Calcium-Regulated Protein CarP Responds to Multiple Host Signals and Mediates Regulation of Pseudomonas aeruginosa Virulence by Calcium.

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca2+) levels increase the production of virulence factors in P. aeruginosa In an effort to characterize the Ca2+ regulatory network, we identified a Ca2+-regulated ß-propeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca2+-regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using transcriptome sequencing (RNA-Seq), reverse transcription (RT)-PCR, quantitative RT-PCR (RT-qPCR), and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca2+ in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe2+ also induces carP The simultaneous addition of Ca2+ and Fe2+ increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca2+ and CarR but remained silent in the triple mutant lacking rhlI, lasI, and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca2+-dependent H2O2 tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments.IMPORTANCEP. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of the P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here, we show that carP plays an important role in P. aeruginosa virulence and is upregulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP Such multifactorial regulation highlights the interconnectedness between regulatory circuits and, together with the pleotropic effect of CarP on virulence, suggests the importance of this protein in P. aeruginosa adaptations to the host.

The decision to germinate is regulated by divergent molecular networks in spores and seeds.

Dispersal is a key step in land plant life cycles, usually via formation of spores or seeds. Regulation of spore- or seed-germination allows control over the timing of transition from one generation to the next, enabling plant dispersal. A combination of environmental and genetic factors determines when seed germination occurs. Endogenous hormones mediate this decision in response to the environment. Less is known about how spore germination is controlled in earlier-evolving nonseed plants. Here, we present an in-depth analysis of the environmental and hormonal regulation of spore germination in the model bryophyte Physcomitrella patens (Aphanoregma patens). Our data suggest that the environmental signals regulating germination are conserved, but also that downstream hormone integration pathways mediating these responses in seeds were acquired after the evolution of the bryophyte lineage. Moreover, the role of abscisic acid and diterpenes (gibberellins) in germination assumed much greater importance as land plant evolution progressed. We conclude that the endogenous hormone signalling networks mediating germination in response to the environment may have evolved independently in spores and seeds. This paves the way for future research about how the mechanisms of plant dispersal on land evolved.

Calcium carbonate mineralization is essential for biofilm formation and lung colonization.

Biofilms are differentiated microbial communities held together by an extracellular matrix. µCT X-ray revealed structured mineralized areas within biofilms of lung pathogens belonging to two distant phyla - the proteobacteria Pseudomonas aeruginosa and the actinobacteria Mycobacterium abscessus. Furthermore, calcium chelation inhibited the assembly of complex bacterial structures for both organisms with little to no effect on cell growth. The molecular mechanisms promoting calcite scaffold formation were surprisingly conserved between the two pathogens as biofilm development was similarly impaired by genetic and biochemical inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. This work offers underexplored druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

Premenstrual symptomatology, alcohol consumption, and family history of alcoholism in women with premenstrual syndrome.

OBJECTIVE: The purpose of this study was to examine the relationship among family history of alcoholism (FH), premenstrual syndrome (PMS) symptoms, and alcohol consumption in women with a PMS diagnosis. METHOD: Participants (N = 46) were predominantly white (73%) women, of whom 17 (37%) reported multigenerational alcoholism on the paternal side (FH positive [FH+]) using the Family Alcohol and Drug Survey. Subjects recorded alcohol consumption and PMS symptoms using a daily record form for 3 consecutive months. RESULTS: Demographics and alcohol consumption during the follicular phase (FOL) and premenstrual phase (PREM) of the menstrual cycle did not differ by FH; however, change in drinking from FOL to PREM was greater in FH+ (mean change = 2.78 drinks/week) versus FH negative (FH-; mean change = -0.72 drinks/week) women. During PREM, FH- women reported more PMS symptomatology compared with FH+ women, and alcohol consumption during PREM was positively correlated with ratings of bloating, craving for alcohol, craving for food, and low energy in FH- but not FH+ women. CONCLUSIONS: Although FH+ women increased their drinking premenstrually, such use was unrelated to PMS symptom severity.

Somatic symptoms and physiologic responses in generalized anxiety disorder and panic disorder: an ambulatory monitor study.

BACKGROUND: Physiologic responses of patients with anxiety disorders to everyday events are poorly understood. OBJECTIVE: To compare self-reports and physiologic recordings in patients with panic disorder (PD), patients with generalized anxiety disorder (GAD), and nonanxious controls during daily activities. DESIGN: Participants underwent four 6-hour recording sessions during daily activities while wearing an ambulatory monitor. Physiologic and subjective data were recorded every 30 minutes and during subject-signaled periods of increased anxiety or tension or panic attack. SETTING: Participants' everyday environment. PARTICIPANTS: Twenty-six patients with PD and 40 with GAD, both without substantial comorbidity, and 24 controls. INTERVENTIONS: Recordings obtained during everyday activities. MAIN OUTCOME MEASURES: Recordings of heart interbeat intervals, skin conductance levels, respirations, motion, and ratings of subjective somatic symptoms and tension or anxiety. RESULTS: Patients with anxiety disorders rated higher on psychic and somatic anxiety symptoms than did controls. Common to both anxiety disorders was diminished autonomic flexibility that manifested itself throughout the day, accompanied by less precise perception of bodily states. The main differences between patients with PD and GAD were a heightened sensitivity to body sensations and more frequent button presses. There also was a trend toward heightened basal arousal in patients with PD, manifesting itself in a faster heart rate throughout the day. CONCLUSIONS: Patients with PD or GAD are more sensitive to bodily changes than nonanxious individuals, and patients with PD are more sensitive than those with GAD. Patients with PD experience more frequent distress than those with GAD and controls, but their physiologic responses are comparable in intensity. The findings suggest that the perception of panic attacks reflects central rather than peripheral responses. The diminished autonomic flexibility observed in both anxiety conditions may result from dysfunctional information processing during heightened anxiety that fails to discriminate between anxiety-related and neutral inputs.

Effect of worry on regional cerebral blood flow in nonanxious subjects.

Several studies suggest that cognitive tasks attenuate activation of the limbic system by emotional stimuli. We investigated the possibility that worry would similarly inhibit the limbic system by examining its effects on regional cerebral blood flow (rCBF). Ten nonanxious volunteers underwent four scans within one session, using positron emission tomography (PET) with H(2)(15)O as tracer. The first two scans recorded emotionally neutral thinking induced after listening to tapes describing neutral statements. Preceding the third and fourth scans, subjects listened to the self-recorded tape describing their individual worries, were instructed to continue to worry, and were scanned 5 min later. Subjects rated themselves as more anxious during the worry scans but showed no significant heart interbeat or skin conductance changes. During worry, rCBF increases were found bilaterally in the medial fronto-orbital gyri and the right thalamus; rCBF decreases were found bilaterally in the hippocampi and amygdalae, in the right insula, the left and right inferior, middle and superior temporal gyri and the occipito-temporal gyri, the right inferior occipital gyrus and the left supramarginal gyrus. Activity of the left orbito-frontal gyrus was negatively correlated with activity of the amygdalae. The results support the hypothesis that worry-induced prefrontal activity suppresses affect-related subcortical regions.

Physiological arousal in females with fragile X or Turner syndrome.

Physiological arousal was measured in 12- to 22-year-old females with either fragile X, Turner syndrome, or neither disorder to explore potential differences in the manifestation of arousal and anxiety in adolescents and young women. Physiological arousal was measured at baseline and during performance on mental arithmetic, divided attention, and risk-taking tasks. Contrary to prediction, females with fragile X rarely exhibited higher arousal than females in either the Turner syndrome or comparison groups. On the Divided Attention Task, both the fragile X and Turner syndrome groups exhibited higher arousal relative to one another based on different physiological indices. Relative to the comparison group, the fragile X group presented with a heightened state of arousal at baseline, based on mean skin conductance range, which may account for little increase in arousal on the cognitive tasks for females with fragile X. Females with Turner syndrome exhibited higher arousal relative to the comparison group on all cognitive tasks, but not for all physiological measures. Factors potentially associated with heightened arousal in fragile X and Turner syndrome are discussed.