Hydroxyapatite-based nano-drug delivery system for nicotinamide mononucleotide (NMN): significantly enhancing NMN bioavailability and replenishing in vivo nicotinamide adenine dinucleotide (NAD+) levels.

OBJECTIVES: This study addresses the bioavailability challenges associated with oral nicotinamide mononucleotide (NMN) administration by introducing an innovative NMN formulation incorporated with hydroxyapatite (NMN-HAP). METHODS: The NMN-HAP was developed using a wet chemical precipitation and physical adsorption method. To assess its superiority over conventional free NMN, we examined NMN, nicotinamide adenine dinucleotide (NAD+), and nicotinamide riboside (NR) levels in mouse plasma and tissues following oral administration of NMN-HAP. KEY FINDINGS: NMN-HAP nanoparticles demonstrated a rod-shaped morphology, with an average size of ~50 nm, along with encapsulation efficiency and drug loading capacity exceeding 40%. In vitro, drug release results indicated that NMN-HAP exhibited significantly lower release compared with free NMN. In vivo studies showed that NMN-HAP extended circulation time, improved bioavailability compared with free NMN, and elevated plasma levels of NMN, NAD+, and NR. Moreover, NMN-HAP administration displayed tissue-specific distribution with a substantial accumulation of NMN, NAD+, and NR in the brain and liver. CONCLUSION: NMN-HAP represents an ideal formulation for enhancing NMN bioavailability, enabling tissue-specific delivery, and ultimately elevating in vivo NAD+ levels. Considering HAP's biocompatible nature and versatile characteristics, we anticipate that this system has significant potential for various future applications.

Bio-Inspired Multiscale Design for Strong and Tough Biological Ionogels.

Structure design provides an effective solution to develop advanced soft materials with desirable mechanical properties. However, creating multiscale structures in ionogels to obtain strong mechanical properties is challenging. Here, an in situ integration strategy for producing a multiscale-structured ionogel (M-gel) via ionothermal-stimulated silk fiber splitting and moderate molecularization in the cellulose-ions matrix is reported. The produced M-gel shows a multiscale structural superiority comprised of microfibers, nanofibrils, and supramolecular networks. When this strategy is used to construct a hexactinellid inspired M-gel, the resultant biomimetic M-gel shows excellent mechanical properties including elastic modulus of 31.5 MPa, fracture strength of 6.52 MPa, toughness reaching 1540 kJ m-3 , and instantaneous impact resistance of 3.07 kJ m-1 , which are comparable to those of most previously reported polymeric gels and even hardwood. This strategy is generalizable to other biopolymers, offering a promising in situ design method for biological ionogels that can be expanded to more demanding load-bearing materials requiring greater impact resistance.

Prediction of Compressive Strength of Fly Ash-Slag Based Geopolymer Paste Based on Multi-Optimized Artificial Neural Network.

The fly ash-slag geopolymer is regarded as one of the new green cementitious materials that can replace cement, but it is difficult to predict its mechanical properties by conventional methods. Therefore, in the present study, the back propagation (BP) artificial neural network technique is used to predict the compressive strength of the fly ash-slag geopolymer. In this paper, data from the published literature were collected as the training set and the experimental results from laboratory experiments were used as the test set. Eight input parameters were determined, as follows: the percentage of fly ash, the percentage of slag, the water-cement ratio, the curing age, the modulus of alkali activator, the mass ratio of NaOH to Na2SiO3 and the moles of Na2O and SiO2 in the alkali activator. Three multilayer artificial neural network models were constructed using the Levenberg-Marquardt (LM), Bayesian regularization (BR) and scaled conjugate gradient (SCG) algorithms to compare the prediction accuracy of the compressive strength of the fly ash-slag geopolymer paste at different ages (3, 7, and 28 d). It was concluded that the training set error of the BR-BP neural network was the smallest. Ultimately, the hyperparameter optimization of the BR-BP neural network was carried out to compare the training set and the test set errors before and after the optimization, and the results show that the BR-BP neural network model with hyperparameter optimization had the highest prediction accuracy.

The role of post-transcriptional modification on a new tRNAIle(GAU) identified from Ganoderma lucidum in its fragments' cytotoxicity on cancer cells.

Ganoderma lucidum (Ganoderma) is a famous Chinese herbal medicine which has been used clinically for thousands of years in China. Despite numerous studies on triterpenes and polysaccharides, the bioactivity of RNAs abundant in Ganoderma remains unknown. Here, based on LC-MS techniques, dihydrouracil, 5-methyluridine (m5U) and pseudouridine were identified at position 19, 52 and 53 of a new tRNAIle(GAU) which was isolated as the most abundant tRNA species in Ganoderma, and is the first purified tRNA from fungus. Cytotoxic screening of tRNA-half (t-half) and tRNA fragment (tRF) derived from this tRNA, as well as their mimics (t-half or tRF as antisense strand), demonstrated that the double-stranded form, i.e., tRF and t-halve mimics, exhibited stronger cytotoxicity than their single-stranded form, and the cytotoxicity of t-half mimic is significantly stronger than that of tRF mimic. Notably, the cytotoxicity of 3'-t-half mimic is not only much more potent than that of taxol, but also is much more potent than that of ganoderic acids, the major bioactive components in Ganoderma. Furthermore, 3'-t-half mimic_M2 (m5U modified) exhibited significantly stronger cytotoxicity than unmodified 3'-t-half mimic, which is consistent with the computational simulation showing that m5U modification enhances the stability of the tertiary structure of 3'-t-half mimic. Overall, the present study not only indicates t-halves are bioactive components in Ganoderma which should not be neglected, but also reveals an important role of post-transcriptional modification on tRNA in its fragments' cytotoxicity against cancer cells, which benefits the design and development of RNAi drugs from natural resource.

A tRNA-derived fragment of ginseng protects heart against ischemia/reperfusion injury via targeting the lncRNA MIAT/VEGFA pathway.

Traditional Chinese medicines (TCMs) have been widely used for treating ischemic heart disease (IHD), and secondary metabolites are generally regarded as their pharmacologically active components. However, the effects of nucleic acids in TCMs remain unclear. We reported for the first time that a 22-mer double-strand RNA consisting of HC83 (a tRNA-derived fragment [tRF] from the 3' end of tRNAGln(UUG) of ginseng) and its complementary sequence significantly promoted H9c2 cell survival after hypoxia/reoxygenation (H/R) in vitro. HC83_mimic could also significantly improve cardiac function by maintaining both cytoskeleton integrity and mitochondrial function of cardiomyocytes. Further in vivo investigations revealed that HC83_mimic is more potent than metoprolol by >500-fold against myocardial ischemia/reperfusion (MI/R) injury. In-depth studies revealed that HC83 directly downregulated a lncRNA known as myocardial infarction-associated transcript (MIAT) that led to a subsequent upregulation of VEGFA expression. These findings provided the first evidence that TCM-derived tRFs can exert miRNA-like functions in mammalian systems, therefore supporting the idea that TCM-derived tRFs are promising RNA drug candidates shown to have extraordinarily potent effects. In summary, this study provides a novel strategy not only for discovering pharmacologically active tRFs from TCMs but also for efficiently exploring new therapeutic targets for various diseases.

A Scalable Bacterial Cellulose Ionogel for Multisensory Electronic Skin.

Electronic skin (e-skin), a new generation of flexible electronics, has drawn interest in soft robotics, artificial intelligence, and biomedical devices. However, most existing e-skins involve complex preparation procedures and are characterized by single-sensing capability and insufficient scalability. Here, we report on a one-step strategy in which a thermionic source is used for the in situ molecularization of bacterial cellulose polymeric fibers into molecular chains, controllably constructing an ionogel with a scalable mode for e-skin. The synergistic effect of a molecular-scale hydrogen bond interweaving network and a nanoscale fiber skeleton confers a robust tensile strength (up to 7.8 MPa) and high ionic conductivity (up to 62.58 mS/cm) on the as-developed ionogel. Inspired by the tongue to engineer the perceptual patterns in this ionogel, we present a smart e-skin with the perfect combination of excellent ion transport and discriminability, showing six stimulating responses to pressure, touch, temperature, humidity, magnetic force, and even astringency. This study proposes a simple, efficient, controllable, and sustainable approach toward a low-carbon, versatile, and scalable e-skin design and structure-performance development.

Antitumor Activities of tRNA-Derived Fragments and tRNA Halves from Non-pathogenic Escherichia coli Strains on Colorectal Cancer and Their Structure-Activity Relationship.

tRNAs purified from non-pathogenic Escherichia coli strains (NPECSs) possess cytotoxic properties on colorectal cancer cells. In the present study, the bioactivity of tRNA halves and tRNA fragments (tRFs) derived from NPECSs are investigated for their anticancer potential. Both the tRNA halves and tRF mimics studied exhibited significant cytotoxicity on colorectal cancer cells, with the latter being more effective, suggesting that tRFs may be important contributors to the bioactivities of tRNAs derived from the gut microbiota. Through high-throughput screening, the EC83 mimic, a double-strand RNA with a 22-nucleotide (nt) 5'-tRF derived from tRNA-Leu(CAA) as an antisense chain, was identified as the one with the highest potency (50% inhibitory concentration [IC50] = 52 nM). Structure-activity investigations revealed that 2'-O-methylation of the ribose of guanosine (Gm) may enhance the cytotoxic effects of the EC83 mimic via increasing the stability of its tertiary structure, which is consistent with the results of in vivo investigations showing that the EC83-M2 mimic (Gm modified) exhibited stronger antitumor activity against both HCT-8 and LoVo xenografts. Consistently, 4-thiouridine modification does not. This provides the first evidence that the bioactivity of tRF mimics would be impacted by chemical modifications. Furthermore, the present study provides the first evidence to suggest that novel tRNA fragments derived from the gut microbiota may possess anticancer properties and have the potential to be potent and selective therapeutic molecules. IMPORTANCE While the gut microbiota has been increasingly recognized to be of vital importance for human health and disease, the current literature shows that there is a lack of attention given to non-pathogenic Escherichia coli strains. Moreover, the biological activities of tRNA fragments (tRFs) derived from bacteria have rarely been investigated. The findings from this study revealed tRFs as a new class of bioactive constituents derived from gut microorganisms, suggesting that studies on biological functional molecules in the intestinal microbiota should not neglect tRFs. Research on tRFs would play an important role in the biological research of gut microorganisms, including bacterium-bacterium interactions, the gut-brain axis, and the gut-liver axis, etc. Furthermore, the guidance on the rational design of tRF therapeutics provided in this study indicates that further investigations should pay more attention to these therapeutics from probiotics. The innovative drug research of tRFs as potent druggable RNA molecules derived from intestinal microorganisms would open a new area in biomedical sciences.

A tRNA-derived fragment from Chinese yew suppresses ovarian cancer growth via targeting TRPA1.

Drug discovery from plants usually focuses on small molecules rather than such biological macromolecules as RNAs. Although plant transfer RNA (tRNA)-derived fragment (tRF) has been associated with the developmental and defense mechanisms in plants, its regulatory role in mammals remains unclear. By employing a novel reverse small interfering RNA (siRNA) screening strategy, we show that a tRF mimic (antisense derived from the 5' end of tRNAHis(GUG) of Chinese yew) exhibits comparable anti-cancer activity with that of taxol on ovarian cancer A2780 cells, with a 16-fold lower dosage than that of taxol. A dual-luciferase reporter assay revealed that tRF-T11 directly targets the 3' UTR of oncogene TRPA1 mRNA. Furthermore, an Argonaute-RNA immunoprecipitation (AGO-RIP) assay demonstrated that tRF-T11 can interact with AGO2 to suppress TRPA1 via an RNAi pathway. This study uncovers a new role of plant-derived tRFs in regulating endogenous genes. This holds great promise for exploiting novel RNA drugs derived from nature and sheds light on the discovery of unknown molecular targets of therapeutics.

Developing cellulosic functional materials from multi-scale strategy and applications in flexible bioelectronic devices.

With the forthcoming of the post-COVID-19 and the ageing era, the novel biomaterials and bioelectronic devices are attracting more and more attention and favor. Cellulose as one of the most globe-abundant natural macromolecules has multiple merits of biocompatibility, processability, carbon neutral feature and mechanical designability. Due to its progressive advancement of multi-scale design from macro to micro followed by new cognitions, cellulose shows a promising application prospect in developing bio-functional materials. In this review, we briefly discuss the role of cellulose from the "top-down" perspective of macro-scale fibers, micro-scale nanofibers, and molecular-scale macromolecular chains for the design of advanced cellulose-based functional materials. The focus then turns to the construction and development of emerging cellulose-based flexible bioelectronic devices including biosensors, biomimetic electronic skins, and biological detection devices. Finally, the dilemma and challenge of cellulose-based bioelectronic materials and their application prospects in basic biology and medical care have been prospected.

A Stiffness-Switchable, Biomimetic Smart Material Enabled by Supramolecular Reconfiguration.

In nature, stiffness-changing behavior is essential for living organisms, which, however, is challenging to achieve in synthetic materials. Here, a stiffness-changing smart material, through developing interchangeable supramolecular configurations inspired from the dermis of the sea cucumber, which shows extreme, switchable mechanical properties, is reported. In the hydrated state, the material, possessing a stretched, double-stranded supramolecular network, showcases a soft-gel behavior with a low stiffness and high pliability. Upon the stimulation of ethanol to transform into the coiled supramolecular configuration, it self-adjusts to a hard state with nearly 500-times enhanced stiffness from 0.51 to 243.6 MPa, outstanding load-bearing capability (over 35 000 times its own weight), and excellent puncture/impact resistance with a specific impact strength of ≈116 kJ m-2 (g cm-3 )-1 (higher than some metals and alloys such as aluminum, and even comparable to the commercially available protective materials such as D3O and Kevlar). Moreover, this material demonstrates reconfiguration-dependent self-healing behavior and designable formability, holding great promise in advanced engineering fields that require both high-strength durability and good formability. This work may open up a new perspective for the development of self-regulating materials from supramolecular-scale configuration regulation.

Alpha-enolase (ENO1), identified as an antigen to monoclonal antibody 12C7, promotes the self-renewal and malignant phenotype of lung cancer stem cells by AMPK/mTOR pathway.

BACKGROUND: Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs. METHODS: Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen. RESULTS: Targeted antigen showed a surface antigen expression pattern, and the 43-55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs. CONCLUSIONS: ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.

Full-Range Profiling of tRNA Modifications Using LC-MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy.

Transfer RNAs (tRNAs) are the most heavily modified RNA species. Liquid chromatography coupled with mass spectrometry (LC-MS/MS) is a powerful tool for characterizing tRNA modifications, which involves pretreating tRNAs with base-specific ribonucleases to produce smaller oligonucleotides amenable to MS. However, the quality and quantity of products from base-specific digestions are severely impacted by the base composition of tRNAs. This often leads to a loss of sequence information. Here, we report a method for the full-range profiling of tRNA modifications at single-base resolution by combining site-specific RNase H digestion with the LC-MS/MS and RNA-seq techniques. The key steps were designed to generate high-quality products of optimal lengths and ionization properties. A linear correlation between collision energies and the m/z of oligonucleotides significantly improved the information content of collision-induced dissociation (CID) spectra. False positives were eliminated by up to 95% using novel inclusion criteria for collecting a census of modifications. This method is illustrated by the mapping of mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC), which were hitherto not investigated. The identities and locations of the five species of modifications on these tRNAs were fully characterized. This approach is universally applicable to any tRNA species and provides an experimentally realizable pathway to the de novo sequencing of post-transcriptionally modified tRNAs with high sequence coverage.

Purification, characterization and cytotoxic activities of individual tRNAs from Escherichia coli.

Transfer RNAs (tRNAs) are the most abundant class in small non-coding RNAs which have been proved to be pharmacologically active. In the present study, we evaluated the potential anticancer activities of tRNAs from Escherichia coli MRE 600 to investigate the relationship between non-pathogenic Escherichia coli strain and colorectal cancer. To purify individual tRNAs, we firstly developed a two-dimensional liquid chromatography (2D-LC) and successfully obtained two pure tRNAs. Nuclease mediated base-specific digestions coupled with UHPLC-MS/MS techniques led to an identification of these two tRNAs as tRNA-Val(UAC) and tRNA-Leu(CAG) with typical cloverleaf-like secondary structure. MTT assay demonstrated that both tRNA-1 and tRNA-2 exhibit strong cytotoxicity with IC50 of 113.0 nM and 124.8 nM on HCT-8 cells in a dose-dependent manner. Further clonogenic assay revealed that the purified tRNAs exhibit significant inhibition in colony formation with survival percentage of 79.0 ±â€¯1.6 and 71.2 ±â€¯2.2 at the concentration of 100 nM. These findings provided evidences of anticancer activities of tRNAs from non-pathogenic Escherichia coli strain, indicating that the pharmacological effects of these neglected biomacromolecules from microorganisms should be emphasized. This study put new insights into the therapeutic effects of intestinal microorganism on human diseases, therefore broadened our knowledge of the biological functions of gut microbiota.

MCOLN1 Promotes Proliferation and Predicts Poor Survival of Patients with Pancreatic Ductal Adenocarcinoma.

BACKGROUND: MCOLN1 (mucolipin subfamily, member 1) was first identified as an autophagic regulator, which was essential for efficient fusion of both autophagosomes and late endosomes with lysosomes. This study is aimed at investigating the role of MCOLN1 in the development of pancreatic ductal adenocarcinoma (PDAC). METHODS: Immunohistochemistry (IHC) assay was conducted to evaluate the expression level of MCOLN1 in 82 human PDAC tumor tissues. Overall survival (OS) and recurrence-free survival (RFS) analysis was performed to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. The expression level of related genes was measured by RT-PCR (reverse transcription polymerase chain reaction) and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis in vivo. RESULTS: The results of IHC showed that a high level of MCOLN1 expression was associated with the poor clinical characteristics of PDAC patients. OS and RFS were significantly worse in patients with high MCOLN1 expression. Silencing of MCOLN1 dramatically blocked the proliferation of PDAC cells. Mechanism studies confirmed that knockdown of MCOLN1 decreased the expression of Ki67 and PCNA (proliferating cell nuclear antigen), two markers of cell proliferation. In vivo, MCOILN1 depletion reduced the formation and growth of tumors in mice. CONCLUSION: The high level of MCOLN1 expression was associated with poor clinical outcomes of PDAC patients. MCOLN1 ablation could inhibit PDAC proliferation of both in vitro and in vivo, which provide a new insight and novel therapeutic target for the treatment of PDAC.

Therapeutic approaches targeting cancer stem cells.

Increasing studies have demonstrated that most tumors consisted a subpopulation of cells with stem cell properties, known as cancer stem cells (CSCs). Accumulating evidence indicated that CSCs may be critical driving force for several types of cancer. Hence, it was necessary to develop therapeutic approaches specifically targeting CSCs. In this review, first, the biological properties of CSCs were introduced, including the self-renewal and differentiation, high tumorigenesis and invasiveness, resistance to chemotherapy and radiotherapy, genetic and epigenetic variations. Meanwhile, CSCs-targeted therapeutic strategies were summarized, including targeting cell surface markers, signaling pathways, CSC niches, differentiation therapy, and drug resistance for CSCs. Furthermore, clinical trials on anti-CSCs therapies supported the efficacy of these therapies, as well as their combination with conventional chemotherapy and radiotherapy. CSCs could be significantly eradicated, eventually resulting in inhibited tumor growth, metastasis, and recurrence. Thus, selectively targeting CSCs with various agents may be a novel and promising therapeutic strategy against cancer.

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library.

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both in vitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7 and 9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.

Quantitative analysis of acankoreoside A and acankoreagenin in the leaves of Schefflera octophylla and Schefflera actinophylla using pressurized liquid extraction and high-performance liquid chromatography coupled with evaporative light scattering detection.

A rapid method based on pressurized liquid extraction followed by high-performance liquid chromatography coupled with evaporative light scattering detection was firstly developed for the quantitative analysis of two bioactive triterpenoids (acankoreoside A and acankoreagenin) in the leaves of Schefflera octophylla and Schefflera actinophylla. The analysis was performed on an Agilent Zorbax SB-Aq column (4.6 × 50 mm, 3.5 µm) with gradient elution of 0.1% formic acid and acetonitrile. Calibration curves of two analytes showed good linearity (R(2) > 0.9990) within the tested ranges. This novel method is simple, rapid and accurate, and the results of quantification showed that contents of each investigated compound is significant high in natural S. octophylla (6.36-14.83%), which indicated that natural S. octophylla as potential medicinal resource. Furthermore, hierarchical clustering analysis based on the typical peaks of acankoreoside A and acankoreagenin from the 17 tested samples showed that natural and cultured Schefflera species were in different clusters, which could provide a means of discriminating between Schefflera species from different origins. Thus, acankoreoside A and acankoreagnin could be selected markers for quality control of S. octophylla and S. actinophylla.