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.

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.

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.

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.

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.

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.

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.

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.

Insight into the Unique Fluorescence Quenching Property of Metal-Organic Frameworks upon DNA Binding.

Metal-organic frameworks (MOFs) have been successfully used as efficient quenchers for fluorescent DNA detection. However, the surface charge property of MOFs can inevitably affect their fluorescence quenching behavior. Herein, nanoscale MOFs (NMOFs), including MOF nanosheets and nanoparticles, have been employed to investigate the relationship between the fluorescence quenching and surface properties of NMOFs. We find that the positively and negatively charged NMOFs exhibited totally opposite fluorescence quenching properties toward negatively charged FAM-labeled double-stranded DNA (dsDNA). On the contrast, they show negligible influence on the sensing of positively charged TAMRA-labeled dsDNA. This study provides a new insight of the fluorescence quenching property of NMOFs and offers a new concept for construction of ratiometric fluorescence DNA biosensors.

Divergence between C. melo and African Cucumis Species Identified by Chromosome Painting and rDNA Distribution Pattern.

The 5S and 45S rDNA sites are useful chromosome landmarks and can provide valuable information about karyotype evolution and species interrelationships. In this study, we employed fluorescence in situ hybridization (FISH) to determine the number and chromosomal location of 5S and 45S rDNA loci in 8 diploid Cucumis species. Two oligonucleotide painting probes specific for the rDNA-bearing chromosomes in C. melo were hybridized to other Cucumis species in order to investigate the homeologies among the rDNA-carrying chromosomes in Cucumis species. The analyzed diploid species showed 3 types of rDNA distribution patterns, which provided clear cytogenetic evidence on the divergence between C. melo and wild diploid African Cucumis species. The present results not only show species interrelationships in the genus Cucumis, but the rDNA FISH patterns can also be used as cytological markers for the discrimination of closely related species. The data will be helpful for breeders to choose the most suitable species from various wild species for improvement of cultivated melon.

Fluorescent Sulfur-Tagged Europium(III) Coordination Polymers for Monitoring Reactive Oxygen Species.

Oxidative stress caused by reactive oxygen species (ROS) is harmful to biological systems and implicated in various diseases. A variety of selective fluorescent probes have been developed for detecting ROS to uncover their biological functions. Generally, the preparation of the fluorescent probes usually undergoes multiple synthetic steps, and the successful fluorescent sensing usually relies on trial-and-error tests. Herein we present a simple way to prepare fluorescent ROS probes that can be used both in biological and environmental systems. The fluorescent europium(III) coordination polymers (CPs) are prepared by simply mixing the precursors [2,2'-thiodiacetic acid and Eu(NO3)3·6H2O] in ethanol. Interestingly, with the increase of reaction temperature, the product undergoes a morphological transformation from microcrystal to nanoparticle while the structure and fluorescent properties retain. The fluorescence of the sulfur-tagged europium(III) CPs can be selectively quenched by ROS, and thus, sensitive and selective monitoring of ROS in aerosols by the microcrystals and in live cells by the nanoparticles has been achieved. The results reveal that the sulfur-tagged europium(III) CPs provide a novel sensor for imaging ROS in biological and environmental systems.

Core-shell Ag@SiO(2) nanoparticles concentrated on a micro/nanofluidic device for surface plasmon resonance-enhanced fluorescent detection of highly reactive oxygen species.

A micro/nanofluidic device integrating a nanochannel in a microfluidic chip was developed for sensitive fluorescent determination of highly reactive oxygen species (hROS) enhanced by surface plasmon resonance-enhanced fluorescence (SPREF). The nanochannel was simply fabricated by polyaniline nanostructures modified on a glass slide. Core-shell Ag@SiO2 nanoparticles were concentrated in front of the nanochannel for fluorescence enhancement based on the SPREF effect. As a demonstration, hROS in the mainstream of cigarette smoke (CS) were detected by the present micro/nanofluidic device. The fluorescent probe for trapping hROS in puffs of CS employed a microcolumn that was loaded with a composite of DNA (conjugated fluorophores, FAM) and Au membrane (coated on cellulose acetate). With a laser-induced fluorescence detection device, hROS was determined on the basis of the amount of FAM groups generated by DNA cleavage. With the optimization of the trapping efficiency, we detected about 4.91 pmol of hROS/puff in the mainstream CS. This micro/nanofluidic-SPREF system promises a simple, rapid, and highly sensitive approach for determination of hROS in CS and other practical systems.

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.

Preparation of bi-layer-polymer coated silica using atom transfer radical polymerization and its application as restricted access phase for HPLC separation of hydrophobic molecules in biological fluids.

A novel restricted access material was prepared by surface initiated atom transfer radical polymerization. The bi-layer-polymer structures were created on the surface of silica layer-by-layer. The inner layer was composed of poly(styrene-co-divinylbenzene), which was grafted first for binding small molecules based on hydrophobic and π-π interactions. The poly(styrene-co-divinylbenzene) bonded silica has good selectivity for aromatic hydrocarbons. It also has hydrophobicity and column efficiency similar to a C(18) bonded silica. The material has shown good ability of protein exclusion after grafting hydrophilic polymer on the external surface while its hydrophobicity and selectivity do not have obvious change. It demonstrated that the material is still qualified for hydrophobic extraction. In the study, the relations between the polymer structures and chromatographic properties of the materials were investigated. The synthetic conditions were optimized. The results have shown that the material prepared in the study has application potential in the HPLC analysis of hydrophobic molecules from biological samples by direct injection. It demonstrated that atom transfer radical polymerization can be used as a method in the preparation of restricted access material.

[FTIR spectral fitting algorithm based on continuous wavelet transform].

An FTIR spectrum fitting algorithm based on continuous wavelet transform is proposed. In calculating the factor of difference spectrum, the algorithm takes into account both the original spectrum and its continuous wavelet transformed spectra, which effectively overcomes the problem of reference peak selection and manual factor selection in most commercial software. The detailed discussions on wavelet scale, order and basis are included. The spectral fitting is performed on six wavelet basis functions and the obtained scale factor is used to quantify the content of liquor, and the corresponding mean absolute error ranges from 0.047 to 0.072, and the standard deviation ranges from 0.056 to 0.091. Experimental results show that the CWT combined with least squares fitting provides an accurate and reliable new method for FTIR spectral subtraction.

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.

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.

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.