Characterization of the gut microbiota and fecal metabolome in the osteosarcoma mouse model.

Previous studies have reported the correlation between gut microbiota (GM), GM-derived metabolites, and various intestinal and extra-intestinal cancers. However, limited studies have investigated the correlation between GM, GM-derived metabolites, and osteosarcoma (OS). This study successfully established a female BALB/c nude mouse model of OS. Mice (n = 14) were divided into the following two groups (n = 7/group): OS group named OG, injected with Saos-2 OS cells; normal control group named NCG, injected with Matrigel. The GM composition and metabolites were characterized using 16S rDNA sequencing and untargeted metabolomics, respectively. Bioinformatics analysis revealed that amino acid metabolism was dysregulated in OS. The abundances of bone metabolism-related genera Alloprevotella, Rikenellaceae_RC9_gut_group, and Muribaculum were correlated with amino acid metabolism, especially histidine metabolism. These findings suggest the correlation between GM, GM-derived metabolites, and OS pathogenesis. Clinical significance: The currently used standard therapeutic strategies for OS, including surgery, chemotherapy, and radiation, are not efficacious. The findings of this study provided novel insights for developing therapeutic, diagnostic, and prognostic strategies for OS.

Exploring dissolved N2O characteristics and unearthing indirect N2O emission factors in the shallow groundwater of paddy and upland fields.

Indirect emissions of nitrous oxide (N2O) stemming from nitrogen (N) leaching in agricultural fields constitute a significant contributor to atmospheric N2O. Groundwater nitrate (NO3--N) pollution is severe in the Ningxia Yellow River Irrigation Area (NYRIA), coupled with high NO3--N leaching, exacerbates the risk of indirect N2O emissions from groundwater. Over two years of field observations, this study investigated the characteristics and interannual variations of dissolved N2O (dN2O) concentrations and indirect N2O emission factors (EF5g) in shallow groundwater. The research focused on three typical farmlands in the NYRIA, each subjected to six levels of N fertilizer application. The mean dN2O concentrations in the groundwater of paddy, corn and vegetable fields were 5.17, 8.40 and 16.35 µg N·L-1, respectively. Notably, the dN2O concentrations in the shallow groundwater of upland fields exceeded those in paddy fields, with maximum levels in vegetable fields nearly an order of magnitude higher. Elevated N application significantly increased dN2O concentrations across various farmlands, showing statistically significant variation. However, differences in EF5g-A and EF5g-B within the same farmland were negligible. Denitrification was the primary process contributing to N2O production in groundwater, with nitrification also played a crucial role in upland fields. Factors such as NO3--N, NH4+-N, dissolved oxygen (DO), and pH critically influenced N2O production. EF5g-B, which considers the NO3--N consumption during denitrification processes in groundwater, was deemed more appropriate than EF5g-A for assessing the indirect N2O emission in the NYRIA. The EF5g of agricultural fields exhibited minimal sensitivity to N input but was significantly affected by other factors, such as the planting pattern. The study revealed the rationality of adopting EF5g-B in assessing indirect N2O emissions, providing valuable insights for N management strategies in regions with high NO3--N leaching. Minimizing N fertilizer application while ensuring crop yield, especially in upland fields, is beneficial for reducing N2O emissions.

MiR-134-5p inhibits the malignant phenotypes of osteosarcoma via ITGB1/MMP2/PI3K/Akt pathway.

Micro RNAs (miRs) have been implicated in various tumorigenic processes. Osteosarcoma (OS) is a primary bone malignancy seen in adolescents. However, the mechanism of miRs in OS has not been fully demonstrated yet. Here, miR-134-5p was found to inhibit OS progression and was also expressed at significantly lower levels in OS tissues and cells relative to normal controls. miR-134-5p was found to reduce vasculogenic mimicry, proliferation, invasion, and migration of OS cells, with miR-134-5p knockdown having the opposite effects. Mechanistically, miR-134-5p inhibited expression of the ITGB1/MMP2/PI3K/Akt axis, thus reducing the malignant features of OS cells. In summary, miR-134-5p reduced OS tumorigenesis by modulation of the ITGB1/MMP2/PI3K/Akt axis, suggesting the potential for using miR-134-5p as a target for treating OS.

Self-Metallized Whole Cell Vaccines Prepared by Microfluidics for Bioorthogonally Catalyzed Antitumor Immunotherapy.

Tumor whole cell, carrying a complete set of tumor-associated antigens and tumor-specific antigens, has shown great potential in the construction of tumor vaccines but is hindered by the complex engineering means and limited efficacy to cause immunity. Herein, we provided a strategy for the self-mineralization of autologous tumor cells with palladium ions in microfluidic droplets, which endowed the engineered cells with both immune and catalytic functions, to establish a bioorthogonally catalytic tumor whole-cell vaccine. This vaccine showed strong inhibition both in the occurrence and recurrence of tumor by invoking the immediate antitumor immunity and building a long-term immunity.

Microfluidic Synthesis of CuH Nanoparticles for Antitumor Therapy through Hydrogen-Enhanced Apoptosis and Cuproptosis.

Cuproptosis has drawn enormous attention in antitumor material fields; however, the responsive activation of cuproptosis against tumors using nanomaterials with high atom utilization is still challenging. Herein, a copper-based nanoplatform consisting of acid-degradable copper hydride (CuH) nanoparticles was developed via a microfluidic synthesis. After coating with tumor-targeting hyaluronic acid (HA), the nanoplatform denoted as HA-CuH-PVP (HCP) shows conspicuous damage toward tumor cells by generating Cu+ and hydrogen (H2) simultaneously. Cu+ can induce apoptosis by relying on Fenton-like reactions and lead to cuproptosis by causing mitochondrial protein aggregation. Besides, the existence of H2 can enhance both cell death types by causing mitochondrial dysfunction and intracellular redox homeostatic disorders. In vivo experimental results further exhibit the desirable potential of HCP for killing tumor cells and inhibiting lung metastases, which will broaden the horizons of designing copper-based materials triggering apoptosis and cuproptosis for better antitumor efficacy.

Self-Generating Gold Nanocatalysts in Autologous Tumor Cells for Targeted Catalytic Immunotherapy.

Employing tumor whole cells for tumor immunotherapy is a promising tumor therapy proposed in the early stage, but its therapeutic efficacy is weakened by the methods of eliminating pathogenicity and the mass ratio of the effective antigen carried by itself. Here, by adding gold ion to live cancer cells in the microfluidic droplets, this work obtains dead tumor whole cells with NIR-controlled catalytic ability whose pathogenicity is removed while plenary tumor antigens, major structure, and homing ability are reserved. The engineered tumor cell (Cell-Au) with the addition of prodrug provides 1O2 in an O2-free Russell mechanism, which serves better in a hypoxic tumor microenvironment. This tumor whole-cell catalytic vaccine (TWCV) promotes the activation of dendritic cells and the transformation of macrophages into tumor suppressor phenotype. In 4T1 tumor-bearing mice, the Cell-Au-based vaccine supports the polarization of cytotoxicity T cells, resulting in tumor eradication and long-term animal survival. Compared with antigen vaccines or adoptive cell therapy which takes months to obtain, this TWCV can be prepared in just a few days with satisfactory immune activation and tumor therapeutic efficacy, which provides an alternative way for the preparation of personalized tumor vaccines across tumor types and gives immunotherapy a new path.

Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy.

Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H2O2-mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H+ and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H2O2-mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.

Erratum: Author correction to "Tumor-microenvironment activated duplex genome-editing nanoprodrug for sensitized near-infrared titania phototherapy" [Acta Pharm Sin B (2022) 4224-4234].

[This corrects the article DOI: 10.1016/j.apsb.2022.06.016.].

Coping with groundwater pollution in high-nitrate leaching areas: The efficacy of denitrification.

The Ningxia Yellow River irrigation area, characterized by an arid climate and high leaching of NO3--N, exhibits complex and unique groundwater nitrate (NO3--N) pollution, with denitrification serving as the principal mechanism for NO3--N removal. The characteristics of N leaching from paddy fields and NO3--N removal by groundwater denitrification were investigated through a two-year field observation. The leaching losses of total nitrogen (TN) and NO3--N accounted for 10.81-27.34% and 7.59-12.74%, respectively, of the N input. The linear relationship between NO3--N leaching and N input indicated that the fertilizer-induced emission factor (EF) of NO3--N leaching in direct dry seeding and seedling-raising and transplanting paddy fields was 8.2% (2021, R2 = 0.992) and 6.7% (2022, R2 = 0.994), respectively. The study highlighted that the quadratic relationship between the NO3--N leaching loss and N input (R2 = 0.999) significantly outperformed the linear relationship. Groundwater denitrification capacity was characterized by monitoring the concentrations of dinitrogen (N2) and nitrous oxide (N2O). The results revealed substantial seasonal fluctuations in excess N2 and N2O concentrations in groundwater, particularly following fertilization and irrigation events. The removal efficiency of NO3--N via groundwater denitrification ranged from 42.70% to 74.38%, varying with depth. Groundwater denitrification capacity appeared to be linked to dissolved organic carbon (DOC) concentration, redox conditions, fertilization, irrigation, and soil texture. The anthropogenic-alluvial soil with limited water retention accelerated the leaching of NO3--N into groundwater during irrigation. This process enhances the groundwater recharge capacity and alters the redox conditions of groundwater, consequently impacting groundwater denitrification activity. The DOC concentration emerged as the primary constraint on the groundwater denitrification capacity in this region. Hence, increasing carbon source concentration and enhancing soil water retention capacity are vital for improving the groundwater denitrification capacity and NO3--N removal efficiency. This study provides practical insights for managing groundwater NO3--N pollution in agricultural areas, optimizing fertilization strategies and improving groundwater quality.

Cisplatin and doxorubicin chemotherapy alters gut microbiota in a murine osteosarcoma model.

The gut microbiota is closely associated with tumor progression and treatment in a variety of cancers. However, the alteration of the gut microbiota during the progression and chemotherapy of osteosarcoma remains poorly understood. This study aimed to explore the relationship between dysbiosis in the gut microbiota during osteosarcoma growth and chemotherapy treatment. We used BALB/c nude mice to establish osteosarcoma xenograft tumor models and administered cisplatin (CDDP) or doxorubicin (DOX) intraperitonially once every 2 days for a total of 5 times to establish effective chemotherapy models. Fecal samples were collected and processed for 16S rRNA sequencing to analyze the composition of the gut microbiota. We observed that the abundances of Colidextribacter, Lachnospiraceae_NK4A136_group, Lachnospiraceae_UCG-010, Lachnospiraceae_UCG-006, and Lachnoclostridium decreased, and the abundances of Alloprevotella and Enterorhabdus increased in the osteosarcoma mouse model group compared to those in the control group. In addition, genera, such as Lachnoclostridium and Faecalibacterium were more abundant in chemotherapy-treated mice than those in saline-treated mice. Additionally, we observed that alterations in some genera, including Lachnoclostridium and Colidextribacter in the osteosarcoma animal model group returned to normal after CDDP or DOX treatment. Furthermore, the function of the gut microbiota was inferred through PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), which indicated that metabolism-related microbiota was highly enriched and significantly different in each group. These results indicate correlations between dysbiosis of the gut microbiota and osteosarcoma growth and chemotherapy treatment with CDDP or DOX and may provide novel avenues for the development of potential adjuvant therapies.

Gut microbial community and fecal metabolomic signatures in different types of osteoporosis animal models.

BACKGROUND: The gut microbiota (GM) constitutes a critical factor in the maintenance of physiological homeostasis. Numerous studies have empirically demonstrated that the GM is closely associated with the onset and progression of osteoporosis (OP). Nevertheless, the characteristics of the GM and its metabolites related to different forms of OP are poorly understood. In the present study, we examined the changes in the GM and its metabolites associated with various types of OP as well as the correlations among them. METHODS: We simultaneously established rat postmenopausal, disuse-induced, and glucocorticoid-induced OP models. We used micro-CT and histological analyses to observe bone microstructure, three-point bending tests to measure bone strength, and enzyme-linked immunosorbent assay (ELISA) to evaluate the biochemical markers of bone turnover in the three rat OP models and the control. We applied 16s rDNA to analyze GM abundance and employed untargeted metabolomics to identify fecal metabolites in all four treatment groups. We implemented multi-omics methods to explore the relationships among OP, the GM, and its metabolites. RESULTS: The 16S rDNA sequencing revealed that both the abundance and alterations of the GM significantly differed among the OP groups. In the postmenopausal OP model, the bacterial genera g__Bacteroidetes_unclassified, g__Firmicutes_unclassified, and g__Eggerthella had changed. In the disuse-induced and glucocorticoid-induced OP models, g__Akkermansia and g__Rothia changed, respectively. Untargeted metabolomics disclosed that the GM-derived metabolites significantly differed among the OP types. However, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that it was mainly metabolites implicated in lipid and amino acid metabolism that were altered in all cases. An association analysis indicated that the histidine metabolism intermediate 4-(ß-acetylaminoethyl) imidazole was common to all OP forms and was strongly correlated with all bone metabolism-related bacterial genera. Hence, 4-(ß-acetylaminoethyl) imidazole might play a vital role in OP onset and progression. CONCLUSIONS: The present work revealed the alterations in the GM and its metabolites that are associated with OP. It also disclosed the changes in the GM that are characteristic of each type of OP. Future research should endeavor to determine the causal and regulatory effects of the GM and the metabolites typical of each form of OP.

Drainage ditches are significant sources of indirect N2O emissions regulated by available carbon to nitrogen substrates in salt-affected farmlands.

Agriculture is a main source of nitrous oxide (N2O) emissions. In agricultural systems, direct N2O emissions from nitrogen (N) addition to soils have been widely investigated, whereas indirect emissions from aquatic ecosystems such as ditches are poorly known, with insufficient data available to refine the IPCC emission factor. In this contribution, in situ N2O emissions from two ditch water‒air interfaces based on a diffusion model were investigated (almost once per month) from June 2021 to December 2022 in an intensive arable catchment with high N inputs and salt-affected conditions in the Qingtongxia Irrigation District, northwestern China. Our results implied that agricultural ditches (mean 148 µg N m-2 h-1) were significant sources for N2O emissions, and were approximately 2.1 times greater than those of the Yellow River directly connected to ditches. Agronomic management strategies increased N2O fluxes in summer, while precipitation events decreased N2O fluxes. Agronomic management strategies, including fertilization (294--540 kg N hm-2) and irrigation on farmland, resulted in enhanced diffuse N loads in drain water, whereas precipitation diluted the dissolved N2O concentration in ditches and accelerated the ditch flow rate, leading to changes in the residence time of N-containing substances in water. The spatial analysis showed that N2O fluxes (202-233 µg N m-2 h-1) in the headstream and upstream regions of ditches due to livestock and aquaculture pollution sources were relatively high compared to those in the midstream and downstream regions (100-114 µg N m-2 h-1). Furthermore, high available carbon (C) relative to N reduced N2O fluxes at low DOC:DIN ratio levels by inhibiting nitrification. Spatiotemporal variations in the N2O emission factor (EF5) across ditches with higher N resulted in lower EF5 and a large coefficient of variation (CV) range. EF5 was 0.0011 for the ditches in this region, while the EF5 (0.0025) currently adopted by the IPCC is relatively high. The EF5 variation was strongly controlled by the DOC:DIN ratio, TN, and NO3--N, while salinity was also a nonnegligible factor regulating the EF5 variation. The regression model incorporating NO3--N and the DOC:DIN ratio could greatly enhance the predictions of EF5 for agricultural ditches. Our study filled a key knowledge gap regarding EF5 from agricultural ditches in salt-affected farmland and offered a field investigation for refining the EF5 currently used by the IPCC.

Ditch level-dependent N removal capacity of denitrification and anammox in the drainage system of the Ningxia Yellow River irrigation district.

Drainage networks, consisting of different levels of ditches, play a positive role in removing reactive nitrogen (N) via self-purification before drainage water returns to natural water bodies. However, relatively little is known about the N removal capacity of irrigation agricultural systems with different drainage ditch levels. In this study, we employed soil core incubation and soil slurry 15N paired tracer techniques to investigate the N removal rate (i.e., N2 flux), denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) rates in the Ningxia Yellow River irrigation district at various ditch levels, including field ditches (FD), paddy field ditches (PFD), lateral ditches (LD1 and LD2), branch ditches (BD1, BD2, BD3), and trunk ditches (TD). The results indicated that the N removal rate ranged from 44.7 to 165.22 nmol N g-1 h-1 in the ditches, in the following decreasing order: trunk ditches > branch ditches > paddy field ditches > lateral ditches > field ditches. This result suggested that the N removal rate in drainage ditches is determined by the ditch level. In addition, denitrification and anammox were the primary pathways for N removal in the ditches, contributing 68.40-76.64 % and 21.55-30.29 %, respectively, to the total N removal. In contrast, DNRA contributed only 0.82-2.15 % to the total nitrate reduction. The N removal rates were negatively correlated with soil EC and pH and were also constrained by the abundances of denitrification functional genes. Overall, our findings suggest that the ditch level should be considered when evaluating the N removal capacity of agricultural ditch systems.

Emerging trends in organ-on-a-chip systems for drug screening.

New drug discovery is under growing pressure to satisfy the demand from a wide range of domains, especially from the pharmaceutical industry and healthcare services. Assessment of drug efficacy and safety prior to human clinical trials is a crucial part of drug development, which deserves greater emphasis to reduce the cost and time in drug discovery. Recent advances in microfabrication and tissue engineering have given rise to organ-on-a-chip, an in vitro model capable of recapitulating human organ functions in vivo and providing insight into disease pathophysiology, which offers a potential alternative to animal models for more efficient pre-clinical screening of drug candidates. In this review, we first give a snapshot of general considerations for organ-on-a-chip device design. Then, we comprehensively review the recent advances in organ-on-a-chip for drug screening. Finally, we summarize some key challenges of the progress in this field and discuss future prospects of organ-on-a-chip development. Overall, this review highlights the new avenue that organ-on-a-chip opens for drug development, therapeutic innovation, and precision medicine.

An Enzyme-Loaded Metal-Organic Framework-Assisted Microfluidic Platform Enables Single-Cell Metabolite Analysis.

Colonization of cancer cells at secondary sites, a decisive step in tumor metastasis, is strongly dependent on the formation of metastatic microenvironments regulated by intrinsic single-cell metabolism traits. Herein, we report a single-cell microfluidic platform for high-throughput dynamic monitoring of tumor cell metabolites to evaluate tumor malignancy. This microfluidic device empowers efficient isolation of single cells (>99 %) in a squashed state similar to tumor extravasation, and employs enzyme-packaged metal-organic frameworks to catalyze tumor cell metabolites for visualization. The microfluidic evaluation was confirmed by in vivo assays, suggesting that the platform allowed predicting the tumorigenicity of captured tumor cells and screening metabolic inhibitors as anti-metastatic drugs. Furthermore, the platform efficiently detected various aggressive cancer cells in unprocessed whole blood samples with high sensitivity, showing potential for clinical application.

In-Tumor Biosynthetic Construction of Upconversion Nanomachines for Precise Near-Infrared Phototherapy.

Targeted construction of therapeutic nanoplatforms in tumor cells with specific activation remains appealing but challenging. Here, we design a cancer-motivated upconversion nanomachine (UCNM) based on porous upconversion nanoparticles (p-UCNPs) for precise phototherapy. The nanosystem is equipped with a telomerase substrate (TS) primer and simultaneously encapsulates 5-aminolevulinic acid (5-ALA) and d-arginine (d-Arg). After coating with hyaluronic acid (HA), it can readily get into tumor cells, where 5-ALA induces efficient accumulation of protoporphyrin IX (PpIX) via the inherent biosynthetic pathway, and the overexpressed telomerase prolonged the TS to form G-quadruplexes (G4) for binding the resulting PpIX as a nanomachine. This nanomachine can respond to near-infrared (NIR) light and promote the active singlet oxygen (1O2) production due to the efficiency of Förster resonance energy transfer (FRET) between p-UCNPs and PpIX. Intriguingly, such oxidative stress can oxidize d-Arg into nitric oxide (NO), which relieves the tumor hypoxia and in turn improves the phototherapy effect. This in situ assembly approach significantly enhances targeting in cancer therapy and might be of considerable clinical value.

Salinity and high pH reduce denitrification rates by inhibiting denitrifying gene abundance in a saline-alkali soil.

Denitrification, as the main nitrogen (N) removal process in farmland drainage ditches in coastal areas, is significantly affected by saline-alkali conditions. To elucidate the effects of saline-alkali conditions on denitrification, incubation experiments with five salt and salt-alkali gradients and three nitrogen addition levels were conducted in a saline-alkali soil followed by determination of denitrification rates and the associated functional genes (i.e., nirK/nirS and nosZ Clade I) via N2/Ar technique in combination with qPCR. The results showed that denitrification rates were significantly decreased by 23.83-50.08%, 20.64-57.31% and 6.12-54.61% with salt gradient increasing from 1 to 3‰, 8‰, and 15‰ under 0.05‰, 0.10‰ and 0.15‰ urea addition conditions, respectively. Similarly, denitrification rates were significantly decreased by 44.57-63.24% with an increase of the salt-alkali gradient from 0.5 to 8‰. The abundance of nosZ decreased sharply in the saline condition, while a high salt level significantly decreased the abundance of nirK and nirS. In addition, the increase of nitrogen concentration attenuated the reduction of nirK, nirS and nosZ gene abundance. Partial least squares regression (PLSR) models demonstrated that salinity, dissolved oxygen (DO) in the overlying water, N concentration, and denitrifying gene abundance were key determinants of the denitrification rate in the saline environment, while pH was an additional determinant in the saline-alkali environment. Taken together, our results suggest that salinity and high pH levels decreased the denitrification rates by significantly inhibiting the abundance of the denitrifying genes nirK, nirS, and nosZ, whereas increasing nitrogen concentration could alleviate this effect. Our study provides helpful information on better understanding of reactive N removal and fertilizer application in the coastal areas.

Metal-Organic Framework-Mediated Bioorthogonal Reaction to Immobilize Bacteria for Ultrasensitive Fluorescence Counting Immunoassays.

Ultrasensitive quantification of protein biomarkers has significant implications in disease diagnosis. Herein, we report a fluorescent bacteria counting immunoassay (FBCIA) strategy for protein biomarker detection based on a cascade signal conversion and amplification strategy including the copper metal-organic framework (Cu-MOF)-mediated Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) for fluorescent bacteria immobilization that converted the concentration of target protein to countable bacterial number and the further self-proliferation of bacteria to amplify the detectable bacterial number. The developed low-background and enzyme-free cascade methodology achieved highly sensitive detection of carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) with detection limits down to 0.8 pg/mL and 64.5 fg/mL, respectively. On top of that, we also developed a smartphone device for visualizing individual bacteria and point-of-care counting of the resulting bacteria for the detection of clinical samples. The good consistency between FBCIA and clinical enzyme-linked immunosorbent assay (ELISA) validated the high reliability and promising potential of our developed platform in clinical applications.

Metal-Organic Framework-DNA Bio-Barcodes Amplified CRISPR/Cas12a Assay for Ultrasensitive Detection of Protein Biomarkers.

CRISPR/Cas12a shows excellent potential in disease diagnostics. However, insensitive signal conversion strategies hindered its application in detecting protein biomarkers. Here, we report a metal-organic framework (MOF)-based DNA bio-barcode integrated with the CRISPR/Cas12a system for ultrasensitive detection of protein biomarkers. In this work, zirconium-based MOF nanoparticles were comodified with antibodies and bio-barcode phosphorylated DNA as an efficient signal converter, which not only recognized the protein biomarker to form the sandwich complex but also released the bio-barcode DNA activators after MOF dissociation to activate the trans-cleavage activity of Cas12a. Due to the obvious advantages, including numerous loaded oligonucleotides, a convenient release process, and the nontoxic release reagent, this MOF-DNA bio-barcode strategy could amplify the CRISPR/Cas12a system to achieve simple and highly sensitive detection of tumor protein biomarkers with detection limits of 0.03 pg/mL (PSA) and 0.1 pg/mL (CEA), respectively. Furthermore, this platform could detect PSA directly in clinical serum samples, offering a powerful tool for early disease diagnosis.

Transcriptomics and metabolomics reveal changes in the regulatory mechanisms of osteosarcoma under different culture methods in vitro.

BACKGROUND: Recently, increasing attention has been drawn to the impact of the tumor microenvironment (TME) on the occurrence and progression of malignant tumors. A variety of 3D culture techniques have been used to simulate TME in vitro. The purpose of this study was to reveal the differences in transcriptional and metabolic levels between osteosarcoma (OS) 2D cells, 3D cells, 3D cell-printed tissue, isolated tissue, and transplanted tumor tissue in vivo. METHODS: We cultured the OS Saos-2 cell line under different culture methods as 2D cells, 3D cells, 3D cell-printed tissue and isolated tissue for 14 days and transplanted tumors in vivo as a control group. Through transcriptomic and metabonomic analyses, we determined the changes in gene expression and metabolites in OS tissues under different culture methods. RESULTS: At the transcriptional level, 166 differentially expressed genes were found, including the SMAD family, ID family, BMP family and other related genes, and they were enriched in the TGF-ß signaling pathway, complement and coagulation cascades, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ferroptosis, cGMP-PKG signaling pathway and other pathways. At the metabolic level, 362 metabolites were significantly changed and enriched in metabolic pathways such as the Fc Epsilon RI signaling pathway, histidine metabolism, primary bile acid biosynthesis, steroid biosynthesis, protein digestion and absorption, ferroptosis, and arachidonic acid metabolism. After integrating the transcriptome and metabolomics data, it was found that 44 metabolic pathways were changed, and the significantly enriched pathways were ferroptosis and pyrimidine metabolism. CONCLUSION: Different culture methods affect the gene expression and metabolite generation of OS Saos-2 cells. Moreover, the cell and tissue culture method in vitro cannot completely simulate TME in vivo, and the ferroptosis and pyrimidine metabolism pathways mediate the functional changes of OS Saos-2 cells in different microenvironments.