Social experiences can have profound effects on an individual's level of anxiety. While various studies have addressed consequences of experiences of a specific type, e.g., social defeat, a recent study in mice investigated the impact of combinations of adverse and beneficial social experiences. Quite surprisingly, mice exposed to benefits during early life phases followed by escapable adversity in adulthood displayed lowest levels of anxiety, even compared to individuals having experienced throughout beneficial conditions. The present study aimed to elucidate whether this phenomenon is restricted to these specific life phases or whether it also exists when all these experiences are made in full adulthood. For this purpose, we compared anxiety-like behavior and stress response of adult male mice exposed to escapable social defeat following beneficial social experiences to that of mice exposed to either throughout adverse or throughout beneficial conditions. More precisely, we performed three established behavioral paradigms measuring anxiety-like behavior and assessed corticosterone metabolites non-invasively via feces sampling. Interestingly, we found no effects of social experience on anxiety-like behavior. In contrast to that, the animals' stress hormone levels were profoundly affected by current social conditions: escapable social defeat (adverse condition) led to an increase in corticosterone metabolite concentrations, whereas living with a female (beneficial condition) led to a decrease. Thus, on the one hand this study suggests the importance of the timing of social experience for affecting an individual's level of anxiety. On the other hand, it demonstrates that anxiety and stress hormone levels can be affected separately by social experience during adulthood.

Humans show a unique capacity to process complex information from multiple sources. Social perception in natural environment provides a good example of such capacity as it typically requires the integration of information from different sensory systems, and also from different levels of sensory processing. Here, instead of studying one isolate system and level of representation, we focused upon a neuroimaging paradigm which allows to capture multiple brain representations simultaneously, i.e., low and high-level processing in two different sensory systems, as well as abstract cognitive processing of congruency. Subjects performed social decisions based on the congruency between auditory and visual processing. Using multivoxel pattern analysis (MVPA) of functional magnetic resonance imaging (fMRI) data, we probed a wide variety of representations. Our results confirmed the expected representations at each level and system according to the literature. Further, beyond the hierarchical organization of the visual, auditory and higher order neural systems, we provide a more nuanced picture of the brain functional architecture. Indeed, brain regions of the same neural system show similarity in their representations, but they also share information with regions from other systems. Further, the strength of neural information varied considerably across domains in a way that was not obviously related to task relevance. For instance, selectivity for task-irrelevant animacy of visual input was very strong. The present approach represents a new way to explore the richness of co-activated brain representations underlying the natural complexity in human cognition.

Working memory (WM) deficit is considered the key cause of learning difficulties (LDs). Studies have shown that WM is plastic and thus can be improved through training. This positive effect is transferable to fluid intelligence and academic performance. This study investigated whether WM updating ability and academic performance in children with LDs could be improved through WM updating training and explored the effects of this training on the children's brain activity. We used a running memory task lasting approximately 40 min per day for 28 days to train a group of 23 children with LDs (TLDs group). We also selected two control groups of 22 children with LDs (CLDs group) and 20 children without LDs (normal control [NC] group). The behavioral results of a pretest indicated that WM updating ability and academic performance in the TLDs and CLDs groups were significantly lower than those in the NC group before training. Compared with the CLDs group, the TLDs group exhibited significant performance improvement in a 2-back WM task, as well as in mathematical ability. Event-related potentials (ERPs) results suggested that the amplitudes of N160 (representative of visual recognition) and P300 (representative of updating processing, which is a valid index for updating WM) in the TLDs and CLDs groups were markedly lower than those in the NC group before training. In the TLDs group, these two components increased considerably after training, approaching levels similar to those in the NC group. The results of this study suggest that WM updating training can improve WM updating ability in children with LDs and the training effect can transfer to mathematical performance in such children. Furthermore, the participants' brain activity levels can exhibit positive changes. This article provides experimental evidence that WM updating training could mitigate the symptoms of LDs to a certain degree.

Ongoing brain dynamics have been proposed as a type of "neuronal noise" that can trigger perceptual switches when viewing an ambiguous, bistable stimulus. However, no prior study has directly quantified how such neuronal noise relates to the rate of percept reversals. Specifically, it has remained unknown whether individual differences in complexity of resting-state oscillations-as reflected in long-range temporal correlations (LRTC)-are associated with perceptual stability. We hypothesized that participants with stronger resting-state LRTC in the alpha band experience more stable percepts, and thereby fewer perceptual switches. Furthermore, we expected that participants who report less discontinuous thoughts during rest, experience less switches. To test this, we recorded electroencephalography (EEG) in 65 healthy volunteers during 5 min Eyes-Closed Rest (ECR), after which they filled in the Amsterdam Resting-State Questionnaire (ARSQ). This was followed by three conditions where participants attended an ambiguous structure-from-motion stimulus-Neutral (passively observe the stimulus), Hold (the percept for as long as possible), and Switch (as often as possible). LRTC of resting-state alpha oscillations predicted the number of switches only in the Hold condition, with stronger LRTC associated with less switches. Contrary to our expectations, there was no association between resting-state Discontinuity of Mind and percept stability. Participants were capable of controlling switching according to task goals, and this was accompanied by increased alpha power during Hold and decreased power during Switch. Fewer switches were associated with stronger task-related alpha LRTC in all conditions. Together, our data suggest that bistable visual perception is to some extent under voluntary control and influenced by LRTC of alpha oscillations.

Background: Transmembrane tumor necrosis factor (TNF) receptors are involved in inflammatory, apoptotic, and proliferative processes. In the bloodstream, soluble TNF receptor II (sTNFR2) can modify the inflammatory response of immune cells and is predictive of cardiovascular disease risk. We hypothesize that sTNFR2 is associated with epigenetic modifications of circulating leukocytes, which may relate to the pathophysiology underlying atherogenic risk. Methods: We conducted an epigenome-wide association study of sTNFR2 levels in the Framingham Heart Study Offspring cohort (examination 8; 2005-2008). sTNFR2 was quantitated by enzyme immunoassay and DNA methylation by microarray. The concentration of sTNFR2 was loge-transformed and outliers were excluded. We conducted linear mixed effects models to test the association between sTNFR2 level and methylation at over 400,000 CpGs, adjusting for age, sex, BMI, smoking, imputed cell count, technical covariates, and accounting for familial relatedness. Results: The study sample included 2468 participants (mean age: 67 ± 9 years, 52% women, mean sTNFR2 level 2661 ± 1078 pg/ml). After accounting for multiple testing, we identified 168 CpGs (P < 1.2 × 10-7) that were differentially methylated in relation to sTNFR2. A substantial proportion (27 CpGs; 16%) are in the major histocompatibility complex region and in loci overrepresented for antigen binding molecular functions (P = 1.7 × 10-4) and antigen processing and presentation biological processes (P = 1.3 × 10-8). Identified CpGs are enriched in active regulatory regions and associated with expression of 48 cis-genes (±500 kb) in whole blood (P < 1.1 × 10-5) that coincide with genes identified in GWAS of diseases of immune dysregulation (inflammatory bowel disease, type 1 diabetes, IgA nephropathy). Conclusion: Differentially methylated loci in leukocytes associated with sTNF2 levels reside in active regulatory regions, are overrepresented in antigen processes, and are linked to inflammatory diseases.

Bleomycin (BLM) is an effective curative option in the management of several malignancies including pleural effusions; but pulmonary toxicity, comprising of pneumonitis and fibrosis, poses challenge in its use as a front-line chemotherapeutic. Although Amifostine has been found to protect lungs from the toxic effects of radiation and BLM, its application is limited due to associated toxicity and unfavorable route of administration. Therefore, there is a need for selective, potent, and safe anti-fibrotic drugs. The current study was undertaken to assess the protective effects of DRDE-30, an analog of Amifostine, on BLM-induced lung injury in C57BL/6 mice. Whole body micro- computed tomography (CT) was used to non-invasively observe tissue damage, while broncheo-alveolar lavage fluid (BALF) and lung tissues were assessed for oxidative damage, inflammation and fibrosis. Changes in the lung density revealed by micro-CT suggested protection against BLM-induced lung injury by DRDE-30, which correlated well with changes in lung morphology and histopathology. DRDE-30 significantly blunted BLM-induced oxidative stress, inflammation and fibrosis in the lungs evidenced by reduced oxidative damage, endothelial barrier dysfunction, Myeloperoxidase (MPO) activity, pro-inflammatory cytokine release and protection of tissue architecture, that could be linked to enhanced anti-oxidant defense system and suppression of redox-sensitive pro-inflammatory signaling cascades. DRDE-30 decreased the BLM-induced augmentation in BALF TGF-β and lung hydroxyproline levels, as well as reduced the expression of the mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the suppression of epithelial to mesenchymal transition (EMT) as one of its anti-fibrotic effects. The results demonstrate that the Amifostine analog, DRDE-30, ameliorates the oxidative injury and lung fibrosis induced by BLM and strengthen its potential use as an adjuvant in alleviating the side effects of BLM.

Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A1, A2A, A2B, and A3. Although adenosine exerts clear neuroprotective effects through A1 receptors during ischemia, the use of selective A1 receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A2B receptors in cerebral ischemia. This study explored the role of adenosine A2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A2Breceptor antagonism significantly prevented astrocyte modifications. Both A2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.

Aim: With the invention of electronic cigarettes (ECIG), many questions have been raised regarding their safety as an alternative to smoking conventional cigarettes. Conventional cigarette smoke contains a variety of toxicants including heavy metals. However, ECIG-generated aerosol contains only trace amounts of metals, adding to the argument for it being a safer alternative. In response to heavy metal exposure, metallothioneins are induced in cells to help store the metal, detoxify the body, and are also known responders to oxidative stress. In an attempt to add to the evaluation of the safety of ECIGs, metallothionein expression was quantified using the nematode Caenorhabditis elegans as an assessment of stress induced cellular damage caused by exposure. Methods: Adult nematodes were exposed to either ECIG aerosol or conventional cigarette smoke at doses of 15, 30, and 45 puffs, the equivalent of one, two, and three cigarettes, respectively. Movement, survival, and stress-induced sleep were assessed for up to 24 h after exposure. Relative expression levels for mtl-1 and mtl-2, C. elegans metallothionein genes, were analyzed after 1, 5, and 24 h post exposure using quantitative RT-PCR. Results: Nematodes exposed to conventional cigarette smoke underwent stress-induced sleep in a dose dependent manner with animals recovering to values within the range of air control after 5 h post exposure. Those exposed to ECIG aerosol did not undergo stress-induced sleep and were indistinguishable from controls. The expression of mtl-1 increased in a dose and time dependent manner in C. elegans exposed to conventional cigarette smoke, with a maximum expression observed at 5 h post exposure of 45 puffs. No induction of mtl-2 was observed in any animals. Additionally, ECIG aerosol did not induce expression of mtl-1 and mtl-2 at levels different than those of untreated. Conclusion: ECIG aerosol failed to induce a stress response in C. elegans. In contrast, conventional cigarette smoke induced the production of mtl-1 in a manner that correlates with the induction of stress-induced sleep suggesting a stress response to damage. The lack of cellular stress response to ECIG aerosol suggests it may be a safer alternative to conventional cigarettes.

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed.

Endurance exercise induces metabolic adaptations and has recently been reported associated with the modulation of a particular class of small noncoding RNAs, microRNAs, that act as post-transcriptional regulators of gene expression. Released into body fluids, they termed circulating miRNAs, and they have been recognized as more effective and accurate biomarkers than classical serum markers. This study examined serum profile of miRNAs through massive parallel sequencing in response to prolonged endurance exercise in samples obtained from four competitive Arabian horses before and 2 h after the end of competition. MicroRNA identification, differential gene expression (DGE) analysis and a protein-protein interaction (PPI) network showing significantly enriched pathways of target gene clusters, were assessed and explored. Our results show modulation of more than 100 miRNAs probably arising from tissues involved in exercise responses and indicating the modulation of correlated processes as muscle remodeling, immune and inflammatory responses. Circulating miRNA high-throughput sequencing is a promising approach for sports medicine for the discovery of putative biomarkers for predicting risks related to prolonged activity and monitoring metabolic adaptations.

Insects have a complex chemosensory system that accurately perceives external chemicals and plays a pivotal role in many insect life activities. Thus, the study of the chemosensory mechanism has become an important research topic in entomology. Spodoptera exigua Hübner (Lepidoptera: Noctuidae) is a major agricultural polyphagous pest that causes significant agricultural economic losses worldwide. However, except for a few genes that have been discovered, its olfactory and gustatory mechanisms remain uncertain. In the present study, we acquired 144,479 unigenes of S. exigua by assembling 65.81 giga base reads from 6 chemosensory organs (female and male antennae, female and male proboscises, and female and male labial palps), and identified many differentially expressed genes in the gustatory and olfactory organs. Analysis of the transcriptome data obtained 159 putative chemosensory genes, including 24 odorant binding proteins (OBPs; 3 were new), 19 chemosensory proteins (4 were new), 64 odorant receptors (57 were new), 22 ionotropic receptors (16 were new), and 30 new gustatory receptors. Phylogenetic analyses of all genes and SexiGRs expression patterns using quantitative real-time polymerase chain reactions were investigated. Our results found that several of these genes had differential expression features in the olfactory organs compared to the gustatory organs that might play crucial roles in the chemosensory system of S. exigua, and could be utilized as targets for future functional studies to assist in the interpretation of the molecular mechanism of the system. They could also be used for developing novel behavioral disturbance agents to control the population of the moths in the future.

Internet gaming disorder (IGD) is characterized by cognitive and emotional deficits. Previous studies have reported the co-occurrence of IGD and depression. However, extant brain imaging research has largely focused on cognitive deficits in IGD. Few studies have addressed the comorbidity between IGD and depression symptoms and underlying neural mechanisms. Here, we systematically investigated this issue by combining a longitudinal survey study, a cross-sectional resting-state functional connectivity (rsFC) study and an intervention study. Autoregressive cross-lagged modeling on a longitudinal dataset of college students showed that IGD severity and depression are reciprocally predictive. At the neural level, individuals with IGD exhibited enhanced rsFC between the left amygdala and right dorsolateral prefrontal cortex (DLPFC), inferior frontal and precentral gyrus, compared with control participants, and the amygdala-frontoparietal connectivity at the baseline negatively predicted reduction in depression symptoms following a psychotherapy intervention. Further, following the intervention, individuals with IGD showed decreased connectivity between the left amygdala and left middle frontal and precentral gyrus, as compared with the non-intervention group. These findings together suggest that IGD may be closely associated with depression; aberrant rsFC between emotion and executive control networks may underlie depression and represent a therapeutic target in individuals with IGD. Registry name: The behavioral and brain mechanism of IGD; URL: https://www.clinicaltrials.gov/ct2/show/NCT02550405; Registration number: NCT02550405.

Introduction: The present study assessed whether motor imagery (MI) produces electromyographic activation in specific muscles of the upper limb during a hand grasping and arm-lifting task in healthy volunteers, patients after stroke, or with Parkinson's disease. Electromyographic (EMG) activation was compared under three conditions: MI, physical execution (PE), and rest. The task is clinically relevant unilateral executed movement using open muscle chains. Methods: In a cross-sectional study EMG activation was measured in four muscles: M. deltoideus pars clavicularis, M. biceps brachii, M. extensor digitorum, M. flexor carpi radialis. MI ability was evaluated with mental rotation, mental chronometry and the Kinaesthetic and Visual Imagery Questionnaire. Cognitive performance was screened with the Mini-Mental State Examination. Results: Twenty-two participants (11 females, age 52.6 ±15.8, age range 21 to 72) were included: ten healthy volunteers, seven patients after stroke (time after stroke onset 16.3 ± 24.8 months), and five patients with Parkinson's disease (disease duration 60.4 ± 24.5 months). Overall Mini-Mental State Examination scores ranged between 27 and 30. An increased EMG activation during MI compared to rest condition was observed in M. deltoideus pars clavicularis and M. biceps brachii across all participants (p-value = 0.001, p = 0.007). Seven participants (two healthy volunteers, three patients after stroke and two patients with Parkinson's disease) showed a EMG activation during MI of the hand grasping and arm-lifting task in at least one of the target muscles. No correlation between EMG activation during MI and scores of three MI ability assessments were found. Conclusions: The findings suggest that MI can yield subliminal EMG activation. However, that might vary on individual basis. It remains unclear what parameters contribute to or inhibit an EMG activation during MI. Future investigations should determine factors that influence EMG activation, e.g. MI instructions, tasks to imagine, amount of MI training, and longitudinal changes after an MI training period.

Objective: The aim of this study was to examine the psychometric properties and factorial structure of the Schema Mode Inventory for Eating Disorders (SMI-ED) in a disordered eating population. Method: 573 participants with disordered eating patterns as measured by the Eating Disorder Examination Questionnaire (EDE-Q) completed the 190-item adapted version of the Schema Mode Inventory (SMI). The new SMI-ED was developed by clinicians/researchers specializing in the treatment of eating disorders, through combining items from the original SMI with a set of additional questions specifically representative of the eating disorder population. Psychometric testing included Confirmatory Factor Analysis (CFA) and internal consistency (Cronbach's α). Multivariate Analyses of Covariance (MANCOVA) was also run to test statistical differences between the EDE-Q subscales on the SMI-ED modes, while controlling for possible confounding variables. Results: Factorial analysis confirmed an acceptable 16-related-factors solution for the SMI-ED, thus providing preliminary evidence for the adequate validity of the new measure based on internal structure. Concurrent validity was also established through moderate to high correlations on the modes most relevant to eating disorders with EDE-Q subscales. This study represents the first step in creating a psychometrically sound instrument for measuring schema modes in eating disorders, and provides greater insight into the relevant schema modes within this population. Conclusion: This research represents an important preliminary step toward understanding and labeling the schema mode model for this clinical group. Findings from the psychometric evaluation of SMI-ED suggest that this is a useful tool which may further assist in the measurement and conceptualization of schema modes in this population.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by interactions between environmental factors and genetic susceptibility. Circadian rhythm dysfunction (CRD) is a significant contributor to neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. However, whether CRD contributes to the progression of ALS remains little known. We performed behavioral and physiological tests on SOD1G93A ALS model mice with and without artificially induced CRD, and on wild-type controls; we also analyzed spinal cord samples histologically for differences between groups. We found that CRD accelerated the disease onset and progression of ALS in model mice, as demonstrated by aggravated functional deficits and weight loss, as well as increased motor neuron loss, activated gliosis, and nuclear factor κB-mediated inflammation in the spinal cord. We also found an increasing abundance of enteric cyanobacteria in the ALS model mice shortly after disease onset that was further enhanced by CRD. Our study provides initial evidence on the CRD as a risk factor for ALS, and intestinal cyanobacteria may be involved.

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.

As we have shown previously, the Cu and Ag concentrations in the sporocarps of Ag-hyperaccumulating Amanita strobiliformis are correlated, and both metals share the same uptake system and are sequestered by the same metallothioneins intracellularly. To further improve our knowledge of the Cu and Ag handling in A. strobiliformis cells, we searched its transcriptome for the P1B-1-ATPases, recognizing Cu+ and Ag+ for transport. We identified transcripts encoding 1097-amino acid (AA) AsCRD1 and 978-AA AsCCC2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of AsCRD1 conferred highly increased Cu and Ag tolerance to metal sensitive yeasts in which the functional AsCRD1:GFP (green fluorescent protein) fusion localized exclusively to the tonoplast, indicating that the AsCRD1-mediated Cu and Ag tolerance was a result of vacuolar sequestration of the metals. Increased accumulation of AsCRD1 transcripts observed in A. strobiliformis mycelium upon the treatments with Cu and Ag (8.7- and 4.5-fold in the presence of 5 μM metal, respectively) supported the notion that AsCRD1 can be involved in protection of the A. strobiliformis cells against the toxicity of both metals. Neither Cu nor Ag affected the levels of AsCCC2 transcripts. Heterologous expression of AsCCC2 in mutant yeasts did not contribute to Cu tolerance, but complemented the mutant genotype of the S. cerevisiae ccc2Δ strain. Consistent with the role of the yeast Ccc2 in the trafficking of Cu from cytoplasm to nascent proteins via post-Golgi, the GFP fluorescence in AsCCC2-expressing ccc2Δ yeasts localized among Golgi-like punctate foci within the cells. The AsCRD1- and AsCCC2-associated phenotypes were lost in yeasts expressing mutant transporter variants in which a conserved phosphorylation/dephosphorylation site was altered. Altogether, the data support the roles of AsCRD1 and AsCCC2 as genuine P1B-1-ATPases, and indicate their important functions in the removal of toxic excess of Cu and Ag from the cytoplasm and charging the endomembrane system with Cu, respectively.

Metagenomics poses opportunities for clinical and public health virology applications by offering a way to assess complete taxonomic composition of a clinical sample in an unbiased way. However, the techniques required are complicated and analysis standards have yet to develop. This, together with the wealth of different tools and workflows that have been proposed, poses a barrier for new users. We evaluated 49 published computational classification workflows for virus metagenomics in a literature review. To this end, we described the methods of existing workflows by breaking them up into five general steps and assessed their ease-of-use and validation experiments. Performance scores of previous benchmarks were summarized and correlations between methods and performance were investigated. We indicate the potential suitability of the different workflows for (1) time-constrained diagnostics, (2) surveillance and outbreak source tracing, (3) detection of remote homologies (discovery), and (4) biodiversity studies. We provide two decision trees for virologists to help select a workflow for medical or biodiversity studies, as well as directions for future developments in clinical viral metagenomics.

Adjustment of cell cycle progression is crucial for bacterial survival and adaptation under adverse conditions. However, the understanding of modulation of cell cycle control in response to environmental changes is rather incomplete. In α-proteobacteria, the broadly conserved cell cycle master regulator CtrA underlies multiple levels of control, including coupling of cell cycle and cell differentiation. CtrA levels are known to be tightly controlled through diverse transcriptional and post-translational mechanisms. Here, small RNA (sRNA)-mediated post-transcriptional regulation is uncovered as an additional level of CtrA fine-tuning. Computational predictions as well as transcriptome and proteome studies consistently suggested targeting of ctrA and the putative cold shock chaperone cspA5 mRNAs by the trans-encoded sRNA (trans-sRNA) GspR (formerly SmelC775) in several Sinorhizobium species. GspR strongly accumulated in the stationary growth phase, especially in minimal medium (MM) cultures. Lack of the gspR locus confers a fitness disadvantage in competition with the wild type, while its overproduction hampers cell growth, suggesting that this riboregulator interferes with cell cycle progression. An eGFP-based reporter in vivo assay, involving wild-type and mutant sRNA and mRNA pairs, experimentally confirmed GspR-dependent post-transcriptional down-regulation of ctrA and cspA5 expression, which most likely occurs through base-pairing to the respective mRNA. The energetically favored secondary structure of GspR is predicted to comprise three stem-loop domains, with stem-loop 1 and stem-loop 3 targeting ctrA and cspA5 mRNA, respectively. Moreover, this work reports evidence for post-transcriptional control of ctrA by CspA5. Thus, this regulation and GspR-mediated post-transcriptional repression of ctrA and cspA5 expression constitute a coherent feed-forward loop, which may enhance the negative effect of GspR on CtrA levels. This novel regulatory circuit involving the riboregulator GspR, CtrA, and a cold shock chaperone may contribute to fine-tuning of ctrA expression.

Members of the genus Bifidobacterium are common inhabitants of the human gastrointestinal tract. Previously it was shown that arabino-oligosaccharides (AOS) might act as prebiotics and stimulate the bifidobacterial growth in the gut. However, despite the rapid accumulation of genomic data, the precise mechanisms by which these sugars are utilized and associated transcription control still remain unclear. In the current study, we used a comparative genomic approach to reconstruct arabinose and AOS utilization pathways in over 40 bacterial species belonging to the Bifidobacteriaceae family. The results indicate that the gene repertoire involved in the catabolism of these sugars is highly diverse, and even phylogenetically close species may differ in their utilization capabilities. Using bioinformatics analysis we identified potential DNA-binding motifs and reconstructed putative regulons for the arabinose and AOS utilization genes in the Bifidobacteriaceae genomes. Six LacI-family transcriptional factors (named AbfR, AauR, AauU1, AauU2, BauR1 and BauR2) and a TetR-family regulator (XsaR) presumably act as local repressors for AOS utilization genes encoding various α- or β-L-arabinofuranosidases and predicted AOS transporters. The ROK-family regulator AraU and the LacI-family regulator AraQ control adjacent operons encoding putative arabinose transporters and catabolic enzymes, respectively. However, the AraQ regulator is universally present in all Bifidobacterium species including those lacking the arabinose catabolic genes araBDA, suggesting its control of other genes. Comparative genomic analyses of prospective AraQ-binding sites allowed the reconstruction of AraQ regulons and a proposed binary repression/activation mechanism. The conserved core of reconstructed AraQ regulons in bifidobacteria includes araBDA, as well as genes from the central glycolytic and fermentation pathways (pyk, eno, gap, tkt, tal, galM, ldh). The current study expands the range of genes involved in bifidobacterial arabinose/AOS utilization and demonstrates considerable variations in associated metabolic pathways and regulons. Detailed comparative and phylogenetic analyses allowed us to hypothesize how the identified reconstructed regulons evolved in bifidobacteria. Our findings may help to improve carbohydrate catabolic phenotype prediction and metabolic modeling, while it may also facilitate rational development of novel prebiotics.