Development of a highly sensitive ultra-small ratiometric fluorescence nanosphere probe for Sunset Yellow detection.

A highly sensitive ultra-small ratiometric fluorescence nanosphere probe was successfully manufactured to detect Sunset Yellow (SY). The probe, CMCS@N, S-CDs/Rh6G, was formed through the encapsulation of N, S-CDs and Rh6G within carboxymethyl chitosan (CMCS) through in situ cross-linking. Remarkably, our nanosphere probe had an average grain diameter of 6.80 nm and exhibited excellent dispersibility without the need for additional solvents. The probe exhibited a strong linear relationship with SY concentration in the range of 0.26-100 µM, with a low detection limit of 0.078 µM. Furthermore, SY demonstrated strong fluorescence quenching capability on our nanosphere probe, with the fluorescence quenching mechanism involving a combined effects of inner filter effect (IFE) and static quenching. Notably, our nanosphere probe retained the bacteriostatic properties of CMCS, with a substantial bacteriostasis rate of 77.58 %, introducing novel potential applications.

Neuroprotective Effects and Therapeutic Potential of Dichloroacetate: Targeting Metabolic Disorders in Nervous System Diseases.

Dichloroacetate (DCA) is an investigational drug used to treat lactic acidosis and malignant tumours. It works by inhibiting pyruvate dehydrogenase kinase and increasing the rate of glucose oxidation. Some studies have documented the neuroprotective benefits of DCA. By reviewing these studies, this paper shows that DCA has multiple pharmacological activities, including regulating metabolism, ameliorating oxidative stress, attenuating neuroinflammation, inhibiting apoptosis, decreasing autophagy, protecting the blood‒brain barrier, improving the function of endothelial progenitor cells, improving mitochondrial dynamics, and decreasing amyloid ß-protein. In addition, DCA inhibits the enzyme that metabolizes it, which leads to peripheral neurotoxicity due to drug accumulation that may be solved by individualized drug delivery and nanovesicle delivery. In summary, in this review, we analyse the mechanisms of neuroprotection by DCA in different diseases and discuss the causes of and solutions to its adverse effects.

Corrigendum: Identification of microRNA-92a-3p as an essential regulator of tubular epithelial cell pyroptosis by targeting Nrf1 via HO-1.

[This corrects the article DOI: 10.3389/fgene.2020.616947.].

Novel N-doped carbon dots derived from citric acid and urea: fluorescent sensing for determination of metronidazole and cytotoxicity studies.

Blue emitting nitrogen-doped carbon dots were synthesized using citric acid and urea through the hydrothermal method, and the fluorescence quantum yield was 35.08%. We discovered that N-CDs featured excellent robust fluorescence stability and chemical resistance. For metronidazole detection, our N-CDs exhibited quick response time, high selectivity and sensitivity, and low cytotoxicity. Specifically, our N-CDs could detect metronidazole in the linear range of 0-179 µM, and the LOD was 0.25 µM. Furthermore, metronidazole efficaciously quenches the fluorescence of N-CDs, possibly owing to the inner filter effect. Lastly, we have employed our N-CDs to detect metronidazole in commercial metronidazole tablets with high accuracy. Overall, the newly prepared fluorescence sensor, N-CDs, demonstrated a huge potential to detect metronidazole in a simple, efficient, sensitive, and rapid manner.

[Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay].

For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 µmol/L Mpro and 5 µmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1, 4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.

[Identifying SARS-CoV-2 main protease inhibitors by a novel sandwich-like fluorescence polarization screening assay].

SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1, 2, 3, 4, 6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.

Preparation of Bi/BiOBr sensitized titania nanorod arrays via a one-pot solvothermal method and construction of kanamycin photoelectrochemical aptasensors.

In this work, a photoelectrochemical (PEC) aptasensor for detecting kanamycin (KAN) was designed based on an aptamer modified Bi/BiOBr/titania nanorod array (TiO2 NRA). Bi/BiOBr was loaded onto the TiO2 NRA via a one-pot solvothermal method using glucose as a reductant. The p-n heterojunction structure constructed from chrysanthemums like BiOBr and the TiO2 NRA improves the electron transfer rate. Combined with metal Bi with the surface plasmon resonance (SPR) effect, it further increases the absorption range of visible light and enhances the light response performance of the PEC aptasensor. The KAN aptamer is fixed to the Bi/BiOBr/TiO2 NRA photoelectric material through the CN structure. Once the aptamer precisely captures KAN molecules, photocurrent changes are generated to realize the detection of KAN. The designed PEC aptasensor shows good detection performance in the linear response range of 1 pM-200 nM, and the detection limit is 0.7 pM (S/N = 3). The aptasensor was applied to the determination of KAN in milk with satisfactory results.

Transforming waste into value: pomelo-peel-based nitrogen-doped carbon dots for the highly selective detection of tetracycline.

Tetracycline (TC) is widely used as a veterinary drug, and its residue in livestock products could enter the human body and cause damage. In this study, we developed an eco-friendly approach that utilized pomelo peel as a carbon source to synthesize new water-soluble N-doped carbon dots (P-NCDs) with blue fluorescence, obtaining a high quantum yield of up to 76.47% and achieving the goal of turning waste into value. Our prepared P-NCDs can selectively recognized TC, and their fluorescence was quenched based on the IFE. P-NCDs could measure the TC concentration in the linear range of 0-100 µmol L-1 with a detection limit (LOD, S/N = 3) as low as 0.045 µmol L-1. Furthermore, we have successfully applied our P-NCDs to the detection of TC in milk samples with convincing results within 90 s. Overall, our newly synthesized fluorescent sensor, P-NCDs, demonstrated huge potential to become an alternative way to detect TC in a simple, efficient, sensitive way without using any special instruments.

Integration detection of mercury(ii) and GSH with a fluorescent "on-off-on" switch sensor based on nitrogen, sulfur co-doped carbon dots.

Using aurine and citric acid as precursors, we have synthesized stable blue-fluorescent nitrogen and sulfur co-doped carbon dots (NS-CDs), with a high quantum yield of up to 68.94% via a thermal lysis method. The fluorescent NS-CDs were employed as a sensitive sensor for the integration detection of Hg2+ and glutathione (GSH). This was attributed to Hg2+ effectively quenching the fluorescence of the NS-CDs by static quenching, and then GSH was able to recover the fluorescence owing to the stronger binding between Hg2+ and the sulfhydryl of GSH. Based on the "on-off-on" tactic, the detection limits of Hg2+ ions and GSH were 50 nM and 67 nM respectively. The fluorescence sensor was successfully applied to detect Hg2+ ions and GSH in actual samples (tap water and fetal bovine serum). Furthermore, we have proved that the sensor had good reversibility. Overall, our NS-CDs can serve as effective sensors for environmental and biological analysis in the future.

Construction of N-CDs and Calcein-Based Ratiometric Fluorescent Sensor for Rapid Detection of Arginine and Acetaminophen.

In our study, a unique ratiometric fluorescent sensor for the rapid detection of arginine (Arg) and acetaminophen (AP) was constructed by the integration of blue fluorescent N-CDs and yellowish-green fluorescent calcein. The N-CD/calcein ratiometric fluorescent sensor exhibited dual emission at 435 and 519 nm under the same excitation wavelength of 370 nm, and caused potential Förster resonance energy transfer (FRET) from N-CDs to calcein. When detecting Arg, the blue fluorescence from the N-CDs of the N-CD/calcein sensor was quenched by the interaction of N-CDs and Arg. Then, the fluorescence of our sensor was recovered with the addition of AP, possibly due to the stronger association between AP and Arg, leading to the dissociation of Arg from N-CDs. Meanwhile, we observed an obvious fluorescence change from blue to green, then back to blue, when Arg and AP were added, exhibiting the "on-off-on" pattern. Next, we determined the detection limits of the N-CD/calcein sensor to Arg and AP, which were as low as 0.08 µM and 0.02 µM, respectively. Furthermore, we discovered that the fluorescence changes of the N-CD/calcein sensor were only responsible for Arg and AP. These results suggested its high sensitivity and specificity for Arg and AP detection. In addition, we have successfully achieved its application in bovine serum samples, indicating its practicality. Lastly, the logic gate was generated by the N-CD/calcein sensor and presented its good reversibility. Overall, we have demonstrated that our N-CD/calcein sensor is a powerful sensor to detect Arg and AP and that it has potential applications in biological analysis and imaging.

A Continuously Tunable Full-Color Emission Nitrogen-Doped Carbon Dots and for Ultrasensitive and Highly Selective Detection of Ascorbic Acid.

Nitrogen-doped carbon dots exhibiting excitation-dependent full-color emissions (F-NCDs) were prepared via the one-step hydrothermal method with citric acid and phenylenediamine. Specifically, the emission wavelength of the F-NCDs tuned from 452 nm to 602 nm due to the introduction of new energy levels by C=O and C=N functional groups. We exploited its stability in illumination, ionic strength, and pH, as well as its specificity, sensitivity, especially in ascorbic acid (AA) detection. F-NCDs could measure the AA concentration in the linear ranges of 0~0.1 and 0.1~1 mmol/L with the detection limit (LOD, S/N = 3) as low as 2.6 nmol/L. Additionally, we successfully detected AA in bovine serum with our F-NCDs and obtained the result within 1 min. Because of full-color emission features, we believe our F-NCDs have a great potential in fluorescent sensor detection.

MicroRNA-200c-3p Negatively Regulates ATP2A2 and Promotes the Progression of Papillary Thyroid Carcinoma.

microRNA-200c-3p (miR-200c-3p) has emerged as an important tumor growth regulator. However, its function in papillary thyroid carcinoma (PTC) is poorly understood. This study was conducted to investigate the role of miR-200c-3p in the progression of human PTC. The miR-200c-3p expression in human PTC tissues and cell lines was evaluated. The target relationship between miR-200c-3p and candidate genes was predicted through bioinformatic analysis and confirmed with a luciferase reporter assay. miRNA or gene expression was altered using transfection, and cell behavior was analyzed using CCK-8, wound healing, Transwell, and colony formation assays. The tumor-promoting effects of miR-200c-3p were evaluated by xenografting tumors with K1 cells in nude mice. The expression level of miR-200c-3p in human PTC tissues and cell lines markedly increased, and this increased expression was significantly associated with a worse overall survival. When inactivated, miR-200c-3p suppressed K1 cells' malignant behaviors, including decreasing proliferation and attenuating colony formation, migration, and invasion. Its inactivation also attenuated the development of xenografted K1 cells in nude mice. The effects of miR-200c-3p mimics on promoting the malignant behaviors of PTC cells were remarkably reversed by the overexpression of ATP2A2, as a downstream target of miR-200c-3p. miR-200c-3p acts as an oncogenic gene and promotes the malignant biological behaviors of human PTC cells, thereby directly targeting ATP2A2. This regulated axis may be used as a potential therapy of PTC.

Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway.

OBJECTIVE: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. METHODS: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. RESULTS: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). CONCLUSION: KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. RESULTS: In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. CONCLUSIONS: This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

Preparation of Eu0.075Tb0.925-Metal Organic Framework as a Fluorescent Probe and Application in the Detection of Fe3+ and Cr2O72.

Luminescent Ln-MOFs (Eu0.075Tb0.925-MOF) were successfully synthesised through the solvothermal reaction of Tb(NO3)3·6H2O, Eu(NO3)3·6H2O, and the ligand pyromellitic acid. The product was characterised by X-ray diffraction (XRD), TG analysis, EM, X-ray photoelectron spectroscopy (XPS), and luminescence properties, and results show that the synthesised material Eu0.075Tb0.925-MOF has a selective ratio-based fluorescence response to Fe3+ or Cr2O72-. On the basis of the internal filtering effect, the fluorescence detection experiment shows that as the concentration of Fe3+ or Cr2O72- increases, the intensity of the characteristic emission peak at 544 nm of Tb3+ decreases, and the intensity of the characteristic emission peak at 653 nm of Eu3+ increases in Eu0.075Tb0.925-MOF. The fluorescence intensity ratio (I653/I544) has a good linear relationship with the target concentration. The detection linear range for Fe3+ or Cr2O72- is 10-100 µM/L, and the detection limits are 2.71 × 10-7 and 8.72 × 10-7 M, respectively. Compared with the sensor material with a single fluorescence emission, the synthesised material has a higher anti-interference ability. The synthesised Eu0.075Tb0.925-MOF can be used as a highly selective and recyclable sensing material for Fe3+ or Cr2O72-. This material should be an excellent candidate for multifunctional sensors.

[Preparation and identification of rat polyclonal antibody against SARS-CoV-2 main protease (Mpro)].

Objective To investigate the immunological functions of SARS-CoV-2 main protease (Mpro) in coronavirus disease 2019 (COVID-19), polyclonal antibody against Mpro was developed. Methods A codon-optimized SARS-CoV-2 Mpro gene was synthesized and ligated into a pET-28a vector for construction of a recombinant plasmid named by pET-28a-Mpro. Subsequently, this plasmid was transformed into E.coli Rosetta (DE3) competent cells for Mpro expression in an optimized condition, and then Mpro was purified using a HisTrap chelating column. The purified Mpro was used as immunogen to inoculate rats and the serum was collected after third immunization cycle. The titer, selectivity and sensitivity of polyclonal antibody against Mpro were analyzed using the ELISA and Western blot analysis. Results An optimized expression condition in E.coli cells for Mpro was determined, and the recombinant Mpro was purified by a HisTrap chelating column. The ELISA and Western blot analysis demonstrated that the highly sensitive polyclonal antibody against Mpro specially recognized the recombinant Mpro, and the titer reached 1:256 000. Conclusion The highly specific polyclonal antibody against SARS-CoV-2 Mpro is successfully prepared, which lays an experimental foundation for investigating the immunological function of Mpro in COVID-19.

Identification of a Seven-lncRNA-mRNA Signature for Recurrence and Prognostic Prediction in Relapsed Acute Lymphoblastic Leukemia Based on WGCNA and LASSO Analyses.

Abnormal expressions of long noncoding RNAs (lncRNAs) and protein-encoding messenger RNAs (mRNAs) are important for the development of childhood acute lymphoblastic leukemia (ALL). This study developed an lncRNA-mRNA integrated classifier for the prediction of recurrence and prognosis in relapsed childhood ALL by using several transcriptome data. Weighted gene coexpression network analysis revealed that green, turquoise, yellow, and brown modules were preserved across the TARGET, GSE60926, GSE28460, and GSE17703 datasets, and they were associated with clinical relapse and death status. A total of 184 genes in these four modules were differentially expressed between recurrence and nonrecurrence samples. Least absolute shrinkage and selection operator analysis showed that seven genes constructed a prognostic signature (including one lncRNA: LINC00652 and six mRNAs: INSL3, NIPAL2, REN, RIMS2, RPRM, and SNAP91). Kaplan-Meier curve analysis observed that patients in the high-risk group had a significantly shorter overall survival than those of the low-risk group. Receiver operating characteristic curve analysis demonstrated that this signature had high accuracy in predicting the 5-year overall survival and recurrence outcomes, respectively. LINC00652 may function by coexpressing with the above prognostic genes (INSL3, SNAP91, and REN) and lipid metabolism-related genes (MIA2, APOA1). Accordingly, this lncRNA-mRNA-based classifier may be clinically useful to predict the recurrence and prognosis for childhood ALL. These genes represent new targets to explain the mechanisms for ALL.

The different interactions of two anticancer drugs with bovine serum albumin based on multi-spectrum method combined with molecular dynamics simulations.

Paclitaxel is the best natural anticancer drug and artemisinin also has anticancer effect. In this study, the interactions between BSA and these two drugs were determined in PBS (pH 7.40) by multi-spectroscopic method and molecular dynamics (MD) simulations. The results showed that paclitaxel and artemisinin could statically quench the BSA fluorescence when the complexes were formed and the stoichiometric ratio of BSA-drugs was 1:1. Particularly, the BSA-paclitaxel complex was more stable than BSA-artemisinin complex. During the binding, the surroundings around Trp residue site was largely affected than Tyr site, especially Trp 214 to a more hydrophobic environment. In addition, the binding processes were mainly spontaneous through electrostatic force interaction. In summary, we concluded that the free drug of paclitaxel in blood was low and duration time of artemisinin was shorter.

MSC-AS1 induced cell growth and inflammatory mediators secretion through sponging miR-142-5p/DDX5 in gastric carcinoma.

Emerging studies have noted that dysregulated lncRNAs are implicated in cancer progression and tumorigenesis. We first showed that MSC-AS1 was overexpressed in gastric cancer (GC) cells (HGC-27, MKN-45, SGC-7901 and MGC-803 cells) compared with GES cells. We observed that MSC-AS1 was upregulated in GC specimens compared with paired normal specimens. MSC-AS1 increased cell growth and cycle progression. Moreover, the overexpression of MSC-AS1 enhanced the secretion of the inflammatory mediators IL-1ß, IL-6 and TNF-α. We found that the overexpression of MSC-AS1 inhibited the expression of miR-142-5p in HGC-27 cells. We noted that DDK5 was a target gene of miR-142-5p. The overexpression of miR-142-5p suppressed the luciferase activity of wild-type DDX5, but the luciferase activity of the mutant DDX5 was not changed. We showed that miR-142-5p was downregulated in GC specimens compared with paired normal specimens. MSC-AS1 expression was inversely correlated with miR-142-5p expression in GC specimens. MSC-AS1 induced cell growth, cell cycle progression and inflammatory mediator secretion by modulating DDX5. These results showed that MSC-AS1 functions as a key oncogene in the development of GC.

Study on peptide-peptide interactions between transmembrane domains of Slc11a1 in model membranes.

In this study, we determined the interaction between TM4 and TM2/TM3 domain of Solute carrier family 11 member 1 (Slc11a1) by circular dichroism (CD) and fluorescence spectrum. The results indicated that, the cation transport process was likely to be accomplished by the collaboration of multiple TM domains rather than by TM4 domain alone. Therefore, this finding suggested possible transportation theory and be helpful to elucidate the mechanism of Slc11a1 in cation transport process.