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The mechanism(s) of how bacteria acquire tolerance and then resistance to antibiotics remains poorly understood. Here, we show that glucose abundance decreases progressively as ampicillin-sensitive strains acquire resistance to ampicillin. The mechanism involves that ampicillin initiates this event via targeting pts promoter and pyruvate dehydrogenase (PDH) to promote glucose transport and inhibit glycolysis, respectively. Thus, glucose fluxes into pentose phosphate pathway to generate reactive oxygen species (ROS) causing genetic mutations. Meanwhile, PDH activity is gradually restored due to the competitive binding of accumulated pyruvate and ampicillin, which lowers glucose level, and activates cyclic adenosine monophosphate (cAMP)/cAMP receptor protein (CRP) complex. cAMP/CRP negatively regulates glucose transport and ROS but enhances DNA repair, leading to ampicillin resistance. Glucose and Mn 2+ delay the acquisition, providing an effective approach to control the resistance. The same effect is also determined in the intracellular pathogen Edwardsiella tarda. Thus, glucose metabolism represents a promising target to stop/delay the transition of tolerance to resistance.

Background Mangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves. Results The protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis. Conclusion Our data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

Antibiotic-resistant bacteria are a serious threat to human and animal health. Metabolite-enabled eradication of drug-resistant pathogens is an attractive strategy, and metabolite adjuvants, such as fumarate, are used for restoring the bactericidal ability of antibiotics. However, we show that metabolites in the TCA cycle increase the viability of Edwardsiella tarda against chloramphenicol (CAP), based on the survival assay of differential metabolites identified by LC-MS/MS. Furthermore, NADPH promotes CAP resistance in the CAP-resistant strain, while oxidants restore the bactericidal ability. Finally, we show that the intracellular redox state determines the sensitivity to CAP, and the total antioxidative capacity is decreased significantly in the antibiotic-resistant strain. Considering that the metabolites promote CAP resistance, metabolite adjuvants should be applied very cautiously. Overall, our research expands on the knowledge that the redox state is related to the bactericidal ability of CAP.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Antibiotic-resistant

Multidrug-resistant

Background WRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata. Results In this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five β-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants. Conclusion These results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.

Photodynamic therapy (PDT) received great attention in cancer therapy due to the advantages of negligible drug resistance, low side effects, and minimal invasiveness. Development of theranostic nanoprobes with specific imaging-guided PDT is of great significance in the field. Herein we report the fabrication of a novel theranostic nanoprobe porphyrin/G-quadruplex conjugated gold/persistent luminescence nanocomposites for imaging-guided PDT. The developed nanoprobe contains NIR-emitting persistent luminescent nanoparticles (PLNP) as the core for autofluorescence-free bioimaging and Au coating on PLNP for facile subsequent DNA conjugation. The DNA sequence is designed to contain G-rich AS1411 aptamer for recognizing the over-expressed cellular nucleolin of cancer cell and forming a G-quadruplex structure to combine with tetrakis (4-carboxyphenyl) porphyrin (TCPP) to realize PDT. The AS1411 aptamer-contained DNA conjugated Au-coated PLNP is rapidly prepared via a freezing method with high content of DNA and good aqueous stability. Meanwhile, TCPP is easily loaded into the G-quadruplex structure formed from G-rich AS1411 aptamer on the surface of Au/PLNP in presence of K+. The theranostic nanoprobe gives integrated merits of PLNP for autofluorescence-free bioimging, TCPP for PDT and AS1411 aptamer-contained DNA for specific binding to cancer cells. This work provides a new specially designed imaging-guided PDT nanoplatform for theranostics.

Antibiotic-resistant bacteria are a serious threat to human and animal health. Metabolite-enabled eradication of drug-resistant pathogens is an attractive strategy, and metabolite adjuvants, such as fumarate, are used for restoring the bactericidal ability of antibiotics. However, we show that metabolites in the TCA cycle increase the viability of

Objective This study was performed to compare the clinical effects of locking plates (LPs) with those of hook plates (HPs) in the treatment of Neer type II distal clavicle fractures. Methods From August 2014 to April 2018, 64 patients with Neer type II distal clavicle fractures were treated in our department. The clinical effects were assessed with respect to the operation time, intraoperative blood loss, incision length, fracture healing, postoperative pain, postoperative complications, and postoperative shoulder joint function. Results There were no significant differences in the healing time, operation time, or intraoperative blood loss between the LP and HP groups. The incision length was significantly shorter in the LP than HP group, and the postoperative complication rate was significantly lower in the LP than HP group. The visual analog scale score, Constant–Murley score, and University of California Los Angeles score were significantly better in the LP than HP group. Conclusions Compared with HPs, the use of LPs involves a smaller incision in the treatment of Neer type II distal clavicle fractures and significantly reduces postoperative pain and complications. Therefore, priority can be given to the use of LPs in the treatment of Neer type II distal clavicle fractures.

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

Antibiotic-resistant Edwardsiella tarda poses a severe challenge to aquaculture. An understanding for antibiotic-resistant mechanisms is crucial to control the disease. The present study characterizes E. tarda ampicillin-stressed proteome and shows the importance of energy metabolism including the TCA cycle and glycolysis/gluconeogenesis in the antibiotic resistance. Further combination with antibiotic measurement develops a new method for identification of antibiotic-binding proteins out of differential abundances of proteins and results in determination of ETAE_0175 and ETAE_3367 as ampicillin-binding proteins in E. tarda . Genes of the two proteins are cloned and recombinant proteins are purified for validation of antibiotic-binding capability. Results show that higher binding capability is detected in ETAE_3367 than ETAE_0175. Out of the two proteins, ETAE_3367 is first reported here to be an antibiotic-binding protein, while ETAE_0175 homology in other bacteria has been shown to bind with other antibiotics. Bioinformatics analysis shows that ETAE_3367 may closely interact with aceF and sucA belonging to the TCA cycle and glycolysis/gluconeogenesis, respectively. These results indicate that energy metabolism contributes to ampicillin resistance in E. tarda and a new method to identify antibiotic-binding proteins is developed. These findings highlight the way to an understanding of antibiotic-resistant mechanisms in content of antibiotic-binding proteins. Biological significance Our data characterizes Edwardsiella tarda ampicillin-stressed proteome and shows the importance of energy metabolism including the TCA cycle and glycolysis/gluconeogenesis in the antibiotic resistance. Furthermore, a new method based 2-DE proteomics is developed for identification of antibiotic-binding proteins, which results in determination of ETAE_0175 and ETAE_3367 as ampicillin-binding proteins in E. tarda . ETAE_3367 is closely interacted with proteins of the TCA cycle and glycolysis/gluconeogenesis, suggesting the drug-resistant mechanism.

SummaryMultidrug-resistant bacteria are an increasingly serious threat to human and animal health. However, novel drugs that can manage infections by multidrug-resistant bacteria have proved elusive. Here we show that glucose and alanine abundances are greatly suppressed in kanamycin-resistant Edwardsiella tarda by GC-MS-based metabolomics. Exogenous alanine or glucose restores susceptibility of multidrug-resistant E. tarda to killing by kanamycin, demonstrating an approach to killing multidrug-resistant bacteria. The mechanism underlying this approach is that exogenous glucose or alanine promotes the TCA cycle by substrate activation, which in turn increases production of NADH and proton motive force and stimulates uptake of antibiotic. Similar results are obtained with other Gram-negative bacteria (Vibrio parahaemolyticus, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacterium (Staphylococcus aureus), and the results are also reproduced in a mouse model for urinary tract infection. This study establishes a functional metabolomics-based strategy to manage infection by antibiotic-resistant bacteria.

We previously showed that glucose potentiated kanamycin to kill multidrug-resistant Edwardsiella piscicida through activation of the TCA cycle. However, whether other regulatory mechanism is involved requires further investigation. By quantitative proteomics technology, iTRAQ, we systematically mapped the altered proteins in the presence of glucose and identified 94 differentially expressed proteins. The analysis of the altered proteins by pathways, amino acid biosynthesis and metabolism were enriched. And the most significantly altered eight amino acids tyrosine, phenylalanine, valine, leucine, isoleucine, glycine, serine and threonine were investigated for their potentiation of kanamycin to kill EIB202, where glycine, serine and threonine showed the strongest efficacy than the others. The combinations of glycine and serine or glucose with glycine, serine or threonine had the best effects. Moreover, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were increased as well as the proton motive force (PMF) and intracellular kanamycin. Finally, inhibitors that disrupt PMF production abolished the potentiation. These results shed light on the mechanism of how glucose promoting the amino acids biosynthesis and metabolism to potentiate kanamycin to kill antibiotic-resistant bacteria. More importantly, our results suggested that adjusting amino acid biosynthesis and metabolism might be a strategy to become phenotypic resistance to antibiotics in bacteria. Significance Tackling antibiotic resistance is an emerging issue in current years. Despite the efforts made toward developing new antibiotics, the progress is still lagged behind expectation. Novel strategies are required. The use of metabolite to revert antibiotic resistant is highly appreciated in recent years due to the less toxicity, more economic and high efficacy. As a continued study of our previous report on glucose potentiating kanamycin to kill antibiotic-resistant bacteria. The current study further expands the previous discovery on the mechanism of how glucose potentiate this effect. This result provides more basis on the action of glucose in reverting antibiotic resistance. And more importantly, we may derive more metabolites other than glucose to manage antibiotic resistance.

Antibiotic-resistant bacteria are an increasingly serious threat to human health and aquaculture. To further explore bacterial antibiotic resistance mechanism, iTRAQ is used to identify a differential proteome in ampicillin-resistant LTB4 (LTB4-RAMP), a strain of Edwardsiella piscicida. A total of 102 differentially proteins with 50 upregulation and 52 downregulation are identified. Since many of these changes are related to metabolism, interactive pathways explorer(iPath) is used to understand a global differentially metabolic response in LTB4-RAMP. This analysis identifies a global depressed metabolic modulation as the most characteristic feature of LTB4-RAMP. Lower membrane potential and ATP in LTB4-RAMP than control support that the central carbon metabolism and energy metabolism are reduced. Since the pyruvate cycle (the P cycle) plays a key role in the central carbon metabolism and energy metabolism, further investigation focuses on the P cycle and shows that expression of genes and activity of enzymes in the P cycle are decreased in LTB4-RAMP. These results support the conclusion that the depressed P cycle contributes to the acquisition of ampicillin resistance in E.piscicida. These findings indicate that the combination of proteomics and iPath analysis can provide a global metabolic profile, which helps us better understand the correlation between ampicillin resistance and cellular metabolism. SIGNIFICANCE: The present study uses iTRAQ to explore ampicillin resistance mechanism in Edwardsiella piscicida and finds many of these differential abundances of proteins are related to metabolism. IPath further identifies a global depressed metabolic modulation and characterizes the reduced pyruvate cycle as the most characteristic feature of the ampicillin-resistant E. piscicida, which is supported by reduced expression of genes and activity of enzymes in the pyruvate cycle. Consisitently, lower membrane potential and ATP are detetced. These results reveal the metabolic mechanism of ampicillin resistance and provide a solid proof to revert the resistance by reprogramming metabolomics.

Canada is the second country after the US to start the shale gas exploration and production in the world. And the Western Canada Sedimentary Basin (WCSB) is known for its rich shale gas reserves. However, with the development of shale gas, it is found that the success of the United States is difficult to replicate. In addition, many oil companies were forced to transfer their focus of exploration and development from shale gas to tight oil for the extended downturn of gas-price in recent years. Afterwards, abundant resources of tight oil were discovered in northeast Alberta located in the Western Canada Sedimentary Basin. This paper studies the developing history and current situation of tight oil, and review the horizontal well drilling technology, stimulated reservoir volume (SRV) fracturing completion technology and multi-well pad factory operation. Meanwhile, some opinions and Suggestions on the tight oil exploration and production in China are proposed.

Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

In general leading medicine, patients takes prescription to drugstore, then a pharmacist get this prescription, seeks gallipot on medicine cabinet. The pharmacist checks each drug, dose distribution, and gives it to patient. But there are lots of drugs and gallipots in large hospitals, the pharmacist must take more times on seeking and checking drug. In this processes, it takes lots of times on checking drug. All of it is safe for patients, but it takes times and person. By law, all of this process can't be automatically by machine. For this case, we try to simplify and shorten process by using mobile application. Thanks to the widespread wireless internet and developed technology products, we use this advantage in this discuss. We try to use mobile devices and mobile application, get the correct gallipots on medicine cabinet fast. The pharmacist only needs take the gallipots, check the drug in gallipots, dose distribution, and give it to patients and tell them how to take. Patients also can get drug information likes how to take, contra-indications listed for dung. It will avoid accidental on take drugs. This discusses will save labor cost and increase medication safety.