These resistant infections arehaving a significant impact on treatment outcomes and contribute toapproximately 25,000 deaths in the U.S. annually. If additional therapeuticoptions are not identified, the number of annual deaths is predicted to rise to317,000 in North America and 10,000,000 worldwide by 2050. Identifyingtherapeutic methodologies that utilize our antibiotic arsenal more effectivelyis one potential way to extend the useful lifespan of our current antibiotics.Recent studies have indicated that modulating metabolic activity is one possiblestrategy that can impact the efficacy of antibiotic therapy. In this review, wewill address recent advances in our knowledge about the impacts of bacterialmetabolism on antibiotic effectiveness and the impacts of antibiotics onbacterial metabolism. We will particularly focus on two studies, Lobritz, Mycobacterium tuberculosis can becomeantibiotic-tolerant by entering a metabolically quiescent, slow-growing state. This isaccomplished by shunting carbon flux away from the tricarboxylic acid (TCA) cycle andinto storage as triacylglycerol instead. If this pathway is inhibited, M.tuberculosis remains susceptible to a variety of antibiotics when placedunder stress, providing a potential therapeutic target to increase the efficacy ofavailable drugs. The TCA cycle is also an important determinant of antibiotic tolerancein the opportunistic pathogen Staphylococcus epidermidis. In S.epidermidis, disabling the TCA cycle has a protective effect againstbeta-lactam antibiotics due to suppression of reactive oxygen species (ROS) generationduring antibiotic exposure, as well as increased tolerance of oxidative stress. TCAcycle defects are also common in clinical isolates of S. epidermidis,suggesting that such mutations may be a common antibiotic defense. In addition, inStaphylococcus aureus and Escherichia coli,antibiotic-tolerant persister and biofilm cells can have reduced proton motive force(PMF), leading to inhibited uptake of aminoglycoside antibiotics . Thistolerance can be reversed by the addition of metabolism-stimulating sugars that boostPMF and the uptake of antibiotics.These two papers build on a significant body of evidence that suggests that reducedmetabolic activity leads to an antibiotic-tolerant state and that elevated metabolicactivity contributes to bactericidal antibiotic toxicity in susceptible bacteria . For example, A growing compendium of evidence suggests that in non-tolerant bacteria, elevated centralmetabolism contributes to bactericidal antibiotic toxicity through the production ofreactive oxidants and other damaging molecules. At low therapeutically-relevantconcentrations, drug-target interactions of bacteriostatic antibiotics induce ROS as theprincipal cause of antibiotic toxicity under oxygenated conditions. At higherconcentrations of antibiotics, ROS and other damaging reactive species appear to play alesser role in toxicity. The link between ROS and antibiotic toxicity has been recentlydisputed, but in general a preponderance of evidence suggests that the induction of ROSand metabolic activity are inexorably linked to bacterial death.et al., which utilized in silico genome scalemetabolic models to identify perturbations that increase intracellular production ofendogenous ROS species. In particular, they found that perturbations to the productionor usage of ATP were associated with predicted increases in ROS. Chiefly, targetingglycolysis, the TCA cycle, and aerobic respiration led to endogenous ROS production andsusceptibility to antibiotic-induced oxidative stress. Subsequent work identifiedexperimentally-tractable futile cycling reactions that decrease net intracellular ATPlevels. Furthermore, actively cycling strains were significantly more susceptible tooxidative stress and certain antibiotics (aminoglycosides). This suggests that thisincrease in sensitivity is attributable to increased damage and/or impaired repairmechanisms caused by perturbations in ROS. Along with other recent studies, this hasforcefully demonstrated that artificially inducing oxidative metabolism can impactantibiotic susceptibility. Under natural conditions, metabolically-costly futile cyclesalso contribute to bactericidal efficacy. Cho et al. demonstrated thatthe bactericidal activity of beta-lactam antibiotics is due, in part, to the inductionof a futile cycle between peptidoglycan cell wall synthesis and degradation. This is ametabolically-costly process that likely contributes considerably to ATP consumption, asseen in previous futile cycle studies. The total body of previous work suggests thatmetabolic activity contributing to ROS production is an integral part of bactericidaltoxicity.Initial evidence about this metabolic link came from a 2013 article by Brynildsenet al.: 8173-8180) and Belenky et al. :968-980) expand on this foundation of work by testing the impacts of bactericidalantibiotics on respiratory metabolism and the toxic consequences of this elevatedmetabolic activity respectively. Lobritz et al. utilized a novelhigh-throughput microplate-based method of analyzing bacterial oxygen consumption . They found that bactericidal antibioticsinduced a rapid induction in oxygen consumption indicative of elevated respiration. On the other hand, the same assay found that treatment with bacteriostaticantibiotics rapidly and permanently eliminated essentially all respiratory activity.They also found that inhibiting respiration eliminated bactericidal activity, whereasincreasing respiration led to boosted toxicity. This indicates that induction ofrespiration is essential for bactericidal antibiotic efficacy. Finally, and mostimportantly, this work identified that many bacteriostatic drugs are antagonistic tobactericidal drugs when used in combinatorial therapy, because thebacteriostatic-dependent inhibition of respiration supersedes the bactericidalrespiration-based benefit. The impact of this observation is that many currentlyutilized empirical therapeutic combinations are detrimental because they may lead todirect inhibition of an essential metabolic burst.The two papers at the heart of this microreview, by Lobritz et al. continued the study of antibiotic-induced metabolicactivity by conducting a total metabolomic profile of antibiotic-treated E.coli. This work found that bactericidal antibiotics - aminoglycosides,fluoroquinolones and beta-lactams - induce a common initial set of metabolicperturbations. These changes included an induction of central carbon metabolites,exemplified by dramatically elevated TCA cycle intermediates, as well as raised levelsof glutathione, which are suggestive of an active antioxidant response. These data areconsistent with the bacteriotoxic metabolic burst hypothesis proposed and tested inLobritz et al. In addition to the TCA cycle and glutathione signatures,Belenky et al. also found metabolic signatures suggestive of nucleotidedamage and perturbed lipid homeostasis. This observation led them to test potentialend-target consequences of metabolically-induced oxidative stress. They found thatbactericidal antibiotics lead to higher levels of malondialdehyde adducts, proteincarbonylation, and 8-oxo-guanine, indicative of lipid, protein, and nucleotide oxidationrespectively . Finally using a fluorescence-based protein assay, they showedthat antibiotic treatment induced double-strand DNA breaks likely due to oxidativedamage.Belenky When taken together, the two papers make a very convincing argument about the importanceof a metabolic burst for the damage of bacterial cells and eventual toxicity duringbactericidal treatment. This perspective comes at a very critical time, when antibioticefficacy is being increasingly compromised by rising rates of resistance. Modulatingmetabolic activity may provide important therapeutic methodologies to extend the usefullife span of our current antibiotics. In addition, the metabolic signature of thisrespiratory burst could be used as a potent readout to aid in the development of newdrugs and for screening of antibiotic resistance in laboratory and clinical settings. Ingeneral, the developing perspective that antibiotic-induced death is not a single,one-step response but rather a programmed set of discernable processes is a wonderfulchange. This new perspective opens up a large set of cellular processes that can bemodulated and utilized to improve therapy. Before this appreciation, antibiotic researchwas very much limited to drug-target interactions, while now the entire cell is open forbusiness.


Reproduction, in mammals, is finely regulated by the hypothalamic-pituitary-gonadal axis (HPG) and driven, by a complex neural network within the hypothalamus that converges onto GnRH-producing neurons, the master regulators of gonadotropin secretion . The corMicroRNAs are small, non-coding RNAs that fine-tune gene expression at the post-transcriptional level. Considered major players in epigenetics, microRNAs, together with transcription factors, form integrated gene regulatory networks that can control complex cellular processes with extreme temporal precision , making Our results show that, during an infantile “critical period” lasting just a few days, a dramatic switch in the microRNA expression pattern of GnRH neurons inverts the balance between inductive and repressive signals, triggering increased hypothalamic GnRH expression and controlling the crucial transition from the early infantile phase, when its levels are low, to the GnRH-fuelled run-up to puberty Figure  2]. Thi. Thi2]. Specifically, we have shown that during this critical period, a switch in the expression of several microRNA species in the hypothalamus in turn flips a switch in a multilayered array of GnRH gene activators and repressors, permitting the sustained increase of the neurohormone required for subsequent sexual maturation . Two micThese results raise the intriguing possibility that the microRNA-dependent epigenetic regulation of GnRH secretion could underlie the pathophysiology of human congenital hypogonadotropic hypogonadism (CHH) when no mutations are found in known CHH genes . ExploriWhile the identification of this unprecedented microRNA-based genetic network controlling GnRH transcription represents a clear advance in our understanding of the molecular basis of puberty, it raises new questions and challenges. What triggers this developmental switch in microRNA expression in GnRH neurons after birth? What roles do hypothalamic wiring; hormones and the environment play in this process? And finally, to what extent can pharmacological interventions modulate this process, and what are their limitations?


Although it is well recognized that autism is associated with altered patterns of over- and under-connectivity, specifics are still a matter of debate. Little has been done so far to synthesize available literature using whole-brain electroencephalography (EEG) and magnetoencephalography (MEG) recordings.1) To systematically review the literature on EEG/MEG functional and effective connectivity in autism spectrum disorder (ASD), 2) to synthesize and critically appraise findings related with the hypothesis that ASD is characterized by long-range underconnectivity and local overconnectivity, and 3) to provide, based on the literature, an analysis of tentative factors that are likely to mediate association between ASD and atypical connectivity .Literature reviews were done using PubMed and PsychInfo databases. Abstracts were screened, and only relevant articles were analyzed based on the objectives of this paper. Special attention was paid to the methodological characteristics that could have created variability in outcomes reported between studies.Our synthesis provides relatively strong support for long-range underconnectivity in ASD, whereas the status of local connectivity remains unclear. This observation was also mirrored by a similar relationship with lower frequencies being often associated with underconnectivity and higher frequencies being associated with both under- and over-connectivity. Putting together these observations, we propose that ASD is characterized by a general trend toward an under-expression of lower-band wide-spread integrative processes compensated by more focal, higher-frequency, locally specialized, and segregated processes. Further investigation is, however, needed to corroborate the conclusion and its generalizability across different tasks. Of note, abnormal lateralization in ASD, specifically an elevated left-over-right EEG and MEG functional connectivity ratio, has been also reported consistently across studies.The large variability in study samples and methodology makes a systematic quantitative analysis  of this body of research impossible. Nevertheless, a general trend supporting the hypothesis of long-range functional underconnectivity can be observed. Further research is necessary to more confidently determine the status of the hypothesis of short-range overconnectivity. Frequency-band specific patterns and their relationships with known symptoms of autism also need to be further clarified. It is well recognized that autism and autism spectrum disorder (ASD)–hereafter used interchangeably–is associated with altered patterns of connectivity, compared to neurotypical (NT) controls. Increased interest in connectivity reflects a shift from understanding the biological basis of autism as focal brain abnormalities affecting specific systems towards an overall pattern of brain reorganization. Moreover, new evidence on early development of white matter tracks suggests that connectivity could be among the earliest markers of autism, with initial signs emerging within the first year of life –3.Although the autistic brain was initially hypothesized as exhibiting a pattern of overall underconnectivity ,5 or by Previous literature reviews have partially addressed questions about connectivity in autism. These reviews have predominantly focused on structural connectivity using diffusion imaging –10 and cstructural/anatomical connectivity), to statistical dependencies  between time series of cerebral activity in different brain regions , or to causal interactions between brain regions (directed/effective connectivity) [Brain connectivity is a broad, multi-faceted concept . In humactivity) . Structuconnectivity is often used interchangeably in the literature to denote any or all of these variants, different measures can show surprisingly little agreement [Although the term greement ,17. Asidgreement ), which Similarly, different modalities of functional connectivity show complex interdependencies. For example, the relationship between electrical and hemodynamic activity has been shown to depend on frequency and spatial scale, with particular EEG rhythms generating region-dependent variations in blood oxygen levels and glucose metabolism ,20. CrosFurthermore, it is worth remembering that functional connectivity is generally based on similarity of signals observed between pair of regions and, in most cases, no control of the potential contribution of a third sources is made. This situation may be improved by multivariate approaches , but it The large amount of literature on connectivity in autism yields multiple distinct hypotheses about the nature of over- and/or under-connectivity. We considered three classes of inter-related hypotheses regarding connection length, topological specificity, and developmental effects.A popular hypothesis considers autism to be characterized by long-range underconnectivity potentially combined with local overconnectivity ,25–27. Tlong-range structural underconnectivity can result from a degradation of fiber bundles ,28–40, wlocal structural overconnectivity can result from a decrease of apoptosis, axonal pruning, and dendritic degradation, and from an increase of neurogenesis .relative number of local connections because of a loss of long-distance connections, but there is no augmentation in their absolute number).However, local connectivity may often be obfuscated by long-distance connections . In such cases, long-range underconnectivity makes local connectivity more clearly observable without meaning that there is a true increase in local connectivity  is confounded with volume conduction . ASD-related abnormalities have been documented in the frontal lobe ,55,56, iAbnormal connectivity between the occipital lobe and the other regions is also often reported; an observation that might be related with structural and functional abnormalities in processing of visual input in ASD –66. In aContrary to hypotheses about deficiency in specific regions, an alternative hypothesis suggests an overall non-topographically-specific alteration of brain connectivity in ASD. Support for this hypothesis is based on evidence of a more randomly connected brain in ASD , which rThe typical developmental course of connectivity remains poorly understood but its determinants  and its indexes  appear to follow an inverted U-shape trajectory during maturation, which progress from posterior to anterior regions and from primary to association cortical areas. These maturational patterns appear to differ in autism from very early in development ,2. DiffuDifferences in developmental trajectories of brain connectivity observed with EEG/MEG might also be exacerbated if excitatory and inhibitory neurons mature at different speeds . Such aRegarding which brain region might show the largest developmental differences, fMRI studies have reported the default mode network as being particularly vulnerable to ASD ,81. The Taken together, these different hypotheses demonstrate the complexity of the impact of ASD on connectivity, which is most probably characterized by both generalized atypicality  and more localized abnormalities  that develops during the first years of life.Although the current body of evidence related to connectivity comes mostly from fMRI –12 or frth 2016 using the research string “ AND connectivity AND autis*” returned 102 and 67 hits respectively. Only journal and conference papers published in English were reviewed.Our review focuses on original research assessing functional and effective connectivity as measured by EEG or MEG. Searches on PubMed and PsychInfo executed on March 6Studies were retained if they presented original experimental findings on participants with an ASD phenotype, including individuals diagnosed at any age as well as studies comparing low versus high-risk infants defined by the presence or absence of an older sibling diagnosed with ASD. One study correlating ASD traits in a neurotypical population with differences in connectivity features was also included.We excluded articles discussing functional or effective brain connectivity without reporting new experimental results on this topic. We also excluded articles reviewing neurofeedback approaches using connectivity measurements, methodological articles proposing new approaches for assessing brain connectivity, and studies inferring on connectivity only through indirect measurements, e.g., EEG/MEG complexity, ERP timing, spectral power, or local inter-trial synchronization.Appraisal of methodological quality of included studies was conducted by one of the authors (COR). This led to exclusion of one study due to probable methodological issues related to multiple comparisons, confounders, and mismatching statistics , and of A total of 52 papers were retained, 31 using EEG –118 and connectivity metrics;recording parameters ;experimental paradigms ;sample characteristics .The wide variability in study characteristics along these methodological dimensions precluded a meta-analysis to address the main objectives of the review. Instead, we synthesized and critically appraised findings of the over-arching hypothesis that ASD is characterized by long-range underconnectivity and local overconnectivity , and systematically reported results related to factors that can impact this hypothesis .We first synthesized included studies across several methodological dimensions that may account for some inconsistencies in including connectivity metrics, recording parameters, experimental paradigm, and sample characteristics. Outcome of this analysis can be found as supplementary documents As reviewed in the introduction, a prominent hypothesis is that autism is characterized by local overconnectivity and long-range underconnectivity ,26. EEG/Overall, the case for long-range underconnectivity is well supported. This is particularly clear for the case of inter-hemispheric connections, for which a decrease in EEG/MEG functional connectivity has been reported in many studies ,114,134.Local overconnectivity in ASD is less robustly established. A few studies report local overconnectivity ,117,139,The hypothesized pattern of long-range underconnectivity and short-range overconnectivity is paralleled by the general trends observed as a function of frequency see . OverallGeneral underconnectivity in lower frequency bands has been observed as a function of the number of autistic traits in a neurotypical sample  as well preferentially associated with more localized processes and activity in lower bands to be preferentially associated with more wide-spread integrative processes. Indeed, integrative top-down processes  involving long-range connections are often associated with slower rhythms , whereas synchronization of local cortical networks through bottom-up processes  tends to be associated with faster frequencies  [Tentative relationships can be proposed between frequency bands, the scale of connectivity, and the degree of connectivity. The modulation of over vs. under connectivity depending on the frequency band is consistent with slower oscillators involving more neurons in larger volumes . Such a , gamma) ,143. Suc, gamma) ). Furthe, gamma) ,134, witEEG and MEG connectivity studies using graph analysis generally report autism to be associated with sub-optimal network properties, such as less clustering, larger characteristic path, reduced eigenvector centrality (a measure of the importance of nodes as communication hubs), and an architecture less typical of small-world networks ,135,136.Neural networks in ASD have been shown to be more resilient because of their greater homogeneity and their more uniform architecture . This, iHowever, these analyses have been performed on graph topologies, which preserve very little relationship with spatial properties of brain activity. Specifically, edges of these graphs are not weighted by the length of the connections and the angles between the edges are unspecified. Therefore, the spatial arrangement of the nodes is lost and the results cannot be reconciled with the findings reviewed above on the impact of connection length on over/underconnectivity.Although the hypothesized pattern of over and under connectivity in autism is a valuable conceptual generalization, many factors may modulate connectivity at a finer scale reflecting context-dependent modulations in connectivity. Below we consider the factors that were prominent in the reviewed of the literature.Many cognitive processes display brain lateralization, language being a classical example. Since symptoms associated with autism are associated with lateralized cognitive functions , many studies have looked for and found atypicalities in lateralization of brain functions in autism . In the For example, studying gamma-band connectivity in relation with face processing, a leftward  lateralization emerging around one year of life was found . The autSimilarly, an elevated long-range fronto-posterior functional connectivity in children with ASD was observed prominently in the left-hemisphere . Such inA rightward reduction of beta-band connectivity (phase lag index) between the occipital lobe and frontal, temporal, and parietal areas in a numerosity task  was also reported in ASD . The autIn an event-related paradigm in which emotional stimuli evoke bilateral activation of the insula in neurotypical adults , a less Using photic stimulation, a leftward predominance in connectivity increased at stimulated frequencies has been observed in ASD, with a larger group difference at higher frequencies ,107. AltDuring the REM phase of sleep, more important increases in coherence were also noted in the left hemisphere, particularly for the delta band . KikuchiIn sum, a consistent pattern of increased left-over-right EEG and MEG connectivity ratio has been observed in ASD. This pattern may well reflect specific perceptual characteristics of this population in which local components are more strongly processed (segregation) relative to global relationships between components . This altered processing style can be hypothesized to be a consequence of the observed drop in long-range connectivity. A lack of proper long-range connectivity may not provide an adequate substrate for the normal development of the rightward more global and integrative style of processing. Compensatory development of more leftward local-processing style may, however, still be possible if shorter-distance connectivity is not as severely affected by the disease. It is also relevant to note that an enhanced EEG power has been consistently reported in the left hemisphere across all frequencies (reviewed in ) which mCognitive processes that are known to be atypical in ASD are, to some extent, localized to specific brain regions. Thus, there is an interest in investigating the topography of connectivity abnormalities to further associate altered patterns of connectivity with observable phenotypes and symptoms. Among the reviewed studies, some discussed general topological differences at sensor-level, whereas others used more precise analyses of cortical sources to localize abnormalities. For example, in MEG, a reduced coherence between the frontal eye field (a region involved in voluntary eye movements) and dorsal anterior cingulate cortex in an ASD population in a saccade/anti-saccade task has been shown . AbnormaThese results are examples showing that more subtle patterns of connectivity are most certainly region- and function-dependent. Consistent with the topological hypotheses reviewed in the introduction, a large number of studies included in Differences in EEG/MEG functional connectivity are emergent rather than static over development ,115. HowDevelopmental change continues to impact connectivity patterns later in development. Using resting-state MEG recordings from 6–21 years old individuals, Kitzbichler et al.  found inOur systematic review of a large body of evidence suggests that ASD is characterized by a pattern of EEG/MEG functional connectivity that is in general more randomly organized, with abnormal connectivity often involving frontal or occipital regions–at least in adult samples–although abnormal connectivity patterns have been reported in almost every region. Abnormal lateralization of activity in resting-state and in specific tasks seems to be typical, with a very systematic report of elevated left-over-right EEG and MEG functional connectivity ratio in ASD. Both abnormal intra- and inter-hemispheric connectivity can be observed with a general trend toward underconnectivity, but with probable local or condition-specific  overconnectivity. Underconnectivity can most reliably be observed in lower frequency bands  whereas a stronger tendency for overconnectivity can be observed see  in high-de novo chromosomal mutation causing agenesis of corpus callosum is another example of a subgroup of individuals with ASD that might be clearly defined from a clinical point of view. These persons reported a normal level of attention to details, with the preserved ability to appreciate the whole rather than a preoccupation with patterns or parts [Despite these general patterns related to categorical diagnosis of ASD, several findings highlight the importance of considering variability in the ASD phenotype related to connectivity. For example, individuals with an Asperger’s diagnosis can be differentiated from those with other ASDs relatively accurately (92.3% in ) based oor parts . Thus, cor parts ) are proFurther, abnormalities in EEG/MEG functional connectivity increase with increasing symptoms severity in autism. Measures of connectivity abnormalities reported in surveyed papers has been correlated with the presence of autistic traits in the NT population  and withThe conclusions derived from our systematic review are limited by a few potential methodological confounders. These are discussed hereafter.The hypothesis underlying every approach used to measure functional connectivity in EEG/MEG is that the estimated connectivity  is proportional to physiological connectivity between the brain areas generating these time series. There are nevertheless pitfalls in interpretation of connectivity measures due to our limited understanding of their underlying physiology, e.g., hidden sources, differential sensor sensitivity for different kind of synaptic activity, etc.The low statistical power in the reviewed studies may be misleading. With a sample size typical of these studies (we take N = 15 per group for this example), the power of a two-tail two-sample t-test with a significance level at 0.05 is only 0.26 for a size-effect considered as medium (Cohen's d at 0.5). That is, three studies out of four will report negative results for medium-sized differences in brain connectivity. Controlling for multiple comparisons further reduces the power of these tests such that only very strong effects may be reliably detected in most studies. When applied to the Publication bias: As mentioned previously, the context of individual studies may impact on obtained results. When results are grouped, these contexts are partly lost and this can produce a bias in the overall conclusions. That is, studies looking for specific information  may report specific findings  which would not be the main outcome if they had investigated a broader context . This creates a potential publication bias where topic of high interest is generally more scrutinized, resulting in the publication of more positive findings.The significant bias in head and brain size between ASD and NT participants may result in biases impacting the distance between EEG sensors and the properties of electrical signal propagation from the cortex to the scalp. Differences in brain size (not head size) may also have some effect on the signal-to-noise ratio in MEG since the cortical surface is in average closer to the sensors for larger brains. This effect is expected to be more important in children since brain size differences between ASD and NT are larger at this age. Entering these factors as co-variates in statistical analyses is advisable to control their impact on group comparisons of brain connectivity. Although brain size might not be readily accessible in studies not including MRI scans, standard metrics for the head size are easily measured .2 is required [We noted that, compared to long-range functional connectivity, studies of local connectivity are more scarce. This pattern might be related to intrinsic limitations of EEG and MEG recording modalities for evaluating short-range connectivity, particularly in coherence analyses performed at sensor level. To record an EEG/MEG potential, a large pool  of synchronously activated cells with parallel apical dendrites  spanning a cortical patch of 40–200 mmrequired . This harequired   sources to the sensors. For EEG, potentials are reaching sensors through omnidirectional volume conduction. For that reason, they spread over larger territories than the initial source area and create severe biases when estimating coherence between close sensors (< 10 cm) . This phOne way to partly mitigate the problem of volume conduction would be to compute connectivity on EEG sources estimated using more accurate electromagnetic model of the propagation of electrical activity. Although the effect of field spread cannot be completely resolved by the current level of sophistication of source-reconstruction algorithms, such an approach should definitely be used to supplement sensor-level analyses as it reduces the impact of volume conduction and helps better link electrical activity with brain regions . FurtherOur review is limited to EEG/MEG functional connectivity in ASD, but future work should also help clarify cross-cutting issue common to multiple neurodevelopmental conditions. It remains to be established if the pattern of connectivity we observed in ASD is specific to the condition or alternatively reflects a more general pattern of changes common to a broad group of neurodevelopmental disorders .This review illustrates the large heterogeneity of both the methods and the results of studies investigating brain electrophysiological connectivity in ASD. Some of this variability might be reduced by further improving the methods adopted .Research on electrophysiological functional connectivity in autism has been pioneered by EEG coherence analyses performed on small sensor grids, which provided crude connectivity assessment between very large brain regions . Currently, high-density grids are available in EEG and are included by default in every MEG system. Furthermore, algorithms for the estimation of cortical  sourcesStudies examining individual variability within ASD and across neurodevelopmental conditions remain very sparse and future studies need to pay more attention to mapping connectivity onto phenotypic differences. The use of functional connectivity features for diagnostic application is also relevant since high accuracy (85–95%) has been reported by many independent research teams ,126,135.A clearer theoretical foundation is necessary to efficiently establishes the role of connectivity length with respect to over/under-connectivity using EEG/MEG functional connectivity. Strong theoretical grounding can help address outstanding questions in Further work is also needed to better understand the complex interactions between frequency bands, brain regions, physiology of EEG/MEG oscillators , and hoFinally, given the nonlinear evolution of brain properties , the developmental evolution of these properties strikes us as a very important area of investigation since these changes are generating confusion in interpretation of the current literature. Aside from helping to reconcile apparent contradictions in the literature, a better understanding of how developmental factors induce ASD-related brain changes early in development would provide invaluable insights on the pathogenesis of ASD. Performing such analyses in a multi-modal framework may also further our understanding of the dynamics of ASD-related abnormalities in brain connectivity and help resolve some of the apparent contradictions arising when comparing results across modalities.S1 File(DOC)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Frontiers in Neuroscience, meant to collect both clinical and preclinical investigations focused on factors associated with illicit drug vulnerability in adolescents, and the long-term consequences of adolescent drug exposure. This Editorial provides an overview of our Special Issue, organized in 3 themes.Adolescence is characterized by risky behavior and impulsive decision making, which often exposes adolescents to psychoactive substances. Meanwhile, neural circuits refine and mature during adolescence, creating a window of opportunity for environmental insults to impact brain maturation and potentially confer susceptibility to life-long drug addiction. Indeed, an early age of first drug use (before 15 years old) is strongly associated with risk for developing a substance use disorder later in life  and report that poor daily executive functioning  significantly relates to increased substance use risk during adolescence. Tervo-Clemmens et al. focused further on the executive function characteristics in adolescents that increase risk for substance use. In a sample of youths from the NCANDA study, Tervo-Clemmens et al. replicated previous findings indicating that poor response inhibition is associated with substance use risk. The nature of this risk was further elucidated using a reward-anti-saccade task in which participants are primed with visual cues during a fixation period. The visual cue provides information as to whether correct performance on the subsequent response trial will be rewarded (monetarily) or not incentivized. During response trials, participants must perform a saccade away from the presented visual stimulus. Although incentivizing accurate responding improved performance in the anti-saccade task, incentives alone were insufficient to modify associations between response inhibition and substance use risk, indicating that deficits in response inhibition and heightened sensitivity to reward are distinct neurocognitive features of adolescent substance use risk.Although many adolescents engage in risky, experimental substance use without developing later-life addiction, certain non-drug-related, functional differences appear to predispose adolescents to increased risk for addiction. Weil and Karelina summarize clinical and preclinical evidence suggesting that traumatic brain injury during adolescence increases risk for alcohol use disorder later in life. For males, this pattern is particularly pertinent, as young men are more likely to suffer from abuse-related head injury, traumatic brain injury, and also alcohol abuse. In preclinical studies, Barfield and Gourley sought to uncover sex-specific behavioral consequences of experiencing elevated levels of the primary stress hormone corticosterone during adolescence. Using an instrumental reversal learning task, Barfield reports that female, but not male, mice exhibit inflexible, perseverative-like response errors following early-adolescent corticosterone. These findings are significant because pre-existing deficits in behavioral flexibility may accelerate the transition from recreational substance use to addiction  and were then sampled again 2 years later. Interestingly, Meda et al. also show that many of the participants who self-reported heavy drinking at the beginning of the study already had smaller frontal cortices, potentially suggestive of a pre-disposition toward substance abuse. Also affected by adolescent substance exposure are the white matter tracts that connect gray matter regions. Li et al. find that adolescent cynomolgus monkeys exposed repeatedly to ketamine have reduced white matter integrity in connections between brain regions involved in executive functions and learning and memory such as the frontal cortex, striatum, thalamus, and hippocampus. Neural changes are also detectable at the molecular level: Liu and Crews report that rats exposed to binge-like ethanol treatment during adolescence exhibit less neurogenesis within the dentate gyrus region of the hippocampus, and also the subventricular zone. Further, Vore et al. report that rats exposed to binge-like ethanol treatment during adolescence have reduced cytokine expression in response to stress and immune challenge.Across multiple mammalian species, exposure to drugs of abuse during adolescence is associated with deficits in neurodevelopment. Using data from the longitudinal Brain and Alcohol Research in College Students (BARCS) study, Sanchez Marin et al. report that adolescent rats exposed to binge-like ethanol treatment not only have aberrant mRNA levels of genes encoding enzymes and receptors in the endocannabinoid system and neuroinflammation-related factors within the frontal cortex, and higher mRNA levels of stress-related genes within the hippocampus, but in addition, develop deficits in the novel object recognition test of learning and memory. Similarly, Marco et al. find that binge-like ethanol exposure not only reduces cannabinoid receptor type 2 protein levels in the frontal cortex, but also causes significant deficits in novel object recognition. Last, Kruse et al. show that, in rats, deficits in risk-based decision making (overly risky behavior), resultant from adolescent ethanol self-administration, are not caused by impulsivity but rather are due to heightened sensitivity to reward-related conditioning stimuli.Damage to specific neurocircuits by adolescent drug exposure is also associated with long-term effects on learning and memory and executive functions. Madayag et al.). Nor does adolescent ethanol exposure energize later-life self-administration in male rats . In several psychosis-related behavioral tasks, Jaehne et al. report that, despite the known actions of methamphetamine on serotonergic neurotransmission, mice lacking the 5-HT1A receptor and exposed to methamphetamine during adolescence are indistinguishable from methamphetamine-exposed wildtype control mice. Finally, Kirschmann et al. present findings that challenge the popular belief that adolescent exposure to marijuana causes long-term deficits in executive functions. Using an operant conditioning procedure that allows adolescent rats to self-administer cannabinoids, Krishmann et al. report that adolescent cannabinoid self-administration in female rats has no detectable effects on spatial or working memory performance. Further, experimenter-administered cannabinoids actually improved spatial and working memory performance in adulthood.The nature of clinical studies often precludes direct cause-and-effect analyses of mechanisms underlying substance use risk. Several studies within this collection use preclinical models to tease out these distinctions, and they report important null and unexpected effects of adolescent drug exposure. For example, in female rats, nicotine exposure during adolescence unexpectedly fails to impair behavioral flexibility or energize ethanol self-administration and reinstatement in adulthood  preclinical studies aimed at elucidating mechanistic factors by which adolescent drug exposure impacts neurocognitive functions, neural structure, and neuronal plasticity during development and into adulthood.AW wrote the first draft of the manuscript. All other authors edited the manuscript and contributed to the final version of the manuscript. All authors approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present work reviewed the connections of changes in consumption of high-fat food with changes in adiposity and lipaemia in adults with overweight or obesity. Hyperlipaemia from higher fat meals and excessive adiposity contributes to atherogenic process. Low-fat diet interventions decrease body fat, lipaemia, and atherosclerosis markers. Inaccuracy of physical estimates of dietary fat intake remains, however, a limit to establishing causal connections. To fill this gap, tracking fat-rich eating episodes at short intervals quantifies the behavioural frequency suggested to measure  direct effects of this eating pattern on adiposity and atherogenic lipaemia. Such evidence will provide the basis for an approach focused on a sustained decrease in frequency of fatty meals or snacks to reduce obesity, hyperlipaemia, and atherosclerosis. Atherosclerosis is globally a leading cause of death . AtherogThe current work articulates research on ingestion of dietary fat as a substantial contributor to the levels of adiposity and lipaemia. First, the physiological events are integrated from high-fat food consumption at eating occasions to infiltration of circulating atherogenic lipoprotein particles into the arterial wall. Then, evidence is presented for changes in adiposity, lipaemia, and markers of atherosclerosis by means of interventions to reduce dietary fat intake. Finally, a missing step for establishing causal links is pointed out together with a proposed approach for filling this gap.The present work considers also the behavioural field to move current research forward. The episodes of fatty meals or snacks are here addressed as a target behaviour pattern . The aimAfter an eating occasion, ingested fat is transported by lipoproteins for deposition as triglycerides within large intracellular lipid droplets in adipocytes without thermogenic cost. That is, lipids are more efficiently stored compared to carbohydrates or proteins. So a pattern of frequently consuming fatty meals or snacks would facilitate the expansion of body fatness. Accordingly, many studies have found association between high-fat diet and increased weight or body mass index , 6. A foLipolysis rate increases in response to excessive deposition of lipids in adipocytes –10. HencThere is an apolipoprotein B (apo B) molecule in the surface of each chylomicron (CM), very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL). Apo B has high molecular weight. Therefore, hyperlipaemia can elevate the blood volume or viscosity by higher number of apo B particles –19. Vascα) [Endothelial failure contributes to slow blood flow as well as hematic stasis at sites of vascular resistance such as coronary arteries . In statα) –30. Macrα) . Such imα) , 32.Decreasing fat intake is a common strategy of dietetic interventions to reduce body fat and lipaemia –37. Nondα, and leukocytes [Prescribing dietary strategies to cut down on fat intake also decreases levels of modified lipoproteins and related immune biomarkers. For instance, reduction in body mass index by lifestyle intervention was associated with decrease in IL-6, protein-C reactive, normal T cell expressed and secreted, and other related markers of immune response and inflammation , 45. Peoukocytes .One of the most studied modified lipoproteins has been the oxidised low-density lipoprotein (ox-LDL) . Like otDespite the well-documented positive association of changes in fat consumption with changes in fat mass and lipaemia, there is a significant bias in the core measure of dietary fat intake, the behavioural dose. Physical estimates of energy and nutrient content of diet are so inaccurate due to underreporting of food amounts that not using them in obesity research is suggested , 61. EstThe effect of low-fat diets is taken for granted from outcome measures. However, even randomised controlled trials are not free of confounding effects from other unmeasured variables . For insIn any case, researchers rarely monitor the behaviour changes during dietary interventions. When change in dietary intake is monitored, it is usually at intervals of several months . The effEvidence from social psychology shows that people are highly accurate in their perception of what they normally do . This psIn a small study to decrease unhealthy eating in adults with overweight or obesity, episodes of fatty meals and snacks were daily tracked during four weeks . ParticiThis preliminary evidence tightens previous research showing that episodes of fatty meals and snacks over time are causally connected with change in adiposity and lipaemia as well as development of atherosclerotic plaque. Moreover, this shows that the effect of eating fatty meals and snacks on the arterial wall occurs within a few weeks.Pursuing this research avenue would contribute to elucidating the causal links of behaviour patterns with obesity, dyslipidaemia, and atherosclerosis. This research line requires, however, substantial development. For instance, future studies could combine the present measure of frequency of an eating pattern alongside amounts of food eaten on each occasion estimated also with novel approaches that overcome limitations in accuracy of conventional dietary evaluations . A charBroad research shows consistently a positive association of fat intake with adiposity and apo B containing lipoproteins as well as with circulating levels of immune cells, cytokines, and modified lipoproteins. However, as gaps in research remain to accurately measure fat intake, there are still no measurements of causal connection between sustained intake of either more or less dietary fat and changes in anthropometry or physiological markers. The innovative use of psychosocial measures to collect records of the timings of the fatty meals and snacks as they are carried out by individuals in their everyday life would greatly benefit research and substantiate the evidence about the impact of dietary fat consumption on body fat, blood lipids, and biomarkers of atherosclerosis.Research is beginning to accumulate showing that a bout of high-fat ingestion rapidly initiates metabolic impairments such as acute inflammation , 14. TheReducing the habitual number of episodes of unhealthy eating may contribute to minimising the physiological disturbances found in people with overweight or obesity. So monitoring fatty meals or snacks provides an approach based on behaviour frequency that readily generates evidence to inform objective recommendations against unhealthy eating. For instance, similar to the recommendation of exercising more than three times per week , a recom


Recent technological advances in EM including serial block-face imaging and focused-ion beam scanning EM have also facilitated automated acquisition of large tissue volumes required to reconstruct neuronal circuits in 3D at nanometer-resolution. These cutting-edge techniques which are now becoming increasingly available will further revolutionize the study of microglia across stages of the lifespan, brain regions, and contexts of health and disease. In this mini-review, we will focus on defining the distinctive ultrastructural features of microglia and the unique insights into their function that were provided by EM.The first electron microscope was constructed in 1931. Several decades later, techniques were developed to allow the first ultrastructural analysis of microglia by transmission electron microscopy (EM). In the 50 years that followed, important roles of microglia have been identified, specifically due to the ultrastructural resolution currently available only with EM. In particular, the addition of electron-dense staining using immunohistochemical EM methods has allowed the identification of microglial cell bodies, as well as processes, which are difficult to recognize in EM, and to uncover their complex interactions with neurons and synapses. The ability to recognize neuronal, astrocytic, and oligodendrocytic compartments in the neuropil without any staining is another invaluable advantage of EM over light microscopy for studying intimate cell–cell contacts. The technique has been essential in defining microglial interactions with neurons and synapses, thus providing, among other discoveries, important insights into their roles in synaptic stripping and pruning  Thes–80 nm seIn 1957, the first ultrastructural image of microglia in the rat parietal cortex was published , and in Microglial cell bodies can be discerned from those of other cell types by their small size (3–6 μm), electron-dense cytoplasm, and characteristically bean-shaped nuclei. They also display a distinct heterochromatin pattern. A thick, dark band of electron-dense heterochromatin is located near the nuclear envelope, with pockets of compact heterochromatin nets throughout the nucleus. These nets are often visualized as small islands of dark heterochromatin within a sea of more loosely packed, lighter euchromatin within the central part of the nucleus , 55. Micin situ. Labeling microglial membranes and cytoplasm, originally with enzymatic reactions and more recently with immunoEM, allowed researchers to investigate microglial processes in animal models and human postmortem tissue  with healthy microglia, yet appear strikingly different under TEM. Their cell bodies can be quickly identified by their condensed, electron-dense cytoplasm that makes them appear as dark as mitochondria. Dark microglia display many signs of cellular stress, including nuclear and chromatin condensation and dilation of their endoplasmic reticulum Figure F. AdditiThe combination of both light microscopy and EM can be used to uncover more information than either technique individually. CLEM was first used in 1969. Silver staining originally described by Río-Hortega was used to identify and investigate microglia in light microscopy. After confirming microglial-specific staining, researchers investigated ultrathin sections under TEM and published the first description of microglial ultrastructure .in situ hybridization, or in situ RT-PCR  in order to determine that demyelination in experimental autoimmune encephalitis is initially performed by invading monocytes, while resident microglia did not contribute to the early stages of inflammation (z-dimension (2) area, researchers may image at 5 nm resolution in x, y, and z  . With thammation . More reimension . By emply, and z .Both SBEM and FIB-SEM are capable of investigating neuronal ultrastructure, and can follow a single process through several microns of neuropil, but FIB-SEM is also capable of resolving synaptic vesicles, lysosomes, and phagosomes in three dimensions. If the FIB-SEM process began within a microglial cell body, researchers could trace fine microglial processes through several microns of neuropil, without having to perform immunoEM. This offers a better chance to investigate lipidic inclusions and other pathological changes in organelles obscured by electron-dense precipitates used in immunoEM.In addition to technological advances in both SEM and TEM, the rapid development of cryoEM techniques described here could uncover native protein structures within microglia. It could additionally pave the way for discoveries into the snapshot of microglial–neuron and microglia–glia interactions without requiring fixatives, and without the corresponding tissue deformation that occurs with rapid fixation currently required to preserve ultrastructure. While fixatives and ultrathin sections required for EM are not compatible with post-imaging analysis of RNA or proteins, future iterations of CLEM (perhaps cryoCLEM) and advances in single-cell mRNA isolation may be able to isolate subcellular tissue fractions for further analysis. Armed with these new tools, biologists may investigate the complex interactions between glia and neurons in a number of diseases. The unique nature of EM allows researchers to characterize unique ultrastructural characteristics of microglia and other immune cells, and uncover possible paths for therapeutic intervention.JS and MET conceived the ideas and drafted the manuscript to which all authors contributed. KP and FG-I created the figures. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors MET.


Insect antenna is a multisensory organ, each modality of which can be modulated by biogenic amines. Octopamine (OA) and its metabolic precursor tyramine (TA) affect activity of antennal olfactory receptor neurons. There is some evidence that dopamine (DA) modulates gustatory neurons. Serotonin can serve as a neurotransmitter in some afferent mechanosensory neurons and both as a neurotransmitter and neurohormone in efferent fibers targeted at the antennal vessel and mechanosensory organs. As a neurohormone, serotonin affects the generation of the transepithelial potential by sensillar accessory cells. Other possible targets of biogenic amines in insect antennae are hygro- and thermosensory neurons and epithelial cells. We suggest that the insect antenna is partially autonomous in the sense that biologically active substances entering its hemolymph may exert their effects and be cleared from this compartment without affecting other body parts. Culex salinarius, which form axo-dendritic synapses with sensory neurons  into the antennal hemolymph under control of dorsal unpaired median (DUM) neurons, originating from the suboesophageal ganglion , triggering different signaling cascades, which lead to a rise or fall in the cAMP level and CaThis review addresses those peripheral effects of biogenic amines—OA, tyramine (TA), serotonin (5-HT), and dopamine (DA)—that are confined to the antenna, the major sensory organ in insects.OA, topically applied or injected into an insect body, evokes pronounced changes in olfactory responses, both behavioral  amplitude. However, it is noteworthy that changes in EAG under the influence of OA and other humoral factors should be interpreted with caution, since EAG is a complex signal rather than just a voltage drop caused by arithmetical summation of leak currents from ORNs. EAG strongly depends on the electrical resistance of the antennal tissue as well as on the position of recording electrodes relative to the responding sensilla Nagai, . Data reRhyparobia (Leucophaea) maderae  receptor knockout  to TA than OA, representing, in fact, a TA receptor. TA activation of this receptor leads to Gi protein-mediated inactivation of adenylate cyclase and a reduction in intracellular cAMP levels  transcripts were detected both in pheromone- and general odor-sensitive antennal sensilla  causes changes in the sugar-sensitive gustatory receptor on the fly labellar sensilla due to increased expression of the Gr64a receptor gene (Nishimura et al., A majority of insects other than flies bear gustatory sensilla on antennae and are likely to use similar dopaminergic regulatory mechanisms. For example, unpaired H-cells with their bodies located in the suboesophageal ganglion of moths and orthopterans (Mesce et al., DA receptors were demonstrated to be expressed in honey bee antennae; moreover, changes in the expression level of one of them, Amdop1, corresponded to the transition from nursing to foraging (McQuillan et al., Aedes aegypti, long-range perception of CO2 changes behavioral responses to a short-range thermal signal (McMeniman et al., Rhodnius prolixus (Bodin et al., Camponotus rufipes (Weidenmüller et al., Bombus terrestris (Westhus et al., The outer layer of the cuticle bears waxes or liquid cuticular hydrocarbons, the repertoire of which may be body part specific (Oppelt and Heinze, The insect antenna is a multisensory organ, and each modality can be modulated by biogenic amines. It appears that insect antenna is partially autonomous in the sense that biologically active substances entering its hemolymph may exert their specific effects and be removed predominantly or even totally inside this compartment without affecting other body parts. OA increases activity of pheromone- and some, but not all, non-pheromone-sensitive antennal ORNs. There is some evidence that DA modulates gustatory receptor neurons. TA, a metabolic OA precursor, also modulates ORNs, usually in an antagonistic manner to OA, but it is unclear if TA and OA receptors are co-localized in receptor neurons. Serotonin can serve as a neurotransmitter in some afferent mechanosensory neurons and both as a neurotransmitter and neurohormone in efferent fibers targeted at the antennal vessel and mechanosensory organs. Aminergic modulation of thermo- and hygrosensory sensilla has not yet been demonstrated, and could potentially be another target for modulation. Functioning of non-sensory antennal tissues in the epithelium, tracheae and hemolympatic vessel may also be under humoral control, including aminergic.MIZ, ADP: manuscript planning, MIZ: draft writing, ADP: editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Climate change is predicted to result in altered precipitation patterns, which may reshape many grassland ecosystems. Rainfall is expected to change in a number of different ways, ranging from periods of prolonged drought to extreme precipitation events, yet there are few community wide studies to accurately simulate future changes. We aimed to test how above- and below-ground grassland invertebrate populations were affected by contrasting future rainfall scenarios. We subjected a grassland community to potential future rainfall scenarios including ambient, increased amount (+50% of ambient), reduced amount (–50% of ambient), reduced frequency  and summer drought . During Austral spring (September 2015), we sampled aboveground invertebrates, belowground macro invertebrates and nematodes. Aboveground communities showed a significant response to altered rainfall regime with the greatest effects observed in summer drought plots. This was mostly due to a large increase in sucking herbivores (658% higher than ambient plots). Plots experiencing summer droughts also had higher populations of parasitoids, chewing herbivores and detritivores. These plots had 92% more plant biomass suggesting that primary productivity increased rapidly following the end of the summer drought 5 months earlier. We interpret these results as supporting the plant vigor hypothesis . While belowground invertebrates were less responsive to altered precipitation, we observed a number of correlations between the abundances of above- and below-ground invertebrate groups under ambient rainfall that dissipated under altered rainfall regimes. Mechanisms underpinning these associations, and reasons for them to become decoupled under altered precipitation regimes (we term this ‘climatic decoupling’), remain speculative, but they provide the basis for formulating hypotheses and future work. In conclusion, we predict that shifts in rainfall patterns, especially summer drought, will likely have large, but probably short-term, impacts on grassland invertebrate communities. In particular, sucking herbivores show sensitivity to precipitation changes, which have the potential to cascade through the food chain and affect higher trophic levels. Invertebrates make up 96% of known terrestrial species, with an estimated contribution to ecosystem services worth $60 billion per year in the US alone . InverteClimate change models predict altered precipitation patterns and an increased number of extreme precipitation events , which wIndirect effects of water stress, such as those mediated by plants, can also affect invertebrates differently . SuckingThe group or guild specific responses of invertebrates to altered precipitation patterns are likely to alter the interactions occurring between species, and subsequently the structure of communities . Yet, reFuture precipitation regimes may therefore have a range of impacts on above- and below-ground invertebrates, and potentially modify linkages and interactions between these two groups. Experiments often use simplified pot and lab experiments, which are useful for teasing apart causal relationships but cannot fully incorporate synergies between the direct and indirect effects of altered precipitation across multiple trophic levels. Above- and below-ground linkages observed in such experiments, for instance, do not necessarily represent what happens in field situations e.g., . CommuniWe aimed to test how above- and below-ground grassland invertebrate populations were affected by contrasting future rainfall scenarios. To achieve this, we used a unique field-based community experiment in southeast Australia that applied ambient levels of precipitation together with four predicted precipitation patterns in a grassland ecosystem. We identified the effects of altered rainfall patterns on the abundance of invertebrate taxonomic groups, feeding guilds and the structure of the community as a whole. Additionally, we explored potential associations between above- and below-ground invertebrate communities and tested whether these were affected by altered precipitation patterns.Drought and Root herbivore Impacts on a Grassland ecosystem) experimental platform for this research. This platform applies different rainfall regimes in a grassland ecosystem based in Richmond, New South Wales at the Western Sydney University Hawkesbury campus . The DRI-Grass experiment consists of 48 permanent rain exclusion shelters  constructed from stainless steel frames and clear acrylic Perspex roofs. These shelters allow five rainfall scenarios to be simulated comprising 12 plots with (i) ambient , (ii) reduced amount (-50% of ambient), (iii) reduced frequency  as well as six plots with, (iv) increased amount (+50% of ambient), and (v) summer drought . Six plots of each of the 12 ambient, reduced amount and reduced frequency plots had been inoculated with scarab larvae as part of a concurrent, but separate, experiment which we accounted for as a covariate factor in our analysis . To assess differences between watering regimes, soil moisture readings are automatically taken every 15 min from TDR probes, and a daily mean was calculated (see We used the DRI-Grass (Axonopus fissifolius (C4 grass), Cymbopogon refractus (C4 grass), Eragrostis curvula (C4 grass), Hypochaeris radicata (forb), Microlaena stipoides (C3 grass), Paspalum dilatatum (C4 grass), Plantago lanceolata (forb) and Setaria parviflora (C4 grass). The soil is characterized as a sandy loam of moderate to low fertility, with a low organic matter content and low water holding capacity .Three sampling methods were used to collect aboveground invertebrates, belowground macro-invertebrates and nematodes during 11–21 September 2015, prior to harvesting aboveground plant material in the following week. Vacuum (‘G-vac’ device) sampling, a proven quantitative technique , was useTwo additional soil cores , one from each side of the plot, were collected for extraction of nematodes, at the same time as sampling belowground macro fauna. These were combined per plot to form a composite sample, gently homogenized and subsampled, with nematodes extracted from 50 g soil (wet weight) using a modified Baermann funnel technique , over 3 car; post hoc comparisons were performed based on visual inspection  for significant factors [glht, multcomp; Statistical analyses were undertaken in R, version 3.2.2 . To confThe effect of rainfall treatment on plant biomass from the October harvest was evaluated with a linear model using a Chi Square test, followed by a Tukey test.sensuMASS; Separate analyses were undertaken for each of the three different sampling methods . Groups of invertebrates (separated by Order or feeding guild identity) that were captured infrequently - i.e., found in <10% of samples or constituting <50 individuals in total - were excluded to permit statistical analysis sensu.The effevegan package; The effect of altered rainfall regimes on the community composition of invertebrates was analyzed using permutational multivariate analysis of variance [PERMANOVA, adonis in the P < 0.01) results from the matrix were investigated further. A mixed model was used to show which of the highly significant correlations appeared to have a linear relationship; those that did not appear to have a linear relationship are not shown. Models used the abundance of two invertebrate groups that were significantly correlated, with the abundance of the aboveground group explained as a function of the abundance of the belowground group, with plot as a random effect. The fit of the model was assessed using residual plots and significance was determined using an ANOVA. Plots treated with additional root herbivory may have altered the relationships between above-below-ground invertebrates, so were not included in the analysis of above-belowground associations.A Pearson’s correlation matrix was used to explore linkages between above- and below-ground invertebrate groups for each rainfall treatment separately. In order to avoid type II errors, only highly statistically significant  . Plant biomass also varied between treatments , with a greater plant biomass in the summer drought plots at the time of sampling, 5 months after the drought period ended . It was also in these plots that soil moisture decreased throughout the drought period, but returned to ambient levels in May, 1 month after the drought period ended .Irrigation regime significantly affected soil moisture which varied by month, as shown by the interaction (FTable 2). Specifically, summer drought increased the abundance of invertebrates in the Orders Hemiptera, Orthoptera, Diptera, and Acari . In terms of functional guilds, summer drought positively affected the abundance of sucking herbivores, parasitoids, chewing herbivores, and detritivores . The abundance of Collembola also varied between rainfall regimes, with a greater number found under the increased amount treatment . A number of taxonomic groups and guilds did not vary in abundance significantly between treatments (Table 2). The community composition of aboveground invertebrates varied significantly between rainfall treatments (Table 3). Despite a number of groups varying in their abundance, sucking herbivores and their associated order Hemiptera largely accounted for the change in community composition because of their very large increase in abundance under summer drought .In total, 6,604 aboveground invertebrates and 3,736 belowground macro invertebrates were counted and identified. A number of aboveground invertebrate taxa varied significantly in abundance between rainfall treatments . The community composition of belowground fauna was not detectably affected by the rainfall treatments (Table 3).Belowground Hymenoptera (Formicidae) and their associated guild, scavengers, were the only belowground groups for which abundance was significantly affected by altered rainfall regimes . The same relationship was observed for their associated guilds, scavengers and root chewers, under ambient rainfall regimes only . A number of other relationships were only observed under ambient rainfall, including positive correlations between aboveground Collembola and belowground Megadrilacea , and aboveground parasitoids and belowground fungal feeding nematodes . Conversely, a negative relationship was found between the abundance of belowground fungal feeding nematodes and root chewers . Additionally, the abundances of aboveground Collembola and belowground Hemiptera were positively correlated under the reduced amount treatment , while a positive relationship was found between the abundance of aboveground detritivores and belowground sucking herbivores under summer drought conditions . The abundance of a number of other aboveground and belowground invertebrates where found to have a strong correlation, however they did not appear to have a linear relationship (see Supplementary Table 3).The abundances of aboveground Acari and belowground Coleoptera were positively correlated under ambient and increased amount rainfall scenarios . When precipitation returned to ambient levels, it is likely that resilient plants could take advantage of reduced competition and display vigorous growth. We propose that these results support the plant vigor hypothesis . In partOur observations of increased abundances of sucking herbivores in plots experiencing summer drought also has compatibilities with the pulsed-stress hypothesis . Their mThe abundance of parasitoids also increased under summer drought, suggesting that parasitoids are tracking their Hemipteran hosts’ population dynamics, as found in The general increase in herbivores under summer drought associated with the higher levels of cool season plant growth may explain the increased abundance of detritivores as well. In particular, the greater quantity of insect cadavers, frass and plant detritus is likely to have increased resources for this group. Increases in fungus gnats (Diptera), rather than Collembola, underpinned the increase in detritivores. Instead, the abundance of Collembola was more sensitive to increases in rainfall as evidenced by their greater numbers under the increased amount treatment compared with reduced frequency and amount plots. The abundance of Acari was reduced within the reduced frequency plots, indicating that this group may be sensitive to reductions in water availability. Both Collembola and Acari could have been negatively affected directly by the low soil moisture in the reduced rainfall plots .In addition to changes in absolute abundance, the community composition of invertebrates also varied between rainfall treatments. Despite a number of aboveground invertebrates increasing in absolute abundance under summer drought plots, the observed changes in community composition aboveground were driven primarily by increases in the relative abundance of sucking herbivores (Hemiptera).Altered rainfall regimes had little measurable effect on the belowground community, with no change in community composition found between treatments. Altered precipitation patterns may have affected belowground invertebrates less because they have a host of adaptations to mitigate changes in their microclimate, such as utilizing metabolic water, moving through the soil profile and constructing earthen chambers . Only HyA number of above- and below-ground linkages were observed under ambient rainfall, but dissipated under altered rainfall regimes. The reasons for such linkages occurring and their decoupling under altered rainfall patterns remain unknown, and we stress that explanations postulated below are highly speculative. They do, however, provide the basis for formulating hypotheses to direct future work.The positive relationship between the abundance of Acari (aboveground) and Coleoptera (belowground), for example, was only observed under ambient and increased amount plots. Root chewing herbivores like Coleopteran larvae can induce plants to reallocate resources aboveground, moving resources away from the site of attack, termed ‘resource sequestration’ . ImproveCollembola (aboveground) and Megadrilacea (belowground) were positively correlated under only ambient rainfall. Both groups are detritivores therefore their abundances may simply reflect similar responses to variations in the detritus inputs in ambient plots, resulting in a correlation. Supplementing detrital material has, for example, been shown to increase detritivore abundance . AlteredThe negative relationship between fungal feeding nematodes (belowground) and chewing herbivores (aboveground) was also only present under ambient rainfall; this finding may possibly be mediated via mycorrhizal communities. Fungal feeding nematodes can indicate the presence of mycorrhizal fungi in the soil . MycorrhThis study indicates that changes in precipitation, specifically changes in the seasonality of rainfall, are likely to cause alterations in the abundance and composition of aboveground, and to a lesser extent belowground, invertebrate communities. In particular, summer drought resulted in outbreaks of sucking herbivores, which probably underpinned the concurrent increase in the abundance of parasitoids. However, this study represents a ‘snapshot’ of impacts on invertebrate communities in spring only and patterns may be different during the growing season. It remains to be determined whether these changes persist over the longer term or whether communities return to a state of equilibrium. These findings, together with the precipitation-sensitive linkages outlined in this study, do, however, lend further empirical support to the idea that climate change will modify grassland invertebrate communities with potential cascading effects. Moreover, linkages between above- and below-ground communities may be modified by climate change , which wSJ conceived the experimental design. MT, KB, UN, SP, and SF collected field data. MT and SF analyzed the data with help from KB and SJ. MT wrote the paper with the assistance of KB, SF, UN, SP, and SJ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Now, the transceptor concept is further enlarged to includemicronutrient sensing via the iron and zinc high-affinity transporters ofSaccharomyces cerevisiae. Interestingly, what seems tounderline the transport and/or sensing function of receptors, transporters andtransceptors is ligand-induced conformational alterations recognized bydownstream intracellular effectors.Cells need to communicate with their environment in order to obtain nutrients,grow, divide and respond to signals related to adaptation in changingphysiological conditions or stress. A very basic question in biology is howcells, especially of those organisms living in rapidly changing habitats, sensetheir environment. Apparently, this question is of particular importance to allfree-living microorganisms. The critical role of receptors, transporters andchannels, transmembrane proteins located in the plasma membrane of all types ofcells, in signaling environmental changes is well established. A relativenewcomer in environment sensing are the so called  Well-studied cases ofenvironmental sensing via typical receptors concern pH 4bona fidae receptors, transportersand channels mediate the uptake of solutes, metabolites, drugs or ions, whichthemselves can act as molecular signals, once intracellularly accumulated. Aprominent case of transport-mediated signaling is described in filamentousascomycetes, particularly in Aspergillus nidulans, where dormantconidiospores transferred to a fresh medium sense their new environment viatransient transcriptional activation of most of their nutrient-related transportersand subsequently adapt their gene expression to the nutrients available Three well-known types of molecular machines are employed for the sensing of theenvironment. Receptors, transporters and channels. All are polytopic transmembraneproteins, monomeric or oligomeric, located in the plasma membrane of all types ofcells. Their importance is reflected by the fact that 10-15% of all genomes encodefor such membrane proteins and by the numerous genetic diseases related to theirmalfunction. Receptors are proteins that interact with an external chemical ligand,which could be any simple molecule, including nutrients, ions, hormones or drugs andxenobiotics. Ligand binding induces a receptor conformational change, which acts asthe primary molecular signal leading to two alternative downstream outcomes. In mostcases, ligand-binding leads to the transduction of the receptor conformationalchange, th Small Meeting of Yeast Transport andEnergetics (SMYTE) held in Cordoba (Spain), Johan Thevelein presented evidence for anew type of sensing mechanism, operating however, through known transporters. Inparticular, he presented evidence that the general amino acid permease Gap1 ofSaccharomyces cerevisiae is necessary for cAMP-independentactivation of the PKA (Protein Kinase A) pathway under conditions of re-addition ofamino acids to cells previously starved for amino acids transceptor (transport andreceptor), and became the founder member ofall transporters classified as transceptors today (see later). The basic originalidea on the role of Gap1 in PKA activation was that signaling to the PKA pathwaydoes not depend on the transport activity of Gap1. In other words, the role of Gap1in activating PKA was independent from its role in amino acid transport. Theevidence that Gap1 acts as a sensor was basically genetic. Strains carrying null orspecific mutations in GAP1 showed no PKA activation or distincteffects on transport and signaling, although these mutants could still transporthigh concentrations of amino acids that are Gap1 substrates via other specificpermeases.In 1999, in the course of the 14Microbial Cell, Thevelein and his colleagues further expand thefamily of transceptors by providing evidence that the iron (Ftr1) and zinc (Zrt1)high-affinity transporters can also signal PKA activation In the years to follow, Thevelein’s group has provided additional evidence supportingthe concept of transceptors 1011121314In summary, highlights of the evidence supporting the idea of transceptors include a)maintenance of signaling in specific transceptor transport-deficient versions, bestexemplified by mutations in residues tentatively involved in proton-coupling in theSul1 transceptor, b) loss of signaling in null mutants of transceptors despiteaccumulation of the relevant substrates by other transporters with similarspecificity, c) identification of competitive and non-competitive inhibitors ofGap1-mediated transport, either with or without agonist action for signaling,including non-transported agonists, d) identification of analogues with distincteffects on Gap1 transport, transporter ubiquitination, endocytosis and signaling(transporter ubiquitination and endocytic turnover in response to substrate excessis a common phenomenon discussed below), and e) involvement of highly induciblescavenging transporters, as expected for proteins acting in signaling.Overall, these results further support the hypothesis that different substrates, orsubstrate analogues, or ligands, bind to partially overlapping binding sites in thesame general substrate-binding pocket of transceptors, triggering divergentconformations, resulting in distinct downstream processes, including ubiquitination,endocytic turnover or signaling A. nidulans or in the Can1arginine transporter in S. cerevisiae) that are active, or evenhyperactive, but still not endocytosed, and some inactive mutants that areendocytosed 18181919In my opinion, the idea of transceptors has gained unexpected conceptual support fromstudies addressing the phenomenon of activity-dependent transporter endocytosis. Infungi and mammalian cells many transporters are down-regulated in response to excesssubstrates by a mechanism involving ubiquitination, followed by immediate endocyticinternalization and subsequent sorting in early endosomes and degradation in thevacuole/lysosome. Importantly, substrate-elicited transporter endocytosis, unlikeendocytosis in response to other signals, such as stress or changes in N or C sourcesupply, is activity-dependent and in most cases requires several transport cycles tobe completed. This conclusion is mostly based on the observation that inactivetransporters are, in most cases, not internalized in the presence of substrates. Inline with the idea of the necessity of full transport cycles to be completed inorder to allow endocytosis to occur, is the observation that substrate analoguesthat act as non-transported ligands do not elicit internalization. However, thereare some exceptions to this rule. For example there are specific transporter mutants.As proposed in signaling through transceptors, transporter activity is thusassociated with a distinct mechanism, endocytosis, via specific conformationalchanges accompanying transport catalysis. In the case of transporters,conformational changes are ‘read’ by Rsp5/HulA/Nedd4 ubiquitin ligase adaptors(α-arrestins), which promote their internalization and turnover. In the case oftransceptors, unknown effectors transduce conformational changes into the PKAsignaling pathway. It thus seems that the cell has the capacity to sense subtleconformational nanochanges in transporters/transceptors, while they function at itsplasma membrane. What is really fascinating about the discovery and the generalconcept of transceptors is that transceptors seem to provide a functionalintermediate link in the apparent evolution of proper receptors from transportersNeurospora crassa andUstilago maydis), plants , protozoa (Leishmania mexicana) and in humancells  and especially with low-affinity substrates. A different butcomplementary approach to direct uptake measurements approach is the use of growthtests in cases where the uptake of a substrate leads to a growth phenotype. Forexample, a strain of S. cerevisiae or A. nidulansexpressing a transporter specific for adenine will grow well in growth mediacontaining this purine as a sole nitrogen source, while it will also be sensitive toadenine toxic analogues, such 8-azaadenine. In contrast, inactivation of the sametransporter will lead to absence of growth on adenine media and resistance to8-azaadenine. Importantly, if similar growth tests are performed with a transporterover-expressed via a strong promoter, they can even allow the detection oftransporters or transport mutants possessing very low activities. In respect to theidentification of substrate analogues that only act as ligands, but still inducetransporter-mediated signaling, rigorous evidence should be obtained that asubstrate analogue is not accumulated, even at very low concentrations, in the cell,by another transporter, or even by the same transporter acting in signaling.In bacteria, yeast, filamentous fungi and other microorganisms, transport activitycan be measured directly using radiolabeled substrates in living cells, a rigorousapproach as the transporter activity is measured in its proper lipid environment.The only limiting factor in this approach might be the simultaneous expression ofseveral transporters with similar or overlapping specificities and/or transportkinetics with the transporter studied. This apparent drawback is however overcomewhen a specific transporter can be studied in a genetically ‘clean’ backgroundlacking, due to null mutations, transporters with similar specificities, as is thecase in the model fungi cis-acting elements arenecessary for signaling in different transceptors? Are the extended cytoplasmicterminal regions of some transceptors  sufficient or critical forsignaling, as it is in proper receptors (e.g. the Ssy1) or more complex synergisticintermolecular interactions determine signaling? And of course, which are thedownstream effectors mediating transceptor signaling? Designing the proper geneticand unbiased screens in model fungi might lead to some answers to thesequestions.Several fascinating questions remain to be answered in respect to the mechanism oftransceptor function. What distinguishes a transporter that acts solely in transportfrom a transporter that also acts as a receptor? Is this difference intrinsicallybased on structural determinants, or is it also a matter of transporter/transceptorlocalization in specific PM microdomains, and thus differentially accessible todownstream cellular effectors? Which


Metformin is the most widely administered anti-diabetic agent worldwide. In patients receiving metformin for metabolic syndrome or diabetes, it reduces the incidence and improves the survival of breast cancer (BC) patients. We have previously shown that metformin is particularly potent against triple negative breast cancer (TNBC), with a reduction of proliferation, oncogenicity and motility, inhibition of pro-oncogenic signaling pathways and induction of apoptosis. These BCs are well recognized to be highly dependent on glucose/glucosamine (metabolized through anaerobic glycolysis) and lipids, which are metabolized for the production of energy and cellular building blocks to sustain a high rate of proliferation. We have previously demonstrated that metformin inhibits lipid metabolism, specifically targeting fatty acid synthase (FASN), cholesterol biosynthesis and GM1 lipid rafts in TNBC. We also reported that glucose promotes phenotypic aggression and reduces metformin efficacy. We now show that metformin inhibits several key enzymes requisite to glucose metabolism in TNBC, providing additional insight into why metformin is especially toxic to this subtype of BC. Our data suggests that the use of metformin to target key metabolic defects in lipid and carbohydrate metabolism in cancer may be broadly applicable, especially against highly aggressive malignant cells. Breast cancer (BC) morbidity and mortality remain stubbornly high worldwide, despite the fact that disease characteristics vary by geography, ethnicity, age, body mass and other factors. While the prognosis for BC patients is better in the US than in much of the world, our incidence of disease is especially high in women with obesity and type II diabetes/metabolic syndrome. Experts anticipate that in 2018, 266,000 new invasive and 64,000 new in situ breast cancer (BC) cases will be diagnosed in the US –5.Gender  is the most potent risk factor for BC . A familA historically attributed mechanism by which obesity  promotes BC is the peripheral conversion of testosterone in adipocytes, leading to increases in circulating, bioavailable oestrogen  . More rede novo fatty acid biosynthesis [With transformation of benign breast epithelial cells to the malignant phenotype, significant changes in fat metabolism and intracytoplasmic fat accumulation are often observed (particularly in TNBC). In fact, all cancer subtypes have shown an enhanced reliance on ynthesis , irrespeynthesis ,24. Thisynthesis ,26. We hynthesis  as well ynthesis . The focMetabolic syndrome and type II diabetes are associated with systemic dysregulation of lipid and carbohydrate metabolism. These changes disrupt a broad array of cell types and put the patient at an increased risk of cardiovascular disease and cancer . Both tyDysregulation of carbohydrate metabolism to preferentially use aerobic glycolysis is a well-recognized hallmark of cancer . This meIn order to meet the need for increased glucose intake from extracellular sources, cancer cells frequently upregulate membrane associated glucose transport proteins known as GLUTs –50, as win vitro models, we seek to define mechanisms of metformin action at the subcellular level.We previously reported that glucose promotes phenotypic cancer aggression and reduces the efficacy of metformin in all molecular subtypes of BC . We havede novo fatty acid synthesis through down-regulation of fatty acid synthase (FASN), via up-regulation of the microRNA 193b [Metformin reduces insulin resistance, promotes glucose and lipid homeostasis, particularly in liver and skeletal muscle. The most widely recognized mechanism of metformin action is through inhibition of the mammalian target of rapamycin complex I (mTORC1) in both AMPK-dependent and independent processes . Less isRNA 193b . MetformRNA 193b .in vitro [in vitro[We have previously shown that glucose promotes BC aggression and reduces metformin efficacy in vitro . Using a[in vitro.Furthermore, we have shown that metformin had significant antitumor effects in this rodent model. These findings suggest a basis for the epidemiological and data indicating that in patients with metabolic syndrome or type II diabetes, metformin treatment reduces cancer incidence and improves survival for patients that develop the disease (in stark contrast to other anti-cancer agents including insulin that increases BC risk) –36. We pGiven that one of the primary metabolic changes observed in BC tumorigenesis is a marked increase in catabolic glucose metabolism, and metformin’s ability to disrupt these shifts, we sought to identify direct targets of the drug amongst glucose transporters and key enzymes of the glycolytic pathway. In brief, TNBC cells MDA-MB-468 and MDA-MB-231 were grown in either 17 mM or 5 mM glucose, with or without metformin. Purified mRNA was used for gene expression profiling , 58. DatIn this experiment, we also showed that metformin attenuated the expression of a number of important genes involved in glucose metabolism. Over 20 genes were shown to be down-regulated, see Metformin also inhibited lactose dehydrogenase (LDH). LDH functions downstream of hexokinase (HK), phosphofructokinse (PFK), and pyruvate kinase (PK). LDHA is a key enzyme that catalyzes the conversion of pyruvate to lactate. Metformin also decreased LDHA, which according to knock-down studies in BC cell lines, may reduce proliferative rates by switching cellular mitochondria to oxidative phosphorylation . AdditioWe postulate that these major shifts in glucose transport and glycolysis are a major mechanism by which metformin reduces growth, oncogenesis, induces apoptosis and may enhance sensitivity to other chemotherapeutic agents. While we have little data from our lab that suggests the latter, data from others provides a rationale for this view. Reduction of LDH by genetic manipulation or the chemical inhibitor oxamate have reversed of taxol-resistance and induced apoptosis in BC cells .Cell line treatment and gene profile array were previously described ,58.Microarray analysis of MDA-MB-468 cells cultured in media with 5 mM glucose in the presence or absence of 10 mM metformin. mRNA from treated cells was purified and analyzed using Affymetrix Human Gene 1.0 ST Array platform. Genes differentially down-regulated by metformin are highlighted in red. A biological triplicate experimental design was used to determine standard error.Statistical considerations and calculations of metformin-mediated inhibition of cholesterol pathway genes were performed using Graph Pad Prism 7 software. Statistical analysis of the experimental data was performed using a 2-sided Student t-test. Significance was set at a P<0.05 values. Gene array samples are representative of biological replicates.Metformin is the only anti-diabetic agent with anti-cancer activity, whereas other agents used in patients with this disease or metabolic syndromes have been shown to increase cancer incidence and reduce cancer-associated survival. Metformin has broad effects on multiple targets of the dysregulated lipid and carbohydrate metabolism associated with BC, and more specifically, TNBC. In this report, we demonstrate that metformin specifically reduces the expression of key glucose transporters in TNBC, including GLUT1. We also show that it reduces transcription of key enzymes in the glycolytic pathway that are critical for cancer replication and survival. Because of the increasing prevalence of obesity, the relationships between excess body weight and cancer development and the underlying biological mechanisms need to be further investigated to prevent and treat BC in the future.