Iconicity mediates semantic networks of sound symbolisma).

One speech sound can be associated with multiple meanings through iconicity, indexicality, and/or systematicity. It was not until recently that this "pluripotentiality" of sound symbolism attracted serious attention, and it remains uninvestigated how pluripotentiality may arise. In the current study, Japanese, Korean, Mandarin, and English speakers rated unfamiliar jewel names on three semantic scales: size, brightness, and hardness. The results showed language-specific and cross-linguistically shared pluripotential sound symbolism. Japanese speakers associated voiced stops with large and dark jewels, whereas Mandarin speakers associated [i] with small and bright jewels. Japanese, Mandarin, and English speakers also associated lip rounding with darkness and softness. These sound-symbolic meanings are unlikely to be obtained through metaphorical or metonymical extension, nor are they reported to colexify. Notably, in a purely semantic network without the mediation of lip rounding, softness can instead be associated with brightness, as illustrated by synesthetic metaphors such as yawaraka-na hizashi /jawaɾakanaçizaɕi/ "a gentle (lit. soft) sunshine" in Japanese. These findings suggest that the semantic networks of sound symbolism may not coincide with those of metaphor or metonymy. The current study summarizes the findings in the form of (phono)semantic maps to facilitate cross-linguistic comparisons of pluripotential sound symbolism.

Iconicity in Ideophones: Guessing, Memorizing, and Reassessing.

Iconicity, or the resemblance between form and meaning, is often ascribed to a special status and contrasted with default assumptions of arbitrariness in spoken language. But does iconicity in spoken language have a special status when it comes to learnability? A simple way to gauge learnability is to see how well something is retrieved from memory. We can further contrast this with guessability, to see (1) whether the ease of guessing the meanings of ideophones outperforms the rate at which they are remembered; and (2) how willing participants' are to reassess what they were taught in a prior task-a novel contribution of this study. We replicate prior guessing and memory tasks using ideophones and adjectives from Japanese, Korean, and Igbo. Our results show that although native Cantonese speakers guessed ideophone meanings above chance level, they memorized both ideophones and adjectives with comparable accuracy. However, response time data show that participants took significantly longer to respond correctly to adjective-meaning pairs-indicating a discrepancy in a cognitive effort that favored the recognition of ideophones. In a follow-up reassessment task, participants who were taught foil translations were more likely to choose the true translations for ideophones rather than adjectives. By comparing the findings from our guessing and memory tasks, we conclude that iconicity is more accessible if a task requires participants to actively seek out sound-meaning associations.

Salmonella Central Carbon Metabolism Enhances Bactericidal Killing by Fluoroquinolone Antibiotics.

The efficacy of killing by bactericidal antibiotics has been reported to depend in large part on the ATP levels, with low levels of ATP leading to increased persistence after antibiotic challenge. Here, we show that an atp operon deletion strain of Salmonella enterica serovar Typhimurium lacking the ATP synthase was at least 10-fold more sensitive to killing by the fluoroquinolone antibiotic ciprofloxacin and yet showed either increased survival or no significant difference compared with the wild-type strain when challenged with aminoglycoside or ß-lactam antibiotics, respectively. The increased cell killing and reduced bacterial survival (persistence) after fluoroquinolone challenge were found to involve metabolic compensation for the loss of the ATP synthase through central carbon metabolism reactions and increased NAD(P)H levels. We conclude that the intracellular ATP levels per se do not correlate with bactericidal antibiotic persistence to fluoroquinolone killing; rather, the central carbon metabolic pathways active at the time of challenge and the intracellular target of the antibiotic determine the efficacy of treatment.

Characterization of the infection process by Peronospora belbahrii on basil by scanning electron microscopy.

Basil downy mildew caused by Peronospora belbahrii is a disease of sweet basil (Ocimum basilicum) production worldwide. In this study, sweet basil was grown in plant growth chambers and inoculated with sporangia of P. belbahrii harvested from previously infected plants. Plants were placed in closed, clear plastic bags and leaves harvested over time and observed using scanning electron microscopy. In most cases, sporangia germinated myceliogenically on abaxial and adaxial leaf surfaces as early as three days after inoculation. Germ tubes and the tips of hyphae ramifying on leaf surfaces directly penetrated basil leaves to initiate the infection process. Hyphal growth was not observed to gain entrance to the interior of leaves through stomata, though growth over these openings was observed. Most frequently, seven days after inoculation, one or more sporangiophores grew through stomata to produce new sporangia on both the abaxial and adaxial surfaces of leaves. Macroscopic signs of infection were visible on both sides of leaves approximately ten days after inoculation under the conditions of this study. These results contribute to a better understanding of the infection process and disease onset of P. belbahrii and should help in the development of more effective measures for reducing basil downy mildew.

The reality of hierarchical morphological structure in multimorphemic words.

This cross-modal priming study is one of the first to empirically test the long-held assumption that individual morphemes of multimorphemic words are represented according to a hierarchical structure. The results here support the psychological reality behind this assumption: Recognition of trimorphemic words (e.g., unkindness or [[un-[kind]]-ness]) was significantly facilitated by prior processing of their substrings when the substrings served as morphological constituents of the target words (e.g., unkind), but not when the substrings were not morphological constituents of the target words (e.g., kindness). This morphological structural priming occurred independently of the linear positions of morphological constituents.

Are tones in the expressive lexicon iconic? Evidence from three Chinese languages.

Recent advances in the literature have focused on sketching phonosemantic mappings of imitative or iconic utterances by relying on vowels and consonants, leaving the suprasegmental information unexplored. To begin bridging this gap, this study looks at the interaction of lexical tone and iconicity by comparing sound symbolic (i.e., mimetic, expressive, ideophonic) strata and general (i.e., arbitrary, prosaic, non-iconic) strata from three Chinese languages (Mandarin, Taiwanese Southern Min, Hong Kong Cantonese) using corpus-based means. For all three languages the distribution of tones in the sound symbolic strata are skewed so that the majority of syllables are largely confined to two tonal categories per language, one of which is high level, while the general strata exhibit no such tonal bias. These results indicate that phonological systematicity at the prosodic level might play an important role in demarcating an iconic class of words. This cross-linguistic tendency towards high tone mappings may be derived from phonotactic strategies to facilitate prosodic foregrounding of iconic utterances as well as an embodiment of expressive voice and marked pitch use like that of Infant Directed Speech.

FVIII proteins with a modified immunodominant T-cell epitope exhibit reduced immunogenicity and normal FVIII activity.

Factor VIII (FVIII)-neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 contains an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells from HA subjects. The aim of this study was to identify amino acid substitutions to deimmunize this epitope while retaining procoagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The shortest DRB1*01:01-binding peptide was FVIII2194-2205, and residues important for affinity were identified as F2196, M2199, A2201, and S2204. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation of clones specific for the WT sequence. T-cell clones that were stimulated by recombinant WT-FVIII-C2 (rWT-FVIII-C2) protein did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic peptide includes a naturally processed T-cell epitope. Additional amino acid substitutions at F2196 and M2199 were evaluated by peptide-MHC class II (MHCII)-binding assays, T-cell proliferation assays, epitope prediction algorithms, and sequence homologies. Six B-domain-deleted (BDD)-FVIII proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W, or M2199R were produced. Proliferation of T-cell clones and polyclonal lines in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A was reduced compared with responses to WT-BDD-FVIII. The BDD-FVIII-F2196K sequence modification appears to be the most promising sequence variant tested here, due to its effectiveness at eliminating DRB1*01:01-restricted immunogenicity, low potential immunogenicity in the context of other MHCII alleles, expression level comparable to WT-BDD-FVIII, and retained procoagulant activity. These results provide proof of principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.

The metabolic pathways utilized by Salmonella Typhimurium during infection of host cells.

Only relatively recently has research on the metabolism of intracellular bacterial pathogens within their host cells begun to appear in the published literature. This reflects in part the experimental difficulties encountered in separating host metabolic processes from those of the resident pathogen. One of the most genetically tractable and thoroughly studied intracellular bacterial pathogens, Salmonella enterica serovar Typhimurium (S. Typhimurium), has been at the forefront of metabolic studies within eukaryotic host cells. In this review, we offer a synthesis of what has been discovered to date regarding the metabolic adaptation of S. Typhimurium to survival and growth within the infected host. We discuss many studies in the context of techniques used, types of host cells, how host metabolites contribute to intracellular survival and proliferation of the pathogen and how bacterial metabolism affects the virulence and persistence of the pathogen.

The role of ATP pools in persister cell formation in (fluoro)quinolone-susceptible and -resistant strains of Salmonella enterica ser. Typhimurium.

In this study, we investigated the reported dependence on the ATP pools for persister cell formation in fluoroquinolone-resistant variants of the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. We compared the generation of persister cell populations after ciprofloxacin challenge of wildtype and a nalidixic acid-resistant variant of S. Typhimurium with reduced ciprofloxacin-susceptibility, as well as strains containing a deletion of the atp operon or harbouring the cloned atp genes. A gyrA mutation (D87Y) was found to contribute to increased stationary phase formation of persister cells in S. Typhimurium. However, in contrast to expectations from prior studies, while treatment with the ATP synthase poison arsenate showed the expected increase in persister cells surviving ciprofloxacin treatment, a more direct approach using a strain of Salmonella deleted for the atp operon showed severe reductions in persister cell formation. Persister cell formation was recovered after introduction of the cloned atp operon which restored the reduced ATP levels. These results suggest either an alternative explanation for previous studies, or that persister cell formation in Salmonella is differently regulated.

Salmonella Enteritidis flagellar mutants have a colonization benefit in the chicken oviduct.

Egg borne Salmonella Enteritidis is still a major cause of human food poisoning. Eggs can become internally contaminated following colonization of the hen's oviduct. In this paper we aimed to analyze the role of flagella of Salmonella Enteritidis in colonization of the hen's oviduct. Using a transposon library screen we showed that mutants lacking functional flagella are significantly more efficient in colonizing the hen's oviduct in vivo. A micro-array analysis proved that transcription of a number of flagellar genes is down-regulated inside chicken oviduct cells. Flagella contain flagellin, a pathogen associated molecular pattern known to bind to Toll-like receptor 5, activating a pro-inflammatory cascade. In vitro tests using primary oviduct cells showed that flagellin is not involved in invasion. Using a ligated loop model, a diminished inflammatory reaction was seen in the oviduct resulting from injection of an aflagellated mutant compared to the wild-type. It is hypothesized that Salmonella Enteritidis downregulates flagellar gene expression in the oviduct and consequently prevents a flagellin-induced inflammatory response, thereby increasing its oviduct colonization efficiency.

Salmonella Co-opts Host Cell Chaperone-mediated Autophagy for Intracellular Growth.

Salmonella enterica are invasive intracellular pathogens that replicate within a membrane-bound compartment inside infected host cells known as the Salmonella-containing vacuole. How Salmonella obtains nutrients for growth within this intracellular niche despite the apparent isolation is currently not known. Recent studies have indicated the importance of glucose and related carbon sources for tissue colonization and intracellular proliferation within host cells during Salmonella infections, although none have been found to be essential. We found that wild-type Salmonella are capable of replicating within infected host cells in the absence of both exogenous sugars and/or amino acids. Furthermore, mutants defective in glucose uptake or dependent upon peptides for growth also showed no significant loss in intracellular replication, suggesting host-derived peptides can supply both carbon units and amino acids. Here, we show that intracellular Salmonella recruit the host proteins LAMP-2A and Hsc73, key components of the host protein turnover pathway known as chaperone-mediated autophagy involved in transport of cytosolic proteins to the lysosome for degradation. Host-derived peptides are shown to provide a significant contribution toward the intracellular growth of Salmonella The results reveal a means whereby intracellular Salmonella gain access to the host cell cytosol from within its membrane-bound compartment to acquire nutrients. Furthermore, this study provides an explanation as to how Salmonella evades activation of autophagy mechanisms as part of the innate immune response.

T cells from hemophilia A subjects recognize the same HLA-restricted FVIII epitope with a narrow TCR repertoire.

Factor VIII (FVIII)-neutralizing antibodies ("inhibitors") are a serious problem in hemophilia A (HA). The aim of this study was to characterize HLA-restricted T-cell responses from a severe HA subject with a persistent inhibitor and from 2 previously studied mild HA inhibitor subjects. Major histocompatibility complex II tetramers corresponding to both of the severe HA subject's HLA-DRA-DRB1 alleles were loaded with peptides spanning FVIII-A2, C1, and C2 domains. Interestingly, only 1 epitope was identified, in peptide FVIII2194-2213, and it was identical to the HLA-DRA*01-DRB1*01:01-restricted epitope recognized by the mild HA subjects. Multiple T-cell clones and polyclonal lines having different avidities for the peptide-loaded tetramer were isolated from all subjects. Only high- and medium-avidity T cells proliferated and secreted cytokines when stimulated with FVIII2194-2213 T-cell receptor ß (TCRB) gene sequencing of 15 T-cell clones from the severe HA subject revealed that all high-avidity clones expressed the same TCRB gene. High-throughput immunosequencing of high-, medium-, and low-avidity cells sorted from a severe HA polyclonal line revealed that 94% of the high-avidity cells expressed the same TCRB gene as the high-avidity clones. TCRB sequencing of clones and lines from the mild HA subjects also identified a limited TCRB gene repertoire. These results suggest a limited number of epitopes in FVIII drive inhibitor responses and that the T-cell repertoires of FVIII-responsive T cells can be quite narrow. The limited diversity of both epitopes and TCRB gene usage suggests that targeting of specific epitopes and/or T-cell clones may be a promising approach to achieve tolerance to FVIII.

A Comparison of the ATP Generating Pathways Used by S. Typhimurium to Fuel Replication within Human and Murine Macrophage and Epithelial Cell Lines.

The metabolism of S. Typhimurium within infected host cells plays a fundamental role in virulence since it enables intracellular proliferation and dissemination and affects the innate immune response. An essential requirement for the intracellular replication of S. Typhimurium is the need to regenerate ATP. The metabolic route used to fulfil this requirement is the subject of the present study. For infection models we used human and murine epithelial and macrophage cell lines. The epithelial cell lines were mICc12, a transimmortalised murine colon enterocyte cell line that shows many of the characteristics of a primary epithelial cell line, and HeLa cells. The model macrophage cell lines were THP-1A human monocyte/macrophages and RAW 264.7 murine macrophages. Using a mutational approach combined with an exometabolomic analysis, we showed that neither fermentative metabolism nor anaerobic respiration play major roles in energy generation in any of the cell lines studied. Rather, we identified overflow metabolism to acetate and lactate as the foremost route by which S. Typhimurium fulfils its energy requirements.

Host Stress Drives Salmonella Recrudescence.

Host stress is well known to result in flare-ups of many bacterial, viral and parasitic infections. The mechanism by which host stress is exploited to increase pathogen loads, is poorly understood. Here we show that Salmonella enterica subspecies enterica serovar Typhimurium employs a dedicated mechanism, driven by the scsA gene, to respond to the host stress hormone cortisol. Through this mechanism, cortisol increases Salmonella proliferation inside macrophages, resulting in increased intestinal infection loads in DBA/2J mice. ScsA directs overall Salmonella virulence gene expression under conditions that mimic the intramacrophagic environment of Salmonella, and stimulates the host cytoskeletal alterations that are required for increased Salmonella proliferation inside cortisol exposed macrophages. We thus provide evidence that in a stressed host, the complex interplay between a pathogen and its host endocrine and innate immune system increases intestinal pathogen loads to facilitate pathogen dispersal.

Transcriptional and Post-Transcriptional Modulation of SPI1 and SPI2 Expression by ppGpp, RpoS and DksA in Salmonella enterica sv Typhimurium.

The expression of genes within Salmonella Pathogenicity Islands 1 and 2 (SPI1, SPI2) is required to facilitate invasion and intracellular replication respectively of S. Typhimurium in host cell lines. Control of their expression is complex and occurs via a variety of factors operating at transcriptional and post-transcriptional levels in response to the environmental stimuli found within the host. Several of the factors that modulate SPI1 and SPI2 expression are involved in the redistribution or modification of RNA polymerase (RNAP) specificity. These factors include the bacterial alarmone, ppGpp, the alternative sigma factor, RpoS, and the RNAP accessory protein, DksA. In this report we show not only how these three factors modulate SPI1 and SPI2 expression but also how they contribute to the 'phased' expression of SPI1 and SPI2 during progress through late-log and stationary phase in aerobic rich broth culture conditions. In addition, we demonstrate that the expression of at least one SPI1-encoded protein, SipC is subject to DksA-dependent post-transcriptional control.

Six amino acid residues in a 1200 Å2 interface mediate binding of factor VIII to an IgG4κ inhibitory antibody.

The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies.

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food--and water--borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

The primary transcriptome of Salmonella enterica Serovar Typhimurium and its dependence on ppGpp during late stationary phase.

We have used differential RNA-seq (dRNA-seq) to characterise the transcriptomic architecture of S. Typhimurium SL1344, and its dependence on the bacterial alarmone, guanosine tetraphosphate (ppGpp) during late stationary phase, (LSP). Under LSP conditions we were able to identify the transcriptional start sites (TSSs) for 53% of the S. Typhimurium open reading frames (ORFs) and discovered 282 candidate non-coding RNAs (ncRNAs). The mapping of LSP TSSs enabled a detailed comparison with a previous dRNA-seq study of the early stationary phase (ESP) transcriptional architecture of S. Typhimurium SL1344 and its dependence on ppGpp. For the purposes of this study, LSP was defined as an aerobic LB culture grown to a later optical density reading (OD600 = 3.6) compared to ESP (OD600 = 2.3). The precise nucleotide positions of the majority of S. Typhimurium TSSs at LSP agreed closely with those identified at ESP. However, the identification of TSSs at different positions, or where additional or fewer TSSs were found at LSP compared to ESP enabled the genome-wide categorisation of growth phase dependent changes in promoter structure, the first time such an analysis has been done on this scale. Comparison of the ppGpp-dependency LSP and ESP TSSs for mRNAs and ncRNAs revealed a similar breadth of ppGpp-activation and repression. However, we note several ncRNAs previously shown to be involved in virulence were highly ppGpp-dependent at LSP. Finally, although SPI1 was expressed at ESP, we found SPI1 was not as highly expressed at LSP, instead we observed elevated expression of SPI2 encoded genes. We therefore also report an analysis of SPI2 transcriptional architecture at LSP resulting in localisation of SsrB binding sites and identification of a previously unreported SPI2 TSS. We also show that ppGpp is required for nearly all of SPI2 expression at LSP as well as for expression of SPI1 at ESP.

Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp.

Pectobacterium atrosepticum (Pca) is a Gram-negative phytopathogen which causes disease by secreting plant cell wall degrading exoenzymes (PCWDEs). Previous studies have shown that PCWDE production is regulated by (i) the intercellular quorum sensing (QS) signal molecule, 3-oxo-hexanoyl-l-homoserine lactone (OHHL), and (ii) the intracellular 'alarmone', (p)ppGpp, which reports on nutrient limitation. Here we show that these two signals form an integrated coincidence circuit which ensures that metabolically costly PCWDE synthesis does not occur unless the population is simultaneously quorate and nutrient limited. A (p)ppGpp null ΔrelAΔspoT mutant was defective in both OHHL and PCWDE production, and nutritional supplementation of wild type cultures (which suppresses (p)ppGpp production) also suppressed OHHL and PCWDE production. There was a substantial overlap in the transcriptome of a (p)ppGpp deficient relA mutant and of a QS defective expI (OHHL synthase) mutant, especially with regards to virulence-associated genes. Random transposon mutagenesis revealed that disruption of rsmA was sufficient to restore PCWDE production in the (p)ppGpp null strain. We found that the ratio of RsmA protein to its RNA antagonist, rsmB, was modulated independently by (p)ppGpp and QS. While QS predominantly controlled virulence by modulating RsmA levels, (p)ppGpp exerted regulation through the modulation of the RsmA antagonist, rsmB.