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Objective. The aim of this study was to compare the effects of multicomponent and strength training programs on dynamic balance, functional capacity, and gait ability in older women. Methods. Thirty individuals (67 ± 4.3 years; 30.6 ± 3.9 kg/m2) were trained for 12 weeks (3 times per week), following multicomponent (MG: exercises focusing on agility, balance, muscle strength, and aerobic) and strength programs (SG: lower limbs strength exercise). Results. Peak torque of hip flexors (p=0.020) and extensors (p=0.009) and knee flexors (p=0.001) of SG was greater than that of MG at posttraining. In addition, both groups increased peak torque of knee extensors (p=0.002) and plantar extensors with higher effect size for SG (d = −0.41 and −0.48), whereas MG presented higher effect size for plantar flexors muscles (d = −0.55). Only the SG improved the rate of torque development of knee extensors (29%; p=0.002), and this variable was also greater to SG than MG at posttraining (106%). The SG and MG improved dynamic balance although SG presented higher effect size (d = 0.61). Both groups improved the performance on 30 s sit to stand test (p=0.010) with higher effect size for MG (d = −0.54). Only the MG improved the stride length (4%; p=0.011) and gait speed (10%; p=0.024). In addition, the groups improved toe clearance (p=0.035) and heel contact (p=0.010) with higher effect sizes for MG (d = −0.066 and 1.07). Conclusion. Strength training should be considered to increase muscle function and dynamic balance in older women, whereas multicomponent training should be considered to increase functional capacity and gait ability in this population.

Microbiome engineering - the targeted manipulation of microbial communities - is considered a promising strategy to restore ecosystems, but experimental support and mechanistic understanding are required. Here, we show that bacterial inoculants for soil microbiome engineering may fail to establish because they inadvertently facilitate growth of native resident microbiomes. By generating soil microcosms in presence or absence of standardized soil resident communities, we show how different nutrient availabilities limit outgrowth of focal bacterial inoculants (three Pseudomonads), and how this might be improved by adding an artificial, inoculant-selective nutrient niche. Through random paired interaction assays in agarose micro-beads, we demonstrate that, in addition to direct competition, inoculants lose competitiveness by facilitating growth of resident soil bacteria. Metatranscriptomics experiments with toluene as selective nutrient niche for the inoculant Pseudomonas veronii indicate that this facilitation is due to loss and uptake of excreted metabolites by resident taxa. Generation of selective nutrient niches for inoculants may help to favor their proliferation for the duration of their intended action while limiting their competitive loss., (© 2024. The Author(s).)

Integrative Conjugative Elements (ICEs) are mosaics containing functional modules allowing maintenance by site-specific integration and excision into and from the host genome and conjugative transfer to a specific host range. Many ICEs encode a range of adaptive functions that aid bacterial survival and evolution in a range of niches. ICEs from the SXT/R391 family are found in γ-Proteobacteria. Over 100 members have undergone epidemiological and molecular characterization allowing insight into their diversity and function. Comparative analysis of SXT/R391 elements from a wide geographic distribution has revealed conservation of key functions, and the accumulation and evolution of adaptive genes. This evolution is associated with gene acquisition in conserved hotspots and variable regions within the SXT/R391 ICEs catalysed via element-encoded recombinases. The elements can carry IS elements and transposons, and a mutagenic DNA polymerase, PolV, which are associated with their evolution. SXT/R391 ICEs isolated from different niches appear to have retained adaptive functions related to that specific niche; phage resistance determinants in ICEs carried by wastewater bacteria, antibiotic resistance determinants in clinical isolates and metal resistance determinants in bacteria recovered from polluted environments/ocean sediments. Many genes found in the element hotspots are undetermined and have few homologs in the nucleotide databases.

Importance: Microbial populations swiftly adapt to changing environments through horizontal gene transfer. While the mechanisms of gene transfer are well known, the impact of environmental conditions on the selection of transferred gene functions remains less clear. We investigated ICEs, specifically the ICE clc -type, in Pseudomonas aeruginosa clinical isolates. Our findings revealed co-evolution between ICEs and their hosts, with ICE transfers occurring within strains. Gene functions carried by ICEs are positively selected, including potential virulence factors and heavy metal resistance. Comparison to publicly available P. aeruginosa genomes unveiled widespread antibiotic-resistance determinants within ICE clc clades. Thus, the ubiquitous ICE clc family significantly contributes to P. aeruginosa 's adaptation and fitness in diverse environments., Competing Interests: The authors declare no conflict of interest.

Strain inoculation (bioaugmentation) is a potentially useful technology to provide microbiomes with new functionalities. However, there is limited understanding of the genetic factors contributing to successful establishment of inoculants. This work aimed to characterize the genes implicated in proliferation of the monoaromatic compound-degrading Pseudomonas veronii 1YdBTEX2 in nonsterile polluted soils. We generated two independent mutant libraries by random minitransposon-delivered marker insertion followed by deep sequencing (Tn-seq) with a total of 5.0 × 10 5 unique insertions. Libraries were grown in multiple successive cycles for up to 50 generations either in batch liquid medium or in two types of soil microcosms with different resident microbial content (sand or silt) in the presence of toluene. Analysis of gene insertion abundances at different time points (passed generations of metapopulation growth), in comparison to proportions at start and to in silico generated randomized insertion distributions, allowed to define ~800 essential genes common to both libraries and ~2,700 genes with conditional fitness effects in either liquid or soil (195 of which resulted in fitness gain). Conditional fitness genes largely overlapped among all growth conditions but affected approximately twice as many functions in liquid than in soil. This indicates soil to be a more promiscuous environment for mutant growth, probably because of additional nutrient availability. Commonly depleted genes covered a wide range of biological functions and metabolic pathways, such as inorganic ion transport, fatty acid metabolism, amino acid biosynthesis, or nucleotide and cofactor metabolism. Only sparse gene sets were uncovered whose insertion caused fitness decrease exclusive for soils, which were different between silt and sand. Despite detectable higher resident bacteria and potential protist predatory counts in silt, we were, therefore, unable to detect any immediately obvious candidate genes affecting P. veronii biological competitiveness. In contrast to liquid growth conditions, mutants inactivating flagella biosynthesis and motility consistently gained strong fitness advantage in soils and displayed higher growth rates than wild type. In conclusion, although many gene functions were found to be important for growth in soils, most of these are not specific as they affect growth in liquid minimal medium more in general. This indicates that P. veronii does not need major metabolic reprogramming for proliferation in soil with accessible carbon and generally favorable growth conditions. IMPORTANCE Restoring damaged microbiomes is still a formidable challenge. Classical widely adopted approaches consist of augmenting communities with pure or mixed cultures in the hope that these display their intended selected properties under in situ conditions. Ecological theory, however, dictates that introduction of a nonresident microbe is unlikely to lead to its successful proliferation in a foreign system such as a soil microbiome. In an effort to study this systematically, we used random transposon insertion scanning to identify genes and possibly, metabolic subsystems, that are crucial for growth and survival of a bacterial inoculant (Pseudomonas veronii) for targeted degradation of monoaromatic compounds in contaminated nonsterile soils. Our results indicate that although many gene functions are important for proliferation in soil, they are general factors for growth and not exclusive for soil. In other words, P. veronii is a generalist that is not a priori hindered by the soil for its proliferation and would make a good bioaugmentation candidate.

The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that is a model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. Previous work showed bimodal ICE activation, but by using single cell time-lapse microscopy coupled to combinations of chromosomally integrated single copy ICE promoter-driven fluorescence reporters, RNA sequencing and mutant analysis, we now describe the complete regulon leading to the arisal of differentiated dedicated transfer competent cells. The regulon encompasses at least three regulatory nodes and five (possibly six) further conserved gene clusters on the ICE that all become expressed under stationary phase conditions. Time-lapse microscopy indicated expression of two regulatory nodes (i.e., bisR and alpA-bisDC) to precede that of the other clusters. Notably, expression of all clusters except of bisR was confined to the same cell subpopulation, and was dependent on the same key ICE regulatory factors. The ICE thus only transfers from a small fraction of cells in a population, with an estimated proportion of between 1.7-4%, which express various components of a dedicated transfer competence program imposed by the ICE, and form the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements., Competing Interests: The authors have declared that no competing interests exist.

Exercise intolerance in chronic obstructive pulmonary disease (COPD) is associated with dyspnea, reduced inspiratory capacity (IC) and occurs with a neuromuscular "power reserve," i.e., an acute ability to increase isokinetic locomotor power. This power reserve is associated with resting forced expiratory volume in 1 s (FEV 1 )/forced vital capacity (FVC) suggesting that treatments to target pulmonary function may protect neuromuscular performance and extend whole body exercise in COPD. We, therefore, tested whether combination long-acting β-agonist and muscarinic antagonist bronchodilator therapy [long-acting muscarinic antagonist (LAMA) + long-acting β-agonist (LABA); Stiolto Respimat] would ameliorate the decline in neuromuscular performance and increase endurance time during constant power cycling at 80% peak incremental power. Fourteen patients with COPD (4 female; 64 [58, 72] yr; FEV 1 67% [56%, 75%] predicted; median [25th, 75th percentile]) participated in a randomized, placebo-controlled crossover trial (NCT02845752). Pulmonary function and cardiopulmonary exercise responses were assessed before and after 1 wk of treatment, with 2 wk washout between conditions. Performance fatigue was assessed using an ∼4-s maximal isokinetic cycling effort at preexercise, isotime, and intolerance. Isotime was the shorter exercise duration of the two treatment conditions. Significance was assessed using ANOVA with treatment as fixed factor and subject as random factor. FEV 1 was greater with LAMA + LABA versus placebo (1.81 [1.58, 1.98] L vs. 1.72 [1.29, 1.99] L; P = 0.006), but IC at isotime, performance fatigue at isotime, and constant power endurance time were not different between conditions (each P > 0.05). A modest (∼95 mL) increase in FEV 1 following 1 wk of combination LAMA + LABA treatment did not alleviate neuromuscular performance fatigue or enhance cycle exercise tolerance in patients with mild-to-severe COPD with largely preserved "static" lung volumes. NEW & NOTEWORTHY Bronchodilation is known to increase forced expiratory volume in 1 s (FEV 1 ) and reduce hyperinflation in COPD. In a randomized controlled trial, we investigated whether combined inhaled long-acting β-agonist and muscarinic antagonist would alleviate maximal voluntary neuromuscular performance fatigue or enhance maximal muscle activation during cycling in patients with COPD. Despite increased FEV 1 , combination bronchodilator therapy did not reduce neuromuscular performance fatigue or enhance muscle activity or exercise tolerance in patients with mild-to-severe COPD.

IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in the Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1's lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters of SGI1 and the helper IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential to elicit the excision and replication of SGI1 and destabilise the helper IncC plasmid., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: The COVID-19 pandemic has shocked the sports world because of the suspension of competitions and the spread of SARS-CoV-2 among athletes. After SARS-CoV-2 infection, cardio-pulmonary complications can occur and, before the resumption of sports competitions, a screening has been recommended. However, few data are available and discrepancies exist in the screening modalities. We conducted this prospective study to investigate the incidence of cardiovascular consequences following SARS-CoV-2 infection in young adult competitive athletes and the appropriate screening strategies for a safe return-to-play., Methods: Ninety competitive athletes (24 ± 10 years) after asymptomatic or mildly symptomatic SARS-CoV-2 infection were screened by physical examination, blood testing, spirometry, 12‑lead resting ECG, 24-h ambulatory ECG monitoring, echocardiogram, and cardiopulmonary exercise testing (CPET)., Results: Sixty-four athletes (71.1%) were male, and most (76.7%) were mildly symptomatic. After SARS-CoV-2 infection, spirometry and resting ECG were normal in all athletes. Ambulatory ECG monitoring demonstrated <50/24 h supraventricular and ventricular premature beats in 53.3% and 52.2% of athletes, respectively, in the absence of malignant arrhythmias. CPET did not demonstrate cardiopulmonary limitations. Echocardiography showed pericardial effusion in 3 athletes (all females) with symptomatic SARS-CoV-2 infection (3.3%; 4.4% in the symptomatic group) with a definitive diagnosis of myopericarditis in 1 athlete (1.1%) and pericarditis in 2 athletes (2.2%)., Conclusions: Cardiac consequences of SARS-CoV-2 infection were found in 3.3% of competitive athletes. An appropriate screening primarily based on the detection of uncommon arrhythmias and cardiac symptoms should be recommended in competitive athletes after SARS-CoV-2 infection to detect a cardiac involvement and guarantee a safe return-to-play., Competing Interests: Declaration of Competing Interest none., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Interspecific interactions are thought to govern the stability and functioning of microbial communities, but the influence of the spatial environment and its structural connectivity on the potential of such interactions to unfold remain largely unknown. Here we studied the effects on community growth and microbial diversity as a function of environmental connectivity, where we define environmental connectivity as the degree of habitat fragmentation preventing microbial cells from living together. We quantitatively compared growth of a naturally-derived high microbial diversity community from soil in a completely mixed liquid suspension (high connectivity) to growth in a massively fragmented and poorly connected environment (low connectivity). The low connectivity environment consisted of homogenously-sized miniature agarose beads containing random single or paired founder cells. We found that overall community growth was the same in both environments, but the low connectivity environment dramatically reduced global community-level diversity compared to the high connectivity environment. Experimental observations were supported by community growth modeling. The model predicts a loss of diversity in the low connectivity environment as a result of negative interspecific interactions becoming more dominant at small founder species numbers. Counterintuitively for the low connectivity environment, growth of isolated single genotypes was less productive than that of random founder genotype cell pairs, suggesting that the community as a whole profited from emerging positive interspecific interactions. Our work demonstrates the importance of environmental connectivity for growth of natural soil microbial communities, which aids future efforts to intervene in or restore community composition to achieve engineering and biotechnological objectives.

The spread of the COVID-19 virus was met by a strict lockdown in many countries around the world, with the closure of all physical activity (PA) facilities and limitations on moving around freely. The aim of the present online survey was to assess the effect of lockdown on physical activity in Italy. Physical activity was assessed using the European Health Interview Survey questionnaire. A total of 1500 datasets were analyzed. Differences between conditions were tested with a chi 2 -based (χ 2 ) test for categorical variables, and with the Student's t -test for paired data. A fixed effects binary logistic regression analysis was conducted to identify relevant predictor variables to explain the compliance with World Health Organisation (WHO) recommendations. We found a substantial decline in all physical activity measures. Mean differences in walking and cycling metabolic equivalent of task minutes per week (METmin/week), respectively, were 344.4 (95% confidence interval (95% CI): 306.6-382.2; p < 0.001) and 148.5 (95% CI: 123.6-173.5; p < 0.001). Time spent in leisure time decreased from 160.8 to 112.6 min/week (mean difference 48.2; 95% CI: 40.4-56.0; p < 0.001). Compliance with WHO recommendations decreased from 34.9% to 24.6% (chi 2 (1, 3000) = 38.306, p < 0.001, V = 0.11). Logistic regression showed a reduced chance (OR 0.640, 95% CI: 0.484-0.845; p = 0.001) to comply with WHO PA recommendations under lockdown conditions. Measures to promote physical activity should be intensified to limit detrimental health effects.

The sewage sludge isolate Pseudomonas nitroreducens HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing revealed three circular DNA replicons: a chromosome and two plasmids. Plasmids were shown to code for putative adaptive functions such as heavy metal resistance, but with unclarified ability for self-transfer. About one-tenth of strain HBP-1's chromosomal genes are likely of recent horizontal influx, being part of genomic islands, prophages and integrative and conjugative elements (ICEs). P. nitroreducens carries two large ICEs with different functional specialization, but with homologous core structures to the well-known ICE clc of Pseudomonas knackmussii B13. The variable regions of ICE Pni 1 (96 kb) code for, among others, heavy metal resistances and formaldehyde detoxification, whereas those of ICE Pni 2 (171 kb) encodes complete meta -cleavage pathways for catabolism of 2-hydroxybiphenyl and salicylate, a protocatechuate pathway and peripheral enzymes for 4-hydroxybenzoate, ferulate, vanillin and vanillate transformation. Both ICEs transferred at frequencies of 10 -6 -10 -8 per P. nitroreducens HBP-1 donor into Pseudomonas putida , where they integrated site specifically into tRNA Gly -gene targets, as expected. Our study highlights the underlying determinants and mechanisms driving dissemination of adaptive properties allowing bacterial strains to cope with polluted environments.

Conjugative transfer of the integrative and conjugative element ICE clc in Pseudomonas requires development of a transfer competence state in stationary phase, which arises only in 3-5% of individual cells. The mechanisms controlling this bistable switch between non-active and transfer competent cells have long remained enigmatic. Using a variety of genetic tools and epistasis experiments in P. putida , we uncovered an 'upstream' cascade of three consecutive transcription factor-nodes, which controls transfer competence initiation. One of the uncovered transcription factors (named BisR) is representative for a new regulator family. Initiation activates a feedback loop, controlled by a second hitherto unrecognized heteromeric transcription factor named BisDC. Stochastic modelling and experimental data demonstrated the feedback loop to act as a scalable converter of unimodal (population-wide or 'analog') input to bistable (subpopulation-specific or 'digital') output. The feedback loop further enables prolonged production of BisDC, which ensures expression of the 'downstream' functions mediating ICE transfer competence in activated cells. Phylogenetic analyses showed that the ICE clc regulatory constellation with BisR and BisDC is widespread among Gamma- and Beta -proteobacteria, including various pathogenic strains, highlighting its evolutionary conservation and prime importance to control the behaviour of this wide family of conjugative elements., Competing Interests: NC, XR, SS, FD, CM, Jv No competing interests declared, (© 2020, Carraro et al.)

The phytohormone auxin plays significant roles in regulating plant growth and development. In Arabidopsis, a subset of ATP-BINDING CASSETTE subfamily B (ABCB) transporters participate in polar movement of auxin by exclusion from and prevention of reuptake at the plasma membrane. A previous analysis identified ABCB21 as a conditional auxin uptake/efflux transporter that regulates cellular auxin levels, but clear physiological roles for ABCB21 in planta remain unknown. Here we show that ABCB21 maintains the acropetal auxin transport stream by regulating auxin levels in the pericycle. Loss of ABCB21 reduces rootward auxin transport and delays lateral root emergence. In seedling shoots, ABCB21 regulates mobilization of auxin from the photosynthetic cotyledons that is important for phototropic bending. In rosette leaves ABCB21 contributes to lateral auxin distribution. These results support a primary role for ABCB21 in regulating auxin distribution supplementary to the primary ABCB auxin transporters ABCB1 and 19.

Bacterial chemotaxis signaling may be interesting for the development of rapid biosensor assays, but is difficult to quantify. Here we explore two potential fluorescent readouts of chemotactically active Escherichia coli cells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of stable or unstable 'split'-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically active E. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise for biosensor assays based on bacterial chemotaxis activity.

Heavy metals (HMs) are compounds that can be hazardous and impair growth of living organisms. Bacteria have evolved the capability not only to cope with heavy metals but also to detoxify polluted environments. Three heavy metal-resistant strains of Mucilaginibacer rubeus and one of Mucilaginibacter kameinonensis were isolated from the gold/copper Zijin mining site, Longyan, Fujian, China. These strains were shown to exhibit high resistance to heavy metals with minimal inhibitory concentration reaching up to 3.5 mM Cu (II) , 21 mM Zn (II) , 1.2 mM Cd (II) , and 10.0 mM As (III) . Genomes of the four strains were sequenced by Illumina. Sequence analyses revealed the presence of a high abundance of heavy metal resistance (HMR) determinants. One of the strain, M. rubeus P2, carried genes encoding 6 putative P IB-1 -ATPase, 5 putative P IB-3 -ATPase, 4 putative Zn (II) /Cd (II) P IB-4 type ATPase, and 16 putative resistance-nodulation-division (RND)-type metal transporter systems. Moreover, the four genomes contained a high abundance of genes coding for putative metal binding chaperones. Analysis of the close vicinity of these HMR determinants uncovered the presence of clusters of genes potentially associated with mobile genetic elements. These loci included genes coding for tyrosine recombinases (integrases) and subunits of mating pore (type 4 secretion system), respectively allowing integration/excision and conjugative transfer of numerous genomic islands. Further in silico analyses revealed that their genetic organization and gene products resemble the Bacteroides integrative and conjugative element CTnDOT. These results highlight the pivotal role of genomic islands in the acquisition and dissemination of adaptive traits, allowing for rapid adaption of bacteria and colonization of hostile environments.

Context: Although metabolic syndrome has been studied in patients with autonomous cortisol secretion, there are limited data for those with nonfunctioning adrenal incidentaloma (NFAI)., Objective: To assess metabolic syndrome frequency in NFAI patients and controls without adrenal adenoma according to World Health Organization (WHO), National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III), American Association of Clinical Endocrinologists/American College of Endocrinology (AACE/ACE) and International Diabetes Federation (IDF) criteria., Design: Retrospective and transversal study., Patients: Seventy-four NFAI and 90 controls were evaluated. NFAI diagnosis was established according to current guidelines. The control group was selected based on normal adrenal imaging examinations., Measurements: Subjects were categorized by metabolic syndrome presence according to WHO, NCEP-ATP III, AACE/ACE and IDF., Results: Age, gender, ethnicity, body mass index, smoking, menopause, statin and fibrate use were comparable between patients and controls. The frequency of prediabetes, dyslipidaemia and hypertension as well as waist circumference were significantly higher in the NFAI patients compared to the controls. The metabolic syndrome frequency in the NFAI group was significantly higher compared to the normal adrenal group: WHO: 69.2% × 31.0% (P < 0.001); NCEP-ATP III: 81.7% × 44.9% (P < 0.001); AACE/ACE: 77.1% × 31.9% (P < 0.001); IDF: 78.6% × 45.5% (P < 0.001). Logistic regression analysis showed that NFAI was a predictor of metabolic syndrome according to WHO (P = 0.001), NCEP-ATP III (P = 0.005) and AACE/ACE (P = 0.007)., Conclusions: Metabolic syndrome is frequently found in patients with NFAI, and this frequency is higher in NFAI patients than in those with normal adrenal imaging., (© 2018 John Wiley & Sons Ltd.)

Escherichia coli , commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars, and peptides. We envisioned modifying the chemotaxis specificity of E. coli by expressing heterologous chemoreceptors from Pseudomonas putida enabling attraction either to toluene or benzoate. The mcpT gene encoding the type 40-helical bundle (40H) methyl-accepting chemoreceptor for toluene from Pseudomonas putida MT53 and the pcaY gene for the type 40H receptor for benzoate and related molecules from P. putida F1 were expressed from the trg promoter on a plasmid in motile wild-type E. coli MG1655. E. coli cells expressing McpT accumulated in chemoattraction assays to sources with 60 to 200 μM toluene, although less strongly than the response to 100 μM serine, but statistically significantly stronger than that to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed in E. coli and yielded weak but distinguishable membranes and polar foci in 1% of cells. E. coli cells expressing PcaY showed weak attraction to 0.1 to 1 mM benzoate, but 50 to 70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in the E. coli chemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing. IMPORTANCE Bacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis of Escherichia coli has been well studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds from Pseudomonas putida at least partly functionally complement the E. coli chemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants, like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds in E. coli ., (Copyright © 2018 American Society for Microbiology.)

The purpose of this review is to provide an update on technology related to Transcranial Color Coded Doppler Examinations. Microvascularization (MicroV) is an emerging Power Doppler technology which can allow visualization of low and weak blood flows even at high depths, thus providing a suitable technique for transcranial ultrasound analysis. With MicroV, reconstruction of the vessel shape can be improved, without any overestimation. Furthermore, by analyzing the Doppler signal, MicroV allows a global image of the Circle of Willis. Transcranial Doppler was originally developed for the velocimetric analysis of intracranial vessels, in particular to detect stenoses and the assessment of collateral circulation. Doppler velocimetric analysis was then compared to other neuroimaging techniques, thus providing a cut-off threshold. Transcranial Color Coded Doppler sonography allowed the characterization of vessel morphology. In both Color Doppler and Power Doppler, the signal overestimated the shape of the intracranial vessels, mostly in the presence of thin vessels and high depths of study. In further neurosonology technology development efforts, attempts have been made to address morphology issues and overcome technical limitations. The use of contrast agents has helped in this regard by introducing harmonics and subtraction software, which allowed better morphological studies of vessels, due to their increased signal-to-noise ratio. Having no limitations in the learning curve, in time and contrast agent techniques, and due to its high signal-to-noise ratio, MicroV has shown great potential to obtain the best morphological definition., (Copyright © 2018 by the American Society of Neuroimaging.)

This substudy of a large, randomized, controlled trial (NCT01072396) examined tiotropium (18 μg qd) effects on dynamic hyperinflation during constant work rate treadmill exercise. Areas-under-the-spontaneous expiratory flow-volume (SEFV)-curves were compared in 20 COPD patients and 16 age-matched untreated controls, using rectangular area ratio (RAR) between peak intrabreath and end-expiratory flow. Seven patients exhibited SEFV curve concavity with RAR ≤ 0.5 (RAR low ) in ≥1 test without tiotropium; (mean ± SD FEV 1 : 1.60 ± 0.59 L; 63.4 ± 14.0%predicted). In RAR low patients, tiotropium increased end-exercise inspiratory capacity (IC, 2.10 ± 0.05 vs. 1.89 ± 0.05 L, tiotropium vs. placebo; p = 0.045) and RAR (0.57 ± 0.02 vs. 0.53 ± 0.02; p < 0.001). Patients without SEFV curve concavity with RAR > 0.5 (n = 13; RAR high ), had higher screening FEV 1 (2.15 ± 0.47 L; 79.6 ± 10.1%predicted) versus RAR low patients and no difference in end-exercise IC and RAR between tiotropium and placebo (IC: 2.24 ± 0.03 vs. 2.17 ± 0.03 L; RAR: 0.63 ± 0.005 vs. 0.62 ± 0.005). RAR and%predicted IC at peak exercise were positively correlated in RAR low patients (R 2  = 0.43, p = 0.0002). Tiotropium increased exercise RAR in GOLD 1-2 patients with SEFV curve concavity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The accurate detection of induced mutations is critical for both forward and reverse genetics studies. Experimental chemical mutagenesis induces relatively few single base changes per individual. In a complex eukaryotic genome, false positive detection of mutations can occur at or above this mutagenesis rate. We demonstrate here, using a population of ethyl methanesulfonate (EMS)-treated Sorghum bicolor BTx623 individuals, that using replication to detect false positive-induced variants in next-generation sequencing (NGS) data permits higher throughput variant detection with greater accuracy. We used a lower sequence coverage depth (average of 7×) from 586 independently mutagenized individuals and detected 5,399,493 homozygous single nucleotide polymorphisms (SNPs). Of these, 76% originated from only 57,872 genomic positions prone to false positive variant calling. These positions are characterized by high copy number paralogs where the error-prone SNP positions are at copies containing a variant at the SNP position. The ability of short stretches of homology to generate these error-prone positions suggests that incompletely assembled or poorly mapped repeated sequences are one driver of these error-prone positions. Removal of these false positives left 1,275,872 homozygous and 477,531 heterozygous EMS-induced SNPs, which, congruent with the mutagenic mechanism of EMS, were >98% G:C to A:T transitions. Through this analysis, we generated a collection of sequence indexed mutants of sorghum. This collection contains 4035 high-impact homozygous mutations in 3637 genes and 56,514 homozygous missense mutations in 23,227 genes. Each line contains, on average, 2177 annotated homozygous SNPs per genome, including seven likely gene knockouts and 96 missense mutations. The number of mutations in a transcript was linearly correlated with the transcript length and also the G+C count, but not with the GC/AT ratio. Analysis of the detected mutagenized positions identified CG-rich patches, and flanking sequences strongly influenced EMS-induced mutation rates. This method for detecting false positive-induced mutations is generally applicable to any organism, is independent of the choice of in silico variant-calling algorithm, and is most valuable when the true mutation rate is likely to be low, such as in laboratory-induced mutations or somatic mutation detection in medicine., (Copyright © 2018 Addo-Quaye et al.)

Integrative and conjugative elements (ICEs) are widespread mobile DNA that transmit both vertically, in a host-integrated state, and horizontally, through excision and transfer to new recipients. Different families of ICEs have been discovered with more or less restricted host ranges, which operate by similar mechanisms but differ in regulatory networks, evolutionary origin and the types of variable genes they contribute to the host. Based on reviewing recent experimental data, we propose a general model of ICE life style that explains the transition between vertical and horizontal transmission as a result of a bistable decision in the ICE-host partnership. In the large majority of cells, the ICE remains silent and integrated, but hidden at low to very low frequencies in the population specialized host cells appear in which the ICE starts its process of horizontal transmission. This bistable process leads to host cell differentiation, ICE excision and transfer, when suitable recipients are present. The ratio of ICE bistability (i.e. ratio of horizontal to vertical transmission) is the outcome of a balance between fitness costs imposed by the ICE horizontal transmission process on the host cell, and selection for ICE distribution (i.e. ICE 'fitness'). From this emerges a picture of ICEs as elements that have adapted to a mostly confined life style within their host, but with a very effective and dynamic transfer from a subpopulation of dedicated cells., (© FEMS 2017.)

Mobile genetic elements are near ubiquitous DNA segments that revealed a surprising variety of strategies for their propagation among prokaryotes and between eukaryotes. In bacteria, conjugative elements were shown to be key drivers of evolution and adaptation by efficiently disseminating genes involved in pathogenicity, symbiosis, metabolic pathways, and antibiotic resistance. Conjugative plasmids of the incompatibility groups A and C (A/C) are important vehicles for the dissemination of antibiotic resistance and the consequent global emergence and spread of multi-resistant pathogenic bacteria. Beyond their own mobility, A/C plasmids were also shown to drive the mobility of unrelated non-autonomous mobilizable genomic islands, which may also confer further advantageous traits. In this commentary, we summarize the current knowledge on different classes of A/C-dependent mobilizable genomic islands and we discuss other DNA hitchhikers and their implication in bacterial evolution. Furthermore, we glimpse at the complex genetic network linking autonomous and non-autonomous mobile genetic elements, and at the associated flow of genetic information between bacteria.

IncC conjugative plasmids and Salmonella genomic island 1 (SGI1) and relatives are frequently associated with multidrug resistance of clinical isolates of pathogenic Enterobacteriaceae. SGI1 is specifically mobilized in trans by IncA and IncC plasmids (commonly referred to as A/C plasmids) following its excision from the chromosome, an event triggered by the transcriptional activator complex AcaCD encoded by these helper plasmids. Although SGI1 is not self-transmissible, it carries three genes, traNS, traHS and traGS, coding for distant homologs of the predicted mating pore subunits TraNC, TraHC and TraGC, respectively, encoded by A/C plasmids. Here we investigated the regulation of traNS and traHGS and the role of these three genes in the transmissibility of SGI1. Transcriptional fusion of the promoter sequences of traNS and traHGS to the reporter gene lacZ confirmed that expression of these genes is inducible by AcaCD. Mating experiments using combinations of deletion mutants of SGI1 and the helper IncC plasmid pVCR94 revealed complex interactions between these two mobile genetic elements. Whereas traNC and traHGC are essential for IncC plasmid transfer, SGI1 could rescue null mutants of each individual gene revealing that TraNS, TraHS and TraGS are functional proteins. Complementation assays of individual traC and traS mutants showed that not only do TraNS/HS/GS replace TraNC/HC/GC in the mating pore encoded by IncC plasmids but also that traGS and traHS are both required for SGI1 optimal transfer. In fact, remodeling of the IncC-encoded mating pore by SGI1 was found to be essential to enhance transfer rate of SGI1 over the helper plasmid. Furthermore, traGS was found to be crucial to allow DNA transfer between cells bearing IncC helper plasmids, thereby suggesting that by remodeling the mating pore SGI1 disables an IncC-encoded entry exclusion mechanism. Hence traS genes facilitate the invasion by SGI1 of cell populations bearing IncC plasmids.

The aim was to investigate the effect of a dietary supplementation on the repeated sprint ability (RSA) performance in recreationally trained team sports athletes. Twelve young men underwent a RSA exercise protocol in five trials, in which participants ingested carbohydrates (CHO) plus caffeine (Caf), CHO plus arginine (Arg), CHO plus branched-chain amino acids (BCAA), CHO plus Caf, Arg, and BCAA (ALL), and CHO only. Heart rate, oxygen saturation, hematic lactate, ratings of perceived exertion, average sprint time, total time, best sprint time, peak power, and average power were taken. Data revealed no significant effects neither on physiological nor performance parameters with any of the supplements.

Background: The vascular contributions to neurodegeneration and neuroinflammation may be assessed by magnetic resonance imaging (MRI) and ultrasonography (US). This review summarises the methodology for these widely available, safe and relatively low cost tools and analyses recent work highlighting their potential utility as biomarkers for differentiating subtypes of cognitive impairment and dementia, tracking disease progression and evaluating response to treatment in various neurocognitive disorders., Methods: At the 9th International Congress on Vascular Dementia (Ljubljana, Slovenia, October 2015) a writing group of experts was formed to review the evidence on the utility of US and arterial spin labelling (ASL) as neurophysiological markers of normal ageing, vascular cognitive impairment (VCI) and Alzheimer's disease (AD). Original articles, systematic literature reviews, guidelines and expert opinions published until September 2016 were critically analysed to summarise existing evidence, indicate gaps in current knowledge and, when appropriate, suggest standards of use for the most widely used US and ASL applications., Results: Cerebral hypoperfusion has been linked to cognitive decline either as a risk or an aggravating factor. Hypoperfusion as a consequence of microangiopathy, macroangiopathy or cardiac dysfunction can promote or accelerate neurodegeneration, blood-brain barrier disruption and neuroinflammation. US can evaluate the cerebrovascular tree for pathological structure and functional changes contributing to cerebral hypoperfusion. Microvascular pathology and hypoperfusion at the level of capillaries and small arterioles can also be assessed by ASL, an MRI signal. Despite increasing evidence supporting the utility of these methods in detection of microvascular pathology, cerebral hypoperfusion, neurovascular unit dysfunction and, most importantly, disease progression, incomplete standardisation and missing validated cut-off values limit their use in daily routine., Conclusions: US and ASL are promising tools with excellent temporal resolution, which will have a significant impact on our understanding of the vascular contributions to VCI and AD and may also be relevant for assessing future prevention and therapeutic strategies for these conditions. Our work provides recommendations regarding the use of non-invasive imaging techniques to investigate the functional consequences of vascular burden in dementia.

Unlabelled: Mobile genetic elements play a pivotal role in the adaptation of bacterial populations, allowing them to rapidly cope with hostile conditions, including the presence of antimicrobial compounds. IncA/C conjugative plasmids (ACPs) are efficient vehicles for dissemination of multidrug resistance genes in a broad range of pathogenic species of Enterobacteriaceae ACPs have sporadically been reported in Vibrio cholerae, the infectious agent of the diarrheal disease cholera. The regulatory network that controls ACP mobility ultimately depends on the transcriptional activation of multiple ACP-borne operons by the master activator AcaCD. Beyond ACP conjugation, AcaCD has also recently been shown to activate the expression of genes located in the Salmonella genomic island 1 (SGI1). Here, we describe MGIVchHai6, a novel and unrelated mobilizable genomic island (MGI) integrated into the 3' end of trmE in chromosome I of V. cholerae HC-36A1, a non-O1/non-O139 multidrug-resistant clinical isolate recovered from Haiti in 2010. MGIVchHai6 contains a mercury resistance transposon and an integron In104-like multidrug resistance element similar to the one of SGI1. We show that MGIVchHai6 excises from the chromosome in an AcaCD-dependent manner and is mobilized by ACPs. Acquisition of MGIVchHai6 confers resistance to β-lactams, sulfamethoxazole, tetracycline, chloramphenicol, trimethoprim, and streptomycin/spectinomycin. In silico analyses revealed that MGIVchHai6-like elements are carried by several environmental and clinical V. cholerae strains recovered from the Indian subcontinent, as well as from North and South America, including all non-O1/non-O139 clinical isolates from Haiti., Importance: Vibrio cholerae, the causative agent of cholera, remains a global public health threat. Seventh-pandemic V. cholerae acquired multidrug resistance genes primarily through circulation of SXT/R391 integrative and conjugative elements. IncA/C conjugative plasmids have sporadically been reported to mediate antimicrobial resistance in environmental and clinical V. cholerae isolates. Our results showed that while IncA/C plasmids are rare in V. cholerae populations, they play an important yet insidious role by specifically propagating a new family of genomic islands conferring resistance to multiple antibiotics. These results suggest that nonepidemic V. cholerae non-O1/non-O139 strains bearing these genomic islands constitute a reservoir of transmissible resistance genes that can be propagated by IncA/C plasmids to V. cholerae populations in epidemic geographical areas as well to pathogenic species of Enterobacteriaceae We recommend future epidemiological surveys take into account the circulation of these genomic islands., (Copyright © 2016 Carraro et al.)

Integrative and conjugative elements (ICEs) are mobile genetic elements encoding their own excision from a replicon of their bacterial host, transfer by conjugation to a recipient bacterium and reintegration for maintenance. The conjugation, recombination and regulation modules of ICEs of the ICE St3 family are grouped together in a region called the ICE 'core region'. In addition to this core region, elements belonging to this family carry a highly variable region including cargo genes that could be involved in bacterial adaptation or in the maintenance of the element. Although ICEs are a major class of mobile elements through bacterial genomes, the functionality of an element encoding only its excision, transfer, integration and regulation has never been demonstrated experimentally. We engineered MiniICE St3 , an artificial ICE derived from ICE St3 , devoid of its cargo genes and thus only harbouring the core region. The functionality of this minimal element was assessed. MiniICE St3 was found to be able to excise at a rate of 3.1 %, transfer with a frequency of 1.0 × 10 - 5 transconjugants per donor cell and stably maintain by site-specific integration into the 3' end of the fda gene, the same as ICE St3 . Furthermore, MiniICE St3 was found in ∼10 copies per chromosome, this multicopy state likely contributing to its stability for >100 generations even in the absence of selection. Therefore, although ICEs were primarily assumed to only replicate along with the chromosome, our results uncovered extrachromosomal rolling-circle replicating plasmid-like forms of MiniICE St3 .

The genetic dissection of root architecture and functions allows for a more effective and informed design of novel root ideotypes and paves the way to evaluate their effects on crop resilience to a number of abiotic stresses. In maize, limited attention has been devoted to the genetic analysis of root architecture diversity at the early stage. The difference in embryonic (including seminal and primary) root architecture between the maize reference line B73 (which mostly develops three seminal roots) and the landrace Gaspé Flint (with virtually no seminal roots) was genetically dissected using a collection of introgression lines grown in paper rolls and pots. Quantitative trait locus (QTL) analysis identified three QTLs controlling seminal root number (SRN) on chromosome bins 1.02, 3.07, and 8.04-8.05, which collectively explained 66% of the phenotypic variation. In all three cases, Gaspé Flint contributed the allele for lower SRN. Primary root dry weight was negatively correlated with SRN (r= -0.52), and QTLs for primary root size co-mapped with SRN QTLs, suggesting a pleiotropic effect of SRN QTLs on the primary root, most probably caused by competition for seed resources. Interestingly, two out of three SRN QTLs co-mapped with the only two known maize genes (rtcs and rum1) affecting the number of seminal roots. The strong additive effect of the three QTLs and the development of near isogenic lines for each QTL in the elite B73 background provide unique opportunities to characterize functionally the genes involved in root development and to evaluate how root architecture affects seedling establishment, early development, and eventually yield in maize., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

PIN auxin efflux carriers and ABCB auxin transporters are important for polar auxin transport, organogenesis and long distance auxin transport. Along with the auxin influx symporter AUX1, they are essential for tropic responses such as gravitropism and phototropism where lateral redistribution of auxin is required for the tropic response to occur. Immunolocalization of plant membrane transporters is an effective technique to determine the transporters' subcellular localization patterns in the tissues of interest, especially when fluorescent protein fusions of the protein of interest are not available. Immunolocalization is also a valuable tool for validation of the localization of fluorescent protein fusions when the fusions are available. Here we describe the procedures to prepare plant tissue samples and fix them for whole mount or embedding and sectioning. We focus on immunolocalizations of PINs and ABCBs in Arabidopsis and maize tissues. In addition, we describe treatments of roots with inhibitors of cellular trafficking: brefeldin A (BFA), a fungal compound that blocks exocytosis; wortmannin, a fungal compound that inhibits phosphatidylinositol 3-kinase and induces fusion of pre-vacuolar compartments and multi-vascular bodies; and oryzalin, a fungal compound that depolymerizes microtubules. Inhibitor treatments are performed prior to fixation and affect the localization patterns of PINs and ABCBs, giving insight into cell type -specific trafficking mechanisms. The procedures described for Arabidopsis and maize can be easily adapted for other herbaceous plants.

Integrative and conjugative elements (ICEs) are mobile genetic elements that play a key role in bacterial adaptation. Such elements are found in almost every bacterial genera and species, and often code for adaptive traits conferring selective advantages to their host. ICEs maintain by integrating into and replicating along with a replicon of the host genome. ICEs can propagate by conjugative transfer toward a recipient cell following excision from the replicon as a circular covalently-closed molecule. For a long time, the excised form of ICEs was assumed to be non-replicative. This assumption predicts that excised ICEs are sensitive to loss during cell division, unless they carry stabilization systems such as addiction modules or antibiotic resistance genes. Over the past few years, growing evidence have been presented that support conditional replication of the circular intermediate as an intrinsic feature of ICEs. We recently confirmed this feature in the large family of SXT/R391 ICEs, which thrive in several species of Enterobacteriaceae and Vibrionaceae . Furthermore, we demonstrated that SXT/R391 ICEs encode a functional plasmid-like type II partition system that enhances their stability, such systems being probably encoded by other ICEs. The lifecycle of ICEs is therefore much more complex than initially thought as many ICEs may use plasmid-like features to improve their stability and dissemination.

The moderating role of academic goal conflict in the relations between action planning (AP) and coping planning (CP) with physical activity was tested using samples of university students concurrently pursuing an academic and a physical activity goal. In Study 1 (N = 317), AP was found to positively relate to physical activity goal progress at low, but not at high, levels of goal conflict. CP trended toward being positively related to goal progress at high, but not at low levels of goal conflict. Study 2 (N = 97), using a 1-week daily diary design and measures of self-reported physical activity behavior and goal progress, showed that daily AP positively related to daily physical activity outcomes on days when students experienced lower, but not higher, levels of goal conflict relative to their average. Conversely, CP positively related to daily physical activity outcomes on days when students experienced higher, but not lower, levels of goal conflict., (© 2015 by the Society for Personality and Social Psychology, Inc.)

Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.

Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

Integrative and Conjugative Elements (ICEs) are bacterial mobile genetic elements that play a key role in bacterial genomes dynamics and evolution. ICEs are widely distributed among virtually all bacterial genera. Recent extensive studies have unraveled their high diversity and complexity. The present review depicts the general conserved features of ICEs and describes more precisely three major families of ICEs that have been extensively studied in the past decade for their biology, their evolution and their impact on genomes dynamics. First, the large SXT/R391 family of ICEs disseminates antibiotic resistance genes and drives the exchange of mobilizable genomic islands (MGIs) between many enteric pathogens such as Vibrio cholerae. Second, ICEBs1 of Bacillus subtilis is the most well understood ICE of Gram-positive bacteria, notably regarding the regulation of its dissemination and its initially unforeseen extrachromosomal replication, which could be a common feature of ICEs of both Gram-positive and Gram-negative bacteria. Finally, ICESt1 and ICESt3 of Streptococcus thermophilus are the prototypes of a large family of ICEs widely distributed among various streptococci. These ICEs carry an original regulation module that associates regulators related to those of both SXT/R391 and ICEBs1. Study of ICESt1 and ICESt3 uncovered the cis-mobilization of related genomic islands (CIMEs) by a mechanism called accretion-mobilization, which likely represents a paradigm for the evolution of many ICEs and genomic islands. These three major families of ICEs give a glimpse about ICEs dynamics and their high impact on bacterial adaptation.

Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.

Auxin is a key regulator of plant growth and development, orchestrating cell division, elongation and differentiation, embryonic development, root and stem tropisms, apical dominance, and transition to flowering. Auxin levels are higher in undifferentiated cell populations and decrease following organ initiation and tissue differentiation. This differential auxin distribution is achieved by polar auxin transport (PAT) mediated by auxin transport proteins. There are four major families of auxin transporters in plants: PIN-FORMED (PIN), ATP-binding cassette family B (ABCB), AUXIN1/LIKE-AUX1s, and PIN-LIKES. These families include proteins located at the plasma membrane or at the endoplasmic reticulum (ER), which participate in auxin influx, efflux or both, from the apoplast into the cell or from the cytosol into the ER compartment. Auxin transporters have been largely studied in the dicotyledon model species Arabidopsis, but there is increasing evidence of their role in auxin regulated development in monocotyledon species. In monocots, families of auxin transporters are enlarged and often include duplicated genes and proteins with high sequence similarity. Some of these proteins underwent sub- and neo-functionalization with substantial modification to their structure and expression in organs such as adventitious roots, panicles, tassels, and ears. Most of the present information on monocot auxin transporters function derives from studies conducted in rice, maize, sorghum, and Brachypodium, using pharmacological applications (PAT inhibitors) or down-/up-regulation (over-expression and RNA interference) of candidate genes. Gene expression studies and comparison of predicted protein structures have also increased our knowledge of the role of PAT in monocots. However, knockout mutants and functional characterization of single genes are still scarce and the future availability of such resources will prove crucial to elucidate the role of auxin transporters in monocots development.

The PIN and ACO gene families present interesting questions about the evolution of plant physiology, including testing hypotheses about the ecological drivers of their diversification and whether unrelated genes have been recruited for similar functions. The PIN-formed proteins contribute to the polar transport of auxin, a hormone which regulates plant growth and development. PIN loci are categorized into groups according to their protein length and structure, as well as subcellular localization. An interesting question with PIN genes is the nature of the ancestral form and location. ACOs are members of a superfamily of oxygenases and oxidases that catalyze the last step of ethylene synthesis, which regulates many aspects of the plant life cycle. We used publicly available PIN and ACO sequences to conduct phylogenetic analyses. Third codon positions of these genes in monocots have a high GC content, which could be historical but is more likely due to a mutational bias. Thus, we developed methods to extract phylogenetic information from nucleotide sequences while avoiding this convergent feature. One method consisted in using only A-T transformations, and another used only the first and second codon positions for serine, which can only take A or T and G or C, respectively. We also conducted tree-searches for both gene families using unaligned amino acid sequences and dynamic homology. PIN genes appear to have diversified earlier than ACOs, with monocot and dicot copies more mixed in the phylogeny. However, gymnosperm PINs appear to be derived and not closely related to those from primitive plants. We find strong support for a long PIN gene ancestor with short forms subsequently evolving one or more times. ACO genes appear to have diversified mostly since the dicot-monocot split, as most genes cluster into a small number of monocot and dicot clades when the tree is rooted by genes from mosses. Gymnosperm ACOs were recovered as closely related and derived.