An electrochemical ratiometric immunosensor for the detection of NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB.

Nuclear matrix protein 22 (NMP22) is one of the most important tumor markers of bladder cancer and is significantly elevated in the urine of bladder cancer patients. Therefore, in this work, a highly sensitive ratiometric electrochemical immunosensor was constructed to detect NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs composites. ZIF-8 had a large surface area and good adsorption ability. Multi-Walled Carbon Nanotubes (MWCNTs) can optimize the electrical conductivity of ZIF-8, so that the electrode surface of ferrocene (Fc) obtains a stable and strong electrochemical signal. In addition, AuPt-MB provided another strong detection signal methylene blue (MB) while immobilizing the secondary antibody (Ab2) through Au-N and Pt-N bonds. A ratiometric electrochemical sensor was formed based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB, which showed a great linear connection between IMB/IFc and the logarithmic concentration of NMP22 with a detection limit of 3.33 fg mL-1 (S/N = 3) under optimized specifications in the concentration interval of 0.01 pg mL-1 to 1000 ng mL-1. In addition, the ratiometric immunosensor showed good selectivity and stability.

Lysosomal-targeted fluorescent probe based pH regulating reactivity for tracking cysteine dynamics under oxidative stress.

The ability to detect and visualize cellular events and associated biological analytes is essential for the understanding of their physiological and pathological functions. Cysteine (Cys) plays a crucial role in biological systems and lysosomal homeostasis. This puts forward higher requirements on the performance of the probe. Herein, we rationally designed a coumarin-based probe for the reversible, specific, sensitive, and rapid detection of Cys based on pH regulating reactivity. The obtained probe (ECMA) introduces a morpholine moiety to target lysosomes, and α,ß-unsaturated-ketone with an electron-withdrawing CN group served as a reversible reaction site for Cys. Importantly, ECMA was successfully applied to the real-time monitoring of Cys dynamics in living cells. Furthermore, cell imaging clearly revealed that exogenous Cys could induce the up-regulation of lysosomal ROS, which provided a powerful tool for investigating the relationship between oxidative stress and lysosomal Cys.

Short-term effect of beinaglutide combined with metformin versus metformin alone on weight loss and metabolic profiles in obese patients with polycystic ovary syndrome: a pilot randomized trial.

Objective: To observe the effect of beinaglutide combined with metformin versus metformin alone on weight loss and metabolic profiles in obese patients with polycystic ovary syndrome(PCOS). Methods: A total of 64 overweight/obese women with PCOS diagnosed via the Rotterdam criteria were randomly assigned to metformin(MET) 850 mg twice a day(BID) or combined MET 850 mg BID with beinaglutide (COMB) starting at 0.1mg three times a day(TID)and increasing to 0.2mg TID two weeks later. The main endpoints were changes in anthropometric measurements of obesity. Glucose and lipid metabolic, gonadal profiles, and antral follicle count changes as secondary outcomes were also observed. Results: 60(93.75%) patients completed the study. In terms of lowering weight, body mass index (BMI),waist circumference(WC) and waist to height ratio(WHtR), COMB treatment outperformed MET monotherapy. Subjects in the COMB arm lost weight 4.54±3.16kg compared with a 2.47±3.59kg loss in the MET arm. In the COMB group, BMI,WC and WHtR were reduced significantly compared with that in the MET group, respectively. COMB therapy is also more favorable in the reduction of fasting insulin(FINS), total testosterone(TT), and homeostasis model assessment-insulin resistance(HOMA-IR) when compared to MET therapy. Antral follicle count and ovarian volume were non-significantly changed in both groups.The most frequent side effects in both groups were mild and moderate digestive symptoms. Itching and induration at the injection site were reported with COMB treatment. Conclusion: Short-term combined treatment with beinaglutide and metformin appears superior to metformin monotherapy in lowering body weight, BMI, WC,WHtR and improving insulin sensitivity and androgen excess in women with PCOS and obesity, with tolerable adverse events. Clinical trial registration: https://www.chictr.org.cn/listbycreater.aspx, identifier ChiCTR2000033741.

Genomic characterization of two new viruses infecting Ageratum conyzoides in China.

Two new RNA viruses were identified in Ageratum conyzoides in China using high-throughput sequencing, and their genome sequences were determined using PCR and rapid amplification of cDNA ends. The new viruses, which have positive-sense, single-stranded RNA genomes, were provisionally named "ageratum virus 1" (AgV1) and "ageratum virus 2" (AgV2). AgV1 has a genome of 3,526 nucleotides with three open reading frames (ORFs) and shares 49.9% nucleotide sequence identity with the complete genome of Ethiopian tobacco bushy top virus (genus Umbravirus, family Tombusviridae). The genome of AgV2 consists of 5,523 nucleotides and contains five ORFs that are commonly observed in members of the genus Enamovirus of the family Solemoviridae. Proteins encoded by AgV2 exhibited the highest amino acid sequence similarity (31.7-75.0% identity) to the corresponding proteins of pepper enamovirus R1 (an unclassified enamovirus) and citrus vein enation virus (genus Enamovirus). Based on their genome organization, sequence, and phylogenetic relationships, AgV1 is proposed to be a new umbra-like virus of the family Tombusviridae, and AgV2 is proposed to be a new member of the genus Enamovirus of the family Solemoviridae.

Autophagy regulates X-ray radiation-induced premature senescence through STAT3-Beclin1-p62 pathway in lung adenocarcinoma cells.

PURPOSE: Ionizing radiation (IR) can induce autophagy and premature senescence; however, the link between them has not been clarified. Our research has shown that X-ray irradiation induces premature senescence in lung adenocarcinoma cells, and its occurrence partially depends on the signal transducer and activator of transcription 3 (STAT3). STAT3 can bind to the promoter region of Beclin1 and regulate its expression. Therefore, it is speculated that there may be a close link between premature senescence and autophagy induced by ionizing radiation in lung adenocarcinoma cells. p62 plays a regulatory role in both autophagy and premature senescence, and it is also an irreplaceable molecule that causes the senescence -associated secretory phenotype (SASP) and a substrate for selective autophagy. This study focused on STAT3, Beclin1 and p62 to clarify the regulatory relationship between IR-induced autophagy and premature senescence. MATERIALS AND METHODS: After exposure to 4 Gy X-rays, a ß-galactosidase staining kit was used to detect the positive rate of premature senescence. STAT3 was overexpressed by pcDNA3.0-STAT3 transfection, and was inhibited by AG490 and rapamycin. Lung adenocarcinoma cells were transduced with the adenovirus vector GV119-Beclin1 to knockdown the expression of Beclin1, or treated with ATM and ATR inhibitors to inhibit premature senescence. Western blotting was used to examine alterations in the radiation response proteins STAT3 and p-STAT3, senescence-related proteins p62 and GATA4, autophagy-related proteins Beclin1, and LC3-II/LC3-I. The mRNA expression levels of SASP factors, including IL-6 and IL-8, were examined by real-time polymerase chain reaction. RESULTS: The activity of SA-ß-gal increased significantly (p < .05), and the expression of p62 decreased significantly at 72 h after 4 Gy X-ray irradiation, accompanied by the increased expression of STAT3, p-STAT3, Beclin1, and the LC3-II/LC3-I ratio. Up- or down-regulation of STAT3 expression was followed by an increase or decrease in Beclin1 expression. After treatment with ATM and ATR inhibitors, there were no significant changes in Beclin1 expression or LC3-II/LC3-I ratio in A549 cells after 4 Gy X-ray irradiation. The p62 expression, the percentage of the SA-ß-gal-positive staining cells, and the expression of IL-6 and IL-8 mRNA in cells transduced with GV119-Beclin1 were also decreased significantly after 4 Gy X-ray irradiation compared with that of the 0 Gy group. CONCLUSION: Radiation induces premature senescence and autophagy in lung adenocarcinoma cells. Autophagy regulates X-ray radiation-induced premature senescence through the STAT3-Beclin1-p62 pathway in lung adenocarcinoma cells.

Development and Validation of a Deep Neural Network for Accurate Identification of Endoscopic Images From Patients With Ulcerative Colitis and Crohn's Disease.

Background and Aim: The identification of ulcerative colitis (UC) and Crohn's disease (CD) is a key element interfering with therapeutic response, but it is often difficult for less experienced endoscopists to identify UC and CD. Therefore, we aimed to develop and validate a deep learning diagnostic system trained on a large number of colonoscopy images to distinguish UC and CD. Methods: This multicenter, diagnostic study was performed in 5 hospitals in China. Normal individuals and active patients with inflammatory bowel disease (IBD) were enrolled. A dataset of 1,772 participants with 49,154 colonoscopy images was obtained between January 2018 and November 2020. We developed a deep learning model based on a deep convolutional neural network (CNN) in the examination. To generalize the applicability of the deep learning model in clinical practice, we compared the deep model with 10 endoscopists and applied it in 3 hospitals across China. Results: The identification accuracy obtained by the deep model was superior to that of experienced endoscopists per patient (deep model vs. trainee endoscopist, 99.1% vs. 78.0%; deep model vs. competent endoscopist, 99.1% vs. 92.2%, P < 0.001) and per lesion (deep model vs. trainee endoscopist, 90.4% vs. 59.7%; deep model vs. competent endoscopist 90.4% vs. 69.9%, P < 0.001). In addition, the mean reading time was reduced by the deep model (deep model vs. endoscopists, 6.20 s vs. 2,425.00 s, P < 0.001). Conclusion: We developed a deep model to assist with the clinical diagnosis of IBD. This provides a diagnostic device for medical education and clinicians to improve the efficiency of diagnosis and treatment.

Dual function metal-organic frameworks based ratiometric electrochemical sensor for detection of doxorubicin.

Cancer is one of the main diseases threatening human health in the world. Doxorubicin (DOX) is a common anti-cancer drug that can be used for chemotherapy to extend a cancer patient's life. It is our common wish that treatment process of cancer is efficient and secure. Therefore, it is of great significance to develop sensitive, rapid and accurate techniques for anti-cancer drug doxorubicin (DOX) detection. Herein, in our work, a ratiometric electrochemical sensor for DOX detection was designed, which based on MB@MWCNTs/UiO-66-NH2 composites. The porous materials UiO-66-NH2 magically shoulder double function in our ratiometric electrochemical strategy, which can reduce the interior error caused by the various complex materials. Specifically, on the one hand, it can be used to catalyze DOX, which can provide a great current signal to be detected, on the other hand, its special property of absorbing molecules was utilized to load materials as internal reference. Consequently, we chose methylene blue (MB) as the substance that can generate an internal reference signal, because it is a specific and stable electroactive substance. Then, we added MWCNTs as a part of the material modified on the ratiometric electrochemical platform to enhance the signal of the target due to its feature of good electrical conductivity. Under the optimized conditions, the ratiometric electrochemical sensor displayed a wide linear detection with the range from 0.1 µM to 75 µM and the limit of detection (LOD) of 0.051 µM. The applicability of this method in the analysis of actual human saliva samples has been confirmed by the results of selectivity, stability, and reproducibility tests, which was prospective in clinical application.

RFWD3 Participates in the Occurrence and Development of Colorectal Cancer via E2F1 Transcriptional Regulation of BIRC5.

Objectives: Colorectal cancer (CRC) is one of the most common human malignancies. It was reported that the alterations in the DNA damage response (DDR) pathways are emerging as novel targets for treatment across different cancer types including CRC. RFWD3 plays a critical role in replication protein A (RPA)-mediated DNA damage in cancer cells. More importantly, RFWD3 can response to DNA damage by positively regulating p53 stability when the G1 cell cycle checkpoint is activated. However, the functional significance of RFWD3 in CRC has not been reported in the existing documents. Materials and Methods: Here, we revealed high expression of RFWD3 in CRC tissues by IHC analysis and The Cancer Genome Atlas (TCGA) database. Besides, overexpression of RFWD3 in CRC cell lines was also confirmed by qRT-PCR and western blot assay. The Celigo cell counting method and wound-healing/transwell migration assay were applied to evaluate CRC cell proliferation and migration. The tumor growth indicators were quantified in nude mice xenografted with shRFWD3 and shCtrl RKO cells. Results: The results indicated that RFWD3 knockdown restricted CRC development in vitro and in vivo. In exploring the downstream mechanism of RFWD3's action, we found that RFWD3 could transcriptionally activate BIRC5 by interacting with E2F transcription factor 1 (E2F1). Accordingly, we identified BIRC5 as a downstream gene of RFWD3 regulating CRC. Subsequent loss- and gain- of function experiments demonstrated that upon overexpressing BIRC5 in RKO cells with down-regulated RFWD3, the inhibitory effects of cell proliferation, migration and colony formation could be reversed, while the capacity of cell apoptosis was ameliorated, suggesting that the effects of RFWD3 depletion was mainly due to BIRC5 suppression. Conclusion: Taken together, this study revealed that RFWD3 participates in the occurrence and development of colorectal cancer via E2F1 transcriptional regulation of BIRC5.

Sulfite as the substrate of C-sulfonate metabolism of α, ß-unsaturated carbonyl containing andrographolide: analysis of sulfite in rats' intestinal tract and the reaction kinetics of andrographolide with sulfite.

One-sixth of the currently known natural products contain α, ß-unsaturated carbonyl groups. Our previous studies reported a rare C-sulfonate metabolic pathway. Sulfonate groups were linked to the ß-carbon of α, ß-unsaturated carbonyl-based natural compounds through this pathway. However, the mechanism of this type of metabolism is still not fully understood, especially whether it is formed through enzyme-mediated biotransformation or direct sulfite addition. In this work, the enzyme-mediated and non-enzymatic pathways were studied. First, the sulfite content in rat intestine was determined by LC-MS/MS. The results showed that the amount of sulfite in rat intestinal contents was from 41.5 to 383 µg·g-1, whereas the amount of sulfite in rat feed was lower than the lower limit of quantitation (20 µg·g-1). Second, the reaction kinetics of sulfite-andrographolide reactions in phosphate buffer solutions (pH 6-8) was studied. The half-lives of andrographolide ranged from minutes to hours. This was suggested that the C-sulfonate reaction of andrographolide was very fast. Third, the C-sulfonate metabolites of andrographolide were both detected when andrographolide and L-cysteine-S-conjugate andrographolide were incubated with the rat small intestine contents or sulfite, indicating that the sulfite amount in rat intestine contents was high enough to react with andrographolide, which assisted a significant portion of andrographolide metabolism. Finally, the comparison of andrographolide metabolite profiles among liver homogenate (with NADPH), liver S9 (with NADPH), small intestine contents homogenate (with no NADPH), and sulfite solution incubations showed that the C-sulfonate metabolites were predominantly generated in the intestinal tract by non-enzymatic pathway. In summary, sulfite can serve as a substrate for C-sulfonate metabolism, and these results identified non-enzymatically nucleophilic addition as the potential mechanism for C-sulfonate metabolism of compounds containing α, ß-unsaturated carbonyl moiety.

Effect of two-step microwave heating on the gelation properties of golden threadfin bream (Nemipterus virgatus) myosin.

The effects of different microwave heating (MH) methods on gelation properties of golden threadfin bream myosin and related mechanism were investigated in this study. Compared with conventional heating and one-step MH methods, myosin gel developed by 100 W coupled with 300 W MH method (MH100 + MH300) had stronger gel strength (p < 0.05) with more immobilized water (p < 0.05). Raman analysis suggested that this two-step method promoted the suitable unfolding of myosin before aggregation formation, and contributed to stabilizing the ordered secondary structure. Confocal laser scanning microscopy images revealed that 100 W microwave followed by 300 W MH produced a compact networked structure with small cavities and a thick cross-linked gel wall. Furthermore, from a perspective of molecular forces, the improvement of gelation properties by the MH100 + MH300 method were mainly involved in the enhancement of regular hydrophobic interaction and stabilization of weak protein-water hydrogenbonds.

Sonchus oleraceus Linn protects against LPS-induced sepsis and inhibits inflammatory responses in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Sonchus oleraceus Linn (SOL) belongs to family of Asteraceae, is a traditional medicinal plant, which has been used to treat tumor, inflammatory diseases, infection and so on in Chinese folk culture. AIM OF THE STUDY: This work investigated the influence of aqueous ethanol extract of whole plant of SOL and contribution of its main components on inflammation METHODS AND RESULTS: Oral administration of SOL (10 mg/kg) to mice reduced the expression of inflammatory cytokines including IL-6, IL-1ß, and TNF-α, in the LPS-induced sepsis mouse model. Major phenolics in SOL were isolated and determined by HPLC. Results indicate that SOL at the concentration range from 25 to 100 µg/mL and its main components, chlorogenic acid, caffeic acid (25-100 µM) significantly reduced the pro-inflammatory cytokine IL-1ß, IL-6, TNF-α, attenuated iNOS and COX-2 expression in LPS-stimulated Macrophages. In addition, western blot analysis showed SOL suppressed inducible nitric oxide synthase (iNOS) protein expression and the phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK). CONCLUSION: The underlying mechanism of anti-inflammation might be in according with the inhibition of MAPKs activation as well as down regulation of iNOS and cyclooxygenase-2 (COX-2).

Structural characterization of a novel neutral polysaccharide from Lentinus giganteus and its antitumor activity through inducing apoptosis.

A novel neutral polysaccharide (LGPS-1), with a molecular weight of 1.547×10(5)Da, was isolated from Lentinus giganteus by precipitation and purification. The monosaccharides included d-mannose (Man), d-glucose (Glc) and d-galactose (Gal) with a molar ratio of 3.0:4.1:7.1. The backbone of LGPS-1 was composed of 1,6-Galp and 1,3,6-Manp whereas the branches were composed of 1,6-Glcp and 1-Glcp. The anticancer efficacy of LGPS-1 was assessed using HepG2 hepatocellular carcinoma cells. The results showed that LGPS-1 inhibited the proliferation of HepG2 cells and also induced the activation of caspase-3, and cleavage of PARP-1. Western blot analysis revealed that LGSP-1 significantly induced a loss of mitochondrial membrane potential (Δym), increased the ratio of Bax/Bcl-2, promoted the release of cytochrome c into cytoplasm as well as inhibited the phosphorylation of Akt in HepG2 cells. These findings suggest that LGPS-1 induced apoptosis in HepG2 cells through intrinsic mitochondrial apoptosis and PI3K/Akt signaling pathways.

Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil.

The effectiveness of ultrasonic-assisted extraction (UAE) of pomegranate seed oil (PSO) was evaluated using a variety of solvents. Petroleum ether was the most effective for oil extraction, followed by n-hexane, ethyl acetate, diethyl ether, acetone, and isopropanol. Several variables, such as ultrasonic power, extraction temperature, extraction time, and the ratio of solvent volume and seed weight (S/S ratio) were studied for optimization using response surface methodology (RSM). The highest oil yield, 25.11% (w/w), was obtained using petroleum ether under optimal conditions for ultrasonic power, extraction temperature, extraction time, and S/S ratio at 140 W, 40 °C, 36 min, and 10 ml/g, respectively. The PSO yield extracted by UAE was significantly higher than by using Soxhlet extraction (SE; 20.50%) and supercriti cal fluid extraction (SFE; 15.72%). The fatty acid compositions were significantly different among the PSO extracted by Soxhlet extraction, SFE, and UAE, with punicic acid (>65%) being the most dominant using UAE.

Tobacco biomass hydrolysate enhances coenzyme Q10 production using photosynthetic Rhodospirillum rubrum.

Coenzyme Q10 (CoQ10), a potent antioxidative dietary supplement, was produced using a photosynthetic bacteria Rhodospirillum rubrum ATCC 25852 by submerged fermentation supplemented with tobacco biomass hydrolysate (TBH) in comparison with media supplemented with hydrolysates prepared with alfalfa (ABH) or spinach (SBH). Growth medium supplemented with 20% (v/v) TBH was found favorable with regard to cell density and CoQ10 concentration. The stimulation effects on cell growth (shortened lag phase, accelerated exponential growth, and elevated final cell concentration) and CoQ10 production (enhanced specific CoQ10 content per unit cell weight) could be attributed to the presence of solanesol, the precursor of CoQ10, in the tobacco biomass. The final yield of CoQ10 reached 20.16 mg/l in the fermentation medium supplemented with 20% TBH.