scDART-seq: Mapping m6A at the single-cell level.

In this issue of Molecular Cell, Tegowski et al. established a single-cell DART-seq (scDART-seq) method for m6A site profiling and further revealed the fundamental features of m6A biology in single cells. The findings improve our understating of the intrinsic regulatory and functional mechanisms of m6A in a single-cell basis.

RNA 5-Methylcytosine Facilitates the Maternal-to-Zygotic Transition by Preventing Maternal mRNA Decay.

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.

High-Throughput Metabolite Analysis of Unicellular Microalgae by Orthogonal Hybrid Ionization Label-Free Mass Cytometry.

Microalgae metabolite analysis is fundamental for the rational design of metabolic engineering strategies for the biosynthesis of high-value products. Mass spectrometry (MS) has been utilized for single-cell microalgae analysis. However, limitations in the detection throughput and polarities of detectable substances make it difficult to realize high-throughput screening of high-performance microalgae. Herein, a plasma-assisted label-free mass cytometry, named as PACyESI-MS, was proposed combining the advantages of orthogonal hybrid ionization and high-throughput MS analysis, which realized rapid metabolite detection of single microalgae. The cell detection throughput of PACyESI-MS was up to 52 cells/min. Dozens of the critical primary and secondary metabolites within single microalgae were detected simultaneously, including pigments, lipids, and energy metabolites. Furthermore, metabolite changes of Chlamydomonas reinhardtii and Haematococcus pluvialis under nitrogen deficiency stress were studied. Discrimination of Chlamydomonas under different nutrient conditions was realized using single-cell metabolite profiles obtained by PACyESI-MS. The relationships between the accumulation of bioactive astaxanthin and changes in functional primary metabolites of Haematococcus were investigated. It was demonstrated that PACyESI-MS can detect the flexible change of metabolites in single microalgae cells under different nutritional conditions and during the synthesis of high-value products, which is expected to become an important tool for the design of metabolic engineering-based high-performance microalgae factories.

Single-Cell Unsaturated Lipid Profiling for Studying Chemoresistance Heterogeneity of Triple-Negative Breast Cancer Cells.

Chemoresistance to triple-negative breast cancer (TNBC) is a critical issue in clinical practice. Lipid metabolism takes a unique role in breast cancer cells; especially, unsaturated lipids involving cell membrane fluidity and peroxidation are highly remarked. At present, for the lack of a high-resolution molecular recognition platform at the single-cell level, it is still hard to systematically study chemoresistance heterogeneity based on lipid unsaturation proportion. By designing a single-cell mass spectrometry workflow based on CyESI-MS, we profiled the unsaturated lipids of TNBC cells to evaluate lipidomic remodeling under platinum stress. Profiling revealed the heterogeneity of the polyunsaturated lipid proportion of TNBC cells under cisplatin treatment. A cluster of cells identified by polyunsaturated lipid accumulation was found to be involved in platinum sensitivity. Furthermore, we found that the chemoresistance of TNBC cells could be regulated by fatty acid supplementation, which determinates the composition of unsaturated lipids. These discoveries provide insights for monitoring and controlling cellular unsaturated lipid proportions to overcome chemoresistance in breast cancer.

Castration reshapes the liver by altering fatty acid composition and metabolism in male mice.

Castration promotes subcutaneous fat deposition that may be associated with metabolic adaptations in the liver. However, fatty acid composition, abundance, and metabolic characteristics of the liver after castration are not fully understood. Our results showed that surgical castration significantly reduced water and food intake, reduced liver weight, and induced liver inflammation in mice. Transcriptome analyses revealed that castration enhanced fatty acid metabolism, particularly that of arachidonic and linoleic acids metabolism. Gas chromatography-mass spectrometry analysis revealed that castration altered the composition and relative abundance of fatty acids in the liver. The relative abundances of arachidonic and linoleic acids were significantly decreased in 4-week-old castrated mice. Analysis of fatty acid synthesis- and metabolism-related genes revealed that castration enhanced the transcription of fatty acid synthesis- and oxidation-related genes. Analyzing the level of key enzymes in the ß-oxidation and tricarboxylic acid cycle pathways of fatty acids in mitochondria, we found that castration enhanced the ß-oxidation of fatty acids in mitochondria, and also enhanced the protein level of the rate-limiting enzyme in the tricarboxylic acid cycle pathway, isocitrate dehydrogenase 2. These results comprehensively clarify metabolic changes in liver fatty acids after castration in mice of different ages and provide a reference for understanding castration-induced fat deposition from the perspective of liver fatty acid metabolism in male mice.

Testosterone regulates thymic remodeling by altering metabolic reprogramming in male rats.

The thymus is an energy-consuming organ, and its metabolism changes with atrophy. Testosterone regulates thymus remodeling (atrophy and regeneration). However, the characteristics of the energy metabolism during testosterone-mediated thymic atrophy and regeneration remain unclear. In this study, we demonstrated that testosterone ablation (implemented by immunocastration and surgical castration) induced global metabolic changes in the thymus. Kyoto Encyclopedia of Genes and Genomes pathway enrichment for differential metabolites and metabolite set enrichment analysis for total metabolites revealed that testosterone ablation affected thymic glycolysis, glutamate metabolism, and fatty acid ß-oxidation. Testosterone ablation-induced thymic regeneration was accompanied by attenuated glycolysis and glutamate metabolism and changed fatty acid composition and content. Testosterone supplementation in immunocastrated and surgically castrated rats enhanced glutaminolysis, reduced the level of unsaturated fatty acids, enhanced the ß-oxidation of unsaturated fatty acids in the mitochondria, boosted the tricarboxylic acid (TCA) cycle, and accelerated thymic atrophy. Overall, these results imply that metabolic reprogramming is directly related to thymic remodeling.

Viola yedoensis Makino alleviates lipopolysaccharide-induced intestinal oxidative stress and inflammatory response by regulating the gut microbiota and NF-κB-NLRP3/ Nrf2-MAPK signaling pathway in broiler.

Viola yedoensis Makino (Vy) is a well-known traditional Chinese medicine widely used to treat inflammatory diseases. However, the regulatory effects of dietary Vy supplementation on lipopolysaccharide (LPS)-induced intestinal damage in broilers and the underlying molecular mechanisms remain unclear. In this study, broilers were intraperitoneally injected with 1 mg/kg LPS on days 17, 19 and 21 to induce intestinal damage. Vy supplementation at 0.5, 1.5 and 4.5 % in the diet was administered separately for 21 days to investigate the potential protective effects of Vy supplementation against LPS-induced intestinal impairment in broilers. Vy supplementation improved intestinal morphology and restored growth performance. Vy supplementation attenuated intestinal inflammation by regulating the nuclear factor kappa B (NF-κB) / NLR family pyrin domain-containing 3 (NLRP3) signaling pathway and inhibited its downstream pro-inflammatory factor levels. In addition, Vy supplementation relieved intestinal oxidative impairment by regulating the nuclear factor erythroid-2 related factor 2 (Nrf2) / mitogen-activated protein kinase (MAPK) signaling pathway and downstream antioxidant enzyme activity. Vy supplementation reduced LPS-induced mitochondrial damage and apoptosis. Furthermore, Vy supplementation alleviated LPS-induced intestinal inflammation and oxidative damage in chickens by increasing the abundance of protective bacteria (Lactobacillus and Romboutsia) and reducing the number of pathogenic bacteria (unclassified_f_Ruminococcaceae, unclassified_f_Oscillospiraceae and norank_f_norank_o_Clostridia_vadinBB60_group). Overall, Vy supplementation effectively ameliorated LPS-induced intestinal damage by regulating the NF-κB-NLRP3/Nrf2-MAPK signaling pathway and maintaining intestinal microbiota balance. Vy supplementation can be used as a dietary supplement to protect broilers against intestinal inflammation and oxidative damage.

Epicoccum sorghinum Causing Leaf Spot on Polygonatum cyrtonema in China.

Polygonatum cyrtonema Hua (family Asparagaceae) is a traditional Chinese medicinal plant that is widely cultivated in various parts of China, including Hunan Province. In summer 2022, a leaf spot disease was observed in 10% of the P. cyrtonema plants (Huang jing) in 18 hectares of this crop in the Hongjiang District (27°18'4″N, 110°11'1″E) of Hunan Province. The initial symptoms of the disease were brown spots on young leaves, and adjacent tissues gradually changed from green to yellow. The entire leaf then became yellow, withered, and eventually exhibited a thn and black appearance. In total, 12 diseased plants from four sampling sites (three plants per site) were collected for laboratory analysis to address the concerns of P. cyrtonema growers. Symptomatic leaf samples were selected, and the leaf fragments containing infected parts of the plants were disinfected with 75% ethanol for 1 min, then immersed in 2.5% hypochlorite for 45 s. After disinfection, symptomatic leaf samples were rinsed three times with sterile water, placed on potato saccharose agar containing 50 µg/ml kanamycin and incubated at 25°C for 2 days. Subsequently, 12 fungal isolates were isolated from various leaf samples through hyphal tip transferring. Ten of the 12 fungal isolates had similar morphological features, and one of them (isolate hjh) was used as the representative isolate for the study. With a growth rate of 6.3 mm per day, its white colonies transformed into red concentric rings in five days; they gradually became black after 10 days of growth. The chlamydospores were round (4.0-9.9 × 3.1-9.3 µm, n = 30), whereas the conidia were ovate (8.0-12.1 × 3.2-6.5 µm, n = 30). The morphological features of the isolate hjh were similar to the features of Epicoccum spp. (Aveskamp et al. 2010). The internal transcribed spacer (ITS) region (including the partial ITS1 sequence and the 5.8S and ITS2 complete sequences), ß-tubulin (tub) gene, and large subunit (LSU) rRNA gene, were amplified from the isolate hjh using the primer pairs ITS5/ITS4, Bt2a/Bt2b, and LROR/LR5, respectively (Taguiam et al. 2021). BLASTn analysis showed that the ITS (OR253745), tub (OR253764), and LSU (OR253746) sequences generated from the isolate hjh were 98-99% similar to the sequences of E. sorghinum strains CBS 179.80 and CBS 627.68. Subsequently, the ITS, tub, and LSU sequences were combined using Sequence Matrix software; phylogenetic analysis via Bayesian and maximum likelihood methods (Vaidya et al. 2011; Li et al. 2021) classified the isolate hjh into the E. sorghinum clade. To fulfill Koch's postulates, pathogenicity tests were conducted on healthy (lesion-free and disease-free) 2-year-old P. cyrtonema plants. Three healthy plants were inoculated by spraying whole plant until run-off with a spore suspension of the isolate hjh (1 × 106 conidia/ml); Three other healthy plants were sprayed with sterile water as controls. The inoculated plants were incubated in a growth chamber at 25 ± 2°C with 85% humidity for 28 days(Chen et al. 2021). Leaves from the inoculated plants gradually became brown within 15 days. Finally, the plants died 28 days after inoculation. The control plants showed no symptoms throughout the experimental period. Isolates (isolate hjh1, hjh2 and hjh3) that were reisolated from the inoculated plants exhibited morphologically similar characteristics and molecularly identical to the original isolate hjh. To our knowledge, this is the first report of E. sorghinum causing leaf spot disease on P. cyrtonema. The results of this study may facilitate the production of P. cyrtonema in China.

Covalent Organic Framework Nanosheets for Fluorescence Quantification of Peptide.

Rapid and sensitive quantification of peptides plays an important role in clinical diagnosis. Fluorescence assay is one of the most promising peptide detection tools, but it relies on intrinsic fluorescence or additional derivatization, resulting in poor versatility. Covalent organic frameworks (COFs) have shown a good application prospect in the field of fluorescence detection, but their application scope is limited to heavy metal ions and some small polar organic molecules. Herein, we report the application of COFs nanosheet for fluorescence detection of peptides. Fluorescent sp2 acrylonitrile-linked COFs nanosheets (TTAN-CON) were prepared by water-assisted ultrasonic exfoliation which performed with excellent fluorescence properties with Stokes shifts of 146 nm and fluorescence quantum yield of up to 24.45%. Compared to the bulk fluorescent COFs, exfoliated CONs films performed with better stability of fluorescence signal in solution. We found the fluorescence of TTAN-CON can be effectively quenched by hydrophobic peptides at a very rapid rate (less than 5 min per sample). TTAN-CON presented good sensitivity and selectivity for hydrophobic peptides detection via the static and dynamic joint quenching mechanism. TTAN-CON was further used to detect NLLGLIEAK and ProGRP31-98, two target peptide fragments of lung cancer biomarker ProGRP. The fluorescence intensities of TTAN-CON were negative linearly correlated with the amounts of hydrophobic NLLGLIEAK over the range of 5-1000 ng/mL with the correlation coefficients over 0.99, and the limit of detection was 1.67 ng/mL, displaying higher sensitivity and convenience than traditional optical methods. What's more, the quantification of ProGRP31-98 was achieved by the quantification of hydrophobic peptides in its enzyme hydrolysis products. We anticipate COFs nanosheets to be a universal fluorescence detection work-box for peptides biomarkers with clinical significance.

Fabrication of a Cation Exchange Membrane with Largely Reduced Anion Permeability for Advanced Aqueous K-ion Battery in an Alkaline-Neutral Electrolyte Decoupling System.

Herein, an efficient method to prepare sulfonated polyether ether ketone (SPEEK) based cation exchange membranes (CEMs) is developed, where polyethersulfone (PES) is used as an additive. The optimized membrane of 30 wt.%PES/SPEEK-M exhibits a rather low anion permeability and a high ionic conductivity of 9.52 mS cm-1 together with low volume swelling in water. Meanwhile, tensile strength of the membrane is as high as 31.4 MPa with a tensile strain of 162%. As separators for aqueous K-ion batteries (AKIBs) with decoupled gel electrolytes (Zn anode in alkaline and Prussian blue (FeHCF) cathode in neutral). Discharge voltage of the AKIB can reach 2.3 V. Meanwhile, Zn dendrites can be effectively suppressed in the gel anolyte. Specific capacities of the FeHCF cathode are 116.7 mAh g-1 at 0.3 A g-1 (close to its theoretical value), and 95.0 mAh g-1 at 1.0 A g-1 , indicating good rate performance. Capacity retention of the cathode is as high as 91.2% after 1000 cycles' cycling owing to the well remained neutral environment of the catholyte. There is almost no pH change for the catholyte after cycling, indicating good anion-blocking or cation-selecting ability of the 30 wt.%PES/SPEEK-M, much better than other membranes.

Predicting post-operative vault and optimal implantable collamer lens size using machine learning based on various ophthalmic device combinations.

BACKGROUND: Implantable Collamer Lens (ICL) surgery has been proven to be a safe, effective, and predictable method for correcting myopia and myopic astigmatism. However, predicting the vault and ideal ICL size remains technically challenging. Despite the growing use of artificial intelligence (AI) in ophthalmology, no AI studies have provided available choices of different instruments and combinations for further vault and size predictions. This study aimed to fill this gap and predict post-operative vault and appropriate ICL size utilizing the comparison of numerous AI algorithms, stacking ensemble learning, and data from various ophthalmic devices and combinations. RESULTS: This retrospective and cross-sectional study included 1941 eyes of 1941 patients from Zhongshan Ophthalmic Center. For both vault prediction and ICL size selection, the combination containing Pentacam, Sirius, and UBM demonstrated the best results in test sets [R2 = 0.499 (95% CI 0.470-0.528), mean absolute error = 130.655 (95% CI 128.949-132.111), accuracy = 0.895 (95% CI 0.883-0.907), AUC = 0.928 (95% CI 0.916-0.941)]. Sulcus-to-sulcus (STS), a parameter from UBM, ranked among the top five significant contributors to both post-operative vault and optimal ICL size prediction, consistently outperforming white-to-white (WTW). Moreover, dual-device combinations or single-device parameters could also effectively predict vault and ideal ICL size, and excellent ICL selection prediction was achievable using only UBM parameters. CONCLUSIONS: Strategies based on multiple machine learning algorithms for different ophthalmic devices and combinations are applicable for vault predicting and ICL sizing, potentially improving the safety of the ICL implantation. Moreover, our findings emphasize the crucial role of UBM in the perioperative period of ICL surgery, as it provides key STS measurements that outperformed WTW measurements in predicting post-operative vault and optimal ICL size, highlighting its potential to enhance ICL implantation safety and accuracy.

3-Bromopyruvate-loaded bismuth sulfide nanospheres improve cancer treatment by synergizing radiotherapy with modulation of tumor metabolism.

BACKGROUND: Radiotherapy (RT) is one of the most mainstream cancer therapeutic modalities. However, due to the lack of specificity of the radiation adopted, both normal and cancerous cells are destroyed indiscriminately. This highlights the crucial need to improve radiosensitization. This study aims to address this issue by constructing a multifunctional nanospheres that can sensitize multiple aspects of radiotherapy. RESULTS: Nanospheres containing high atomic element Bi can effectively absorb ionizing radiation and can be used as radiosensitizers. Cell viability after Bi2S3 + X-ray treatment was half that of X-ray treatment alone. On the other hand, exposed 3-bromopyruvate (3BP) could reduce the overactive oxygen (O2) metabolism of tumor cells and alleviate tumor hypoxia, thereby promoting radiation-induced DNA damage. The combination index (CI) of 3BP and Bi2S3-based RT in Bi2S3-3BP + X-ray was determined to be 0.46 with the fraction affected (fa) was 0.5 via Chou-Talalay's isobolographic method, which indicated synergistic effect of 3BP and Bi2S3-based RT after integration into Bi2S3-3BP + X-ray. Under the combined effect of 3BP and RT, autophagy was over-activated through starvation-induced and redox homeostasis dysregulation pathways, which in turn exhibited pro-death effects. In addition, the prepared nanospheres possess strong X-ray attenuation and high near-infrared (NIR) optical absorption, thus eliminating the need for additional functional components and could serve as bimodal contrast agents for computed tomography/photoacoustic (CT/PA) imaging. CONCLUSIONS: The rational design of multifunctional nanospheres with the unique properties provided a novel strategy to achieving high therapeutic efficacy in RT. This was accomplished through simultaneous activation of multiple sensitization pathways by increasing ionizing radiation, reducing tumor oxygen consumption, inducing pro-death autophagy, and providing multiple-imaging guidance/monitoring.

Mechanism of action of quercetin in rheumatoid arthritis models: meta-analysis and systematic review of animal studies.

Quercetin, a typical flavonoid derived from a common natural plant, has multiple biological activities. Previous research in animal models has demonstrated the effectiveness of quercetin in treating rheumatoid arthritis (RA). The pharmacological effects and probable mechanisms of quercetin were evaluated in this study. Three databases, PubMed, Web of Science, and Embase, were searched for relevant studies from the creation of the databases to November 2022. Methodological quality was assessed using the SYRCLE risk of bias tool. STATA 15.1 was used to perform the statistical analysis. This research included 17 studies involving 251 animals. The results indicated that quercetin was able to reduce arthritis scores, paw swelling, histopathological scores, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), C-reactive protein (CRP), malondialdehyde (MDA), reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), nuclear factor kappa B (NF-kB) and increase interleukin-10 (IL-10), catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), glutathione (GSH), and heme oxygenase-1 (HO-1). These may be related to quercetin's potential anti-inflammatory, anti-oxidative stress, and osteoprotective properties. However, more high-quality animal studies are needed to assess the effect of quercetin on RA. Additionally, the safety of quercetin requires further confirmation. Given the importance of the active ingredient, dose selection and the improvement of quercetin's bioavailability remain to be explored.

Monomerization of abscisic acid receptors through CARKs-mediated phosphorylation.

Cytosolic ABA Receptor Kinases (CARKs) play a pivotal role in abscisic acid (ABA)-dependent pathway in response to dehydration, but their regulatory mechanism in ABA signaling remains unexplored. In this study, we showed that CARK4/5 of CARK family physically interacted with ABA receptors (RCARs/PYR1/PYLs), including RCAR3, RCAR11-RCAR14, while CARK2/7/11 only interacted with RCAR11-RCAR14, but not RCAR3. It indicates that the members in CARK family function redundantly and differentially in ABA signaling. RCAR12 can form heterodimer with RCAR3 in vitro and in vivo. Moreover, the members of CARK family can form homodimer or heterodimer in a kinase activity dependent manner. ITC (isothermal titration calorimetry) analysis demonstrated that the phosphorylation of RCAR12 by CARK1 enhanced the ABA binding affinity. The phosphor-mimic RCAR12T105D significantly displayed ABA-induced inhibition of the phosphatase ABI1 (ABA insensitive 1) activity, leading to upregulation of ABA-responsive genes RD29A and RD29B in cark157:RCAR12T105D transgenic plants, which exhibited ABA hypersensitive phenotype. The transcription factor ABI5 (ABA insensitive 5) activates the transcriptions of CARK1 and CARK3 by binding to ABA-response elements (ABREs) of their promoters. Collectively, our data imply that the dimeric CARKs phosphorylate homodimer or heterodimer ABA receptors, leading to monomerization for triggering ABA responses in Arabidopsis.

Paeonol Suppresses Vasculogenesis Through Regulating Vascular Smooth Muscle Phenotypic Switching.

OBJECTIVE: Smooth muscle cell (SMC) phenotypic switching is associated with development of a variety of occlusive vascular diseases. Paeonol has been reported to be involved in suppressing SMC proliferation. However, it is still unknown whether paeonol can regulate SMC phenotypic switching, and which eventually result in suppressing vasculogenesis. METHODS: Murine left common carotid artery was injured by completely ligation, and paeonol was administrated by intraperitoneal injection. Hematoxylin and eosin (H&E) staining was performed to visualize vascular neointima formation. Rat aortic SMCs were used to determine whether paeonol suppresses cell proliferation and migration. And murine hind limb ischemia model was performed to confirm the function role of paeonol in suppressing vasculogenesis. RESULTS: Complete ligation of murine common carotid artery successfully induced neointima formation. Paeonol treatment dramatically reduced the size of injury-induced neointima. Using rat aortic primary SMC, we identified that paeonol strongly suppressed cell proliferation, migration, and decreased extracellular matrix deposition. And paeonol treatment dramatically suppressed vasculogenesis after hind limb ischemia injury. CONCLUSION: Paeonol could regulate SMC phenotypic switching through inhibiting proliferation and migration of SMC, which results in inhibiting ischemia-induced vasculogenesis.

The heterogeneity of oxidized lipids in individual tumor cells reveals NK cell-mediated cytotoxicity by label-free mass cytometry.

NK cell-mediated immunotherapy has received increasing attention in the past decade due to its efficacy and bio-safety. The composition and content of lipids in individual cells are closely related to NK cell-mediated cytotoxicity, especially polyunsaturated fatty acids (PUFA) which are oxidized during NK cell-mediated apoptosis. Here we investigated the changes of lipids in single HepG2 cells by label-free mass cytometry and obtained information on 53 lipids and 13 oxidized lipids after the interaction with NK92 MI cells. We found that the contents of lipids and oxidized lipids of HepG2 cells changed obviously during the NK cell-mediated apoptosis. The HepG2 cells could be classified into two phenotypes after co-culturing with NK92 MI cells based on the ratio of PC(38:6-2OH)/PC(38:6) in individual cells, which may serve as a feature to evaluate NK cell-mediated cytotoxicity. The present work used the lipids and oxidized lipids of individual cells to reveal the heterogeneity in NK cell-mediated apoptosis which would be a powerful method for evaluating the cytotoxicity of NK cells at the single-cell level.

Low-intensity pulsed ultrasound/nanomechanical force generators enhance osteogenesis of BMSCs through microfilaments and TRPM7.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) has been reported to accelerate fracture healing, but the mechanism is unclear and its efficacy needs to be further optimized. Ultrasound in combination with functionalized microbubbles has been shown to induce local shear forces and controllable mechanical stress in cells, amplifying the mechanical effects of LIPUS. Nanoscale lipid bubbles (nanobubbles) have high stability and good biosafety. However, the effect of LIPUS combined with functionalized nanobubbles on osteogenesis has rarely been studied. RESULTS: In this study, we report cyclic arginine-glycine-aspartic acid-modified nanobubbles (cRGD-NBs), with a particle size of ~ 500 nm, able to actively target bone marrow mesenchymal stem cells (BMSCs) via integrin receptors. cRGD-NBs can act as nanomechanical force generators on the cell membrane, and further enhance the BMSCs osteogenesis and bone formation promoted by LIPUS. The polymerization of actin microfilaments and the mechanosensitive transient receptor potential melastatin 7 (TRPM7) ion channel play important roles in BMSCs osteogenesis promoted by LIPUS/cRGD-NBs. Moreover, the mutual regulation of TRPM7 and actin microfilaments promote the effect of LIPUS/cRGD-NBs. The extracellular Ca2 + influx, controlled partly by TRPM7, could participated in the effect of LIPUS/cRGD-NBs on BMSCs. CONCLUSIONS: The nanomechanical force generators cRGD-NBs could promote osteogenesis of BMSCs and bone formation induced by LIPUS, through regulation TRPM7, actin cytoskeleton, and intracellular calcium oscillations. This study provides new directions for optimizing the efficacy of LIPUS for fracture healing, and a theoretical basis for the further application and development of LIPUS in clinical practice.

Two Synthetic Peptides Corresponding to the Human Follicle-Stimulating Hormone ß-Subunit Promoted Reproductive Functions in Mice.

A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-ß-(34−37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-ß-(37−49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-ß-(37−49) and hFSH-ß-(34−49), created by merging hFSH-ß-(34−37) and hFSH-ß-(37−49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 µg/g BW ip of hFSH-ß-(37−49) or hFSH-ß-(34−49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 µg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-ß-(34−49) was a more potent (p < 0.05) reproductive modulator than hFSH-ß-(37−49), both in vivo and in vitro. We concluded that hFSH-ß-(37−49) and especially hFSH-ß-(34−49), have the potential for reproductive modulation.

Synthesis, Configurational Analysis, Molecular Recognition and Chirality Sensing of Methylene-Bridged Naphthotubes.

Macrocyclic hosts with a well-defined cavity are particularly appealing for supramolecular chemistry, but they are still rare. In this research, we reported a new class of macrocyclic hosts, namely methylene-bridged naphthotubes, with well-defined cavities. They were synthesized through TFA-catalyzed Friedel-Crafts reactions between alkoxy-substituted bisnaphthalenes and paraformaldehyde. A configurational selection was observed. Three-membered macrocycles possess a single cavity, while four-membered macrocycles adopt a double-cavity conformation or a self-filling conformation depending on the alkoxy length. The small homologue shows quite strong binding affinities (up to 108  M-1 ) to organic cations, which is better than structurally similar but flexible macrocycles. This demonstrates the advantage of a well-defined cavity in molecular recognition. Moreover, these naphthotubes can be used as chirality sensors for chiral organic cations through different chirality transfer mechanisms.

Discriminating Leukemia Cellular Heterogeneity and Screening Metabolite Biomarker Candidates using Label-Free Mass Cytometry.

Discriminating various leukocyte subsets with specific functions is critical due to their important roles in the development of many diseases. Here, we proposed a general strategy to unravel leukocytes heterogeneity and screen differentiated metabolites as biomarker candidates for leukocyte subtypes using the label-free mass cytometry (CyESI-MS) combined with a homemade data processing workflow. Taking leukemia cells as an example, metabolic fingerprints of single leukemia cells were obtained from 472 HL-60, 416 THP-1, 313 U937, 356 Jurkat, and 366 Ramos cells, with throughput up to 40 cells/min. Five leukemia subtypes were clearly distinguished by unsupervised learning t-SNE analysis of the single-cell metabolic fingerprints. Cell discrimination in the mixed leukemia cell samples was also realized by supervised learning of the single-cell metabolic fingerprints with high recovery and good repetition (98.31 ± 0.24%, -102.35 ± 4.82%). Statistical analysis and metabolite assignment were carried out to screen characteristic metabolites for discrimination and 36 metabolites with significant differences were annotated. Then, differentiated metabolites for pairwise discrimination of five leukemia subtypes were further selected as biomarker candidates. Furthermore, discriminating cultured leukemia cells from human normal leukocytes, separated from fresh human peripheral blood, was performed based on single-cell metabolic fingerprints as well as the proposed biomarker candidates, unveiling the potential of this strategy in clinical research. This work makes efforts to realize high-throughput single-leukocyte metabolic analysis and metabolite-based discrimination of leukocytes. It is expected to be a powerful means for the clinical molecular diagnosis of hematological diseases.