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The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229 Mycobacterium tuberculosis ( Mtb ) clinical isolates has identified an essential transcriptional regulator, Rv1830 , herein called resR for resilience regulator, as a frequent target of positive (adaptive) selection. resR mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable Mtb to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of Mtb . Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

The ability of bacterial pathogens to regulate growth is crucial to control homeostasis, virulence, and drug response. Yet, we do not understand the growth and cell cycle behaviors ofMycobacterium tuberculosis(Mtb), a slow-growing pathogen, at the single-cell level. Here, we use time-lapse imaging and mathematical modeling to characterize these fundamental properties of Mtb. Whereas most organisms grow exponentially at the single-cell level, we find that Mtb exhibits a unique linear growth mode. Mtb growth characteristics are highly variable from cell-to-cell, notably in their growth speeds, cell cycle timing, and cell sizes. Together, our study demonstrates that growth behavior of Mtb diverges from what we have learned from model bacteria. Instead, Mtb generates a heterogeneous population while growing slowly and linearly. Our study provides a new level of detail into how Mtb grows and creates heterogeneity, and motivates more studies of growth behaviors in bacterial pathogens.

The remarkable ability of Mycobacterium tuberculosis to survive attacks from the host immune response and drug treatment is due to the resilience of a few bacilli rather than a result of survival of the entire population. Maintenance of mycobacterial subpopulations with distinct phenotypic characteristics is key for survival in the face of dynamic and variable stressors encountered during infection. Mycobacterial populations develop a wide range of phenotypes through an innate asymmetric growth pattern and adaptation to fluctuating microenvironments during infection that point to heterogeneity being a vital survival strategy. In this Review, we describe different types of mycobacterial heterogeneity and discuss how heterogeneity is generated and regulated in response to environmental cues. We discuss how this heterogeneity may have a key role in recording memory of their environment at both the single-cell level and the population level to give mycobacterial populations plasticity to withstand complex stressors.

While previous investigations on online machine translation (MT) in language learning have analyzed how second language (L2) learners use and post-edit MT output, no study as of yet has investigated how the learners process MT errors and what factors affect this process using response and reading times. The present study thus investigates L2 processing of MT errors that are caused by syntactic, morphological, and semantic differences between the source and target language and also examines how L2 proficiency and visual display affect this process. Forty-seven Korean learners of English participated in an acceptability judgment task in which they read a Korean sentence and then its translated counterpart in English and had to judge the accuracy of the translated sentence on a four-point scale. The response latencies for the accuracy judgment as well as the total reading times of source and target sentences were measured. The results revealed that (1) learners generally find it harder to reject mistranslations than to accept correct translations, (2) high vs. low proficiency learners focus on different aspects of language when processing translated output, and (3) constant visual access to the source text does not facilitate but rather interferes with processing MT errors.

Many studies that have investigated the educational value of online machine translation (MT) in second language (L2) writing generally report significant improvements after MT use, but no study as ...

The present study examines how proficiency in the second language (L2) affects language learners’ ability to post-edit texts that are machine-translated from the learners’ native language (L1) to L2. More specifically, the study investigates how L2 proficiency level affects the degree to which Korean learners of English can discern the accuracy of L1-to-L2 machine-translated (MT) text and whether it affects the level of errors (word, phrase, clause, sentence) that are corrected in the post-editing process. Fifty-nine Korean university students’ proficiency was measured using a cloze test and a writing test. They were then given a source text (Korean) and a machine-translated text (English) and were asked to detect and correct as many errors as they can. The overall results of the study showed that L2 proficiency does have a significant effect on how language learners post-edit machine-translated output. With increasing proficiency, the number of corrections increased especially above the word-level, and significant group differences could be found in post-editing patterns of the MT text. The findings have important pedagogical implications for integrating MT activities in L2 classrooms with learners of different proficiency levels.

Increased O-GlcNAcylation can confer neuroprotective effects against necroptosis in the context of Alzheimer’s disease., O-GlcNAcylation (O-linked β-N-acetylglucosaminylation) is notably decreased in Alzheimer’s disease (AD) brain. Necroptosis is activated in AD brain and is positively correlated with neuroinflammation and tau pathology. However, the links among altered O-GlcNAcylation, β-amyloid (Aβ) accumulation, and necroptosis are unclear. Here, we found that O-GlcNAcylation plays a protective role in AD by inhibiting necroptosis. Necroptosis was increased in AD patients and AD mouse model compared with controls; however, decreased necroptosis due to O-GlcNAcylation of RIPK3 (receptor-interacting serine/threonine protein kinase 3) was observed in 5xFAD mice with insufficient O-linked β-N-acetylglucosaminase. O-GlcNAcylation of RIPK3 suppresses phosphorylation of RIPK3 and its interaction with RIPK1. Moreover, increased O-GlcNAcylation ameliorated AD pathology, including Aβ burden, neuronal loss, neuroinflammation, and damaged mitochondria and recovered the M2 phenotype and phagocytic activity of microglia. Thus, our data establish the influence of O-GlcNAcylation on Aβ accumulation and neurodegeneration, suggesting O-GlcNAcylation–based treatments as potential interventions for AD.

The present study investigates native (L1) and second language (L2) processing of scope ambiguities in English sentences containing the universal quantifier every in subject NP and negation. Previous studies in L1 and L2 processing of scope ambiguities have found speakers to generally employ a ‘minimal effort’ principle that highly prefers the surface scope reading regardless of contextual support because accessing the inverse scope reading incurs significant processing cost. The present study compared L1 and L2 scope judgments and processing strategies of sentences such as Every horse didn’t jump over the fence and examined whether the two groups differ in their speed and manner of analysis. Thirty native English speakers and 42 Korean learners of English participated in a self-paced reading/interpretation task that has context (ambiguous vs. unambiguous) and scope reading (surface vs. inverse) as variables. The results revealed significant differences in scope endorsement rates with L2 learners arriving at the surface scope as the dominant reading and L1 learners’ judgments being highly dependent on contextual ambiguity. Moreover, L1 vs. L2 differences in processing strategies were found: L2 learners exhibited a strong tendency to arrive at the most economical interpretation while L1 speakers consulted detailed syntactic and semantic rules of computation.

This study examines whether the mental representation of sentences contains a morphological representation of case marking, using a priming technique....

Purpose. It is known that the arnB (or pmrH) gene encoding uridine 5′-(beta-1-threo-pentapyranosyl-4-ulose diphosphate) aminotransferase plays a critical role in colistin resistance in Pseudomonas aeruginosa through the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A. In this study, we attempted to obtain a colistin-resistant mutant from an arnB-deleted mutant through exposure to colistin. Methodology. We constructed an arnB deletion mutant (P5ΔarnB :: nptIII) from a colistin-susceptible strain (P5) by allelic replacement mutagenesis, and colistin-resistant mutants were selected in vitro using P5 and P5ΔarnB :: nptIII. The growth rate, lipid A structure, biofilm-forming activity and cell viability in diverse stressful conditions (osmotic, oxidative, acidic and heat stress) were investigated. Expression of phoP, pmrA, parR, and cprR was evaluated by qRT-PCR. Results. An arnB deletion mutant was shown to develop colistin resistance through the addition of l-Ara4N to lipid A, despite a low survival rate (over 1000-fold lower than that of the wild-type strain) in the media with colistin. Two colistin-resistant mutants showed higher survival rates than colistin-susceptible strains against 5 % NaCl. In the presence of acidic and heat stress, P5ΔarnB :: nptIII-CstR exhibited higher survival rates during conditions of 1 % HCl and 42 °C than the other strains. Both phoP and pmrA genes were overexpressed significantly in both colistin-resistant mutants, but parR and cprR genes were not. Conclusion. We revealed that colistin resistance could be developed despite arnB deletion in P. aeruginosa through the addition of l-Ara4N to lipid A, which was accompanied by diverse physiological changes.

The primary objective of this paper is to investigate how early and late bilinguals attain implicit knowledge of Korean case drop that necessitates integration of multiple levels of information. An oral picture description task and a written forced-choice elicitation task were developed to investigate how different populations employ the relevant factors in Korean case drop and if certain types of cues are more accessible than others. The results reveal qualitative differences in the underlying linguistic knowledge of early vs. late bilinguals with early bilinguals achieving a higher level of mastery than late bilinguals in both oral and written tasks. The results underline the importance of age, context, and mode of acquisition and suggest that bilingual difficulty in the present phenomenon mainly arises from learners heavily relying on cues that are readily available to them in their respective context of acquisition and failing to effectively coordinate multiple constraints.

In this study, we compared the virulence-associated factors of Acinetobacter baumannii complex species. Sixty-three isolates of five A. baumannii complex species, including 19 A. baumannii, 15 A. nosocomialis, 13 A. seifertii, 13 A. pittii, and 3 A. calcoaceticus isolates, were included in this study. For all isolates, biofilm formation, A549 cell adherence, resistance to normal human serum, and motility were evaluated. A. baumannii complex isolates showed diversity in biofilm formation, A549 cell adherence, and serum resistance, and no strong positive relationships among these virulence characteristics. However, A. seifertii showed relatively consistent virulence-associated phenotypes. In addition, A. baumannii clone ST110 exhibited consistently high virulence-associated phenotypes. Motility was observed in seven isolates, and all four A. baumannii ST110 isolates showed twitching motility. Although some inconsistencies in virulence-associated phenotypes were seen, high virulence characteristics were observed in A. seifertii, which has been mainly reported in Korea and shows high rates of colistin resistance.

Objectives Persister cells following antibiotic exposure may cause failure of antibiotic treatment. The synergistic effects of antibiotic combinations with respect to eliminating persister cells were investigated based on their characteristics. Methods For Acinetobacter baumannii clinical isolates, persister assays were performed using colistin, amikacin, imipenem and ciprofloxacin in various ways, including exposure to antibiotics in combination and sequentially. Persister phenotypes were observed through analysis of ATP concentration, membrane potential and transmission electron microscopy. Results Each A. baumannii isolate showed a specific survival rate of persister cells against each antibiotic. The persister cells were eradicated effectively by exposure to the combination of colistin and amikacin, especially in the sequential order of colistin then amikacin. While the persister cells were not identified after 6 h when exposed to the antibiotics in the order colistin then amikacin, they remained at 0.016% when antibiotic exposure was done in the order amikacin then colistin. Although membrane potential was low in both colistin and amikacin persisters, depletion of the intracellular ATP concentration was only observed in colistin persisters. In addition, transmission electron microscopy analysis showed that colistin persisters have a unique morphology with a rough and rippled membrane and many outer membrane vesicles. Empty pore-like structures surrounded by cracks were also observed. Conclusions In A. baumannii, the combination of colistin and amikacin was most effective for eradication of persister cells, probably due to different mechanisms of persister cell formation between antibiotics. It was also identified that the sequential order of colistin followed by amikacin was important to eradicate the persister cells.

We recently demonstrated a high rate of colistin dependence in Acinetobacter baumannii isolates exposed to colistin in vitro. In the present study, we obtained a colistin-resistant (H08-391R) and colistin-dependent mutant (H08-391D) from a colistin-susceptible parental strain (H08-391). We found that the colistin-dependent mutant converted into a stable colistin-resistant mutant (H08-391D-R) in vitro after four serial passages without colistin. H08-391D and H08-391D-R were both found to harbor defective lipid A, as indicated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis. Additionally, both contained an ISAba1 insertion in lpxC, which encodes a lipid A biosynthetic enzyme. Further, membrane potential measurements using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide (DiOC2[3]) showed that the membrane potential of H08-391D and H08-391D-R was significantly decreased as compared to that of the parental strain, H08-391. Moreover, these mutant strains exhibited increased susceptibilities to antibiotics other than colistin, which may be attributed to their outer membrane fragility. Such phenomena were identified in other A. baumannii strains (H06-855 and its derivatives). Taken together, our study reveals that the colistin-dependent phenotype is a transient phenotype that allows A. baumannii to survive under colistin pressure, and can transition to the extremely resistant phenotype, even in an antibiotic-free environment.

Objectives Colistin susceptibility in Pseudomonas aeruginosa is associated with a lipopolysaccharide (LPS) structure that is controlled by the modulation of several two-component regulatory systems. In this study, we attempted to elucidate the role of these two-component systems in the development of colistin resistance in P. aeruginosa. Methods pmrA-, phoP-, parR- or cprR-inactivated mutants were constructed from a colistin-susceptible P5 strain. Colistin-resistant mutants (P5R, P5ΔpmrA-R, P5ΔphoP-R, P5ΔparR-R and P5ΔcprR-R) were developed in vitro from a wild-type strain (P5) and pmrA-, phoP-, parR- or cprR-inactivated mutants by serial passage in colistin-containing media. Expression levels of the pmrA, phoP, parR, cprR and arnB genes were determined and amino acid alterations of two-component regulatory systems during development of colistin resistance were also investigated. Results While P5ΔpmrA-R, P5ΔparR-R and P5ΔcprR-R showed elevated expression of the phoP gene, the expression levels of the pmrA, parR and cprR genes were not different between gene-inactivated mutants and the adapted colistin-resistant mutants. P5ΔphoP-R showed no significant elevation in expression of any of the pmrA, parR or cprR genes. The arnB gene was overexpressed in all in vitro-selected colistin-resistant mutants compared with colistin-susceptible wild-type and gene-inactivated mutants. Three amino acid alterations in PhoQ and three in ParS were identified in induced colistin-resistant mutants. Conclusions Our data suggest that individual two-component systems may not be essential for the acquisition of colistin resistance in P. aeruginosa. The PhoPQ two-component system may play a major role in the development of colistin resistance in our strains, but alternative or compensatory pathways may exist.

We investigated variations in the rate of persister cell formation against meropenem in 68 Klebsiella pneumoniae isolates from blood. The persister cell formation rates varied markedly but were not significantly different between the patient survival group and death group at 30 days. In addition, they were not associated with the patients' underlying diseases. However, the isolates of CC15 and CC23 showed higher survival rates against 10× MIC of meropenem than CC11. The survival rate of persister cells was less for amikacin and colistin than that for ciprofloxacin. When combinations of meropenem and other antibiotics were administered, persister formation rates decreased compared with those against only meropenem. However, no synergistic effect to remove persister cells was observed. Further investigation is needed to understand persister cell formation in K. pneumoniae with respect to the mechanism involved and clinical implications and that diverse strategies should be explored to remove persister cells.

Scientific Reports 6: Article number: 25543; published online: 06 May 2016; updated: 25 July 2016 In this Article, the amino acid sequences for strain P5R have been omitted from Figure 3A. The correct Figure 3A appears below as Figure 1.

The increased reliance on colistin for treating multidrug-resistant Gram-negative bacterial infections has resulted in the emergence of colistin-resistant Pseudomonas aeruginosa. We attempted to identify genetic contributors to colistin resistance in vitro evolved isogenic colistin-resistant and -susceptible strains of two P. aeruginosa lineages (P5 and P155). Their evolutionary paths to acquisition and loss of colistin resistance were also tracked. Comparative genomic analysis revealed 13 and five colistin resistance determinants in the P5 and P155 lineages, respectively. Lipid A in colistin-resistant mutants was modified through the addition of 4-amino-L-arabinose; this modification was absent in colistin-susceptible revertant strains. Many amino acid substitutions that emerged during the acquisition of colistin resistance were reversed in colistin-susceptible revertants. We demonstrated that evolved colistin resistance in P. aeruginosa was mediated by a complicated regulatory network that likely emerges through diverse genetic alterations. Colistin-resistant P. aeruginosa became susceptible to the colistin upon its withdrawal because of genetic reversion. The mechanisms through which P. aeruginosa acquires and loses colistin resistance have implications on the treatment options that can be applied against P. aeruginosa infections, with respect to improving bactericidal efficacy and preventing further resistance to antibiotics.

Second language (L2) learners have been found to experience difficulty in tasks that require the integration of discourse–pragmatic properties with syntactic and semantic properties (Sorace and Serratrice, 2009; Tsimpli and Sorace, 2006; Valenzuela, 2006). The present article investigates the sources of L2 difficulty in a phenomenon where multiple components of linguistic analysis are involved. The study examines L1-Korean L2-English adult learners’ interpretation of English sentences with the universally quantified object NP ( every NP) and negation using an offline contextualized acceptability judgment task. The results suggest that the sources of L2 variability reflect L2 adults’ focus on content and meaning access and L1 transfer of interpretive preferences in initial to intermediate stages of acquisition but are replaced by the difficulty of integrating L2 pragmatic properties with other aspects of linguistic analysis in the advanced stages, a difficulty which can nevertheless be overcome in ultimate attainment. The study posits pragmatic information to be one of the last considerations for L2 learners when multiple types of information need to be integrated to calculate meaning despite the fact that L2 adults possess mature cognitive mechanisms and pragmatic abilities.

Background Biliary tract cancers have limitations in information about different location-related pathogenesis and clinico-pathological characteristics. The goal of this study was to investigate anatomical site-related similarities and differences in biliary tract cancers and to assess the expression and clinical significance of functional proteins such as p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1. Methods One hundred and sixty-one patients with biliary tract adenocarcinomas, who underwent curative or palliative surgery in a single institution between October 1994 and December 2003 were evaluated, retrospectively. The level of protein expression of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 was assessed by immunohistochemistry. Results With respect to clinico-pathological characteristics, gallbladder cancer was more frequent in women, and bile duct cancer was more common in men. Perineural invasion was more common in bile duct cancer. Recurrence as a distant metastasis was more common in gallbladder cancer. Immunohistochemical analysis revealed that thymidine phosphorylase expression was significantly higher in gallbladder cancer than in bile duct cancer. Positive thymidine phosphorylase and p53 staining were associated with an advanced stage. Differentiation, vascular invasion, perineural invasion, lymphatic invasion, lymph node metastasis, and TNM stage independently predicted poor prognosis in biliary tract cancer. These correlations were seen more clearly in gallbladder cancer. The immunohistochemical staining patterns of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 showed no prognostic significance in biliary tract cancers. Conclusions We concluded that gallbladder and bile duct cancers are considered to be separate diseases with different clinico-pathological characteristics and prognostic factors. In addition, we hypothesize that high expression of thymidine phosphorylase by gallbladder cancer results in a higher response rate to capecitabine by gallbladder cancer than bile duct cancer.