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The apple is a significant global fruit cultivated extensively worldwide. Fire blight, caused by Erwinia amylovora (Ea), poses a significant threat to global apple production. To control this disease, characterizing the virulence mechanisms/factors is imperative. Carbamoyl phosphate synthase is an essential enzyme in the biosynthesis of arginine and pyrimidine. However, the functions of this protein in Ea remains poorly understood. This study aimed to investigate the functions of the carbamoyl phosphate synthase large subunit in Ea (CarBEa). In a virulence assay using fruitlets, an Ea strain lacking CarBEa exhibited significantly reduced virulence on fruitlets. In the auxotrophy assay, this mutant failed to grow in minimal media lacking both arginine and pyrimidine, but growth was restored when both compounds were supplemented. The comparative proteomic analysis suggests that CarBEa is involved in diverse biological processes, including amino acid and nucleotide metabolism, and inorganic ion transport. Finally, we demonstrated that CarBEa is related to siderophore secretion/production by the chrome azurol S agar plate assay. This report provides valuable insights into the functions of carbamoyl phosphate synthase large subunit, which serves as a potential target for developing efficient anti-virulence substances to control fire blight.

Acidovorax citrulli (Ac) is a phytopathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops, including watermelon. However, there are no effective methods to control this disease. YggS family pyridoxal phosphate-dependent enzyme acts as a coenzyme in all transamination reactions, but its function in Ac is poorly understood. Therefore, this study uses proteomic and phenotypic analyses to characterize the functions. The Ac strain lacking the YggS family pyridoxal phosphate-dependent enzyme, AcΔyppAc(EV), virulence was wholly eradicated in geminated seed inoculation and leaf infiltration. AcΔyppAc(EV) propagation was inhibited when exposed to L-homoserine but not pyridoxine. Wild-type and mutant growth were comparable in the liquid media but not in the solid media in the minimal condition. The comparative proteomic analysis revealed that YppAc is primarily involved in cell motility and wall/membrane/envelop biogenesis. In addition, AcΔyppAc(EV) reduced biofilm formation and twitching halo production, indicating that YppAc is involved in various cellular mechanisms and possesses pleiotropic effects. Therefore, this identified protein is a potential target for developing an efficient anti-virulence reagent to control BFB.

Acidovorax citrulli (Ac) is a causal agent of watermelon bacterial fruit blotch (BFB) disease. Because resistance cultivars/lines have not yet been developed, it is imperative to elucidate Ac’s virulence factors and their mechanisms to develop resistant cultivars/lines in different crops, including watermelon. The glucose-6-phosphate isomerase (GPI) is a reversible enzyme in both glycolysis and gluconeogenesis pathways in living organisms. However, the functions of GPI are not characterized in Ac. In this study, we determined the roles of GpiAc (GPI in Ac) by proteomic and phenotypic analyses of the mutant lacking GPI. The mutant displayed significantly reduced virulence to watermelon in two different virulence assays. The mutant’s growth patterns were comparable to the wild-type strain in rich medium and M9 with glucose but not with fructose. The comparative proteome analysis markedly identified proteins related to virulence, motility, and cell wall/membrane/envelope. In the mutant, biofilm formation and twitching halo production were reduced. We further demonstrated that the mutant was less tolerant to osmotic stress and lysozyme treatment than the wild-type strain. Interestingly, the tolerance to alkali conditions was remarkably enhanced in the mutant. These results reveal that GpiAc is involved not only in virulence and glycolysis/gluconeogenesis but also in biofilm formation, twitching motility, and tolerance to diverse external stresses suggesting the pleiotropic roles of GpiAc in Ac. Our study provides fundamental and valuable information on the functions of previously uncharacterized glucose 6-phosphate isomerase and its virulence mechanism in Ac.

Erwinia amylovora, the causal agent of fire-blight disease in apple and pear trees, was first isolated in South Korea in 2015. Although numerous studies, including omics analyses, have been conducted on other strains of E. amylovora, studies on South Korean isolates remain limited. In this study, we conducted a comparative proteomic analysis of the strain TS3128, cultured in three media representing different growth conditions. Proteins related to virulence, type III secretion system, and amylovoran production, were more abundant under minimal conditions than in rich conditions. Additionally, various proteins associated with energy production, carbohydrate metabolism, cell wall/membrane/envelope biogenesis, and ion uptake were identified under minimal conditions. The strain TS3128 expresses these proteins to survive in harsh environments. These findings contribute to understanding the cellular mechanisms driving its adaptations to different environmental conditions and provide proteome profiles as reference for future studies on the virulence and adaptation mechanisms of South Korean strains.

Cryptojacking is often used by attackers as a means of gaining profits by exploiting users’ resources without their consent, despite the anticipated positive effect of browser-based cryptomining. Previous approaches have attempted to detect cryptojacking websites, but they have the following limitations: (1) they failed to detect several cryptojacking websites either because of their evasion techniques or because they cannot detect JavaScript-based cryptojacking and (2) they yielded several false alarms by focusing only on limited characteristics of cryptojacking, such as counting computer resources. In this paper, we propose CIRCUIT, a precise approach for detecting cryptojacking websites. We primarily focuse on the JavaScript memory heap, which is resilient to script code obfuscation and provides information about the objects declared in the script code and their reference relations. We then extract a reference flow that can represent the script code behavior of the website from the JavaScript memory heap. Hence, CIRCUIT determines that a website is running cryptojacking if it contains a reference flow for cryptojacking. In our experiments, we found 1,813 real-world cryptojacking websites among 300K popular websites. Moreover, we provided new insights into cryptojacking by modeling the identified evasion techniques and considering the fact that characteristics of cryptojacking websites now appear on normal websites as well.

Acidovorax citrulli (Ac) is a gram-negative bacterium that causes bacterial fruit blotch (BFB) disease in cucurbit crops including watermelon. However, despite the great economic losses caused by this disease worldwide, Ac-resistant watermelon cultivars have not been developed. Therefore, characterizing the virulence factors/mechanisms of Ac would enable the development of effective control strategies against BFB disease. The 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (BdpM) is known to participate in the glycolysis and gluconeogenesis pathways. However, the roles of the protein have not been characterized in Ac. To elucidate the functions of BdpmAc (Bdpm in Ac), comparative proteomic analysis and diverse phenotypic assays were conducted using a bdpmAc knockout mutant (bdpmAc:Tn) and a wild-type strain. The virulence of the mutant to watermelon was remarkably reduced in both germinated seed inoculation and leaf infiltration assays. Moreover, the mutant could not grow with fructose or pyruvate as a sole carbon source. However, the growth of the mutant was restored to levels similar to those of the wild-type strain in the presence of both fructose and pyruvate. Comparative proteomic analyses revealed that diverse proteins involved in motility and wall/membrane/envelop biogenesis were differentially abundant. Furthermore, the mutant exhibited decreased biofilm formation and twitching halo size. Interestingly, the mutant exhibited a higher tolerance against osmotic stress. Overall, our findings suggest that BdpmAc affects the virulence, glycolysis/gluconeogenesis, biofilm formation, twitching halo size, and osmotic tolerance of Ac, suggesting that this protein has pleiotropic properties. Collectively, our findings provide fundamental insights into the functions of a previously uncharacterized phosphoglycerate mutase in Ac.

Dapagliflozin (DAP), which improves glycemic control in patients with type 2 diabetes mellitus, has poor physical properties against heat and moisture, thus hindering its manufacturing potential. The superior physicochemical properties of a recently developed cocrystal of DAP and citric acid (DAP cocrystal) in comparison with those of DAP and Forxiga®, a patented solvate form with propandiol monohydrate, were identified via structural analysis and moisture sorption isotherm. For the first time, the formulation, manufacturability, and in vivo bioavailability of DAP cocrystals were successfully investigated to develop oral dosage forms that substitute Forxiga®. The intrinsic dissolution rate of DAP cocrystal was controlled by varying particle size distribution. Unlike the direct compression (DC), roller compaction (RC) was more preferable to obtain good flowability of dry granules for a continuous manufacturing system. The cocrystal structure was maintained throughout the stability assessment period. In Vitro dissolution pattern differences of the optimized DAP cocrystal tablet with RC and the reference tablet, Forxiga® 10 mg, were pharmaceutically equivalent within 5% in four different media. Furthermore, comparative pharmacokinetic analysis confirmed that a 10 mg DAP cocrystal tablet with RC was bioequivalent to a 10 mg Forxiga® tablet, as assessed in beagle dogs and human volunteers.

Recently, the area of sea ice is rapidly decreasing due to global warming, and since the Arctic sea ice has a great impact on climate change, interest in this is increasing very much all over the world. In fact, the area of sea ice reached a record low in September 2012 after satellite observations began in late 1979. In addition, in early 2018, the glacier on the northern coast of Greenland began to collapse. If we are interested in record values of sea ice area, modeling relationships of these values and predicting future record values can be a very important issue because the record values that consist of larger or smaller values than the preceding observations are very closely related to each other. The relationship between the record values can be modeled based on the pivotal quantity and canonical and drawable vine copulas, and the relationship is called a dependence structure. In addition, predictions for future record values can be solved in a very concise way based on the pivotal quantity. To accomplish that, this article proposes an approach to model the dependence structure between record values based on the canonical and drawable vine. To do this, unknown parameters of a probability distribution need to be estimated first, and the pivotal-based method is provided. In the pivotal-based estimation, a new algorithm to deal with a nuisance parameter is proposed. This method allows one to reduce computational complexity when constructing exact confidence intervals of functions with unknown parameters. This method not only reduces computational complexity when constructing exact confidence intervals of functions with unknown parameters, but is also very useful for obtaining the replicated data needed to model the dependence structure based on canonical and drawable vine. In addition, prediction methods for future record values are proposed with the pivotal quantity, and we compared them with a time series forecasting method in real data analysis. The validity of the proposed methods was examined through Monte Carlo simulations and analysis for Arctic sea ice data.

In this paper, we describe a new method for the selective extraction and quantification of glutathione (GSH) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and maleimide-presenting gold nanoparticles (Mal-AuNPs). Our strategy utilizes the Michael addition to selectively extract GSH, from chosen samples, onto the maleimide of Mal-AuNPs. After the extraction step, the GSH bound to the AuNPs was analyzed by MALDI-TOF MS in the presence of an internal standard which was prepared by reacting Mal-AuNPs with isotope-labeled GSH (GSH*). The GSH* has the same structure as GSH but a higher molecular weight, and therefore, enables absolute quantification of GSH by comparing the mass signal intensities of the GSH- and GSH*-conjugated alkanethiols. Our strategy was verified by analyzing GSH- spiked fetal bovine serum and NIH 3T3 cells. [ABSTRACT FROM AUTHOR]

The laser shock cleaning (LSC) method has recently attracted substantial attention since it can remove micro/nano-scale contaminant particles from a solid surface without direct exposure of the surface to laser irradiation. However, despite the importance of the particle detachment and redeposition mechanisms in the LSC process, the behavior of the particles during the cleaning process has never been analyzed experimentally. In this work, the motion of the micrometer-scale particles detached by a laser-induced plasma/shock wave is visualized by a photoluminescence imaging technique. The technique yields time-resolved particle trajectories under typical conditions of the LSC process, with and without a gas jet blowing. Discussions are made on the behavior of the detached particles and redeposition mechanisms. [ABSTRACT FROM AUTHOR]