[Efficacy of Bright Greenish-Yellow Fluorescence Sorting on Mycotoxin-Contaminated Nutmeg Selection and Characterization of Fungal Flora Related to Mycotoxin Production].

Spices have been known to be highly contaminated commodities with mycotoxins. The Codex Alimentarius reports that nutmeg is particularly contaminated with aflatoxins (AFs) and ochratoxin A (OTA). To eliminate contaminated commodities, visual sorting and bright greenish-yellow fluorescence (BGYF) sorting are used as low-cost technologies in production engineering. In Indonesia, nutmeg is mainly sorted by visual sorting and classified into three grades according to the Indonesian national standards, with importers further defining their own brand as imported products. In this study, we evaluate the efficacy of BGYF sorting as a further selection method to reduce AFs and OTA using the importer's own brand. Further, the level of these mycotoxins and the relationship between fungal flora and mycotoxin contamination were examined. These results showed that BGYF sorting effectively reduces AFs as well as OTA. In addition, BGYF-positive groups were infected by Aspergillus sections Flavi, Nigri, and Circumdati.

A bifunctional selectable marker for wheat transformation contributes to the characterization of male-sterile phenotype induced by a synthetic Ms2 gene.

KEY MESSAGE: An engineered selectable marker combining herbicide resistance and yellow fluorescence contributes to the characterization of male-sterile phenotype in wheat, the severity of which correlates with expression levels of a synthetic Ms2 gene. Genetic transformation of wheat is conducted using selectable markers, such as herbicide and antibiotic resistance genes. Despite their proven effectiveness, they do not provide visual control of the transformation process and transgene status in progeny, which creates uncertainty and prolongs screening procedures. To overcome this limitation, this study developed a fusion protein by combining gene sequences encoding phosphinothricin acetyltransferase and mCitrine fluorescent protein. The fusion gene, introduced into wheat cells by particle bombardment, enabled herbicide selection, and visual identification of primary transformants along with their progeny. This marker was then used to select transgenic plants containing a synthetic Ms2 gene. Ms2 is a dominant gene whose activation in wheat anthers leads to male sterility, but the relationship between the expression levels and the male-sterile phenotype is unknown. The Ms2 gene was driven either by a truncated Ms2 promoter containing a TRIM element or a rice promoter OsLTP6. The expression of these synthetic genes resulted in complete male sterility or partial fertility, respectively. The low-fertility phenotype was characterized by smaller anthers than the wild type, many defective pollen grains, and low seed sets. The reduction in the size of anthers was observed at earlier and later stages of their development. Consistently, Ms2 transcripts were detected in these organs, but their levels were significantly lower than those in completely sterile Ms2TRIM::Ms2 plants. These results suggested that the severity of the male-sterile phenotype was modulated by Ms2 expression levels and that higher levels may be key to activating total male sterility.

Intraoperative application of yellow fluorescence in resection of intramedullary spinal canal ependymoma.

BACKGROUND: Spinal ependymoma is the most common intramedullary tumor in adults. This study was performed to evaluate whether intraoperative yellow fluorescence use enhances our ability to identify the tumor margin and residual tumor tissue in intramedullary spinal cord ependymoma resection. We also evaluated patients' clinical conditions at a 3-month follow-up. METHODS: We retrospectively evaluated 56 patients with intramedullary ependymoma. Thirty minutes before anesthesia, the patients received intravenous sodium fluorescein injections. Tumor resection was performed under two illumination modes, traditional white light and yellow fluorescence, and the residual tumor tissue was detected. Magnetic resonance imaging was performed 3 months postoperatively to observe the tumor resection outcome and residual tumor tissue. The McCormick spinal cord function grade was evaluated preoperatively and 3 months postoperatively. RESULTS: The total resection rate was 100.0% in all patients. Nine patients had no significant fluorescence imaging. After 3 months, patients with a spinal function grade of I to IV showed significant spinal function improvement. Magnetic resonance imaging showed no residual tumor tissue or recurrence. CONCLUSION: Sodium fluorescein aids in total excision of intramedullary spinal cord ependymoma and intraoperative residual tumor tissue identification. At the 3-month follow-up, the patients' functional outcome in the fluorescein group was good.

Thiourea-functionalized graphene aerogel for the aqueous phase sensing of toxic Pb(II) metal ions and H2O2.

A simpler approach of functionalization for the fabrication of thiourea-functionalized-Graphene Aerogel (t-GA) is described here. Graphene Aerogel (GA) was synthesized from bio-mass, which on a simpler oxidative treatment get converted to its water-soluble version due to the impregnation of several oxygenous functionalities like carboxylic, hydroxyl, etc. Further, these carboxylated groups have been functionalized with the molecules of thiourea using the long known dicyclohexylcarbodiimide (DCC) as a coupling agent. The as-synthesized t-GA shows bright yellow fluorescence with a quantum yield of ~3% and holds the high-aqueous solubility and photostability. The fluorescence property of t-GA has been used here for the specific and selective sensing of toxic lead (Pb(II)) metal ions from the used many other metal ions via the fluorescence quenching and showed a limit of detection ~7.3 nM. Further, the mechanism for selective sensing was studied in detail and found to be preferable via ligand to metal charge transfer quenching mechanism. The cyclic voltammetry studies supported the selective sensing of Pb(II). Moreover, t-GA has also been studied for the sensing of hydrogen peroxide and as a yellow fluorescent ink.

Preparation of yellow-emitting carbon dots and their bifunctional detection of tetracyclines and Al3+ in food and living cells.

A novel bifunctional carbon dot (CD)-based sensing platform was constructed for detection of tetracyclines (TCs) and Al3+. The fluorescence CDs were fabricated by hydrothermal method using phenylenediamine (p-PD) and ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) as precursors. The obtained prepared CDs show bright yellow fluorescence (y-CDs, EX = 400 nm and Em = 556 nm), high fluorescence quantum yield (QY = 21.55 ± 0.06%), and preferable optical stability. TCs can directly quench the fluorescence of y-CDs based on static quenching characteristics and a small internal filtration effect (IEF). By adding Al3+ to the y-CDs + TCs system, the fluorescence is partly recovered because TCs escape from the surface of the y-CDs and form a more stable chelate with Al3+. The sensing platform displays good selectivity and high sensitivity to TCs and Al3+ with low detection limits of 0.057-0.23 µM and 0.091 µM, respectively. Importantly, this sensing platform has enabled the detection of TCs and Al3+ in milk samples with satisfactory recoveries and RSDs, confirming the reliability and feasibility of this method. Combining with low toxicity and preferable biocompatibility, the y-CDs are extended to cellular imaging and detection of CTC and Al3+ in A549 cells.

Relationship between the sodium fluorescein yellow fluorescence boundary and the actual boundary of high-grade gliomas during surgical resection.

OBJECTIVE: Resection of high-grade glioma with sodium fluorescein can improve the resection rate of the glioma and improve survival. However, it is unclear whether the yellow fluorescence boundary of the high-grade glioma is consistent with the actual boundary of the tumor. This study explores the yellow fluorescence boundary and the actual tumor boundary in high-grade glioma surgery. METHODS: This is a retrospective analysis of 10 patients with high-grade gliomas who underwent tumor visualization with sodium fluorescein. After staining of the tumor, random selections of both developed and non-developed yellow fluorescent border tissue at the fluorescence chromogenic boundary were made, followed by pathological examination. Claudin-5, an important component of the tight connections between vascular endothelial cells, was assessed by immunohistochemistry and qRT-PCR in the tumor and surrounding tissues in order to determine the tumor cell content of the tissue, blood-brain barrier damage, and vascular proliferation. The yellow fluorescence boundary was compared with the actual tumor boundary and the results analyzed. RESULTS: Tumor cells were still detected outside the yellow fluorescence boundary during high-grade glioma surgery (P < 0.05). Claudin-5 expression was higher in high-grade gliomas than in adjacent normal tissues (P < 0.05), while disconnected Claudin-5 expression was associated with intraoperative yellow fluorescence imaging (r = 0.67). CONCLUSIONS: There is a difference between the yellow fluorescence boundary and the actual boundary of the tumor in high-grade glioma, and there are glioma cell infiltrations in the brain tissue of the undeveloped yellow fluorescent border. To ensure patient recovery and function, it is recommended that tumor resection be expanded based on yellow fluorescence visualization. Claudin-5 is overall up-regulated in high-grade gliomas, but some Claudin-5 expression is disconnected. This Claudin-5 expression pattern may be related to the development of yellow fluorescence.

A fluorometric and colorimetric dual-readout nanoprobe based on Cl and N co-doped carbon quantum dots with large stokes shift for sequential detection of morin and zinc ion.

A novel visual nanoprobe was developed for the sequential detection of morin and zinc ion (Zn2+) based on Cl and N co-doped carbon quantum dots (ClNCQDs) via a fluorometric and colorimetric dual-readout mode. The yellow fluorescence ClNCQDs was synthesized by the one-step hydrothermal treatment of o-chlorobenzoic acid and p-phenylenediamine. The most distinctive property of the ClNCQDs is the large stokes shift (177 nm), which is significantly higher than other reported CQDs. The fluorescence of the ClNCQDs can be effectively quenched by morin based on the synergistic effect of IFE, electrostatic interaction, and dynamic quenching process, and recovered upon the addition of Zn2+ due to strong interaction between morin and Zn2+. The nanoprobe exhibited favorable selectivity and sensitivity toward morin and Zn2+ with detection limits of 0.09 µM and 0.17 µM, respectively. Simultaneously, the color of the ClNCQDs solution was changed (light-pink â†’ faint-yellow â†’ dark-yellow) along with the variation of the fluorescence signal of the ClNCQDs. This proposed nanoprobe was successfully applied for morin and Zn2+ analyses in actual samples and live cells with high accuracy. The results of this study demonstrate the great application prospects of the ClNCQDs for morin and Zn2+ detection in complex actual samples and biosystems.

Yellow emissive Se,N-codoped carbon dots toward sensitive fluorescence assay of crystal violet.

Crystal violet (CV), a hazardous dye, poses a serious threat to the environment and human health. This motivates us to develop a facile method for its sensitive detection. Herein, we demonstrate a rapid sensing of CV using a novel fluorescent nanomaterial, yellow emissive Se,N-codoped carbon dots (CDs). CDs with an intense photoluminescence peak at 566 nm are synthesized by a hydrothermal technique using selenourea and o-phenylenediamine as precursors. This material shows a high quantum yield of up to 16.7 %. It is found that the yellow fluorescence of CDs can be selectively quenched by CV, which makes them promising for CV sensing. The linearity is obeyed in the range of 0.02-1.60 µM, and the limit of detection is as low as 7.3 nM. After detailed investigations, the inner filter effect is proposed to be the sensing mechanism. For practical usage, the newly built method is applied to determine the trace amount of CV in fish tissue samples, and satisfactory results are obtained.

Whole Genome Sequencing and Root Colonization Studies Reveal Novel Insights in the Biocontrol Potential and Growth Promotion by Bacillus subtilis MBI 600 on Cucumber.

Bacillus spp. MBI 600 is a gram-positive bacterium and is characterized as a PGPR strain involved in plant growth promotion and control of various plant pathogens which has recently been introduced into the agricultural practice. In this study we performed a Next Generation Sequencing analysis, to analyze the full genome of this microorganism and to characterize it taxonomically. Results showed that MBI 600 strain was phylogenetically close to other Bacillus spp. strains used as biocontrol agents and identified as B. subtilis. GOG analysis showed clusters contributed to secondary metabolites production such as fengycin and surfactin. In addition, various genes which annotated according to other plant-associated strains, showed that play a main role in nutrient availability from soil. The root colonization ability of MBI 600 strain was analyzed in vivo with a yellow fluorescence protein (yfp) tag. Confocal laser scanning microscopy of cucumber roots treated with yfp-tagged MBI 600 cells, revealed that the strain exhibits a strong colonization ability of cucumber roots, although it is affected significantly by the growth substrate of the roots. In vitro and in planta experiments with MBI 600 strain and F. oxysporum f.sp. radicis cucumerinum and P. aphanidernatum, showed a high control ability against these soilborne pathogens. Overall, our study demonstrates the effectiveness of MBI 600 in plant growth promotion and antagonism against different pathogens, highlighting the use of this microorganism as a biocontrol agent.

Yellow-emissive carbon dots as a fluorescent probe for chromium(VI).

The authors describe a one-step method for the preparation of yellow fluorescent carbon dots (CDs) starting from 4-aminoacetanilide hydrochloride and 4-acetamidobenzaldehyde. The CDs have excitation/emission peaks at 470/550 nm, good water solubility, salt-tolerance and photostability. Their fluorescence is quenched by hexavalent chromium [Cr(VI)] via static quenching. Fluorescence intensity drops linearly in the 1 to 400 µM Cr(VI) concentration range, and the limit of detection is 0.13 µM. This method is selective for Cr(VI) over potential metal ion interferences and was successfully applied to the detection of Cr(VI) in spiked water and biological tissue samples. Recoveries from spiked samples ranged from 97.7% to 103.8%. Graphical abstract Schematic presentation of (a) the preparation of the CD fluorescent probe and (b), the principle of Cr(VI) determination.

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates.

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4's specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.

OPTIMIZATION OF THREE-CUBE FRET ASSAY AND APPLICATION IN THE ANALYSIS OF SUBUNIT ASSEMBLING OF iGluR RECEPTORS IN LIVING CELLS / 解剖学报

Objective To establish and optimize three-cube FRET assay in living cells and analyze subunit assembling of iGluR receptors. Methods Taking HEK293 cells cotransfed with pECFP and pEYFP as negative control, and those transfected with pECFP-YFP as positive control,different calculation methods using fluorescence microscopy were compared. Results These calculation methods were all suitable for FRET measurement in the system. but the measurement results were affected by the ratio of Donor/Acceptor (D/A) in some degree,and different calculation methods have different optimized conditions. FRET measurement using FR value showed subunit specific assembly of iGluR subtypes.Conclusion There are different optimized conditions for these different calculation methods in the three-cube FRET measurement system,and a further evidence is provided for subunit specific assembling of iGluR subtypes from the FRET assay.