DNA adductomics aided rapid screening of genotoxic impurities using nucleosides and 3D bioprinted human liver organoids.

Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.

Visual and Electrochemical Detection of let-7a: A Tumor Suppressor and Biomarker.

Let-7a, a type of low-expressed microRNAs in cancer cells, has been investigated as a promising biomarker and therapeutic target for tumor suppression. Developing simple and sensitive detection methods for let-7a is important for cancer diagnosis and treatment. In this work, the hybridization chain reaction (HCR) was initiated by let-7a via two hairpin primers (H1 and H2). After the HCR, the remaining hairpin H1 was further detected by lateral flow assay (LFA) and electrochemical impedance spectroscopy. For LFA, biotin-modified H1(bio-H1) and free H2 were used for HCR. With the decrease of let-7a concentration, the color of T line gradually increased. As for electrochemical methods, the H1'-AuNP-modified electrode was used for detection of bio-H1 based on the difference of impedance (ΔRct) detected without and with different concentrations of let-7a participating in the HCR. This method could detect let-7a in the range of 10.0 fM and 1.0 nM with detection limits of 4.2 fM.

Fluorescent intracellular imaging of reactive oxygen species and pH levels moderated by a hydrogenase mimic in living cells.

The catalytic generation of H2 in living cells provides a method for antioxidant therapy. In this study, an [FeFe]-hydrogenase mimic [Ru + Fe2S2@F127(80)] was synthesized by self-assembling polymeric pluronic F-127, catalytic [Fe2S2] sites, and photosensitizer Ru(bpy)3 2+. Under blue light irradiation, hydrated protons were photochemically reduced to H2, which increased the local pH in living cells (HeLa cells). The generated H2 was subsequently used as an antioxidant to decrease reactive oxygen species (ROS) levels in living cells (HEK 293T, HepG2, MCF-7, and HeLa cells). Our findings revealed that the proliferation of HEK 293T cells increased by a factor of about six times, relative to that of other cells (HepG2, MCF-7, and HeLa cells). Intracellular ROS and pH levels were then monitored using fluorescent cell imaging. Our study showed that cell imaging can be used to evaluate the ability of Ru + Fe2S2@F127 to eliminate oxidative stress and prevent ROS-related diseases.

Oral Metal-Organic Gel Protected Whole-Cell Biosensor for in Situ Monitoring Nitrosamines in the Gastrointestinal Tract.

Nitrite, a type of food additive, has been proved convertible to genotoxic nitrosamines in the gastrointestinal tract by intestinal flora. There is no appropriate method for in situ detection of nitrosamines. Herein, plasmid-introduced Saccharomyces cerevisiae, which can respond to nitrosamine-induced DNA damage and activate pMAG1-based DNA damage repair (DDR), was designed as whole-cell biosensors (WCBs) for monitoring the in situ generated nitrosamines by a reporter gene expressing enhanced green fluorescent protein (EGFP). In order to protect the validity of WCBs (pMAG1 yeast) from the gastric acid environment, a type of metal-organic gel (MOG), coordinated by Fe3+ and 2,2'-thiodiacetic acid (TDA), was prepared to embed the WCBs. The MOG(Fe-TDA) is gastric acid resistant and can deliver the pMAG1 yeast to the gut without compromising the performance of pMAG1 yeast to detect in situ generated nitrosamines. The genotoxicity of nitrosamines converted from nitrite was successfully detected in the gastrointestinal tract of mice.

Lanthanide Complexes for Tumor Diagnosis and Therapy by Targeting Sialic Acid.

Sialic acid (SA) is overexpressed on cell membranes of tumor cells, and increased serum SA concentration has been observed in tumor-bearing patients. Herein, a series of lanthanide-containing bimetallic complexes (TDA-M-Lns) for targeting SA were prepared via coordination among luminescent lanthanide ions (Ln3+ = Tb3+, Eu3+, Dy3+, or Sm3+), metal ion quenchers (M2+ = Cu2+ or Co2+), and the organic ligand 2,2'-thiodiacetic acid (TDA). SA can competitively coordinate with Ln3+, resulting in the "signal-on" of the Ln3+. Therefore, the TDA-M-Lns can be simply used for cost-saving detection of SA in the blood samples. Among the TDA-M-Lns, TDA-Co-Eu showed the highest sensitivity to detect SA in the blood of tumor-bearing mice. Furthermore, the TDA-Co-Eu was successfully used to target SA and deposit Eu3+ on the surfaces of tumor cells for the inhibition of tumor cell growth and migration. The therapeutic effect of TDA-Co-Eu on a Balb/c mouse liver tumor model was evaluated. It was proved that TDA-Co-Eu can be applied for SA detection as well as for inhibiting tumor growth.

Enzyme-Immobilized Metal-Organic Frameworks: From Preparation to Application.

As a class of widely used biocatalysts, enzymes possess advantages including high catalytic efficiency, strong specificity and mild reaction condition. However, most free enzymes have high requirements on the reaction environment and are easy to deactivate. Immobilization of enzymes on nanomaterial-based substrates is a good way to solve this problem. Metal-organic framework (MOFs), with ultra-high specific surface area and adjustable porosity, can provide a large space to carry enzymes. And the tightly surrounded protective layer of MOFs can stabilize the enzyme structure to a great extent. In addition, the unique porous network structure enables selective mass transfer of substrates and facilitates catalytic processes. Therefore, these enzyme-immobilized MOFs have been widely used in various research fields, such as molecule/biomolecule sensing and imaging, disease treatment, energy and environment protection. In this review, the preparation strategies and applications of enzyme-immobilized MOFs are illustrated and the prospects and current challenges are discussed.

Cancer drug resistance related microRNAs: recent advances in detection methods.

Drug resistance is a significant factor that hinders the success of cancer chemotherapy. The widely recognized mechanisms of drug resistance include changes to cell proliferation, cycle/apoptosis, drug metabolism/transport, DNA damage and the epithelial to mesenchymal transition. MicroRNAs (miRNAs), short non-coding RNAs with lengths of approximately 19-25 nucleotides, are related to cancer drug resistance, which is regulated by the aforementioned mechanisms. Based on the importance of miRNAs in regulating drug resistance, it is also necessary to take appropriate miRNA detection methods into consideration. To date, a number of advanced miRNA detection methods with high specificity and sensitivity have been developed, such as isothermal amplification-based methods, nanomaterial-based methods, chromatography-based methods, mass spectrometry-based methods and so on. Herein, biogenesis of miRNAs, the relationship between miRNAs and cancer drug resistance, and miRNA detection methods are introduced and discussed to facilitate the development of non-invasive diagnosis and inhibition of cancer drug resistance.

Adherence to Oral Targeted Anti-Lung Cancer Therapy: A Qualitative Interview Study.

Objective: Oral targeted antineoplastic drugs (OTADs) are becoming more and more acceptable for lung cancer treatment due to their advantages such as the convenience of administration and milder side effects. However, medication adherence represents a major issue for prolonged OTAD treatment. In this study, the factors associated with treatment adherence to OTAD were explored through the Adherence Influencing Factor Framework suggested by WHO. Based on these results, we further examined the potential factors related to social psychological cognition in OTAD adherence in patients with lung cancer. Methods: This qualitative study was conducted in public hospitals in Henan, China. Data were collected through semi-structured interviews with selected lung cancer patients. Face-to-face interviews were audio-recorded and transcribed for thematic analysis. Results: Of the 21 patients interviewed, 17 were males and 4 were females. The analysis of the data led to four themes, ie, patient-related factors (medication-taking introspection, family structure, weigh the pros and cons of OTAD treatment), medication-related factors (medication experience, adverse reactions, information access), physician/nurse-related factors (shared decision making, doctor's reaction, nurse's inquiry) and society-related factors (fear, stigma). Conclusion: Family structure, weigh the pros and cons of OTAD treatment, information access, shared decision making, nurse's inquiry are potential factors affecting OTAD adherence in lung cancer patients. Providing drug information support to patients, inviting patients to join in shared decision-making and strengthening doctor-patient-nurse cooperation are important for improving medication adherence. Further research should be conducted to help healthcare providers to promote the medication adherence of lung cancer patients to OTAD treatment.

Molecular mapping for fruit-related traits, and joint identification of candidate genes and selective sweeps for seed size in melon.

Melon is a popular fruit vegetable crop worldwide with diverse morphological variation. We report a high-density genetic map of melon and nine major QTLs with physical region ranging from 43.47 kb to 1.89 Mb. Importantly, two seed-related trait QTLs were repeatedly detected in two environments, and the mapping region was narrowed to 522 kb according to a regional linkage analysis. A total of 40 annotated genes were screened for nonsynonymous variations, of which EVM0009818, involved in cytokinin-activated signaling, was differentially expressed in the young fruits of parents based on RNA-seq. Selective sweep analysis identified 152 sweep signals for seed size, including the two seed-related QTLs and nine homologs that have been verified to regulate seed size in Arabidopsis or rice. This work illustrates the power of a joint analysis combining resequencing-based genetic map for QTL mapping and a combination of KASP genotyping and RNA-seq analysis to facilitate QTL fine mapping.

Targeted creating new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants.

Fruits and vegetables in the Cucurbitaceae family contribute greatly to the human diet, for example, cucumber, melon, watermelon and squash. The widespread use of genome editing technologies has greatly accelerated the functional characterization of genes as well as crop improvement. However, most economically important cucurbit plants, including melon and squash, remain recalcitrant to standard Agrobacterium tumefaciens-mediated transformation, which limits the effective use of genome editing technology. In this study, we describe the "optimal infiltration intensity" strategy to establish an efficient genetic transformation system for melon and squash. We harnessed the power of this method to target homologs of the ERECTA family of receptor kinase genes and created alleles resulting in a compact plant architecture with shorter internodes in melon, squash and cucumber. The optimized transformation method presented here allows stable CRISPR/Cas9-mediated mutagenesis and will lay a solid foundation for functional gene manipulation in cucurbit crops.

Surface functionalized biomimetic bioreactors enable the targeted starvation-chemotherapy to glioma.

Altering the glucose supply and the metabolic pathways would be an intriguing strategy in starvation therapy toward cancers. Nevertheless, starvation therapy alone could be inadequate to eliminate tumor cells completely. Herein, a multifunctional bioreactor was fabricated for synergistic starvation-chemotherapy through embedding glucose oxidase (GOx) and doxorubicin (DOX) in the tumor targeting ligands (RGD) modified red blood cell membrane camouflaged metal-organic framework (MOF) nanoparticle (denoted as RGD-mGZD). Owing to the remarkable biointerfacing property, the designed RGD-mGZD could not only possess enhanced blood retention time inherited from red blood cells, but also preferentially target the tumor site after the modification with RGD peptide. Once the bioreactor reached the desired region, GOx promptly consumed the intratumoral glucose and oxygen to starve cancer cells for robust starvation therapy. More importantly, the aggravated acidic microenvironment at the tumor region was found to induce the decomposition of the MOF structure, thus triggering the release of DOX for reinforced chemotherapy. This bioreactor would further prompt the development of synergistic patterns toward cancer treatment in a spatiotemporally controlled manner.

Fine-mapping and identification of a candidate gene controlling seed coat color in melon (Cucumis melo L. var. chinensis Pangalo).

KEY MESSAGE: MELO3C019554 encoding a homeobox protein (PHD transcription factor) is a candidate gene that involved in the formation of seed coat color in melon. Seed coat color is related to flavonoid content which is closely related to seed dormancy. According to the genetic analysis of a six-generation population derived from two parents (IC2508 with a yellow seed coat and IC2518 with a brown seed coat), we discovered that the yellow seed coat trait in melon is controlled by a single dominant gene, named CmBS-1. Bulked segregant analysis sequencing (BSA-Seq) revealed that the gene is located at 11,860,000-15,890,000 bp (4.03 Mb) on Chr 6. The F2 population was genotyped using insertion-deletions (InDels), from which cleaved amplified polymorphic sequence (dCAPS) markers were derived to construct a genetic map. The gene was then fine-mapped to a 233.98 kb region containing 12 genes. Based on gene sequence analysis with two parents, we found that the MELO3C019554 gene encoding a homeobox protein (PHD transcription factor) had a nonsynonymous single nucleotide polymorphism (SNP) mutation in the coding sequence (CDS), and the SNP mutation resulted in the conversion of an amino acid (A → T) at residue 534. In addition, MELO3C019554 exhibited lower relative expression levels in the yellow seed coat than in the brown seed coat. Furthermore, we found that MELO3C019554 is related to 12 flavonoid metabolites. Thus, we predicted that MELO3C019554 is a candidate gene controlling seed coat color in melon. The study lays a foundation for further cloning projects and functional analysis of this gene, as well as marker-assisted selection breeding.

Anti-cancer adjuvant drug screening via epithelial-mesenchymal transition-related aptamer probe.

Epithelial-mesenchymal transition (EMT) is implicated in the pathological processes of cancer metastasis and drug resistance. Anti-cancer drugs may also potentially lead to EMT, resulting in their reduced therapeutic effect. Therefore, the combination of these anti-cancer drugs with anti-EMT agents has been promoted in clinic. Screening anti-EMT drugs and evaluation of EMT process are highly dependent on EMT biomarkers on cell membrane. At present, the detection of EMT biomarker is mainly by Western blot method, which is time-consuming and complicated. In this work, for effectively screening anti-EMT drugs by evaluation of the EMT process, a type of aptamer probe based on aggregation-induced emission (AIE) was designed. The aptamer SYL3C was employed to target the EMT biomarker EpCAM on cell membrane. Two fluorophores, FAM and tetraphenylethene (TPE, an AIE dye), were modified at the two ends of SYL3C, respectively. This aptamer probe (TPE-SYL3C-FAM) can monitor the EpCAM expression, which can be recovered by anti-EMT drugs. By observation of the change in TPE emission intensity, the anti-EMT effect of drugs can be evaluated. The FAM emission was used as internal reference to reduce environmental interferences. This probe can be potentially used to screen anti-EMT agents as anti-cancer adjuvant drugs with high throughput.

In situ activation of therapeutics through bioorthogonal catalysis.

Bioorthogonal chemistry refers to any chemical reactions that can occur inside of living systems without interfering with native biochemical processes, which has become a promising strategy for modulating biological processes. The development of synthetic metal-based catalysts to perform bioorthogonal reactions has significantly expanded the toolkit of bioorthogonal chemistry for medicinal chemistry and synthetic biology. A wide range of homogeneous and heterogeneous transition metal catalysts (TMCs) have been reported, mediating different transformations such as cycloaddition reactions, as well as bond forming and cleaving reactions. However, the direct application of 'naked' TMCs in complex biological media poses numerous challenges, including poor water solubility, toxicity and catalyst deactivation. Incorporating TMCs into nanomaterials to create bioorthogonal nanocatalysts can solubilize and stabilize catalyst molecules, with the decoration of the nanocatalysts used to provide spatiotemporal control of catalysis. This review presents an overview of the advances in the creation of bioorthogonal nanocatalysts, highlighting different choice of nano-scaffolds, and the therapeutic and diagnostic applications.

GFP-fused yeast cells as whole-cell biosensors for genotoxicity evaluation of nitrosamines.

Nitrosamine compounds, represented by N-nitrosodimethylamine, are regarded as potentially genotoxic impurities (PGIs) due to their hazard warning structure, which has attracted great attention of pharmaceutical companies and regulatory authorities. At present, great research gaps exist in genotoxicity assessment and carcinogenicity comparison of nitrosamine compounds. In this work, a collection of GFP-fused yeast cells representing DNA damage repair pathways were used to evaluate the genotoxicity of eight nitrosamine compounds (10-6-105 µg/mL). The high-resolution expression profiles of GFP-fused protein revealed the details of the DNA damage repair of nitrosamines. Studies have shown that nitrosamine compounds can cause extensive DNA damage and activate multiple repair pathways. The evaluation criteria based on the total expression level of protein show a good correlation with the mammalian carcinogenicity data TD50, and the yeast cell collection can be used as a potential reliable criterion for evaluating the carcinogenicity of compounds. The assay based on DNA damage pathway integration has high sensitivity and can be used as a supplementary method for the evaluation of trace PGIs in actual production. KEY POINTS: • The genotoxicity mechanism of nitrosamines was systematically studied. • The influence of compound structure on the efficacy of genotoxicity was explored. • GFP-fused yeast cells have the potential to evaluate impurities in production.

Comprehensive Analysis of Transcriptome and Metabolome Reveals the Flavonoid Metabolic Pathway Is Associated with Fruit Peel Coloration of Melon.

The peel color is an important external quality of melon fruit. To explore the mechanisms of melon peel color formation, we performed an integrated analysis of transcriptome and metabolome with three different fruit peel samples (grey-green 'W', dark-green 'B', and yellow 'H'). A total of 40 differentially expressed flavonoids were identified. Integrated transcriptomic and metabolomic analyses revealed that flavonoid biosynthesis was associated with the fruit peel coloration of melon. Twelve differentially expressed genes regulated flavonoids synthesis. Among them, nine (two 4CL, F3H, three F3'H, IFS, FNS, and FLS) up-regulated genes were involved in the accumulation of flavones, flavanones, flavonols, and isoflavones, and three (2 ANS and UFGT) down-regulated genes were involved in the accumulation of anthocyanins. This study laid a foundation to understand the molecular mechanisms of melon peel coloration by exploring valuable genes and metabolites.

Circulating tumor DNA analysis for tumor diagnosis.

Tumor is a kind of abnormal organism generated by the proliferation and differentiation of cells in the body under the action of various initiating and promoting factors, which seriously threatens human life and health. Tumorigenesis is a gradual process that involves multistage reactions and the accumulation of mutations. Gene mutation usually occurs during tumorigenesis, and can be used for tumor diagnosis. Early diagnosis is the most effective way to improve the cure rate and reduce the mortality rate. Among the peripheral blood circulating tumor DNA (ctDNA), gene mutation in keeping with tumor cells can be detected, which can potentially replace tumor tissue section for early diagnosis. It has been considered as a liquid biopsy marker with good clinical application prospect. However, the high fragmentation and low concentration of ctDNA in blood result in the difficulty of tumor stage determination. Therefore, high sensitive and specific mutation detection methods have been developed to detect trace mutant ctDNA. At present, the approaches include digital PCR (dPCR), Bead, Emulsion, Amplification and Magnetic (BEAMing), Next Generation Sequencing (NGS), Amplification Refractory Mutation System (ARMS), etc. In this paper, the principle, characteristics, latest progress and application prospects of these methods are reviewed, which will facilitate researchers to choose appropriate ctDNA detection approaches.

QTLs and candidate genes analyses for fruit size under domestication and differentiation in melon (Cucumis melo L.) based on high resolution maps.

BACKGROUND: Melon is a very important horticultural crop produced worldwide with high phenotypic diversity. Fruit size is among the most important domestication and differentiation traits in melon. The molecular mechanisms of fruit size in melon are largely unknown. RESULTS: Two high-density genetic maps were constructed by whole-genome resequencing with two F2 segregating populations (WAP and MAP) derived from two crosses (cultivated agrestis × wild agrestis and cultivated melo × cultivated agrestis). We obtained 1,871,671 and 1,976,589 high quality SNPs that show differences between parents in WAP and MAP. A total of 5138 and 5839 recombination events generated 954 bins in WAP and 1027 bins in MAP with the average size of 321.3 Kb and 301.4 Kb respectively. All bins were mapped onto 12 linkage groups in WAP and MAP. The total lengths of two linkage maps were 904.4 cM (WAP) and 874.5 cM (MAP), covering 86.6% and 87.4% of the melon genome. Two loci for fruit size were identified on chromosome 11 in WAP and chromosome 5 in MAP, respectively. An auxin response factor and a YABBY transcription factor were inferred to be the candidate genes for both loci. CONCLUSION: The high-resolution genetic maps and QTLs analyses for fruit size described here will provide a better understanding the genetic basis of domestication and differentiation, and provide a valuable tool for map-based cloning and molecular marker assisted breeding.

Multiple adsorption properties of aptamers on metal-organic frameworks for nucleic acid assay.

Enrichment and detection of circulating free nucleic acids in biological samples have gained great attention for disease diagnosis or prognostic evaluation. Nanoscale metal-organic frameworks (NMOFs) have been used for aptamer-based nucleic acid sensing. In this work, different NMOFs, including ZIF-8, MIL-88, MIL-100, MIL-101, as well as Eu-TDA and Tb-TDA [prepared by the coordination of 2,2'-thiodiacetic acid (TDA) and Eu3+ or Tb3+], were investigated in nucleic acid sensing by employing their aptamer adsorption ability and fluorescence quenching capacity for the labeled dyes. Two types of dye aptamer, FAM-labeled aptamer (FAM-Ap) and TexasRedaptamer (TexasRed-Ap) were designed, and their adsorption properties on NMOFs-were compared. It was found that the TexasRed-Ap can be well used for nucleic acid (miR-21) extraction and sensing by linking with a pH-responsive nucleotide chain (TexasRed-Ap-pH) or with an additional random chain ssDNA-1' (TexasRed-Ap-a). After interacted with the target miR-21 in biosamples, the TexasRed-dsDNA + NMOFs composites can be collected, and the formed TexasRed-dsDNA can be released by changing pH value or addition of ssDNA-1, which is matched with ssDNA-1'. A linear relationship from 0.1 to 200 pM for miR-21 detection was obtained. The results show that the NMOFs can be used as promising platforms for nucleic acid extraction and fluorescent sensing.

Colorable Zeolitic Imidazolate Frameworks for Colorimetric Detection of Biomolecules.

We report a series of colorable zeolitic imidazolate framework (ZIF)-based nanomaterials prepared by encapsulating starches (amylopectin, dextrin, or amylose) or tannic acid in the frameworks of ZIFs and first applied them in colorimetric assay of microRNA/DNA by adding I2/KI or FeCl3 solutions as chromogenic reagents. We found that iodine molecules can lead to rapid degradation of the ZIF-8 framework, while ZIF-90 remains stable. Therefore, ZIF-90 was selected for encapsulating the starches or tannic acid, and then assembled with polyethylenimine (PEI) and aptamers of microRNA/DNA. After interacting with the target microRNA/DNA, the aptamers (Ap) move away from the surface of the prepared Ap-starch@ZIF-90 or Ap-tan@ZIF-90, and the I2/KI or FeCl3 solution is added into the system to interact the starches (amylopectin, dextrin, or amylose) or tannic acid to generate different colors. According to the absorbance spectra, good linear correlations between the logarithm of absorbance intensity and the concentration of microRNA (1-180 nM) can be observed, and the naked eye can distinguish the change from ∼60 to ∼180 nM with a concentration gradient of 20 nM. A similar colorimetric assay ability for pathogenic bacteria can also be realized by detecting the gene fragments IS200 and eaeA. The detection limits can be potentially optimized by changing the amount of adsorbed PEI and aptamers on the surface of Ap-starch@ZIF-90 (or Ap-tan@ZIF-90) nanoparticles. This method could be a promising alternative for simple and cost-effective assay of microRNA/DNA.