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Beside the well‐documented involvement of secondary somatosensory area, the cortical network underlying late somatosensory evoked potentials (P60/N60 and P100/N100) is still unknown. Electroencephalogram and magnetoencephalogram source imaging were performed to further investigate the origin of the brain cortical areas involved in late somatosensory evoked potentials, using sensory inputs of different strengths and by testing the correlation between cortical sources. Simultaneous high‐density electroencephalograms and magnetoencephalograms were performed in 19 participants, and electrical stimulation was applied to the median nerve (wrist level) at intensity between 1.5 and 9 times the perceptual threshold. Source imaging was undertaken to map the stimulus‐induced brain cortical activity according to each individual brain magnetic resonance imaging, during three windows of analysis covering early and late somatosensory evoked potentials. Results for P60/N60 and P100/N100 were compared with those for P20/N20 (early response). According to literature, maximal activity during P20/N20 was found in central sulcus contralateral to stimulation site. During P60/N60 and P100/N100, activity was observed in contralateral primary sensorimotor area, secondary somatosensory area (on both hemispheres) and premotor and multisensory associative cortices. Late responses exhibited similar characteristics but different from P20/N20, and no significant correlation was found between early and late generated activities. Specific clusters of cortical activities were activated with specific input/output relationships underlying early and late somatosensory evoked potentials. Cortical networks, partly common to and distinct from early somatosensory responses, contribute to late responses, all participating in the complex somatosensory brain processing. [ABSTRACT FROM AUTHOR]

Wireless communication has become an integral part of our lives. The growing number of antennas in our environment and the expanding use of mobile phones (MPs) are increasing the population's exposure to electromagnetic fields. The present study aimed to examine the potential impact of MPs radiofrequency electromagnetic fields (RF-EMF) exposure on the brainwaves of the resting electroencephalogram (EEG) in humans. Twenty-one healthy volunteers were exposed to Global System for Mobile communications (GSM) signal at 900 MHz MP RF-EMF. The maximum specific absorption rate (SAR) of the MP averaged on 10 g tissue and 1 g tissue were measured at 0.49 W/kg, 0.70 W/kg, respectively. Results showed that while delta and beta rhythms of resting EEG were not affected, theta brainwaves were significantly modulated during exposure to RF-EMF related to MPs. For the first time, it was shown that this modulation is dependent on the eye condition, i.e. closed or open. This study strongly suggests that acute exposure to RF-EMF alters the EEG theta rhythm at rest. Long-term exposure studies are required to explore the effect of this disruption in high-risk or sensitive populations. [ABSTRACT FROM AUTHOR]

Proteomic analysis is intrinsically an iterative, incremental process. Information is usually acquired gradually by researchers, and in different projects. At the same time, there are relatively few examples of biological data management systems which take into account this reality, most of them usually treat the experiment generated data as static and unchangeable: data are never reconsidered, or seldom, whereas technology becomes more powerful or that other researchers have brought information on data correction. And yet, post-planned analysis [21] which involves multiple iterations and subsequent re-investigations of previously prepared data might bring tremendous benefits.

In gram-positive bacteria, oligopeptide transport systems, called Opp or Ami, play a role in nutrition but are also involved in the internalization of signaling peptides that take part in the functioning of quorum-sensing pathways. Our objective was to reveal functions that are controlled by Ami via quorum-sensing mechanisms in Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria. Using a label-free proteomic approach combining one-dimensional electrophoresis with liquid chromatography- tandem mass spectrometry analysis, we compared the proteome of the S. thermophilus LMD-9 to that of a mutant deleted for the subunits C, D, and E of the ami operon. Both strains were grown in a chemically defined medium (CDM) without peptides. We focused our attention on proteins that were no more detected in the ami deletion mutant. In addition to the three subunits of the Ami transporter, 17 proteins fulfilled this criterion and, among them, 7 were similar to proteins that have been identified as essential for transformation in S. pneumoniae. These results led us to find a condition of growth, the early exponential state in CDM, that allows natural transformation in S. thermophilus LMD-9 to turn on spontaneously. Cells were not competent in M17 rich medium. Furthermore, we demonstrated that the Ami transporter controls the triggering of the competence state through the control of the transcription of comX, itself controlling the transcription of late competence genes. We also showed that one of the two oligopeptide-binding proteins of strain LMD-9 plays the predominant role in the control of competence.

Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate ([V.sub.max]/[K.sub.m] ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted D-2-hydroxyacids but not L-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the D-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the [NAD.sup.+] necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

The effect of top–down attention on stimulus‐evoked responses and alpha oscillations and the association between arousal and pupil diameter are well established. However, the relationship between these indices, and their contribution to the subjective experience of attention, remains largely unknown. Participants performed a sustained (10–30 s) attention task in which rare (10%) targets were detected within continuous tactile stimulation (16 Hz). Trials were followed by attention ratings on an 8‐point visual scale. Attention ratings correlated negatively with contralateral somatosensory alpha power and positively with pupil diameter. The effect of pupil diameter on attention ratings extended into the following trial, reflecting a sustained aspect of attention related to vigilance. The effect of alpha power did not carry over to the next trial and furthermore mediated the association between pupil diameter and attention ratings. Variations in steady‐state amplitude reflected stimulus processing under the influence of alpha oscillations but were only weakly related to subjective ratings of attention. Together, our results show that both alpha power and pupil diameter are reflected in the subjective experience of attention, albeit on different time spans, while continuous stimulus processing might not contribute to the experience of attention. [ABSTRACT FROM AUTHOR]

With the extensive use of mobile phone (MP), several studies have been realized to investigate the effects of radiofrequency electromagnetic fields (RF-EMF) exposure on brain activity at rest via electroencephalography (EEG), and the most consistent effect has been seen on the alpha band power spectral density (PSD). However, some studies reported an increase or a decrease of the PSD, while others showed no effect. It has been suggested that these differences might partly be due to a variability of the physiological state of the brain between subjects. So, the aim of this study was to investigate the alpha band modulation, exploring the impact of the alpha band frequency ranges applied in the PSD analysis. Twenty-one healthy volunteers took part to the study with a double-blind, randomized and counterbalanced crossover design, during which eyes-open (EO) and eyes-closed (EC) resting-state EEG was recorded. The exposure system was a sham or a real GSM (global system for mobile) 900 MHz MP (pulse modulated at 217 Hz, mean power of 250 mW and 2 W peak, with a maximum specific absorption rate of 0.70 W/kg on 1 g tissue). The experimental protocol presented a baseline recording phase without MP exposure, an exposure phase during which the exposure system was placed against the left ear, and the post-exposure phase without MP. EEG data from baseline and exposure phases were analyzed and PSD was computed for the alpha band in the fixed range of 8–12 Hz and for the individual alpha band frequency range (IAF). Results showed a trend in decrease or increase of EEG power of both alpha oscillations during exposure in relation to EC and EO recording conditions, respectively, but not reaching statistical significance. Findings did not provide evidence for a different sensitivity to RF-EMF MP related to individual variability in the frequency of the alpha band. In conclusion, these results did not show alpha band activity modulation during resting-state under RF-EMF. It might be argued the need of a delay after the exposure in order to appreciate an EEG spectral power modulation related to RF-EMF exposure. [ABSTRACT FROM AUTHOR]

A study is conducted to characterize the proteomic profile of Lactococcus lactis during its development in milk. The proteomic analysis provides the role of glutamine synthetase and further genetic experiments highlights the key role of this enzyme in the development of Lactococcus lactis in dairy environments.

The outer membrane protein (Omp) A is a major constituent of the outer membrane of Escherichia coli. This protein has been used in several vaccine development studies, but seldom with a view to vaccinating against mastitis. The objective of this study was to investigate the immunogenicity of E. coli OmpA and its vaccine potential for cows. Both the humoral and cellular immune responses were investigated. The gene for OmpA of the mastitis-causing strain P4 was cloned and expressed, and the recombinant protein (rEcOmpA) purified. Cows were immunized twice with rEcOmpA with adjuvant one month apart by the systemic route. Before immunization, few antibodies to rEcOmpA were detected, and there was little production of IL-17A in a whole blood stimulation assay (WBA) with rEcOmpA. Antibodies to rEcOmpA were induced by immunization. These antibodies were not able to react with E. coli P4, but reacted with a rough P4 mutant prepared by inactivating the rfb locus. This suggests that the complete LPS O-chain precluded the accessibility of antibodies to their target at the outer membrane. The cellular immune response appeared to be biased towards a Th17-type, as more IL-17A than IFN-γ was produced in the OmpA-specific WBA. There was a good correlation between antibody titers and the production of IL-17A in the WBA. The intramammary instillation of rEcOmpA elicited a slight local inflammatory response which was not related to the WBA. Overall, the interest of OmpA as vaccine immunogen was not established, although other experimental conditions (dose, adjuvant, route) need to be investigated to conclude definitively. The study pointed to several important issues such as the accessibility of OmpA to antibodies and the weakness of Th1-type response induced by OmpA. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus is the archetype of lactose-adapted bacterium and so far, its sugar metabolism has been mainly investigated in vitro. The objective of this work was to study the impact of lactose and lactose permease on S. thermophilus physiology in the gastrointestinal tract (GIT) of gnotobiotic rats. We used rats mono-associated with LMD-9 strain and receiving 4.5% lactose. This model allowed the analysis of colonization curves of LMD-9, its metabolic profile, its production of lactate and its interaction with the colon epithelium. Lactose induced a rapid and high level of S. thermophilus in the GIT, where its activity led to 49 mM of intra-luminal L-lactate that was related to the induction of mono-carboxylic transporter mRNAs (SLC16A1 and SLC5A8) and p27Kip1 cell cycle arrest protein in epithelial cells. In the presence of a continuous lactose supply, S. thermophilus recruited proteins involved in glycolysis and induced the metabolism of alternative sugars as sucrose, galactose, and glycogen. Moreover, inactivation of the lactose transporter, LacS, delayed S. thermophilus colonization. Our results show i/that lactose constitutes a limiting factor for colonization of S. thermophilus, ii/that activation of enzymes involved in carbohydrate metabolism constitutes the metabolic signature of S. thermophilus in the GIT, iii/that the production of lactate settles the dialogue with colon epithelium. We propose a metabolic model of management of carbohydrate resources by S. thermophilus in the GIT. Our results are in accord with the rationale that nutritional allegation via consumption of yogurt alleviates the symptoms of lactose intolerance. [ABSTRACT FROM AUTHOR]

Background: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the LAB model Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. Results: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. Conclusion: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms. [ABSTRACT FROM AUTHOR]

This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms. [ABSTRACT FROM AUTHOR]

In streptococci, ComX is the alternative sigma factor controlling the transcription of the genes encoding the genetic transformation machinery. In Streptococcus thermophilus, comX transcription is controlled by a complex consisting of a transcriptional regulator of the Rgg family, ComR, and a signaling peptide, ComS, which controls ComR activity. Following its initial production, ComS is processed, secreted, and imported back into the cell by the Ami oligopeptide transporter. We characterized these steps and the partners interacting with ComS during its extracellular circuit in more detail. We identified the mature form of ComS and demonstrated the involvement of the membrane protease Eep in ComS processing. We found that ComS was secreted but probably not released into the extracellular medium. Natural competence was first discovered in a chemically defined medium without peptides. We show here that the presence of a high concentration of nutritional peptides in the medium prevents the triggering of competence. In milk, the ecological niche of S. thermophilus, competence was found to be functional, suggesting that the concentration of nutritional peptides was too low to interfere with ComR activation. The kinetics of expression of the comS, comR, and comX genes and of a late competence gene, dprA, in cultures inoculated at different initial densities revealed that the activation mechanism of ComR by ComS is more a timing device than a quorum-sensing mechanism sensu stricto. We concluded that the ComS extracellular circuit facilitates tight control over the triggering of competence in S. thermophilus. [ABSTRACT FROM AUTHOR]

The thermophilic lactic acid bacterium Streptococcus thermophilus is widely and traditionally used in the dairy industry. Despite the vast level of consumption of S. thermophilus through yogurt or probiotic functional food, very few data are available about its physiology in the gastrointestinal tract (GIT). The objective of the present work was to explore both the metabolic activity and host response of S. thermophilus in vivo. Our study profiles the protein expression of S. thermophilus after its adaptation to the GIT of gnotobiotic rats and describes the impact of S. thermophilus colonization on the colonic epithelium. S. thermophilus colonized progressively the GIT of germ-free rats to reach a stable population in 30 days (108 cfu/g of feces). This progressive colonization suggested that S. thermophilus undergoes an adaptation process within GIT. Indeed, we showed that the main response of S. thermophilus in the rat's GIT was the massive induction of the glycolysis pathway, leading to formation of lactate in the cecum. At the level of the colonic epithelium, the abundance of monocarboxylic acid transporter mRNAs (SLC16A1 and SLC5A8) and a protein involved in the cell cycle arrest (p27kip1) increased in the presence of 5, thermophilus compared with germ-free rats. Based on different mono-associated rats harboring two different strains of S. thermophilus (LMD-9 or LMG 18311) or weak lactate-producing commensal bacteria (Bacteroides thetaiotaomicron and Ruminococcus gnavus), we propose that lactate could be a signal produced by S. thermophilus and modulating the colon epithelium. [ABSTRACT FROM AUTHOR]

Escherichia coli is the leading cause of severe mastitis in dairy farms. As E. coli mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough E. coli J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated E. coli Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough E. coli but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough E. coli strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated E. coli We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to E. coli In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve E. coli mastitis vaccines. IMPORTANCE Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated E. coli strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria., (Copyright © 2021 Rainard et al.)

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14., Competing Interests: The authors have declared that no competing interests exist.

Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis.

Current vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.

We characterized the insoluble proteome of Lactococcus lactis using 1D electrophoresis-LC-MS/MS and identified 313 proteins with at least two different peptides. The identified proteins include 89 proteins having a predicted signal peptide and 25 predicted to be membrane-located. In addition, 67 proteins had alkaline isoelectric point values. Using spectra and peptide counts, we compared protein abundances in two different conditions: growth in rich medium, and after transit in the mouse digestive tract. We identified the large mechanosensitive channel and a putative cation transporter as membrane markers of bacterial adaptation to the digestive tract.