Metagenomics next-generation sequencing (mNGS) reveals emerging infection induced by Klebsiella pneumoniaeniae.

OBJECTIVES: The increasing emergence of hypervirulent Klebsiella pneumoniae (hv-Kp) and carbapenem-resistant K. pneumoniae (CR-Kp) is a serious and substantial public health problem. The use of the last resort antimicrobials, tigecycline and polymyxin to combat infections is complicated by the expanding repertoire of newly-identified CR-hvKp. The transmission and co-occurrence of the corresponding antimicrobial resistance and virulence determinants are largely unknown. The aim of this study was to investigate the dissemination and dynamics of CR-Kp and its antibiotic resistance in a hospitalised patient. METHODS: Metagenomic next-generation sequencing (mNGS) was conducted for different specimens collected from an elderly male hospitalised patient. CR-Kp strains were examined using antibiotic susceptibility and string testing. Antimicrobial and virulence genes were annotated using whole-genome sequencing (WGS). RESULTS: A clinical case of a patient infected with a variety of CR-Kp isolates was reported. The co-occurrence of KPC-2 and NDM-1 in the patient was revealed. The CR-Kp isolates, such as BALF2, and Sputum T1 and T3, were classified into ST11 and ST147, respectively. The genetic signature (iuc operon) of hypervirulence was identified in strain T1, although string testing indicated its intermediate virulence. CONCLUSIONS: In this study, multiple infections of CR-Kp isolates were revealed by mNGS, and their dissemination was attributed to plasmid variations, mgrB inactivation and integrative conjugative elements (ICEs). Furthermore, the finding indicated one likely convergence to form CR-hvKp, different from acquisition of carbapenem-resistance determinants in hvKp. A combination of mNGS and WGS is beneficial for clinical diagnosis and anti-infection therapy, and facilitates a better understanding of genetic variants conferring antimicrobial and virulence properties.

[Advances in genomics of multi-drug resistant Stenotrophomonas].

Stenotrophomonas species are non-fermentative Gram-negative bacteria that are widely distributed in environment and are highly resistant to numerous antibiotics. Thus, Stenotrophomonas serves as a reservoir of genes encoding antimicrobial resistance (AMR). The detection rate of Stenotrophomonas is rapidly increasing alongside their strengthening intrinsic ability to tolerate a variety of clinical antibiotics. This review illustrated the current genomics advances of antibiotic resistant Stenotrophomonas, highlighting the importance of precise identification and sequence editing. In addition, AMR diversity and transferability have been assessed by the developed bioinformatics tools. However, the working models of AMR in Stenotrophomonas are cryptic and urgently required to be determined. Comparative genomics is envisioned to facilitate the prevention and control of AMR, as well as to gain insights into bacterial adaptability and drug development.

[Microorganisms capable of degrading neonicotinoids and their metabolic pathways: a review].

Neonicotinoid compounds are usually considered harmless and eco-friendly in terms of their targeted toxicity compared to that of pyrethroids and phosphorus-containing pesticides. However, overuse of neonicotinoid insecticides resulted in the accumulation of its residuals or intermediates in soil and water, which consequently affected beneficial insects as well as mammals, yielding pollution and secondary risks. This review summarized the recent advances in neonicotinoid degrading microorganisms and their metabolic diversity, with the aim to address the urgent need for degrading these insecticides. These advances may facilitate the development of controllable and reliable technologies for efficiently transforming neonicotinoid insecticides into value-added products by synthetic biology and metagenomics.

Crop pattern optimization for the coordination between economy and environment considering hydrological uncertainty.

With the rapid growth of population and economy, shortage and mismatch of land and water resources have deepened the need for cropping pattern optimization. In the context of the sustainable development of agriculture, cropping pattern optimization should not only pursue economic benefits, but the consequent environmental effects also deserve equal attention. Meanwhile, climate change increases the complexity of balancing conflicts of economic-environmental system by cropping pattern optimization. Therefore, this paper builds a multi-objective programming model for Economic-Environmental Synergistic Optimization for Cropping Pattern under Climate Change (EESO-CP-CC) model, with the goals of economic benefit increment and environmental pollutants emission reduction. The EESO-CP-CC model couples a non-point source pollution input-output model, a one-dimensional water quality model and an economic benefit function into an integrated framework. Fuzzy method was used to solve the optimization model, and the stochastic uncertainty of water supply under climate change was quantified by the integration of Bayesian approach and interval linear regression. The model was applied to Jinxi Irrigation District (JXID) in Heilongjiang Province, northeast of China. Results show that by adjusting the acreage of rice, corn and soybean, the harmony degree of economy-society-environment system increased by 10.7% compared to the current situation, indicating that the model tends to achieve the best possible economic benefits while ensuring the environmental effects. Compared with actual cropping pattern, the pollutants emissions reduced by 24.7% and 3% from corn and soybean, respectively. However, this led to a decrease of economic benefit by 8% in exchange, showing the trade-off between environmental pollution reduction and economic benefits improvement. The output coefficients of nitrogen and phosphorus pollutants were optimized, with the optimal output reducing by 20% compared to the standard. Cropping pattern and water resources allocation vary with different climate change conditions, however, the amplitude of variation is modest, indicating that the model can cope well with the changing environment. The developed model can help achieve synergistic development of economic benefits and environmental effects, and thus promote sustainable development of irrigation areas, and improve the coping capacity of agricultural water and land under climate change, by cropping pattern optimization and planning.

Elucidating controls on cyanobacteria bloom timing and intensity via Bayesian mechanistic modeling.

The adverse impacts of harmful algal blooms (HABs) are increasing worldwide. Lake Erie is a North American Great Lake highly affected by cultural eutrophication and summer cyanobacterial HABs. While phosphorus loading is a known driver of bloom size, more nuanced yet crucial questions remain. For example, it is unclear what mechanisms are primarily responsible for initiating cyanobacterial dominance and subsequent biomass accumulation. To address these questions, we develop a mechanistic model describing June-October dynamics of chlorophyll a, nitrogen, and phosphorus near the Maumee River outlet, where blooms typically initiate and are most severe. We calibrate the model to a new, geostatistically-derived dataset of daily water quality spanning 2008-2017. A Bayesian framework enables us to embed prior knowledge on system characteristics and test alternative model formulations. Overall, the best model formulation explains 42% of the variability in chlorophyll a and 83% of nitrogen, and better captures bloom timing than previous models. Our results, supported by cross validation, show that onset of the major midsummer bloom is associated with about a month of water temperatures above 20 °C (occurring 19 July to 6 August), consistent with when cyanobacteria dominance is usually reported. Decreased phytoplankton loss rate is the main factor enabling biomass accumulation, consistent with reduced zooplankton grazing on cyanobacteria. The model also shows that phosphorus limitation is most severe in August, and nitrogen limitation tends to occur in early autumn. Our results highlight the role of temperature in regulating bloom initiation and subsequent loss rates, and suggest that a 2 °C increase could lead to blooms that start about 10 days earlier and grow 23% more intense.

Structural basis of the mechanism of ß-methyl epimerization by enzyme MarH.

Diverse derivatives of amino acids with different steric configurations are important biosynthetic building blocks. In biology, epimerization is an important way to generate steric diversity. MarH catalyzes the epimerization of the ß-position of (3R)-ß-methyl-indolepyruvate (MeInPy), forming (3S)-ß-MeInPy. Both compounds are derivatives of l-tryptophan (l-Trp) and are important precursors of bioactive natural products. Here, we report the crystal structures of MarH and the NMR structure of its complex with l-Trp, an analogue of its native substrate, (3R)-ß-MeInPy. Structural analysis and mutagenesis studies indicated that His25 acts as a base to remove Hß and generate a planar carbanion intermediate, which is then putatively reprotonated on the opposite face by a water molecule to form (3S)-ß-MeInPy in a stereospecific manner. The details of ß-site isomerization at the atomic level provide deeper insights into the epimerization mechanism of MarH and will facilitate further enzyme design to extend the substrate scope.

A space-time geostatistical model for probabilistic estimation of harmful algal bloom biomass and areal extent.

Harmful algal blooms (HABs) have been increasing in intensity worldwide, including the western basin of Lake Erie. Substantial efforts have been made to track these blooms using in situ sampling and remote sensing. However, such measurements do not fully capture HAB spatial and temporal dynamics due to the limitations of discrete shipboard sampling over large areas and the effects of clouds and winds on remote sensing estimates. To address these limitations, we develop a space-time geostatistical modeling framework for estimating HAB intensity and extent using chlorophyll a data sampled during the HAB season (June-October) from 2008 to 2017 by five independent monitoring programs. Based on the Bayesian information criterion for model selection, trend variables explain bloom northerly and easterly expansion from Maumee Bay, wind effects over depth, and variability among sampling methods. Cross validation results demonstrate that space-time kriging explains over half of the variability in daily, location-specific chlorophyll observations, on average. Conditional simulations provide, for the first time, comprehensive estimates of overall bloom biomass (based on depth-integrated concentrations) and surface areal extent with quantified uncertainties. These new estimates are contrasted with previous Lake Erie HAB monitoring studies, and deviations among estimates are explored and discussed. Overall, results highlight the importance of maintaining sufficient monitoring coverage to capture bloom dynamics, as well as the benefits of the proposed approach for synthesizing data from multiple monitoring programs to improve estimation accuracy while reducing uncertainty.

Cationic Albumin Encapsulated DNA Origami for Enhanced Cellular Transfection and Stability.

DNA nanostructures, owing to their controllable and adaptable nature, have been considered as highly attractive nanoplatforms for biomedical applications in recent years. However, their use in the biological environment has been restricted by low cellular transfection efficiency in mammalian cells, weak stability under physiological conditions, and endonuclease degradation. Herein, we demonstrate an effective approach to facilitate fast transfection of DNA nanostructures and enhance their stability by encapsulating DNA origami with a biocompatible cationic protein (cHSA) via electrostatic interaction. The coated DNA origami is found to be stable under physiological conditions. Moreover, the cHSA coating could significantly improve the cellular transfection efficiency of DNA origami, which is essential for biological applications.

Space-Time Geostatistical Assessment of Hypoxia in the Northern Gulf of Mexico.

Nearly every summer, a large hypoxic zone forms in the northern Gulf of Mexico. Research on the causes and consequences of hypoxia requires reliable estimates of hypoxic extent, which can vary at submonthly time scales due to hydro-meteorological variability. Here, we use an innovative space-time geostatistical model and data collected by multiple research organizations to estimate bottom-water dissolved oxygen (BWDO) concentrations and hypoxic area across summers from 1985 to 2016. We find that 27% of variability in BWDO is explained by deterministic trends with location, depth, and date, while correlated stochasticity accounts for 62% of observational variance within a range of 185 km and 28 days. Space-time modeling reduces uncertainty in estimated hypoxic area by 30% when compared to a spatial-only model, and results provide new insights into the temporal variability of hypoxia. For years with shelf-wide cruises in multiple months, hypoxia is most severe in July in 59% of years, 29% in August, and 12% in June. Also, midsummer cruise estimates of hypoxic area are only modestly correlated with summer-wide (June-August) average estimates ( r2 = 0.5), suggesting midsummer cruises are not necessarily reflective of seasonal hypoxic severity. Furthermore, summer-wide estimates are more strongly correlated with nutrient loading than midsummer estimates.

Backbone and side-chain chemical shift assignments of MarH, a critical intermediary epimerase for biosynthesis of Maremycins in Streptomyces.

MarH is an essential epimerase that catalyzes the isomerization of 3R-ß-methyl-indolepyruvate (ß-MeInPy) to 3S-ß-MeInPy, which is the important precursor for biosynthesis of Maremycins. Biophysical study of the structure of MarH would be informative for better understanding of its catalytic mechanism and feasible application of the enzyme in isomerization reaction. Here, we report the backbone and side-chain NMR chemical shift assignments of MarH, which lays a foundation for further structural and mechanical study of the enzyme.

Simultaneous quantification of endogenous and exogenous plasma glucose by isotope dilution LC-MS/MS with indirect MRM of the derivative tag.

Quantification of endogenous and exogenous plasma glucose can help more comprehensively evaluate the glucose metabolic status. A ratio-based approach using isotope dilution liquid chromatography tandem mass spectrometry (ID LC-MS/MS) with indirect multiple reaction monitoring (MRM) of the derivative tag was developed to simultaneously quantify endo-/exogenous plasma glucose. Using diluted D-[13C6] glucose as tracer of exogenous glucose, 12C6/13C6 glucoses were first derivatized and then data were acquired in MRM mode. The metabolism of exogenous glucose can be tracked and the concentration ratio of endo/exo-genous glucose can be measured by calculating the endo-/exo-genous glucose concentrations from peak area ratio of specific daughter ions. Joint application of selective derivatization and MRM analysis not only improves the sensitivity but also minimizes the interference from the background of plasma, which warrants the accuracy and reproducibility. Good agreement between the theoretical and calculated concentration ratios was obtained with a linear correlation coefficient (R) of 0.9969 in the range of D-glucose from 0.5 to 20.0 mM, which covers the healthy and diabetic physiological scenarios. Satisfactory reproducibility was obtained by evaluation of the intra- and inter-day precisions with relative standard deviations (RSDs) less than 5.16%, and relative recoveries of 85.96 to 95.92% were obtained at low, medium, and high concentration, respectively. The method was successfully applied to simultaneous determination of the endo-/exogenous glucose concentration in plasma of non-diabetic and type II diabetic cynomolgus monkeys. Graphical Abstract The scheme of the proposed ratio-based approach using isotope dilution LC-MS/MS with indirect MRM of the derivative tag for simultaneous quantification of endogenous and exogenous plasma glucose.

Potential Anti-inflammatory Sesquiterpene Lactones from Eupatorium lindleyanum.

Eupatorium lindleyanum has traditionally been used as folk medicine in Asian countries for its therapeutic effects on tracheitis and tonsillitis. Investigation of the anti-inflammatory active constituents from E. lindleyanum led to the isolation of two novel sesquiterpene lactones, named eupalinolide L (1: ) and eupalinolide M (2: ), and seven known sesquiterpene lactones (3: -9: ). The structures and configurations of the new compounds were determined on the basis of spectroscopic analysis, especially 2D NMR techniques. In vivo experiments showed that the sesquiterpenes fraction significantly reduced mouse ear edema induced by xylene (18.6%, p < 0.05). In in vitro assays, compounds 1: -9: showed excellent anti-inflammatory activities, as they lowered TNF-α and IL-6 levels in lipopolysaccharide-stimulated murine macrophage RAW 264.7 cells (p < 0.001). The above results suggest that the sesquiterpene lactones from E. lindleyanum can be developed as novel potential natural anti-inflammatory agents.

Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctium lappa (L.), which has been found to exert a neuroprotective effect on scopolamine­induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol­induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10 µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10 µM ATG had a protective effect on ethanol­induced injury in PC12 cells.

Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry.

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H2 O, CO, and CH2 O2 losses, together with retro-Diels-Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C-ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C4 H8 ) and 68 Da (C5 H8 ) were yielded from a prenyl chain; neutral losses of 42 (C3 H6 ), 54 (C4 H6 ), and 70 Da (C4 H6 O) were generated by a pyran ring. Particularly, neutral losses of 18 (H2 O), 16 (CH4 ), 112 (C8 H16 ), and 98 Da (C7 H14 ) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.

Beneficial effects of chlorogenic acid on alcohol-induced damage in PC12 cells.

As one of the most commonly abused psychotropic substances, ethanol exposure has deleterious effects on the central nervous system (CNS). The most detrimental results of ethanol exposure during development are the loss of neurons in brain regions such as the hippocampus and neocortex, which may be related to the apoptosis and necrosis mediated by oxidative stress. Recent studies indicated that a number of natural drugs from plants play an important role in protection of nerve cells from damage. Among these, it has been reported that chlorogenic acid (CA) has neuroprotective effects against oxidative stress. Thus, it may play some beneficial effects on ethanol-induced neurotoxicity. However, the effects of CA on ethanol-induced nerve damage remain unclear. In order to investigate the protective effects of CA on alcohol-induced apoptosis in rat pheochromocytoma PC12 cells, in the present study, cell viability and the optimal dosage of CA were first quantified by MTT assay. Then, the cell apoptosis and cell cycle were respectively investigated by Hoechst 33258 staining and flow cytometer (FCM). To further clarify the possible mechanism, followed with the test of mitochondria transmembrane potential with Rhodamine 123 (Rho 123) staining, the expression of Bcl-2, Capase-3 and growth associated protein-43 (GAP-43) were analyzed by immunofluorescence assay separately. The results showed that treatment with 500 mM alcohol decreased the cell viability and then significantly induced apoptosis in PC12 cells. However, when pretreated with different concentrations of CA (1, 5, 10, 50 µM), cell viability increased in different degree. Comparatively, CA with the concentration of 10 µM most effectively promoted the proliferation of damaged cells, increased the distribution ratio of the cells at the G2/M and S phases, and enhanced mitochondria transmembrane potential. This appears to be in agreement with up-regulation of the expression of Bcl-2 and GAP-43, and down-regulation of the expression of Capsae-3. Taken together, CA can increase cell viability and promote cell differentiation by preventing alcohol-induced cell from apoptosis. The mechanism may be related to the enhancement of the expression of GAP-43 and the inhibition of mitochondrial apoptotic pathway including promotion of mitochondria transmembrane potential, up-regulation of the expression of Bcl-2, and down-regulation of the expression of Capsae-3.

Hepatoprotective triterpene saponins from the roots of Glycyrrhiza inflata.

Two novel oleanane-type triterpene saponins, licorice-saponin P2 (1) and licorice-saponin Q2 (3), together with nine known compounds 2, 4-11, have been isolated from the water extract of the roots of Glycyrrhiza inflata. The structures of these compounds were elucidated on the basis of spectroscopic analysis, including 2D-NMR experiments (1H-1H COSY, HSQC, HMBC and ROESY). In in vitro assays, compounds 2-4, 6 and 11 showed significant hepatoprotective activities by lowering the ALT and AST levels in primary rat hepatocytes injured by D-galactosamine (D-GalN). In addition, compounds 2-4, 6, 7 and 11 were found to inhibit the activity of PLA2 with IC50 values of 6.9 µM, 3.6 µM, 16.9 µM, 27.1 µM, 32.2 µM and 9.3 µM, respectively, which might be involved in the regulation of the hepatoprotective activities observed.