Environmentally relevant exposure to cotinine induces neurobehavioral toxicity in zebrafish (Danio rerio): A study using neurobehavioral and metabolomic approaches.

As an important psychoactive substance, cotinine is ubiquitous in aquatic environment and poses a threat to aquatic organisms. However, the mechanism of its adverse health impacts remains unclear. We evaluated the effects of cotinine exposure at environmentally relevant concentrations on the development and locomotor behavior of zebrafish (Danio rerio) larvae using neurotransmitters and whole endogenous metabolism. Mild developmental toxicity and significant neurobehavior disorder, such as spontaneous movement (1-1000 µg/L), 48 hpf tactile response (50, 100, and 1000 µg/L), and 144 hpf swimming speed (1, 10, 100, 500, and 1000 µg/L), were observed in zebrafish. Exposure to cotinine led to significant alterations in 11 neurotransmitters, including homogentisic acid, serotonin, glutamic acid and aspartic acid, etc. 298 metabolites were identified and two pathways - linoleic acid metabolism and taurine and hypotaurine metabolism - were delineated. In addition, amino acid neurotransmitters were significantly correlated with metabolites such as arachidonic acid as well as its derivatives, steroidal compounds, and amino acids. Serotonin demonstrates a noteworthy correlation with 31 out of 40 differentially expressed neurotransmitters, encompassing lipids, amino acids, and other compounds. These novel findings contribute to a comprehensive understanding of the ecological risks associated with cotinine contamination in surface waters.

Identification of key factors affecting neonicotinoid residues in crops and risk of dietary exposure.

Neonicotinoids, widely used on farmland, are ubiquitous in food; however, their distribution among various crops and associated exposure risks at the provincial level in China remain unclear. We collected 19 types of crop samples (fruits, vegetables, and tea) from farmland in nine prefectural cities in Zhejiang Province, China. We analyzed nine commonly used neonicotinoids in the edible portions of these crops. A notable detection rate (42.1 %-82.9 %) and high residual neonicotinoid concentrations (278 ± 357 ng/g) were observed. Tea exhibited the highest residue, followed by fruits, and vegetables showed the lowest (P < 0.05). Neonicotinoid ratios in crops to soil (R_C/S) and soil to water (R_S/W) were defined to discern insecticide distribution across different environments. Increased water solubility leads to increased migration of neonicotinoids (R_S/W) from agricultural soils to water through runoff, thereby increasing the relative contribution of nitenpyram and dinotefuran in water. In comparison with other studied compounds, all crops demonstrated the strongest soil uptake of thiamethoxam, denoted by the highest R_C/S value. Elevated R_C/S values in tea, pickled cabbage, and celery suggest increased susceptibility of these crops to neonicotinoid absorption from the soil (P < 0.05). Estimated dietary intake for teenagers, adults and elders was 8.9 ± 0.5, 8.9 ± 0.6, and 8.8 ± 0.3 µg/kg/d, respectively, below the reference dose (57 µg/kg/d). Teenagers, compared to adults and elders, exhibited significantly higher neonicotinoid exposure through fruit consumption, emphasizing the need for increased attention to neonicotinoid exposure among vulnerable populations.

The incorporation of Mg2+ ions into aragonite during biomineralization: Implications for the dolomitization of aragonite.

Bacteria can facilitate the increase of Mg2+ content in biotic aragonite, but the molecular mechanisms of the incorporation of Mg2+ ion into aragonite facilitated by bacteria are still unclear and the dolomitization of aragonite grains is rarely reported. In our laboratory experiments, the content of Mg2+ ions in biotic aragonite is higher than that in inorganically-precipitated aragonite and we hypothesize that the higher Mg content may enhance the subsequent dolomitization of aragonite. In this study, biotic aragonite was induced by Bacillus licheniformis Y1 at different Mg/Ca molar ratios. XRD data show that only aragonite was precipitated in the media with Mg/Ca molar ratios at 6, 9, and 12 after culturing for 25 days. The EDS and atomic absorption results show that the content of Mg2+ ions in biotic aragonite increased with rising Mg/Ca molar ratios. In addition, our analyses show that the EPS from the bacteria and the organics extracted from the interior of the biotic aragonite contain the same biomolecules, including Ala, Gly, Glu and hexadecanoic acid. The content of Mg2+ ions in the aragonite precipitates mediated by biomolecules is significantly higher than that in inorganically-precipitated aragonite. Additionally, compared with Ala and Gly, the increase of the Mg2+ ions content in aragonite promoted by Glu and hexadecanoic acid is more significant. The DFT (density functional theory) calculations reveal that the energy needed for Mg2+ ion incorporation into aragonite mediated by Glu, hexadecanoic acid, Gly and Ala increased gradually, but was lower than that without acidic biomolecules. The experiments also show that the Mg2+ ion content in the aragonite significantly increased with the increasing concentration of biomolecules. In a medium with high Mg2+ concentration and with bacteria, after 2 months, micron-sized dolomite rhombs were precipitated on the surfaces of the aragonite particles. This study may provide new insights into the important role played by biomolecules in the incorporation of the Mg2+ ions into aragonite. Moreover, these experiments may contribute towards our understanding of the dolomitization of aragonite in the presence of bacteria.

Genome-wide association mapping identifies an SNF4 ortholog that impacts biomass and sugar yield in sorghum and sugarcane.

Sorghum is a feed/industrial crop in developed countries and a staple food elsewhere in the world. This study evaluated the sorghum mini core collection for days to 50% flowering (DF), biomass, plant height (PH), soluble solid content (SSC), and juice weight (JW), and the sorghum reference set for DF and PH, in 7-12 testing environments. We also performed genome-wide association mapping with 6 094 317 and 265 500 single nucleotide polymorphism markers in the mini core collection and the reference set, respectively. In the mini core panel we identified three quantitative trait loci for DF, two for JW, one for PH, and one for biomass. In the reference set panel we identified another quantitative trait locus for PH on chromosome 6 that was also associated with biomass, DF, JW, and SSC in the mini core panel. Transgenic studies of three genes selected from the locus revealed that Sobic.006G061100 (SbSNF4-2) increased biomass, SSC, JW, and PH when overexpressed in both sorghum and sugarcane, and delayed flowering in transgenic sorghum. SbSNF4-2 encodes a γ subunit of the evolutionarily conserved AMPK/SNF1/SnRK1 heterotrimeric complexes. SbSNF4-2 and its orthologs will be valuable in genetic enhancement of biomass and sugar yield in plants.

Development and Internal Validation of a Preoperative Prediction Model for Sentinel Lymph Node Status in Breast Cancer: Combining Radiomics Signature and Clinical Factors.

PURPOSE: To develop and internally validate a nomogram combining radiomics signature of primary tumor and fibroglandular tissue (FGT) based on pharmacokinetic dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and clinical factors for preoperative prediction of sentinel lymph node (SLN) status in breast cancer patients. METHODS: This study retrospectively enrolled 186 breast cancer patients who underwent pretreatment pharmacokinetic DCE-MRI with positive (n = 93) and negative (n = 93) SLN. Logistic regression models and radiomics signatures of tumor and FGT were constructed after feature extraction and selection. The radiomics signatures were further combined with independent predictors of clinical factors for constructing a combined model. Prediction performance was assessed by receiver operating characteristic (ROC), calibration, and decision curve analysis. The areas under the ROC curve (AUCs) of models were corrected by 1,000-times bootstrapping method and compared by Delong's test. The added value of each independent model or their combinations was also assessed by net reclassification improvement (NRI) and integrated discrimination improvement (IDI) indices. This report referred to the "Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis" (TRIPOD) statement. RESULTS: The AUCs of the tumor radiomic model (eight features) and the FGT radiomic model (three features) were 0.783 (95% confidence interval [CI], 0.717-0.849) and 0.680 (95% CI, 0.604-0.757), respectively. A higher AUC of 0.799 (95% CI, 0.737-0.862) was obtained by combining tumor and FGT radiomics signatures. By further combining tumor and FGT radiomics signatures with progesterone receptor (PR) status, a nomogram was developed and showed better discriminative ability for SLN status [AUC 0.839 (95% CI, 0.783-0.895)]. The IDI and NRI indices also showed significant improvement when combining tumor, FGT, and PR compared with each independent model or a combination of any two of them (all p < 0.05). CONCLUSION: FGT and clinical factors improved the prediction performance of SLN status in breast cancer. A nomogram integrating the DCE-MRI radiomics signature of tumor and FGT and PR expression achieved good performance for the prediction of SLN status, which provides a potential biomarker for clinical treatment decision-making.

Selective Adsorption of Amino Acids in Crystals of Monohydrocalcite Induced by the Facultative Anaerobic Enterobacter ludwigii SYB1.

The morphology, crystal structure, and elemental composition of biominerals are commonly different from chemically synthesized minerals, but the reasons for these are not fully understood. A facultative anaerobic bacterium, Enterobacter ludwigii SYB1, is used in experiments to document the hydrochemistry, mineral crystallization, and cell surface characteristics of biomineralization. It was found that carbonate anhydrase and ammonia production were major factors influencing the alkalinity and saturation of the closed biosystem. X-ray diffraction (XRD) spectra showed that calcite, monohydrocalcite (MHC), and dypingite formed in samples with bacterial cells. It was also found that the (222) plane of MHC was the preferred orientation compared to standard data. Scanning transmission electron microscopy (STEM) analysis of cell slices provides direct evidence of concentrated calcium and magnesium ions on the surface of extracellular polymeric substances (EPS). In addition, high-resolution transmission electron microscopy (HRTEM) showed that crystallized nanoparticles were formed within the EPS. Thus, the mechanism of the biomineralization induced by E. ludwigii SYB1 can be divided into three stages: (i) the production of carbonate anhydrase and ammonia increases the alkalinity and saturation state of the milieu, (ii) free calcium and magnesium ions are adsorbed and chelated onto EPS, and (iii) nanominerals crystallize and grow within the EPS. Seventeen kinds of amino acids were identified within both biotic MHC and the EPS of SYB1, while the percentages of glutamic and aspartic acid in MHC increased significantly (p < 0.05). Furthermore, the adsorption energy was calculated for various amino acids on seven diffracted crystal faces, with preferential adsorption demonstrated on (111) and (222) faces. At the same time, the lowest adsorption energy was always that of glutamic and aspartic acid for the same crystal plane. These results suggest that aspartic and glutamic acid always mix preferentially in the crystal lattice of MHC and that differential adsorption of amino acids on crystal planes can lead to their preferred orientation. Moreover, the mixing of amino acids in the mineral structure may also have a certain influence on the mineral lattice dislocations, thus enhancing the thermodynamic characteristics.

An efficient sorghum transformation system using embryogenic calli derived from mature seeds.

Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The nptII and gus gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T1 seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.

An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9.

Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×106 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.