YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of satellite cells.

Skeletal muscle satellite cells (SCs) are adult muscle stem cells responsible for muscle regeneration after acute or chronic injuries. The lineage progression of quiescent SC toward activation, proliferation, and differentiation during the regeneration is orchestrated by cascades of transcription factors (TFs). Here, we elucidate the function of TF Yin Yang1 (YY1) in muscle regeneration. Muscle-specific deletion of YY1 in embryonic muscle progenitors leads to severe deformity of diaphragm muscle formation, thus neonatal death. Inducible deletion of YY1 in SC almost completely blocks the acute damage-induced muscle repair and exacerbates the chronic injury-induced dystrophic phenotype. Examination of SC revealed that YY1 loss results in cell-autonomous defect in activation and proliferation. Mechanistic search revealed that YY1 binds and represses mitochondrial gene expression. Simultaneously, it also stabilizes Hif1α protein and activates Hif1α-mediated glycolytic genes to facilitate a metabolic reprogramming toward glycolysis which is needed for SC proliferation. Altogether, our findings have identified YY1 as a key regulator of SC metabolic reprogramming through its dual roles in modulating both mitochondrial and glycolytic pathways.

RNA-targeted small-molecule drug discoveries: a machine-learning perspective.

In the past two decades, machine learning (ML) has been extensively adopted in protein-targeted small molecule (SM) discovery. Once trained, ML models could exert their predicting abilities on large volumes of molecules within a short time. However, applying ML approaches to discover RNA-targeted SMs is still in its early stages. This is primarily because of the intrinsic structural instability of RNA molecules that impede the structure-based screening or designing of RNA-targeted SMs. Recently, with more studies revealing RNA structures and a growing number of RNA-targeted ligands being identified, it resulted in an increased interest in the field of drugging RNA. Undeniably, intracellular RNA is much more abundant than protein and, if successfully targeted, will be a major alternative target for therapeutics. Therefore, in this context, as well as under the premise of having RNA-related research data, ML-based methods can get involved in improving the speed of traditional experimental processes. [Figure: see text].

Photodynamic treatment with purpurin 18 effectively inhibits triple negative breast cancer by inducing cell apoptosis.

This study aims to evaluate the photodynamic efficacy of purpurin 18 (pu-18) on triple negative breast cancer both in vitro and in vivo. Two states of 4T1 cells, 2D culture and 3D spheroids, were used to evaluate the photodynamic action of pu-18 in vitro. The in vitro study results indicated that for the 4T1 2D cell culture, the photodynamic therapy (PDT) treatment showed significant photocytotoxicity at low pu-18 concentrations following light irradiation. Pu-18 was found to distribute on the lysosomes, mitochondria, Golgi apparatus, and endoplasmic reticulum. After irradiation, pu-18 can generate ROS to destroy the mitochondrial membrane potential (MMP) and eventually induce apoptosis in the 2D 4T1 cells. Light-activated pu-18 could also induce the destruction of the 3D 4T1 cell spheroids. The in vivo study was conducted by using a subcutaneous 4T1 breast cancer animal model. The results demonstrated that pu-18 could remain in the tumor for more than 4 days by direct intra-tumoral injection. The PDT treatment was performed every 2 days for a total of 3 times. The results showed that PDT treatment could significantly inhibit tumor growth in vivo, indicating a good photodynamic efficacy of pu-18 in the mouse breast cancer model, without influencing weight and major organ function. The survival pattern results showed that PDT treatment could largely extend the survival time of mice with breast cancer. The preliminary conclusion is that photodynamic treatment using pu-18 is effective at preventing the growth of triple negative breast cancer cells both in vitro and in vivo. A combination of light irradiation and pu-18 could therefore be a worthwhile approach for the treatment of triple negative breast cancer.

Artificial Intelligence in Aptamer-Target Binding Prediction.

Aptamers are short single-stranded DNA, RNA, or synthetic Xeno nucleic acids (XNA) molecules that can interact with corresponding targets with high affinity. Owing to their unique features, including low cost of production, easy chemical modification, high thermal stability, reproducibility, as well as low levels of immunogenicity and toxicity, aptamers can be used as an alternative to antibodies in diagnostics and therapeutics. Systematic evolution of ligands by exponential enrichment (SELEX), an experimental approach for aptamer screening, allows the selection and identification of in vitro aptamers with high affinity and specificity. However, the SELEX process is time consuming and characterization of the representative aptamer candidates from SELEX is rather laborious. Artificial intelligence (AI) could help to rapidly identify the potential aptamer candidates from a vast number of sequences. This review discusses the advancements of AI pipelines/methods, including structure-based and machine/deep learning-based methods, for predicting the binding ability of aptamers to targets. Structure-based methods are the most used in computer-aided drug design. For this part, we review the secondary and tertiary structure prediction methods for aptamers, molecular docking, as well as molecular dynamic simulation methods for aptamer-target binding. We also performed analysis to compare the accuracy of different secondary and tertiary structure prediction methods for aptamers. On the other hand, advanced machine-/deep-learning models have witnessed successes in predicting the binding abilities between targets and ligands in drug discovery and thus potentially offer a robust and accurate approach to predict the binding between aptamers and targets. The research utilizing machine-/deep-learning techniques for prediction of aptamer-target binding is limited currently. Therefore, perspectives for models, algorithms, and implementation strategies of machine/deep learning-based methods are discussed. This review could facilitate the development and application of high-throughput and less laborious in silico methods in aptamer selection and characterization.

Structural Biology for the Molecular Insight between Aptamers and Target Proteins.

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer-target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer-protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.

A PD-L1 Aptamer Selected by Loss-Gain Cell-SELEX Conjugated with Paclitaxel for Treating Triple-Negative Breast Cancer.

BACKGROUND The clinical challenges of triple-negative breast cancer (TNBC) includes the lack of targeted therapy and chemoresistance. TNBC has relatively high PD-L1 expression, and PD-L1 antibody in combination with nab-paclitaxel has been approved by FDA for TNBC treatment. Aptamers, also termed chemical antibody, are widely used in targeted drug delivery. The present study aimed to select a DNA aptamer that could specifically bind and deliver drugs to TNBC cells. MATERIAL AND METHODS An innovative loss-gain cell-SELEX strategy was used to select DNA aptamer for PD-L1 protein. Construction of PD-L1 knock-out and over-expression MDA-MB-231 cell lines were conducted through transfection and confirmed by western blot and flow cytometry. Confocal microscopy and flow cytometry were used to analyze the binding ability of aptamer with TNBC cells. The cytotoxicity of aptamer-paclitaxel complex against TNBC cells was evaluated by Cell Counting Kit-8 assay. The reactivation of the T cell function by aptamer was measured by IL-2 enzyme-linked immunosorbent assay after T cells co-cultured with tumor cells. RESULTS In this work, using an innovative loss-gain cell-SELEX strategy, we screened a PD-L1-targeting aptamer. PD-L1 aptamer selectively bound to PD-L1 over-expressed TNBC cells with a dissociation constant in the nanomolar range. PD-L1 aptamer could also inhibit PD-1/PD-L1 interaction and restore the function of T cells. Moreover, we developed a PD-L1 aptamer-paclitaxel conjugate which showed improved cellular uptake and anti-proliferation efficacy in PD-L1 over-expressed TNBC cells. CONCLUSIONS In summary, these findings suggest that the selected PD-L1 aptamer might have potential implication in immune modulation and targeted therapy against TNBC.

Long Noncoding RNA lncMUMA Reverses Established Skeletal Muscle Atrophy following Mechanical Unloading.

Reversing established muscle atrophy following mechanical unloading is of great clinical challenge. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in myogenesis. Here we identified a lncRNA (mechanical unloading-induced muscle atrophy-related lncRNA [lncMUMA]) enriched in muscle, which was the most downregulated lncRNA during muscle atrophy development in hindlimb suspension (HLS) mice. The in vitro and in vivo data demonstrated that the decreased expression levels of lncMUMA closely associated with a reduction of myogenesis during mechanical unloading. Mechanistically, lncMUMA promoted myogenic differentiation by functioning as a miR-762 sponge to regulate the core myogenic regulator MyoD in vitro. The enforced expression of lncMUMA relieved the decreases in MyoD protein and muscle mass in miR-762 knockin mice. Therapeutically, the enforced expression of lncMUMA improved the in vitro myogenic differentiation of myoblasts under microgravity simulation, prevented the muscle atrophy development, and reversed the established muscle atrophy in HLS mice. These findings identify lncMUMA as an anabolic regulator to reverse established muscle atrophy following mechanical unloading.

Molecular Communication from Skeletal Muscle to Bone: A Review for Muscle-Derived Myokines Regulating Bone Metabolism.

Besides the mechanical loading-dependent paradigm, skeletal muscle also serves as an endocrine organ capable of secreting cytokines to modulate bone metabolism. In this review, we focused on reviewing the myokines involved in communication from skeletal muscle to bone, i.e. (1) myostatin and myostatin-binding proteins including follistatin and decorin, (2) interleukins including interleukin-6 (IL-6), interleukin-7 (IL-7) and interleukin-15 (IL-15), (3) insulin-like growth factor 1 (IGF-1) and its binding proteins, (4) other myokines including PGC-1α-irisin system and osteoglycin (OGN). To better understand the molecular communication from skeletal muscle to bone, we have summarized the recent advances in muscle-derived cytokines regulating bone metabolism in this review.

Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine.

Aptamers are short DNA/RNA oligonucleotides capable of binding to target molecules with high affinity and specificity. The process of selecting an aptamer is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Thanks to the inherit merits, aptamers have been used in a wide range of applications, including disease diagnosis, targeted delivery agents and therapeutic uses. To date, great achievements regarding the selection, modifications and application of aptamers have been made. However, few aptamer-based products have already successfully entered into clinical and industrial use. Besides, it is still a challenge to obtain aptamers with high affinity in a more efficient way. Thus, it is important to comprehensively review the current shortage and achievement of aptamer-related technology. In this review, we first present the limitations and notable advances of aptamer selection. Then, we compare the different methods used in the kinetic characterization of aptamers. We also discuss the impetus and developments of the clinical application of aptamers.

Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization.

Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg(-1)·day(-1)) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques.

Bioinformatics and Microarray Analysis of miRNAs in Aged Female Mice Model Implied New Molecular Mechanisms for Impaired Fracture Healing.

Impaired fracture healing in aged females is still a challenge in clinics. MicroRNAs (miRNAs) play important roles in fracture healing. This study aims to identify the miRNAs that potentially contribute to the impaired fracture healing in aged females. Transverse femoral shaft fractures were created in adult and aged female mice. At post-fracture 0-, 2- and 4-week, the fracture sites were scanned by micro computed tomography to confirm that the fracture healing was impaired in aged female mice and the fracture calluses were collected for miRNA microarray analysis. A total of 53 significantly differentially expressed miRNAs and 5438 miRNA-target gene interactions involved in bone fracture healing were identified. A novel scoring system was designed to analyze the miRNA contribution to impaired fracture healing (RCIFH). Using this method, 11 novel miRNAs were identified to impair fracture healing at 2- or 4-week post-fracture. Thereafter, function analysis of target genes was performed for miRNAs with high RCIFH values. The results showed that high RCIFH miRNAs in aged female mice might impair fracture healing not only by down-regulating angiogenesis-, chondrogenesis-, and osteogenesis-related pathways, but also by up-regulating osteoclastogenesis-related pathway, which implied the essential roles of these high RCIFH miRNAs in impaired fracture healing in aged females, and might promote the discovery of novel therapeutic strategies.

Adaptive responses of TRPC1 and TRPC3 during skeletal muscle atrophy and regrowth.

INTRODUCTION: We assessed the time-dependent changes of transient receptor potential canonical type 1 (TRPC1) and TRPC3 expression and localization associated with muscle atrophy and regrowth in vivo. METHODS: Mice were subjected to hindlimb unloading for 7 or 14 days (7U, 14U) followed by 3, 7, or 14 days of reloading (3R, 7R, 14R). RESULTS: Soleus muscle mass and tetanic force were reduced significantly at 7U and 14U and recovered by 14R. Recovery of muscle fiber cross-sectional area was observed by 28R. TRPC1 mRNA was unaltered during the unloading-reloading period. However, protein expression remained depressed through 14R. Decreased localization of TRPC1 to the sarcolemma was observed. TRPC3 mRNA and protein expression levels were decreased significantly during the early phase of reloading. CONCLUSIONS: Given the known role of these channels in muscle development, changes observed in TRPC1 and TRPC3 may relate closely to muscle atrophy and remodeling processes.

Experimental and numerical study of physiological responses in hot environments.

This paper proposed a multi-node human thermal model to predict human thermal responses in hot environments. The model was extended based on the Tanabe's work by considering the effects of high temperature on heat production, blood flow rate, and heat exchange coefficients. Five healthy men dressed in shorts were exposed in thermal neutral (29 °C) and high temperature (45 °C) environments. The rectal temperatures and skin temperatures of seven human body segments were continuously measured during the experiment. Validation of this model was conducted with experimental data. The results showed that the current model could accurately predict the skin and core temperatures in terms of the tendency and absolute values. In the human body segments expect calf and trunk, the temperature differences between the experimental data and the predicted results in high temperature environment were smaller than those in the thermally neutral environment conditions. The extended model was proved to be capable of predicting accurately human physiological responses in hot environments.

Discovery of the small molecular inhibitors against sclerostin loop3 as potential anti-osteoporosis agents by structural based virtual screening and molecular design.

Sclerostin is a secreted glycoprotein that expresses predominantly in osteocytes and inhibits bone formation by antagonizing the Wnt/ß-catenin signaling pathway, and the loop3 region of sclerostin has recently discovered as a novel therapeutic target for bone anabolic treatment without increasing cardiovascular risk. Herein, we used a structural based virtual screening to search for small molecular inhibitors selectively targeting sclerostin loop3. A novel natural product hit ZINC4228235 (THFA) was identified as the sclerostin loop3-selective inhibitor with a Kd value of 42.43 nM against sclerostin loop3. The simplification and derivation of THFA using molecular modeling-guided modification allowed the discovery of an effective and loop3-selective small molecular inhibitor, compound (4-(3-acetamidoprop-1-yn-1-yl)benzoyl)glycine (AACA), with improved binding affinity (Kd = 15.4 nM) compared to the hit THFA. Further in-vitro experiment revealed that compound AACA could attenuate the suppressive effect of transfected sclerostin on Wnt signaling and bone formation. These results make AACA as a potential candidate for development of anti-osteoporosis agents without increasing cardiovascular risk.

Aptamer functionalized hypoxia-potentiating agent and hypoxia-inducible factor inhibitor combined with hypoxia-activated prodrug for enhanced tumor therapy.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. Hypoxia-activated prodrugs (HAPs) have shown promise as potential therapeutic agents for TNBC. While increasing hypoxia levels may promote the HAP activation, it raises concerns regarding HIF1α-dependent drug resistance. It is desirable to develop a targeted approach that enhances tumor hypoxia for HAP activation without promoting HIF1α-dependent drug resistance in TNBC treatment. Herein, we proposed a multi-responsive carrier-free self-assembled nanomedicine named AQ4N@CA4T1ASO. This nanomedicine first targeted tumors by the TNBC-targeting aptamers (T1), and then disassembled in the reductive and acidic conditions within tumors. The released Combretastatin 4 (CA4) could exacerbate hypoxia, thereby promoting the conversion of inactive Banoxantrone (AQ4N) to its active form, AQ4. Simultaneously, the released antisense oligonucleotide (ASO) could attenuate hypoxia-induced HIF1α mRNA expression, thereby sensitizing the tumor to chemotherapy. Overall, this smart nanomedicine represents a profound targeted therapy strategy, combining "hypoxia-potentiating, hypoxia-activated, chemo-sensitization" approaches for TNBC treatment. In vivo study demonstrated significant suppression of tumor growth, highlighting the promising potential of this nanomedicine for future clinical translation.

Unique quinoline orientations shape the modified aptamer to sclerostin for enhanced binding affinity and bone anabolic potential.

Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility and bone formation. Sclerostin could negatively regulate bone formation by antagonizing the Wnt signal pathway, whereas it imposes severe cardiac ischemic events in clinic. Our team has screened an aptamer that could promote bone anabolic potential without cardiovascular risk. However, the affinity of the aptamer is lower and needs to be improved. In the study, hydrophobic quinoline molecule with unique orientations (seven subtypes) were incorporated into key sites of a bone anabolic aptamer against sclerostin to form a modified aptamer library. Among all the quinoline modifications, 5-quinoline modification could shape the molecular recognition of modified aptamers to sclerostin to facilitate enhancing its binding to sclerostin toward the highest affinity by interacting with newly participated binding sites in sclerostin. Further, 5-quinoline modification could facilitate the modified aptamer attenuating the suppressed effect of the transfected sclerostin on both Wnt signaling and bone formation marker expression levels in vitro, promoting bone anabolism in OI mice (Col1a2+/G610C). The proposed quinoline-oriented modification strategy could shape the molecular recognition of modified aptamers to proteins to facilitate enhancing its binding affinity and therapeutic potency.

Nucleic acid amphiphiles: Synthesis, properties, and applications.

Nucleic acid amphiphiles, referring to nucleic acids modified with large hydrophobic groups, have been widely used in programmable bioengineering. Since nucleic acids are intrinsically hydrophilic, the hydrophobic groups endow nucleic acid amphiphiles with unique properties, such as self-assembling, interactions with artificial or biological membranes, and transmembrane transport. Importantly, the hybridization or target binding capability of oligonucleotide itself supplies nucleic acid amphiphiles with excellent programmability. As a result, this type of molecule has attracted considerable attention in academic studies and has enormous potential for further applications. For a comprehensive understanding of nucleic acid amphiphiles, we review the reported research on nucleic acid amphiphiles from their molecular design to final applications, in which we summarize the synthetic strategies for nucleic acid amphiphiles and draw much attention to their unique properties in different contexts. Finally, a summary of the applications of nucleic acid amphiphiles in drug development, bioengineering, and bioanalysis are critically discussed.

Chemically modified aptamers for improving binding affinity to the target proteins via enhanced non-covalent bonding.

Nucleic acid aptamers are ssDNA or ssRNA fragments that specifically recognize targets. However, the pharmacodynamic properties of natural aptamers consisting of 4 naturally occurring nucleosides (A, G, C, T/U) are generally restricted for inferior binding affinity than the cognate antibodies. The development of high-affinity modification strategies has attracted extensive attention in aptamer applications. Chemically modified aptamers with stable three-dimensional shapes can tightly interact with the target proteins via enhanced non-covalent bonding, possibly resulting in hundreds of affinity enhancements. This review overviewed high-affinity modification strategies used in aptamers, including nucleobase modifications, fluorine modifications (2'-fluoro nucleic acid, 2'-fluoro arabino nucleic acid, 2',2'-difluoro nucleic acid), structural alteration modifications (locked nucleic acid, unlocked nucleic acid), phosphate modifications (phosphorothioates, phosphorodithioates), and extended alphabets. The review emphasized how these high-affinity modifications function in effect as the interactions with target proteins, thereby refining the pharmacodynamic properties of aptamers.

The role of sclerostin in lipid and glucose metabolism disorders.

Lipid and glucose metabolism are critical for human activities, and their disorders can cause diabetes and obesity, two prevalent metabolic diseases. Studies suggest that the bone involved in lipid and glucose metabolism is emerging as an endocrine organ that regulates systemic metabolism through bone-derived molecules. Sclerostin, a protein mainly produced by osteocytes, has been therapeutically targeted by antibodies for treating osteoporosis owing to its ability to inhibit bone formation. Moreover, recent evidence indicates that sclerostin plays a role in lipid and glucose metabolism disorders. Although the effects of sclerostin on bone have been extensively examined and reviewed, its effects on systemic metabolism have not yet been well summarized. In this paper, we provide a systemic review of the effects of sclerostin on lipid and glucose metabolism based on in vitro and in vivo evidence, summarize the research progress on sclerostin, and prospect its potential manipulation for obesity and diabetes treatment.

A bimolecular modification strategy for developing long-lasting bone anabolic aptamer.

The molecular weight of nucleic acid aptamers (20 kDa) is lower than the cutoff threshold of the renal filtration (30-50 kDa), resulting in a very short half-life, which dramatically limits their druggability. To address this, we utilized 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-hydroxy-2-oxo-2H-chromen-6-yl)propenamide (HC) and 12-((2,5-dioxopyrrolidin-1-yl)oxy)-12-oxododecanoic acid (DA), two newly designed coupling agents, for synergistic binding to human serum albumin (HSA). Both HC and DA are conjugated to a bone anabolic aptamer (Apc001) against sclerostin to form an Apc001OC conjugate with high binding affinity to HSA. Notably, HC and DA could synergistically facilitate prolonging the half-life of the conjugated Apc001 and promoting its bone anabolic potential. Using the designed blocking peptides, the mechanism studies indicate that the synergistic effect of HC-DA on pharmacokinetics and bone anabolic potential of the conjugated Apc001 is achieved via their synergistic binding to HSA. Moreover, biweekly Apc001OC at 50 mg/kg shows comparable bone anabolic potential to the marketed sclerostin antibody given weekly at 25 mg/kg. This proposed bimolecular modification strategy could help address the druggability challenge for aptamers with a short half-life.