tRNAs Are Stable After All: Pitfalls in Quantification of tRNA from Starved Escherichia coli Cultures Exposed by Validation of RNA Purification Methods.

tRNAs and ribosomal RNAs are often considered stable RNAs. In contrast to this view, we recently proposed that tRNAs are degraded during amino acid starvation and drug-induced transcription inhibition. However, reevaluation of our experimental approach revealed that common RNA extraction methods suffer from alarming extraction and size biases that can lead to gross underestimation of RNA levels in starved Escherichia coli populations. Quantification of tRNAs suffers additional biases due to differing fractions of tRNAs with base modifications in growing versus starved bacteria. Applying an improved methodology, we measured tRNA levels after starvation for amino acids, glucose, phosphate, or ammonium and transcription inhibition by rifampicin. We report that tRNA levels remain largely unaffected in all tested conditions, including several days of starvation. This confirms that tRNAs are remarkably stable RNAs and serves as a cautionary tale about quantification of RNA from cells cultured outside the steady-state growth regime. rRNA, conversely, is extensively degraded during starvation. Thus, E. coli downregulates the translation machinery in response to starvation by reducing the ribosome pool through rRNA degradation, while a high concentration of tRNAs available to supply amino acids to the remaining ribosomes is maintained. IMPORTANCE We show that E. coli tRNAs are remarkably stable during several days of nutrient starvation, although rRNA is degraded extensively under these conditions. The levels of these two major RNA classes are considered to be strongly coregulated at the level of transcription. We demonstrate that E. coli can control the ratio of tRNAs per ribosome under starvation by means of differential degradation rates. The question of tRNA stability in stressed E. coli cells has become subject to debate. Our in-depth analysis of RNA quantification methods reveals hidden technical pitfalls at every step of the analysis, from RNA extraction to target detection and normalization. Most importantly, starved E. coli populations were more resilient to RNA extraction than unstarved populations. The current results underscore that the seemingly trivial task of quantifying an abundant RNA species is not straightforward for cells cultured outside the exponential growth regime.

Screening for Highly Transduced Genes in Staphylococcus aureus Revealed Both Lateral and Specialized Transduction.

Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the packaging of bacterial DNA by viruses and enables the transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, ϕ11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that the screen had selected for just 1 or 2 transposon mutant(s) within each pool of 96 mutants. Subsequent analysis showed that the position of the transposon, rather than the inactivation of bacterial genes, was responsible for the phenotype. Interestingly, from multiple rounds, we identified a pattern of transduction that encompassed mobile genetic elements as well as chromosomal regions both upstream and downstream of the phage integration site. The latter was confirmed by DNA sequencing of purified phage lysates. Importantly, transduction frequencies were lower for phage lysates obtained by phage infection rather than induction. Our results confirmed previous reports of lateral transduction of bacterial DNA downstream of the integrated phage but also indicated a novel form of specialized transduction of DNA upstream of the phage. These findings illustrated the complexity of transduction processes and increased our understanding of the mechanisms by which phages transfer bacterial DNA. IMPORTANCE Horizontal transfer of DNA between bacterial cells contributes to the spread of virulence and antibiotic resistance genes in human pathogens. For Staphylococcus aureus, bacterial viruses play a major role in facilitating the horizontal transfer. These viruses, termed bacteriophages, can transfer bacterial DNA between cells by a process known as transduction, which despite its importance is only poorly characterized. Here, we employed a transposon mutant library to investigate transduction in S. aureus. We showed that the genomic location of bacterial DNA relative to where bacteriophages integrated into that bacterial genome affected how frequently that DNA was transduced. Based on serial transduction of transposon mutant pools and direct sequencing of bacterial DNA in bacteriophage particles, we demonstrated both lateral and specialized transduction. The use of mutant libraries to investigate the genomic patterns of bacterial DNA transferred between cells could help us understand how horizontal transfer influences virulence and resistance development.

Gastrocolic Reflex Is Delayed and Diminished in Adults with Type 1 Diabetes.

BACKGROUND: Constipation is a prevalent gastrointestinal complication in diabetes. The pathophysiology may include neural dysfunction and impaired gastrocolic reflex; however, investigation of the latter has been limited in diabetes. Using the wireless motility capsule, we investigated whether the gastrocolic reflex was impaired in adults with type 1 diabetes compared to healthy. METHODS: One hundred and four adults with type 1 diabetes underwent investigation with the wireless motility capsule and recorded sleep cycle, eating habits, and bowel movements in a diary. Colonic motility index, contraction amplitudes, time-to-peak, peak motility, and colonic transit time were investigated directly in response to a meal. Diagnosis of peripheral (nerve conduction) and autonomic (orthostatic hypotension) polyneuropathy was verified. RESULTS: In comparison with health, people with diabetes had at the time of ingestion decreased motility index and contraction amplitudes (p < 0.001), prolonged time-to-peak (p = 0.01), and borderline decreased peak motility (p = 0.06), which taken together indicate impaired coordination of the gastrocolic reflex. These features were most prominent in those with concomitant peripheral or autonomic neuropathy. Additionally, they were associated with prolonged colonic transit time (p > 0.01). CONCLUSIONS: In type 1 diabetes, the gastrocolic reflex was delayed and diminished and further associated with the presence of neuropathy and constipation. These results suggest that impaired reflex is part of the underlying pathogenesis in the development of constipation.

The day-night pattern of colonic contractility is not impaired in type 1 diabetes and distal symmetric polyneuropathy.

Colonic contractility normally shows circadian variability regulated by sleep and especially food intake. However, individuals with type 1 diabetes have a reduced or even absent gastrocolic response to a meal, indicating that colonic contractility may be affected by the disease. We hypothesized that individuals with type 1 diabetes and distal symmetric polyneuropathy (DSPN) have decreased motility (expressed as the motility index) and contractility of the colon and a reduced increase in motility index from night to morning compared to healthy controls and individuals with type 1 diabetes without DSPN. Cohorts of 35 individuals with type 1 diabetes and DSPN, 40 individuals with type 1 diabetes without DSPN, and 28 healthy controls were included in this post-hoc, cross-sectional analysis. We investigated, using a wireless motility capsule that measures pH, temperature, and pressure throughout the gastrointestinal tract, whether individuals with type 1 diabetes with and without DSPN, compared to healthy controls, exhibit altered colonic contractility in the evening, night, and morning. Max amplitude, mean peak amplitude, mean contraction, and motility index of the colon were calculated at the afore-designated times. Motility index of the colon tended to be higher in individuals with type 1 diabetes and DSPN compared to controls in the evening (P = .064), but the effect size was small (1.74%). There was no difference in motility index between the groups in the morning or evening. Furthermore, there was no difference in max amplitude, mean peak amplitude, or mean contraction between groups in the morning, evening, and night. As expected, overall contractility increased from night to morning in all groups, but there was no difference between groups in the increase in contractility from night to morning. Colonic contractility generally peaked in the morning, decreased in the evening, and was almost absent at night. Type 1 diabetes and/or DSPN did not impair contractility of the colon at any time point. Contractility and motility increased from morning to night unaffected by type 1 diabetes and/or DSPN.

Mechanisms Preserving Insulin Action during High Dietary Fat Intake.

Prolonged intervention studies investigating molecular metabolism are necessary for a deeper understanding of dietary effects on health. Here we provide mechanistic information about metabolic adaptation to fat-rich diets. Healthy, slightly overweight men ingested saturated or polyunsaturated fat-rich diets for 6 weeks during weight maintenance. Hyperinsulinemic clamps combined with leg balance technique revealed unchanged peripheral insulin sensitivity, independent of fatty acid type. Both diets increased fat oxidation potential in muscle. Hepatic insulin clearance increased, while glucose production, de novo lipogenesis, and plasma triacylglycerol decreased. High fat intake changed the plasma proteome in the immune-supporting direction and the gut microbiome displayed changes at taxonomical and functional level with polyunsaturated fatty acid (PUFA). In mice, eucaloric feeding of human PUFA and saturated fatty acid diets lowered hepatic triacylglycerol content compared with low-fat-fed control mice, and induced adaptations in the liver supportive of decreased gluconeogenesis and lipogenesis. Intake of fat-rich diets thus induces extensive metabolic adaptations enabling disposition of dietary fat without metabolic complications.

Prevalent reliance of bacterioplankton on exogenous vitamin B1 and precursor availability.

Vitamin B1 (B1 herein) is a vital enzyme cofactor required by virtually all cells, including bacterioplankton, which strongly influence aquatic biogeochemistry and productivity and modulate climate on Earth. Intriguingly, bacterioplankton can be de novo B1 synthesizers or B1 auxotrophs, which cannot synthesize B1 de novo and require exogenous B1 or B1 precursors to survive. Recent isolate-based work suggests select abundant bacterioplankton are B1 auxotrophs, but direct evidence of B1 auxotrophy among natural communities is scant. In addition, it is entirely unknown if bulk bacterioplankton growth is ever B1-limited. We show by surveying for B1-related genes in estuarine, marine, and freshwater metagenomes and metagenome-assembled genomes (MAGs) that most naturally occurring bacterioplankton are B1 auxotrophs. Pyrimidine B1-auxotrophic bacterioplankton numerically dominated metagenomes, but multiple other B1-auxotrophic types and distinct uptake and B1-salvaging strategies were also identified, including dual (pyrimidine and thiazole) and intact B1 auxotrophs that have received little prior consideration. Time-series metagenomes from the Baltic Sea revealed pronounced shifts in the prevalence of multiple B1-auxotrophic types and in the B1-uptake and B1-salvaging strategies over time. Complementarily, we documented B1/precursor limitation of bacterioplankton production in three of five nutrient-amendment experiments at the same time-series station, specifically when intact B1 concentrations were ≤3.7 pM, based on bioassays with a genetically engineered Vibrio anguillarum B1-auxotrophic strain. Collectively, the data presented highlight the prevalent reliance of bacterioplankton on exogenous B1/precursors and on the bioavailability of the micronutrients as an overlooked factor that could influence bacterioplankton growth and succession and thereby the cycling of nutrients and energy in aquatic systems.

A growing microcolony can survive and support persistent propagation of virulent phages.

Bacteria form colonies and secrete extracellular polymeric substances that surround the individual cells. These spatial structures are often associated with collaboration and quorum sensing between the bacteria. Here we investigate the mutual protection provided by spherical growth of a monoclonal colony during exposure to phages that proliferate on its surface. As a proof of concept we exposed growing colonies of Escherichia coli to a virulent mutant of phage P1. When the colony consists of less than [Formula: see text]50,000 members it is eliminated, while larger initial colonies allow long-term survival of both phage-resistant mutants and, importantly, colonies of mostly phage-sensitive members. A mathematical model predicts that colonies formed solely by phage-sensitive bacteria can survive because the growth of bacteria throughout the colony exceeds the killing of bacteria on the surface and pinpoints how the critical colony size depends on key parameters in the phage infection cycle.

In silico Evolution of Lysis-Lysogeny Strategies Reproduces Observed Lysogeny Propensities in Temperate Bacteriophages.

Bacteriophages are the most abundant organisms on the planet and both lytic and temperate phages play key roles as shapers of ecosystems and drivers of bacterial evolution. Temperate phages can choose between (i) lysis: exploiting their bacterial hosts by producing multiple phage particles and releasing them by lysing the host cell, and (ii) lysogeny: establishing a potentially mutually beneficial relationship with the host by integrating their chromosome into the host cell's genome. Temperate phages exhibit lysogeny propensities in the curiously narrow range of 5-15%. For some temperate phages, the propensity is further regulated by the multiplicity of infection, such that single infections go predominantly lytic while multiple infections go predominantly lysogenic. We ask whether these observations can be explained by selection pressures in environments where multiple phage variants compete for the same host. Our models of pairwise competition, between phage variants that differ only in their propensity to lysogenize, predict the optimal lysogeny propensity to fall within the experimentally observed range. This prediction is robust to large variation in parameters such as the phage infection rate, burst size, decision rate, as well as bacterial growth rate, and initial phage to bacteria ratio. When we compete phage variants whose lysogeny strategies are allowed to depend upon multiplicity of infection, we find that the optimal strategy is one which switches from full lysis for single infections to full lysogeny for multiple infections. Previous attempts to explain lysogeny propensity have argued for bet-hedging that optimizes the response to fluctuating environmental conditions. Our results suggest that there is an additional selection pressure for lysogeny propensity within phage populations infecting a bacterial host, independent of environmental conditions.

Negative feedback loops involving small regulatory RNAs precisely control the Vibrio harveyi quorum-sensing response.

Quorum-sensing (QS) bacteria assess population density through secretion and detection of molecules called autoinducers (AIs). We identify and characterize two Vibrio harveyi negative feedback loops that facilitate precise transitions between low-cell-density (LCD) and high-cell-density (HCD) states. The QS central regulator LuxO autorepresses its own transcription, and the Qrr small regulatory RNAs (sRNAs) posttranscriptionally repress luxO. Disrupting feedback increases the concentration of AIs required for cells to transit from LCD to HCD QS modes. Thus, the two cooperative negative feedback loops determine the point at which V. harveyi has reached a quorum and control the range of AIs over which the transition occurs. Negative feedback regulation also constrains the range of QS output by preventing sRNA levels from becoming too high and preventing luxO mRNA levels from reaching zero. We suggest that sRNA-mediated feedback regulation is a network design feature that permits fine-tuning of gene regulation and maintenance of homeostasis.

Gene dosage compensation calibrates four regulatory RNAs to control Vibrio cholerae quorum sensing.

Quorum sensing is a mechanism of cell-to-cell communication that allows bacteria to coordinately regulate gene expression in response to changes in cell-population density. At the core of the Vibrio cholerae quorum-sensing signal transduction pathway reside four homologous small RNAs (sRNAs), named the quorum regulatory RNAs 1-4 (Qrr1-4). The four Qrr sRNAs are functionally redundant. That is, expression of any one of them is sufficient for wild-type quorum-sensing behaviour. Here, we show that the combined action of two feedback loops, one involving the sRNA-activator LuxO and one involving the sRNA-target HapR, promotes gene dosage compensation between the four qrr genes. Gene dosage compensation adjusts the total Qrr1-4 sRNA pool and provides the molecular mechanism underlying sRNA redundancy. The dosage compensation mechanism is exquisitely sensitive to small perturbations in Qrr levels. Precisely maintained Qrr levels are required to direct the proper timing and correct patterns of expression of quorum-sensing-regulated target genes.

A small-RNA-mediated negative feedback loop controls quorum-sensing dynamics in Vibrio harveyi.

The bioluminescent marine bacterium Vibrio harveyi uses a cell-to-cell communication process called quorum sensing (QS) to co-ordinate behaviours in response to changes in population density. QS is accomplished through the secretion and detection of extracellular signalling molecules called autoinducers. At the centre of the V. harveyi QS circuit are five small regulatory RNAs called Qrr1-5 which destabilize the mRNA of luxR, encoding LuxR, the master transcriptional regulator of QS target genes. Here we show that LuxR directly activates transcription of qrr2, qrr3 and qrr4, leading to the rapid downregulation of luxR. The LuxR-binding sites in the promoters of qrr2, qrr3 and qrr4 were identified and mutated to determine the consequences of this regulatory loop on QS dynamics. Disruption of the loop delays the transition from high to low cell density, and more significantly, decreases the cell density at which the population reaches a quorum. Our results suggest that feedback is essential for optimizing the dynamics of the transitions between individual and group behaviours.

A negative feedback loop involving small RNAs accelerates Vibrio cholerae's transition out of quorum-sensing mode.

Quorum sensing is a cell-to-cell communication process that allows bacteria to measure their population numbers and to synchronously alter gene expression in response to changes in cell population density. At the core of the Vibrio cholerae quorum-sensing signal transduction pathway lie four redundant small RNAs (sRNAs), named the Quorum Regulatory RNAs (Qrr1-4). Expression of qrr1-4 is cell population density-dependent due to a requirement for the quorum-sensing controlled phosphorylated response regulator LuxO-P, which is abundant only at low cell population density. When expressed, Qrr1-4 repress translation of HapR, the "master" quorum-sensing transcription factor. Here we show a negative feedback loop in which HapR activates transcription of the qrr genes, which indirectly leads to hapR repression. Efficient feedback activation of the qrr genes requires the simultaneous presence of LuxO-P (present only at low cell population density) and HapR (present only at high cell population density). For this reason, the feedback loop does not influence quorum sensing at steady-state low or high cell population density. However, LuxO-P and HapR are simultaneously present immediately following the switch from high to low cell density conditions. In this state, the HapR feedback loop dramatically accelerates V. cholerae's transition from the high to the low cell density mode.

On the role of Cro in lambda prophage induction.

The lysogenic state of bacteriophage lambda is exceptionally stable yet the prophage is readily induced in response to DNA damage. This delicate epigenetic switch is believed to be regulated by two proteins; the lysogenic maintenance promoting protein CI and the early lytic protein Cro. First, we confirm, in the native configuration, the previous observation that the DNA loop mediated by oligomerization of CI bound to two distinct operator regions (O(L) and O(R)), increases repression of the early lytic promoters and is important for stable maintenance of lysogeny. Second, we show that the presence of the cro gene might be unimportant for the lysogenic to lytic switch during induction of the lambda prophage. We revisit the idea that Cro's primary role in induction is instead to mediate weak repression of the early lytic promoters.

Yohimbine challenge in children with anxiety disorders.

OBJECTIVE: The authors evaluated the neurohormonal and subjective mood response of children with anxiety disorders who were challenged with yohimbine. METHOD: Seventeen children with DSM-IV diagnoses of anxiety disorders and 15 normal comparison children were given yohimbine orally (0.1 mg/kg). Neurohormonal measures and visual analog self-reports of tenseness were recorded over a 150-minute period. RESULTS: Yohimbine was uniformly well tolerated, and it behaviorally differentiated children with anxiety disorders from normal comparison children with higher maximum change (Deltamax) ratings of anxiety in the patients (mean=17.4 mm, SD=29.8) than in the comparison subjects (mean=0.3 mm, SD=4.4). Yohimbine-stimulated Deltamax growth hormone (GH) for children with anxiety disorders (mean=-1.5 ng/ml, SD=5.9) was significantly reduced compared to that of normal comparison children (mean=2.7 ng/ml, SD=4.5). CONCLUSIONS: Yohimbine selectively elevates self-rated anxiety in children with anxiety disorders and is associated with the blunting of GH in those children relative to that of comparison children. Presence of a blunted GH response to yohimbine in children with anxiety disorders is reminiscent of findings in adults with anxiety disorders, particularly panic disorder. These findings support enhanced central adrenergic sensitivity in children with anxiety disorders, as demonstrated by yohimbine-exacerbated anxiety. The findings should be reconciled with the absence of clonidine-related GH blunting in the same cohort.

Tourette's syndrome improvement with pergolide in a randomized, double-blind, crossover trial.

OBJECTIVE: To determine whether pergolide, a mixed D1-D2-D3 dopamine agonist, is efficacious and safe in the treatment of children with Tourette's syndrome. BACKGROUND: Neuroleptics, which block dopamine transmission, are currently used for treatment of children with severe tics, but major side effects and limited efficacy reduce clinical utility. Prior open-label reports of pergolide suggest potential benefit. METHODS: The authors enrolled 24 children age 7 to 17 years with Tourette's disorder, chronic motor tic disorder, or chronic vocal tic disorder by Diagnostic and Statistical Manual of Mental Disorders (4th ed.) criteria, plus severity criteria on the Yale Global Tic Severity Scale (YGTSS) of > or =20, in a double-blind, placebo-controlled, crossover study. Children were randomized to receive either placebo or up to 300 microg/day pergolide for the first 6-week treatment period, with a 2-week placebo washout, followed by crossover to the alternate treatment. The primary outcome measure was tic severity assessed by YGTSS. RESULTS: Compared with placebo treatment, pergolide treatment was associated with significantly lower YGTSS scores (42.0 +/- 20.4 versus 23.5 +/- 18.7; F = 12.0, df = 1, 17, p = 0.0011). No patient had a serious adverse event and pergolide was well tolerated. CONCLUSIONS: In this randomized, placebo-controlled, crossover trial, pergolide appeared to be a safe and efficacious treatment for Tourette's syndrome in children.

Clonidine challenge in childhood anxiety disorder.

OBJECTIVE: To evaluate the neurohormonal and subjective mood response of children with anxiety disorder to clonidine challenge METHOD: Children with DSM-IV diagnoses of anxiety disorder (ANX) (n = 24) and normal controls (n = 15) were given a challenge of intravenous clonidine (1.3 micrograms/kg) and provided neurohormonal and mood self-report assessment over a 180-minute period. RESULTS: The ANX group differed from normal controls in Hamilton Anxiety Rating, Revised Children's Manifest Anxiety Scale score, and maximum change from baseline (delta max) in growth hormone (GH). Clonidine-stimulated GH concentration of the ANX group was significantly elevated compared with that of controls but returned to baseline within 2 hours. A subgroup with obsessive-compulsive disorder (OCD) (n = 9) had significantly higher delta max GH (17.5 +/- 10.1 ng/mL) than the group with other anxiety disorders (ANX-OCD) (9.1 +/- 5.8 ng/mL) and controls (5.7 +/- 4.1 ng/mL). CONCLUSION: GH response to clonidine challenge is not blunted in ANX subjects. This finding is in contrast to adult disorder and suggests that adrenergic postsynaptic receptor down-regulation is not a feature of childhood anxiety. These findings suggest enhanced central adrenergic sensitivity in ANX which is most pronounced in OCD and argue against a neurobiological continuum from childhood to adult anxiety disorder.

Relative efficacy of haloperidol and pimozide in children and adolescents with Tourette's disorder.

OBJECTIVE: The authors evaluated the relative efficacy and safety of pimozide and haloperidol in the treatment of Gilles de la Tourette's syndrome in children and adolescents. METHOD: A double-blind, 24-week, placebo-controlled double crossover study of equivalent dose formulations of haloperidol and pimozide was conducted with 22 subjects, aged 7-16 years, with Tourette's disorder who were randomly assigned to first one active drug treatment and then the other. Biweekly assessment and flexible dose titration mimicked clinical practice. The primary outcome variable was total score on the Tourette Syndrome Global Scale. Final outcome was determined after 6 weeks of each treatment (placebo, pimozide, haloperidol), with a 2-week placebo baseline period and intervening 2-week placebo washout periods between treatments. RESULTS: Pimozide proved significantly different from placebo in affecting the primary outcome variable, whereas haloperidol failed to have a significant effect. Haloperidol exhibited a threefold higher frequency of serious side effects and significantly greater extrapyramidal symptoms relative to pimozide. Haloperidol-associated treatment-limiting adverse events were experienced by 41% of the patients. The therapeutic doses of pimozide and haloperidol were equivalent (mean = 3.4 mg/day, SD = 1.6, and mean = 3.5 mg/day, SD = 2.2, respectively). CONCLUSIONS: At equivalent doses, pimozide is superior to haloperidol for controlling symptoms of Tourette's disorder in children and adolescents.

Eye movement desensitization and reprocessing: treating trauma and substance abuse.

Eye Movement Desensitization and Reprocessing (EMDR) is a new psychological methodology that has been applied to a wide range of psychological disorders. Clinical reports over the past three years indicate that it is an important addition to the treatment of substance abuse. EMDR offers a structured, client-centered model that integrates key elements of intrapsychic, behavioral, cognitive, body-oriented, and interactional approaches. Treatment effects are quite rapid and, during an individual session, the therapist may witness accelerated processing of information involving a shift of cognitive structures (including the assimilation of positive beliefs) along with the desensitization of attendent traumata. The application of EMDR apparently stimulates an inherent physiological processing system that allows dysfunctional information to be adaptively resolved, resulting in increased insight and more functional behavior. The judicious use of EMDR includes a comprehensive client history and extensive preparation, allowing the client to deal with the high levels of disturbance often engendered by the treatment itself. After the inauguration of a sufficient therapeutic alliance, adequately addressing potential issues of secondary gain, and appropriate client stabilization, EMDR may be used to ameliorate the effects of earlier memories that contribute to the dysfunction, potential relapse triggers, and physical cravings. In addition, EMDR is used to incorporate new coping skills and assist in learning more adaptive behaviors. Other potential targets for reprocessing include treatment noncompliance, ambivalence about abstinence, and present crises. Finally, EMDR should be used on this clinical population only by a trained clinician who is educated and experienced with this problem area.