Rapid and sensitive detection of Pseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection.

CRISPR based technologies have been used for fast and sensitive detection of pathogens. To test the possibility of CRISPR based detection strategy in Pseudomonas aeruginosa infections, a combined method of recombinase polymerase amplification followed by Cas12a-mediated detection via fluorescence reader or lateral flow biosensor (named Cas12a-RCFL) has been established in this study. The Cas12a-RCFL can detect as low as 50 CFU/mL Pseudomonas aeruginosa. The whole detection process can be finished within one hour with satisfied detection specificity. Cas12a-RCFL also shows good sensitivity of detecting Pseudomonas aeruginosa inStaphylococcus aureus and Acinetobacter baumannii contaminated samples. For the detection of 22 clinical samples, Cas12a-RCFL matches with PCR sequencing result exactly without DNA purification. This Cas12a-RCFL is rapid and sensitive with low cost, which shows good quality to be adopted as a point-of-care testing method.

Discrimination of Chinese green tea according to tea polyphenols using fluorescence sensor array based on Tb (III) and Eu (III) doped Zr (IV) metal-organic frameworks.

A facile and rapid fluorescence sensor array based on Tb (III) and Eu (III) doped Zr (IV) metal-organic frameworks was proposed for Chinese green tea discrimination. According to large porosity of Tb@UiO-66-(COOH)2 and Eu@UiO-66-(COOH)2, phenolic hydroxyl groups of tea polyphenols could coordinate with free carboxylic acid groups and was captured into the pores, which led to the disturbance of electronic structure of ligand and inhibited the energy transfer efficiency from ligand to Tb (III) and Eu (III) center, causing the fluorescence quenching effect. Based on Hierarchy Cluster Analysis and Linear Discrimination Analysis, the fluorescence sensor array was employed for successful tea polyphenols classification through the analysis of different fluorescence quenching effect to tea polyphenols. Green tea samples within different categories and grades were also successfully discriminated using this assay according to tea polyphenols, providing a new method for Chinese green tea identification.

Characterization of a novel polysaccharide from red ginseng and its ameliorative effect on oxidative stress injury in myocardial ischemia.

BACKGROUND: Red ginseng (RG) was widely used as traditional Chinese medicine (TCM) or dietary supplement. However, few researches had been reported on the red ginseng polysaccharide (RGP). METHODS: In this study, a novel heteropolysaccharide named RGP1-1 was fractionated sequentially by DEAE-52 column and Sephadex G-100 gel column. The primary structure of RGP1-1, including glycosyl linkages, molecular weight, monosaccharide composition, morphology and physicochemical property were conducted by nuclear magnetic resonance (NMR), gas chromatography-mass spectrometer (GC-MS), atomic force microscope (AFM), scanning electron microscope (SEM), differential scanning calorimetry-thermogravimetric analysis (DSC-TG) and so on. The effect of RGP1-1 in preventing and treating myocardial ischemia was evaluated by an animal model isoprenaline (ISO) induced mice. RESULTS: RGP1-1, with a homogeneous molecular weight of 5655 Da, was composed of Glc and Gal in the ratio of 94.26:4.92. The methylation and NMR analysis indicated the backbone was composed of → 1)-Glcp-(4 → and → 1)-Galp-(4 →, branched partially at O-4 with α-D-Glcp-(1 → residue. Morphology and physicochemical property analysis revealed a triple-helical conformation, flaky and irregular spherical structure with molecule aggregations and stable thermal properties of RGP1-1. And it contained 6.82 mV zeta potential, 117.4 nm partical size and polymerization phenomenon. Furthermore, RGP1-1 possessed strong antioxidant activity in vitro and in vivo, RGP1-1 could decrease cardiomyocyte apoptosis and myocardium fibrosis of mice in histopathology and it could decrease significantly the serum levels of cardiac troponin (cTnI), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), malondialdehyde (MDA). Western blot analysis showed that RGP1-1 can increase the expression of main protein Nuclear factor E2-related factor 2(Nrf2), NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase-1(HO-1) and kelch-like ECH-associated protein1(keap1) in oxidative stress injure progress, and therefore regulate the pathway of Nrf2/HO-1. CONCLUSION: The above findings indicated that RGP1-1 had an improving effect on ISO-induced myocardial ischemia injury in mice, as novel natural antioxidant and heart-protecting drugs.

A review on the applications of Traditional Chinese medicine polysaccharides in drug delivery systems.

Traditional Chinese medicine polysaccharides (TCMPs) are plentiful and renewable resources with properties such as biocompatibility, hydrophilicity, biodegradability, and low cytotoxicity. Because the polysaccharide molecular chain contains a variety of active groups, different polysaccharide derivatives can be easily produced through chemical modification. They have been increasingly used in drug delivery systems (DDS). However, the potential of polysaccharides is usually ignored due to their structural complexity, poor stability or ambiguity of mechanisms of actions. This review summarized the applications of TCMPs in DDS around four main aspects. The general characteristics of TCMPs as drug delivery carriers, as well as the relationships between structure and function of them were summarized. Meanwhile, the direction of preparing multifunctional drug delivery materials with synergistic effect by using TCMPs was discussed. This review aims to become a reference for further research of TCMPs and their derivatives, especially applications of them as carriers in pharmaceutical preparation industry.

Factors associated with the time to return negative RT-PCR from COVID-19 in paediatric patients: a retrospective cohort study.

OBJECTIVE: This study aimed to describe the epidemiological and clinical features and potential factors related to the time to return negative reverse transcriptase (RT)-PCR in discharged paediatric patients with COVID-19. DESIGN: Retrospective cohort study. SETTING: Unscheduled admissions to 12 tertiary hospitals in China. PARTICIPANTS: Two hundred and thirty-three clinical charts of paediatric patients with confirmed diagnosis of COVID-19 admitted from 1 January 2020 to 17 April 2020. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcome measures: factors associated with the time to return negative RT-PCR from COVID-19 in paediatric patients. SECONDARY OUTCOME MEASURES: epidemiological and clinical features and laboratory results in paediatric patients. RESULTS: The median age of patients in our cohort was 7.50 (IQR: 2.92-12.17) years, and 133 (57.1%) patients were male. 42 (18.0%) patients were evaluated as asymptomatic, while 162 (69.5%) and 25 (10.7%) patients were classified as mild or moderate, respectively. In Cox regression analysis, longer time to negative RT-PCR was associated with the presence of confirmed infection in family members (HR (95% CI): 0.56 (0.41 to 0.79)). Paediatric patients with emesis symptom had a longer time to return negative (HR (95% CI): 0.33 (0.14 to 0.78)). During hospitalisation, the use of traditional Chinese medicine (TCM) and antiviral drugs at the same time is less conducive to return negative than antiviral drugs alone (HR (95% CI): 0.85 (0.64 to 1.13)). CONCLUSIONS: The mode of transmission might be a critical factor determining the disease severity of COVID-19. Patients with emesis symptom, complications or confirmed infection in family members may have longer healing time than others. However, there were no significant favourable effects from TCM when the patients have received antiviral treatment.

MoS2 QDs-Based sensor for measurement of fluazinam with triple signal output.

In this study, direct detection of fluazinam was realized using a fluorescent sensor using disulfide quantum dots (MoS2 QDs) via inner filter effect (IFE). The maximum excitation of as-prepared MoS2 QDs presented a complementary spectral-overlap with the maximum absorption of fluazinam. Thus the occurrence of inner filter effect led to the significant fluorescence quenching of MoS2 QDs. Additionally, fluorescent quenching efficiency of MoS2 QDs could be enhanced by the effects of π-π stacking, hydrogen bond and electrostatic interaction between fluazinam and MoS2 QDs, and these non-chemical bond responses also promoted the selectivity for fluazinam detection. Under the optimum conditions, the IFE-based fluorescent sensor exhibited a relative wide linear range from 50 nM to 25 µM with the LOD of 2.53 nM (S/N = 3). In addition, a paper-based sensor was established by cross-linking the MoS2 QDs into cellulose membrane for naked-eyed detection and digital analysis of fluazinam. The paper-based sensor presented a liner range from 10 µM to 800 µM for fluazinam detection with the LOD of 2.26 µM. Additionally, the acceptable recoveries were obtained for fluazinam detection in the spiked samples of tomato, potato and cucumber, indicating that the proposed method provided an effective sensing platform for real applications of fluazinam detection in food safety.

Effects of an exopolysaccharide from Lactococcus lactis Z-2 on innate immune response, antioxidant activity, and disease resistance against Aeromonas hydrophila in Cyprinus carpio L.

Microbial exopolysaccharides (EPS) from Lactococcus have been found to have an important role in the probiotic activity of this bacterium; however, the immunomodulatory and antioxidant activities have not been fully explored in aquaculture. In the present study, we investigated EPS-2 from Lactococcus lactis Z-2, isolated from healthy common carp, for its immunomodulatory and antioxidant effects and disease resistance against Aeromonas hydrophila in Cyprinus carpio L. We found that the molecular weight of EPS-2 was 18.65 KDa. The monosaccharide composition of this polymer was rhamnose, xylose, mannose, glucose, and galactose at a molar percentage of 13.3%, 14.1%, 18.5%, 27.4%, and 26.7%, respectively. EPS-2 treatment could modulate the immune responses in vitro and in vivo. In vitro tests showed that EPS-2 could significantly enhance the proliferation and phagocytosis activities (P < 0.05) as well as induce the production of nitic oxide (NO), pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), and anti-inflammatory cytokines (IL-10, TGF-ß) (P < 0.05) in head kidney cells. When the fish were gavaged with three different concentrations of EPS-2 (250, 500, 1000 µg/mL) for 7 days and infected with A. hydrophila, different expression patterns of the NO, cytokines, lysozyme (LZM), and alkaline phosphatase (AKP) in the serum and of antioxidants (T-AOC, SOD, CAT, GSH, GSH-Px and MDA) in hepatopancreas were observed. Before infection with A. hydrophila, EPS-2 supplementation significantly up-regulated the NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), LZM and AKP activities, and levels of antioxidant molecules compared to those in the negative (G1) group (P < 0.05), whereas levels of NO and pro-inflammatory cytokines and LZM and AKP activities were significantly lower than those in the positive (G2) group after infection (P < 0.05). However, whether infected or not, the expression levels of anti-inflammatory cytokines (IL-10, TGF-ß) were significantly increased in the EPS-2 treatment groups (P < 0.05). These results indicate that EPS-2 has immunomodulatory and antioxidant effects on common carp, both in vitro and/or in vivo, and can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.

Pharmacological Properties of the Medical Maggot: A Novel Therapy Overview.

In the last decade, maggot has been hailed as the miraculous "medicinal maggot" for its diverse properties, including antimicrobial, antibiofilm, anti-inflammatory, and wound healing activities. The fact that maggots show so many beneficial properties has increased the interest in these tiny larvae dramatically. Whilst there is relatively abundant clinical evidence to demonstrate the success of maggots as debridement agents, not so much emphasis has been placed on the basic science evidence, which was a combination of physical and biochemical actions. This review differs from those earlier works in that it is undertaken to provide an update of the latest scientific basis published on maggot, particularly active ingredients within maggot excretions/secretions (ES). Further investigations should focus on the isolation, identification, recombination, transgenosis, and mass production of the beneficial molecules within maggots.

Hydroclimate changes across the Amazon lowlands over the past 45,000 years.

Reconstructing the history of tropical hydroclimates has been difficult, particularly for the Amazon basin-one of Earth's major centres of deep atmospheric convection. For example, whether the Amazon basin was substantially drier or remained wet during glacial times has been controversial, largely because most study sites have been located on the periphery of the basin, and because interpretations can be complicated by sediment preservation, uncertainties in chronology, and topographical setting. Here we show that rainfall in the basin responds closely to changes in glacial boundary conditions in terms of temperature and atmospheric concentrations of carbon dioxide. Our results are based on a decadally resolved, uranium/thorium-dated, oxygen isotopic record for much of the past 45,000 years, obtained using speleothems from Paraíso Cave in eastern Amazonia; we interpret the record as being broadly related to precipitation. Relative to modern levels, precipitation in the region was about 58% during the Last Glacial Maximum (around 21,000 years ago) and 142% during the mid-Holocene epoch (about 6,000 years ago). We find that, as compared with cave records from the western edge of the lowlands, the Amazon was widely drier during the last glacial period, with much less recycling of water and probably reduced plant transpiration, although the rainforest persisted throughout this time.

The Asian monsoon over the past 640,000 years and ice age terminations.

Oxygen isotope records from Chinese caves characterize changes in both the Asian monsoon and global climate. Here, using our new speleothem data, we extend the Chinese record to cover the full uranium/thorium dating range, that is, the past 640,000 years. The record's length and temporal precision allow us to test the idea that insolation changes caused by the Earth's precession drove the terminations of each of the last seven ice ages as well as the millennia-long intervals of reduced monsoon rainfall associated with each of the terminations. On the basis of our record's timing, the terminations are separated by four or five precession cycles, supporting the idea that the '100,000-year' ice age cycle is an average of discrete numbers of precession cycles. Furthermore, the suborbital component of monsoon rainfall variability exhibits power in both the precession and obliquity bands, and is nearly in anti-phase with summer boreal insolation. These observations indicate that insolation, in part, sets the pace of the occurrence of millennial-scale events, including those associated with terminations and 'unfinished terminations'.

Increased therapeutic efficacy of combination of azithromycin and ceftazidime on Pseudomonas aeruginosa biofilm in an animal model of ureteral stent infection.

BACKGROUND: Infection caused by ureteral stent indwelling is one of the most difficult medical problems, since once bacteria reside in biofilms they are extremely resistant to antibiotics as well as to the host immune defences. In this study we assessed the in vitro and in vivo efficacy of azithromycin and ceftazidime in preventing ureteral stent infection by Pseudomonas aeruginosa. RESULTS: The susceptibility testing with adherent bacteria showed that the biofilm was strongly inhibited by azithromycin treatment, ceftazidime against adherent bacteria in the presence of azithromycin showed the minimum inhibitory concentrations (MICs) and minimum bacteriocidal concentrations (MBCs) dramatically lower than those obtained in the absence of azithromycin. Moreover, ceftazidime plus azithromycin reduced twitching motility and production of rhamnolipid. For the single-treatment groups, in vivo intravenous injection of ceftazidime showed the highest inhibitory effect on bacterial load. Azithromycin prophylactic injection combined with ceftazidime showed increased inhibitory effect on bacterial load than that of each single antibiotic. CONCLUSIONS: Combination of azithromycin and ceftazidime effectively prevent the formation of biofilm and reduced bacteria load of Pseudomonas aeruginosa compared to separate treatment of either of these two antibiotics. This combined treatment option have the potential to contribute to the success of Pseudomonas biofilm elimination in the clinical environment.

Developmental exposure to the organophosphorus flame retardant tris(1,3-dichloro-2-propyl) phosphate: estrogenic activity, endocrine disruption and reproductive effects on zebrafish.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant that is detectable in the environment and biota, prompting concern over its risk to wildlife and human health. Our objective was to investigate whether long-term exposure to low concentrations of TDCPP can affect fish reproduction. Zebrafish embryos were exposed to low concentrations (0, 4, 20 and 100µg/L) of TDCPP from 2h post-fertilization until sexual maturation. Exposure to TDCPP significantly increased plasma estradiol and testosterone levels in females, but had no effect in males. TDCPP exposure also caused a significant reduction in fecundity as indicated by decreased egg production. Real-time PCR was performed to examine selected genes in the hypothalamic-pituitary-gonadal (HPG) axis and liver. Principle component analysis (PCA) showed that sex hormone levels and fecundity were related to the mRNA level of several genes in the HPG axis. Furthermore, hepatic vitellogenin (vtg1 and vtg3) expression was upregulated in both females and males, suggesting TDCPP has estrogenic activity. Histological examination revealed promotion of oocyte maturation in the females, but retardation of spermiation in males. Reduced egg quality (e.g., egg diameter) and increased malformation rates were observed in the F1 generation. Chemical analysis showed significant levels of TDCPP and its metabolite bis(1,3-dichloro-2-propyl) phosphate in the gonads of males and females. In conclusion, long-term exposure to low concentrations of TDCPP impairs fish reproduction.

[Fourth-line and beyond therapy in advanced non-small cell lung cancer: a retrospective analysis].

BACKGROUND AND OBJECTIVE: A common consensus has been reached regarding first- and second-line therapies for advanced non-small cell lung cancer (NSCLC). The newest guideline from National Comprehensive Cancer Network (NCCN) also provides recommendations for third-line therapy. This study focused on fourth-line and enhanced treatments for advanced NSCLC. Treatment results and impact factors were analyzed and discussed. METHODS: A total of 140 patients with advanced NSCLC were selected and their data were analyzed. Response rate, survival rate, and prognostic factors were evaluated. RESULTS: Among the 140 patients, 12.9% (18 cases) showed partial response, 25.7% (36 cases) suffered from a stable disease, and 38.6% (54 cases) exhibited a disease control rate. Median overall survival (OS) and fourth-line therapy OS were 31 months and 10.1 months, respectively. The median progression free survival (PFS) of fourth-line therapy was 2.6 months. Univariate and multivariate analyses indicated different therapy regimens and suggested whether or not patients should undergo follow-up treatments. These parameters were independent prognostic factors of the OS of the fourth-line therapy; by contrast, no independent impact factor of PFS was found. Chemotherapy resulted in better median OS in fourth-line therapy than in targeted therapy (11.7 months vs 7.1 months, P=0.013). Considering the median OS of fourth-line therapy, we observed that single agent therapy did not significantly differ from double agent therapy; likewise, we found that first-time usage did not significantly differ from multiple usage of epidermal growth factor receptor tyrosine kinase inhibitor. CONCLUSIONS: Fourth-line therapy is recommended to increase the survival of advanced NSCLC patients. Nevertheless, the role of fourth-line therapy in advanced NSCLC should be further assessed in clinical trials.

Bioactivity-guided fractionation of physical fatigue-attenuating components from Rubus parvifolius L.

Alleviation of fatigue has been emerging as a serious issue that requires urgent attention. Health professionals and sports physiologists have been looking for active natural products and synthetic compounds to overcome fatigue in humans. This study was designed to define the anti-fatigue property of Rubus parvifolius L. (RPL) by characterization of active constituents using a mouse forced swimming test model. Four RPL fractions with different polarities containing anti-fatigue activity were sequentially isolated from the n-butanol RPL extract, followed by elution of 50% ethanol-water fraction from D101 macroporous resin chromatography to obtain nigaichigoside F1, suavissimoside R1 and coreanoside F1. Active constituents of the 50% ethanol-water eluate of RPL were total saponins. The fractions were examined based on the effect on weight-loaded swimming capacity of mice. Serum levels of urea nitrogen (SUN), triglyceride fatty acids (TG), lactate dehydrogenase (LDH), lactic acid (LA), ammonia and hepatic glycogen (HG) were also examined for potential mechanisms underlying the anti-fatigue effect of RPL extracts. During the experiment, two inflammatory markers, interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in serum, were measured. We found that total saponins from RPL possess potent capabilities to alleviate mouse fatigue induced by forced swimming and that nigaichigoside F1 was responsible for the pharmacological effect. The underlying mechanisms include delays of SUN and LA accumulation, a decrease in TG level by increasing fat consumption, increases in HG and LDH so that lactic acid accumulation and ammonia in the muscle were reduced, and suppression of increased immune activation and inflammatory cytokine production. Our findings will be helpful for functional identification of novel anti-fatigue components from natural medicinal herbs.

Omega-3 polyunsaturated fatty acids antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway.

Macrophages play a key role in obesity-induced inflammation. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert anti-inflammatory functions in both humans and animal models, but the exact cellular signals mediating the beneficial effects are not completely understood. We previously found that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. Here we aim to determine whether ω-3 PUFAs antagonize macrophage inflammation via activation of AMPK/SIRT1 pathway. Treatment of ω-3 PUFAs suppresses lipopolysaccharide (LPS)-induced cytokine expression in macrophages. Luciferase reporter assays, electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) assays show that treatment of macrophages with ω-3 PUFAs significantly inhibits LPS-induced NF-κB signaling. Interestingly, DHA also increases expression, phosphorylation and activity of the major isoform α1AMPK, which further leads to SIRT1 over-expression. More importantly, DHA mimics the effect of SIRT1 on deacetylation of the NF-κB subunit p65, and the ability of DHA to deacetylate p65 and inhibit its signaling and downstream cytokine expression require SIRT1. In conclusion, ω-3 PUFAs negatively regulate macrophage inflammation by deacetylating NF-κB, which acts through activation of AMPK/SIRT1 pathway. Our study defines AMPK/SIRT1 as a novel cellular mediator for the anti-inflammatory effects of ω-3 PUFAs.

Characterization of the antibacterial activity and the chemical components of the volatile oil of the leaves of Rubus parvifolius L.

Rubus parvifolius L. (Rp) is a medicinal herb that possesses antibacterial activity. In this study, we extracted the volatile oil from the leaves of Rp to assess its antibacterial activity and analyze its chemical composition. A uniform distribution design was used to optimize the extraction procedure, which yielded 0.36% (w/w) of light yellowish oil from the water extract of Rp leaves. We found that the extracted oil effectively inhibited the growth of a wide range of Gram positive and negative bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii, Bacillus cloacae, and Klebsiella pneumoniae. We further analyzed the components contained in the hydro-distillated Rp volatile oil by gas chromatography-mass spectroscopy. Twenty nine compounds were identified, including 4-hydroxy-3-methoxystyrene (66%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (10%) and 4-tert-butylbenzoic acid (2%). Our results suggest that one or multiple constituents contained in Rp volatile oil may account for its antibacterial activity.