNA (ncRNA) in bone-forming osteoblasts. However, the role of MACF1 in targeting the competing endogenous RNA (ceRNA) network to regulate osteoblast differentiation remains poorly understood. Here, we profiled messenger RNA (mRNA), microRNA (miRNA), and long ncRNA (lncRNA) expression in MACF1 knockdown MC3TC­E1 pre­osteoblast cells. RESULTS: In total, 547 lncRNAs, 107 miRNAs, and 376 mRNAs were differentially expressed. Significantly altered lncRNAs, miRNAs, and mRNAs were primarily found on chromosome 2. A lncRNA-miRNA-mRNA network was constructed using a bioinformatics computational approach. The network indicated that mir-7063 and mir-7646 were the most potent ncRNA regulators and mef2c was the most potent target gene. Pathway enrichment analysis showed that the fluid shear stress and atherosclerosis, p53 signaling, and focal adhesion pathways were highly enriched and contributed to osteoblast proliferation. Importantly, the fluid shear stress and atherosclerosis pathway was co-regulated by lncRNAs and miRNAs. In this pathway, Dusp1 was regulated by AK079370, while Arhgef2 was regulated by mir-5101. Furthermore, Map3k5 was regulated by AK154638 and mir-466q simultaneously. AK003142 and mir-3082-5p as well as Ak141402 and mir-446 m-3p were identified as interacting pairs that regulate target genes. CONCLUSION: This study revealed the global expression profile of ceRNAs involved in the differentiation of MC3TC­E1 osteoblasts induced by MACF1 deletion. These results indicate that loss of MACF1 activates a comprehensive ceRNA network to regulate osteoblast proliferation.