Oligodendrocytes (OLGs) are generated late in development and myelination is thus a tardive event in the brain developmental process. It is however maintained whole life long at lower rate, and myelin sheath is crucial for proper signal transmission and neuronal survival. Unfortunately, OLGs present a high susceptibility to oxidative stress, thus demyelination often takes place secondary to diverse brain lesions or pathologies. OLGs can also be the target of immune attacks, leading to primary demyelination lesions. Following oligodendrocytic death, spontaneous remyelination may occur to a certain extent. In this review, we will mainly focus on the adult brain and on the two main sources of progenitor cells that contribute to oligodendrogenesis: parenchymal oligodendrocyte precursor cells (OPCs) and subventricular zone (SVZ)-derived progenitors. We will shortly come back on the main steps of oligodendrogenesis in the postnatal and adult brain, and summarize the key factors involved in the determination of oligodendrocytic fate. We will then shed light on the main causes of demyelination in the adult brain and present the animal models that have been developed to get insight on the demyelination/remyelination process. Finally, we will synthetize the results of studies searching for factors able to modulate spontaneous myelin repair. OLGs are the last cells to be generated after neurons and astrocytes during development. In the developing forebrain, several waves of OPC production and migration generate the entire OLG population. At E12.5 in mice, a first ventral wave of OPC production originates in the ganglionic eminence  possesses specialized properties regulating stem cell proliferation and commitment toward specific lineages    is immediately followed by micro hemorrhages and focal impairment of the cerebral blood flow that trigger secondary inflammatory and degenerative processes involving NO, glutamate and free radicals production  occurs in immuno-deficient people and is caused by an ubiquitous neurotropic virus: the JC virus  which is a recurrent progressive disease. It is estimated that approximately 2.5 million people worldwide suffer from MS , viral models may provide relevant information on the interactions between the immune and nervous systems in this pathology. However, these models are rarely used to dissect mechanisms of remyelination due to the complex interplay between immune and nervous systems. The most common viral models in rodents are Semliki Forest virus (SFV), Theiler's murine encephalomyelitis virus (TMEV) and mouse hepatitis virus (MHV). Virus may directly target neurons, thus triggering primary neuron loss and secondary demyelination (“inside out models” such as TMEV) or infect OLGs with primary demyelination and secondary axon loss (“outside in” models such as MHV) or act in an indirect manner provoking immune-mediated attack of the CNS  and ethidium bromide are widely used to trigger focal demyelination. A comparative study of these different approaches showed that all compounds triggered little axonal damage  but large demyelination lesions that underwent spontaneous remyelination with slightly different kinetics, remyelination being faster in rats treated with LPC compared to ethidium bromide  triggers an ellipsoid demyelinated lesion over few mm2. Demyelination can be observed as soon as 3 days after LPC injection, reaches its maximum at day 7 and then the remyelination process takes over. The remyelination is complete after 14–21 days. Although this model is usually considered as non-inflammatory, it has been shown that cytokines such as MCP1, MIP1a, and TNFα are secreted after LPC injection and attract lymphocytes and macrophages that contribute to demyelination , or more generalized white matter disease . This is respectively obtained by focal vasoconstriction   is expressed in the adult SVZ and plays a major role in the maintenance of NSCs  can also induce proliferation of adult stem cells and neural progenitors in the adult brain  also signals through the EgfR ErbB. During development, Nrg-1-ErbB signaling is a key regulator of axon ensheathment and myelin thickness, regulating OLG differentiation, survival, maturation, and myelination   and leukemia inhibitory factor (LIF) share some common protective roles in axons and oligodendrocytes. LIF exerts protective functions on oligodendrocytes and regulates positively many steps of the repair process  or triiodothyronine (T3)-treated mice exhibit enhanced OPC proliferation and survival after cuprizone intoxication  communication. Ligands/ integrins complex are known to play a critical role in white matter development by promoting survival of newly formed OLGs that establish appropriate contacts with axons to be myelinated  and LINGO-1 si-RNA, Mi and collaborators have shown that LINGO-1 is an important negative regulator of myelination during development  acts as an inhibitor of myelination, presumably by preventing myelin-forming cells from attaching to the axon (Doherty et al., In vitro data have suggested that PSA-NCAM could be specifically involved in establishing the directionality of OPC migration in response to a concentration gradient of PDGF (Zhang et al., In vivo, the deletion of St8siaIV, one of the two polysialyltransferases enzymes responsible for the PSA synthesis, leads to an earlier remyelination in cuprizone mouse model (Koutsoudaki et al., Despite recent progress in treating the inflammatory component of MS, current therapies have no clear impact on progression of disability related to myelin and axon injury. As described above, various factors can specifically regulate the cellular processes involved in remyelination including OPC migration, survival and proliferation and OLG maturation. Among these factors, those that have been shown to control OPC differentiation and could regulate the late phase of the repair process have received considerable attention and some are now promising candidate medicaments. For example, one approach currently in phase 1 clinical trial is the use of a neutralizing antibody targeting LINGO-1 since antagonism of LINGO-1 promotes OPC differentiation and leads to remyelination in mouse models of MS (Mi et al., Our knowledge of oligodendrogenesis has a great deal evolved over these past 10 years, with increasing number of research teams getting interested in postnatal production of OLGs and myelin repair. Interestingly, progenitor cells including parenchymal OPCs and SVZ cells represent more than 5% of the total cell number in the adult brain parenchyma. Such an important pool of progenitors may relate to high plasticity properties. Indeed, as mentioned in this review, oligodendrogenesis carries on throughout life, contributing to myelination and/or myelin remodeling, a process that may be important for functional plasticity, learning and memory. In parallel, many OPCs remain as quiescent or cycling very slowly progenitors, but with the ability to reactivate as soon as a demyelination insult occurs. Spontaneous myelin repair is therefore a strikingly common event, very efficient in most rodent models of demyelination, but quite variable in human patients suffering from autoimmune disease such as MS. The interplay between immune and nervous system is very complex in this pathology; inflammation acts like doctor Jekill and Mr. Hide: it is necessary to trigger OPC mobilization and differentiation, but deleterious for cell survival when it is prolonged. Because of this prevalent inflammatory component, existing treatments mainly exclusively target inflammation, with low efficiency on the progression of invalidity on the long-term. Neuroprotection, and thus remyelination needs to be promoted in conjunction to these treatments. A better knowledge of all the cell populations and factors involved in the repair process will undoubtedly allow the development of new therapeutic strategies.The Guest Associate Editor Antoine De Chevigny declares that, despite being affiliated to the same institution as authors El Waly, Macchi, Cayre and Durbec, the review process was handled objectively and no conflict of interest exists. 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.


Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in  RNA performs a broad variety of fundamental cellular functions, many of which depend on its ability to actively fold into numerous conformational structures. These RNA structures can form motifs, which are frequently bound by an assortment of RNA-binding proteins (RBPs). In eukaryotic organisms, this provides a means to couple transcription with various levels of post-transcriptional gene regulation such as splicing, nuclear export, RNA localization, turnover, and translation . Conventional models of miRNA regulation are based on perfect or imperfect linear hybridization to mRNAs, which results in the targeted mRNA being degraded and/or inhibited at the translation level. The former's mechanism is well explained by direct cleavage of the mRNA, however the latter's is not yet perfectly elucidated. It is possible that in searching for a mechanism similar to the originally identified destructive pathway, scope was artificially limited to cases with reduced translation of targeted mRNAs. However, as an mRNA cannot be “slightly cleaved,” it is logical to assume that repressive effects of imperfect miRNA hybridization result from a wholly separate, second process rather than simply an attenuated version of the first. If the second process is more generic, it could conceivably manage multiple downstream responses, perhaps via affecting third party elements such as RBPs. In fact, examples of increased translation of a miRNA targeted mRNA have been reported  as part of a RIP. This has become increasingly true as more comprehensive genomic tools, such as tiling-arrays and next generation sequencing technologies have been used. Although experimentally challenging to definitively prove, at least a portion of this ncRNA appears to be cis within the message  are common subdomains critical to the function of numerous structured RNAs  and form when the RNA folds to produce three separate helices meeting around a central unpaired region  and for general hybridization characteristics .To find potential examples of our model, we used the well-characterized HSL motif and determined the potential for miRNAs to target this structure and form a 3WJ. We first identified the set of HSL containing human histone mRNAs present in the Refseq hg19 annotation by using the PatSearch program , it is yet to be shown that these HSL-sxRNAs naturally form or if they play a functional role in histone metabolism. However, histone metabolism is a very elegant and tightly regulated essential cellular process that has many layers, any of which could use the type of post-transcriptional regulation we have predicted Figure . For exam Figure .We have identified similarly predicted sxRNAs for the iron-response element (IRE), which is the target of the IRE-binding proteins that regulate the post-transcriptional expression of genes involved in iron metabolism. A dramatic example of this is found in the erythroid-specific ALA synthase gene (ALAS2), a critical protein whose misregulated activity is associated with both sideroblastic anaemia  and this may be the single most biologically important attribute of RNA. This property permits RNA to serve both as an intermediate for conveying the genetic code while also creating unique surfaces for performing protein-like enzymatic functions in the cell. This also allows mRNA to simultaneously encode for a protein as well as regulate where, when and how much of the mRNA will be translated into this protein. Accordingly, eukaryotic genes are not exclusively transcriptionally regulated, but are also highly regulated at the post-transcriptional level, typically mediated by RBPs, which have repeatedly been shown to facilitate the expression of different mRNA isoforms, localize specific mRNAs and influence variable mRNA translation, and it is reasonable to assume that at least some will be found to play a regulatory role  manner, by eliminating the RBP binding motif, or a positive (inducer) mode by assisting an RBP in binding. Additionally, the miRNA could even help form the RBP motif by actually comprising a portion of it  An RBP whose binding site is hidden by an alternate structure. In the presence of the associated trans-acting ncRNA, the site is revealed. (B) An RBP that recognizes a double stranded binding site that only forms via interaction with a separate ncRNA. (C) An RBP whose binding site must be presented in a particular orientation, with respect to its flanking sequence, that is facilitated by a 3WJ. (D) An RBP whose binding site is hidden by its sequence hybridizing with an ncRNA. (E) An RBP whose binding site structure is weak and may sample alternate structures over time and an sxRNA stabilizes the active conformation, allowing the protein to bind. This model allows a single mRNA transcript variant to exhibit multiple behaviors amongst cell types via the combinatorial absence and presence (in varying concentrations) of both the relevant RBPs and, more specifically, ncRNAs. It is these latter that may facilitate a distinct regulation for sub-populations of expressed mRNAs, where a much broader group may hold the potential for control by the same RBP.Figure An additional level of sxRNA regulation could occur by indirectly influencing tertiary structure to reveal (or hide) complex regulatory motifs that must be properly oriented in space. Figure A) interact with nascent pre-mRNA to affect alternative splicing, perhaps by affecting splicing factor recognition sites or (as depicted here) by binding to and “joining” exon regions on both sides of a particular splice junction, (B) affect the structural conformation of an miRNA primary transcript, determining its availability for trimming to pre-miRNA and nuclear export, (C) reinforce a stem loop binding motif for a localization RBP, thereby determining mRNA shuttling destination, (D) further lessen translation via iron regulatory protein by stabilizing a 5′ IRE in particular mRNAs, (E) alternatively, enhance mRNA stabilization via iron regulatory protein by stabilizing a 3′ IRE in particular mRNAs (F) activate structure dependent ribozymes, (G) act in a combinatorial fashion with multiple signals in a single mRNA.We have envisioned a number of hypothetical biological effects of sxRNA regulation, which are depicted in Figure trans as opposed to a small molecule or metabolite regulating cis structural changes (Serganov and Nudler, This additional role for miRNA we have suggested could explain a variety of observed downstream effects and underscores the importance of ncRNA as a major post-transcriptional gene regulator. This model could also allow for the regulated masking or revealing of many conformational dependent mRNA regulatory elements beyond RBP binding motifs. By stabilizing or disrupting these regulatory elements, miRNAs, and possibly many other ncRNAs, could perform diverse functions with respect to individual or multiple mRNAs. In some aspects, this model is analogous to the behavior observed for riboswitches but here a miRNA is influencing shape in trans-acting post-transcriptional regulatory network in which various sxRNA arrangements could sequentially influence multiple RBP regulatory elements or vice versa. Analogous to the classical attenuator regulatory sequence found in some prokaryotic operons, structural changes to one or more sxRNA could then result in the conformational alterations of the structure of the mRNA such that a second RBP regulatory element can now be revealed or masked, and this could continue in a manner that would enable the post-transcriptional regulation of multi-functional genes via combinations of regulatory elements embedded in the UTRs or even the coding region of the mRNA. The sxRNA model proposed here resonates well with an earlier prosed idea of ncRNAs acting as intelligent scaffolds for the dynamic regulation of the information landscapes (Laurent et al., Additionally, and perhaps most biologically compelling, is the potential for nature to use sxRNA interactions to form an intricate combinatorial Despite compelling informatic support, the sxRNA model we have proposed will need substantial supporting experimental data before it can be recognized as credible and we encourage interested researchers to participate in this process. We have successfully RIPed informatically predicted sxRNA complexes using a bacteriophage MS2-tagged-RNA construct (Yoon et al., Francis Doyle and Scott A. Tenenbaum contributed equally to this manuscript.Scott A. Tenenbaum and Francis Doyle are inventors on sxRNA intellectual property that is owned by SUNY-Research Foundation and may stand to profit in accord with the SUNY-Research Foundation Patents and Inventions Policy. The SUNY-Research Foundation has entered into a licensing agreement with HocusLocus, LLC to develop and market the sxRNA technology.


Prior studies have noted significant health disadvantages experienced by LGBT  populations in the US. While several studies have identified that fears or experiences of stigma and disclosure of sexual orientation and/or gender identity to health care providers are significant barriers to health care utilization for LGBT people, these studies have concentrated almost exclusively on urban samples. Little is known about the impact of stigma specifically for rural LGBT populations, who may have less access to quality, LGBT-sensitive care than LGBT people in urban centers.LBGT individuals residing in rural areas of the United States were recruited online to participate in a survey examining the relationship between stigma, disclosure and “outness,” and utilization of primary care services. Data were collected and analyzed regarding LGBT individuals’ demographics, health care access, health risk factors, health status, outness to social contacts and primary care provider, and anticipated, internalized, and enacted stigmas.Higher scores on stigma scales were associated with lower utilization of health services for the transgender & non-binary group, while higher levels of disclosure of sexual orientation were associated with greater utilization of health services for cisgender men.The results demonstrate the role of stigma in shaping access to primary health care among rural LGBT people and point to the need for interventions focused towards decreasing stigma in health care settings or increasing patients’ disclosure of orientation or gender identity to providers. Such interventions have the potential to increase utilization of primary and preventive health care services by LGBT people in rural areas. Healthy People 2020  identifiThis augmented prevalence of risk factors is confounded by LGBT people’s decreased access to care and lower rates of screenings for multiple preventable or treatable diseases . LGBT adLower rates of primary care utilization among LGBT populations may be founded on expectations and experiences of stigma based on sexuality or gender identity. This stigma can be defined as “the negative regard, inferior status, and the relative powerlessness that society collectively accords to any non-heterosexual [or non-cisnormative] behavior, identity, relationship, or community” . SexualiStigma can be understood as having three domains: anticipated stigma (concern for possible future instance of discrimination), internalized stigma , and enacted stigma  . Each tyStigma also interacts with and effects “outness,” or disclosure of one’s sexual orientation and/or gender identity to social contacts. Increasing levels of stigma are correlated with decreased levels of disclosure of sexual orientation and/or gender identity to health care providers . Prior sHistorically, the majority of studies of LGBT populations have been conducted in urban centers due to ease of access to high concentrations of LGBT people in social spaces catering to this community. Though little data exists thus far on the rural LGBT population, at first glance rural areas appear less hospitable, in part due to increased stigma and social isolation . Even noIn order to facilitate increased access to rural LGBT populations, the internet has emerged as an effective method of study recruitment. Although racial and socio-economic status-based biases remain concerns in internet surveying, these concerns are diminishing as internet access becomes more widespread . A recenParticipants were recruited to the online survey primarily via banner ads on Facebook during a 17 day period in August 2014. Ads were targeted towards Facebook users age 18+ with LGBT-related interests who reported residence in rural zip codes  determined eligibility for the study.“Do you have one location where you typically go to receive primary care services?” and “Do you have one person you think of as your primary doctor or health care provider for checkups, medication refills, or treatment for non-emergent illnesses?”). Participants were asked follow-up questions about how they chose their PCP and how LGBT-friendly they perceived their provider to be. For participants who indicated that they did not have a PCP, they were asked to respond to questions with regards to the provider they had seen the most often in the last year.Healthcare utilization was assessed by asking the frequency of visits  , health excellent, very good, good, fair, or poor)  health” and 27% said it was “none of [the provider’s] business.” Thus, vocalizing and providing openly available materials regarding the benefits of disclosure and the relationship between sexual behavior and overall health , 76–78, Degree of rurality is another factor that requires further study and explanation. In our sample, transgender & non-binary respondents were more likely to travel longer distances to receive care, possibly related to their propensity to prioritize and seek out more LGBT-specific care . Many of the vaccination and screening recommendations require separate or repeated visits to a health care provider . Long travel times may reduce the rural LGBT individual’s ability to utilize primary care clinics, and this effect seemed to be exaggerated in our transgender & non-binary population.Insurance status surely impacts access to care in the general population, and that held true for our sample of rural cisgender men and women. However, insurance status did not significantly correlate with greater utilization of primary care for our sample of rural transgender & non-binary people. This may be because provider choice was important to this group, and some insurance plans limit provider choice. In fact, of the 15% of our sample that reported changing their insurance or gaining new insurance under the Affordable Care Act, one in five said that their new insurance allowed them to choose a provider that was more LGBT-friendly. As the insurance structure of this country continues to change, we may see changes in the interaction between insurance status and health care utilization.It is important to note several limitations of the current study. First, although rural areas nationwide are 78% white , this sIn addition, the current survey collected data on how respondents identified their own sexual orientation and gender identity, not on their sexual behavior. Thus, the sample excluded individuals who may engage in same-sex sexual behavior but do not identify as part of the LGBT community. In addition, this survey did not collect data on gender expression or how others perceived respondents’ gender. It is likely that individuals who do not conform to traditional gender roles in their everyday appearance experience more stigma, and rates of disclosure vary for these individuals as well . Relatedth percentile, approximately 25th-75th percentile, and approximately 75th percentile and above). Although this method was devised as a manner for determining whether a dose-response relationship was present between stigma scores and health outcomes, the determination of correlations between those variables that lie outside of the present division was restricted.Further, data analysis deviated from common practice by dividing stigma scores into domains .The study findings overall paint a picture of how stigma and outness influence rural LGBT people’s utilization of health care, beyond simply insurance status and geographic constraints. Our findings that stigma and outness are both related to utilization of care support the trend of increasing LGBT-health competency among current providers and incorporating these aspects of health care into medical education. By increasing knowledge and by promoting LGBT-inclusion within health care spaces, especially related to transgender health, stigma can be overcome and barriers to optimal primary care utilization can be dismantled. Other areas identified for further study include provider education on creating a safe environment for LGBT patients, patient education on the benefits of disclosure of sexual orientation and/or gender identity, and the relationship between risk factors, chronic disease, and poor prevention behaviors in LGBT populations.


Conjugative plasmids play a vital role in bacterial adaptation through horizontal gene transfer. Explaining how plasmids persist in host populations however is difficult, given the high costs often associated with plasmid carriage. Compensatory evolution to ameliorate this cost can rescue plasmids from extinction. In a recently published study we showed that compensatory evolution repeatedly targeted the same bacterial regulatory system, GacA/GacS, in populations of plasmid-carrying bacteria evolving across a range of selective environments. Mutations in these genes arose rapidly and completely eliminated the cost of plasmid carriage. Here we extend our analysis using an individual based model to explore the dynamics of compensatory evolution in this system. We show that mutations which ameliorate the cost of plasmid carriage can prevent both the loss of plasmids from the population and the fixation of accessory traits on the bacterial chromosome. We discuss how dependent the outcome of compensatory evolution is on the strength and availability of such mutations and the rate at which beneficial accessory traits integrate on the host chromosome. In the absence of Hg(II) however, plasmid carriage reduces the fitness of the host by ∼25%. Plasmid containing bacteria were experimentally evolved under 6 Hg(II) concentrations: from no Hg(II), where the plasmid is purely parasitic, to high levels of Hg(II) where the relationship is mutualistic.Conjugative plasmids are vehicles for horizontal gene transfer, a key process in bacterial adaptation.mer transposon onto the chromosome accompanied by plasmid loss under Hg(II) selection. Genome sequencing of evolved plasmid-containing clones revealed that mutations were exclusive to the bacterial chromosome. No mutations were found on evolved plasmids, with the exception of hypermutator strains that had acquired mutations in their mismatch repair pathway, and one clone with a transposon duplication. On the bacterial chromosome however we observed highly parallel evolution of the bacterial GacA/GacS 2-component response regulator. Excluding hypermutators, ∼80% of sequenced clones carried mutations in either gacA or gacS, with several clones sharing the exact nucleotide change and thus exhibiting parallelism at both the locus and nucleotide levels. In contrast, no gacA or gacS mutations were observed in plasmid-free control clones. Tracking the frequency of a subset of these gacA and gacS mutations through time revealed that they arose early and rapidly invaded the plasmid-containing fraction of the population  and transposition of the pulation . Knock-ogacA/gacS mutations had relieved this translational burden.Finally we examined the impact of plasmid acquisition and compensatory evolution on gene regulation. In the ancestral strain, plasmid carriage resulted in the up-regulation of ∼17% of the bacterial genome, including many genes associated with protein production processes, such as ribosome biogenesis and protein folding. These data indicate that plasmid carriage places a strong translational burden on the ancestral host, driving greater investment in the machinery of protein production. Following compensatory evolution, however, bacterial gene expression resembled the plasmid-free ancestor, suggesting that The GacA/GacS system positively regulates the translation of a suite of bacterial secondary metabolites and secretions.mer that had lost the plasmid arose in all Hg(II) environments. These genotypes were detectable at the first sampling point in 16 out of 36 populations despite relatively low resolution of sampling, unlikely to detect genotypes at frequencies below 10%. Thus by reducing purifying selection acting on the plasmid backbone, compensatory mutations were able to hinder the spread of these alternative genotypes and preserve the mobility of mercury resistance genes which may otherwise become lost or rendered non-conjugative on the bacterial chromosome.Our study demonstrates the capacity for compensatory evolution to rescue a costly plasmid from extinction across a broad range of environments. Importantly, plasmids were maintained despite the early appearance of genotypes predicted to outcompete plasmid-carriers, specifically, plasmid free cells rapidly arose in the absence of mercury, and genotypes with chromosomal Pseudomonas sp. PCL1171 suggests that gacA/gacS loci may in fact have elevated mutation rates in pseudomonads, estimated to be ∼100 times that of the genome-wide mutation rate.In the study described, compensatory evolution was characterized by highly repeatable, rapid evolution of a global bacterial regulator, GacA/GacS. Knockout mutants demonstrate that loss of function in either gene is able to completely ameliorate the cost of the plasmid allowing strong selection on single mutations. In addition, evidence from gacA/gacS loci (∼5×10−5) compensatory mutations arose in time to stabilize plasmid prevalence before the plasmid could be lost from the population. At lower mutation rates however the plasmid was generally lost before compensatory mutations were able to arise and invade (Supp figure).We next investigated the impact of mutation rate on the outcome of bacteria-plasmid dynamics. As observed in our empirical system, complete compensatory mutations were able to rescue plasmids from extinction even in the absence of positive selection for the plasmid . Howevermer operon maintained plasmids in the population in the short-term but, in the absence of compensatory mutations, plasmid carriers were ultimately out competed by plasmid free, mer carrying individuals, which do not pay the cost of plasmid carriage −7) were chromosomal mer genotypes able to become established in the population  toxicity, positive selection for the carriage . Our modpulation . This diThe persistence of plasmids in bacterial populations has been challenging to explain because, while mechanisms to increase plasmid stability are widespread, they are far from universalOur model also highlights the role for positive selection acting on plasmid-borne traits in expanding the conditions under which compensatory evolution is likely to occur. As plasmid loss is contingent first on the movement of accessory genes onto the bacterial chromosome the resulting delay allows even rare amelioration mutations to appear and spread within the plasmid population. Indeed even very transient positive selection, by increasing plasmid population size, can facilitate the establishment of compensatory mutations and stabilization of initially costly plasmids in bacterial populations.gacA/gacS mutations observed in our empirical study, bacterial genomes can be highly flexible. The GacA/GacS system has been suggested to act as a contingency loci for environmental adaptation, where high mutation rates allow for phenotypic switching by mutation and reversion.In the absence of positive selection however, we find that the conditions for plasmid maintenance are surprisingly stringent; amelioration mutations must have both large fitness effects and occur at a high rate in order to stabilize plasmids in bacterial populations. That such large-effect, high-frequency mutations appeared in our experimental system may therefore merely be good fortune, but it is possible that such mutations are more readily available than might be expected. At present we have a limited understanding of what causes the costs of plasmid carriage and thus it is hard to predict which mutations might be available to ameliorate their cost. Recent evidence however suggests that, in addition to more general costs of plasmid maintenance , plasmid costs are frequently associated with specific, acute maladaptations, for instance strong epistatic interactions between bacterial and plasmid bound genes allowing single mutations to have large fitness effects.To understand the dynamics of plasmids in bacterial populations we must therefore view bacteria-plasmid associations through both an ecological and evolutionary lens. While the initial invasion of plasmids into bacterial populations may be dependent on the specifics of the bacteria x plasmid x environment interaction, compensatory evolution allows plasmids to be stably maintained in the absence of positive selection, preserving a reservoir of mobile accessory genes and allowing potential for adaptation by horizontal gene transfer. The implications of these evolutionary changes for bacterial and plasmid populations are a rich subject for future investigations. As yet we know very little about the types of mechanisms that underlie the costs of plasmid carriage and consequently the avenues to their amelioration. Do compensatory mutations lead to tradeoffs in bacterial fitness in other environments, as might be expected in the case of the GacA/GacS loci? Are these adaptations specific to the coevolving bacteria-plasmid association? Or do compensatory mutations in the host ameliorate the cost of other plasmids in the community, producing more generalist hosts? Understanding what consequences compensatory evolution might have for both bacteria and plasmids will therefore be key in understanding the persistence of plasmids in natural microbial communities.


As tThe advantages that Tex present for the delivery of tumorigenic signaling molecules in relation to passive release into the medium are clear, especially for transports across long distances in the body. Firstly, encapsulation of labile molecules such as RNAs and proteins within lipid bilayers offers them protection to degradation; secondly, the surface landscape of the vesicle allows for the possibility of tissue or cell specific targeting and thirdly they are suited for simultaneous multiple message delivery allowing horizontal transfer of complex information from cancer to healthy cells.In addition to attenuation of antitumor immunity, Tex stimulate angiogenesis, modulate stromal cells activity, and help on the extracellular matrix remodeling contributing to the establishment of a premetastatic niche and generating suitable microenvironments at distant metastatic sites . For exain vitro culture conditions. In particular, dendritic cell (DC)-derived exosomes (dexosomes) have been shown to carry NK cell activating ligands and can be loaded with antigens to activate invariant NKT cells to induce antigen-specific T and B cell responses. Tumor-antigen-derived dexosomes have been investigated as therapeutic agents against cancer in two phase I clinical trials, with a phase II clinical trial currently ongoing. The results show that although dexosomes were well tolerated, the therapeutic success and immune activation were limited -loaded Tex occurred when hepatocarcinoma cells were treated with chemotherapeutics such as paclitaxel or carboplatin. These Tex released by treated cancer cells conferred superior immunogenicity in inducing HSPs-specific NK cell responses . Exosome limited , 15. Mul limited . Because limited .Although immune cells, and probably dexosomes, can be found in primary tumor lesions as infiltrating components playing favorable prognostic role , in metaReached this point, the key question for cancer therapeutics is: will patients benefit by the presence of Tex or effective removal of Tex should be recommended?The answer does not seem to be straight forward but recent modeling for putative therapeutic implications of exosome exchange between tumor and immune cells may throw some light on it . The autAn important observation that should be included in Lu et al. model is that exosome release is controlled by feedback regulatory loops that involve not only cancer cells but also exosome production by normal cells. In this sense, it has been reported that exosome release from normal human mammary epithelial cells and breast cancer cells is not only regulated by the presence of exosomes derived from their own cells but also exosomes from normal mammary epithelial cells inhibit exosome secretion by breast cancer cells in a tissue specific manner, implicating a dynamic equilibrium between exosome release of normal and tumor cells .in vivo  , we can Deregulation of splicing is associated to acquisition of cancer advantages, aggressiveness of the tumor, and drug resistance events –29. For Tex cancer-associated exon sequences could be used for immunotherapy, to boost DC dexosome release and, at the same time, as targets for Tex inactivation and/or sequestration. If only inactivation/sequestration is applied, the patient would miss the benefit of the immune response against the tumor. On the other side, if the unloading of Tex is not prevented, their cargo will work toward advancement of the disease. This double-approach consisting on blocking Tex function while using their specific cancer-associated exon sequences to induce tumor-immune response could complement current tumor treatments to reduce relapses and to limit metastatic events in immuno-competent patients. In addition, this directional targeting strategy would leave exosome-dependent physiological functions unaffected.In summary, the study of Tex surface landscape and the detection of cancer-specific exons on Tex derived from alternative splicing events results essential for the development of Tex targeted therapies. These qualitative switches should be considered in the context of patient immunological and cancer load states for the modeling and the development of improved, personalized cancer therapeutic programs.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Langan et al.). For example, among older adults greater brain activation levels associated with better motor performance, which suggest over-activation of the brain (compared to young adults) serves to compensate for age-related deterioration of brain function . As certain age-related neurophysiological changes may be beneficial, it is important to understand how age-related changes in the nervous system correlate with behavioral outcomes. These studies further depend on identification of behavior and neurophysiological measures that are sensitive to aging.Most older adults self-report disability for daily activities using their hands. Scientists have attempted to characterize specific behavioral markers of sensorimotor dysfunction, identify the changes in central and peripheral nervous systems, and establish a link between behavioral and neurophysiological alterations with aging. Neuroimaging studies have reported associations between changes in the brain and performance on cognitive and motor tasks in older adults  or bimanual tasks .Studies published as a part of this research topic used innovative approaches to assess behavioral and neurophysiological deficits in older adults. These approaches ranged from the utilization of behavioral paradigms to neuroimaging tools that focused on unimanual motor tasks . This finding was suggested to arise from older adults' inability to handle additional attention load, in the presence of an underlying age-related increased cost of processing proprioceptive signals. Further, older adults were impaired when the target limb was placed farther away, compared to near targets (Boisgontier and Swinnen). For the farthest targets older adults most likely experienced an increase in proprioceptive signal processing load and/or an increase in noise in the joint proprioceptor firing rate. The authors suggested that these factors might have saturated the reserve for proprioceptive processing in those older adults who were dependent on compensatory mechanisms for task performance, thus impairing performance on the joint matching task.Bimanual behavioral paradigms allow characterization of complex interaction between two sides of the brain during performance of a motor task. Bhakuni and Mutha; Hoff et al.). Older adults were found to match their younger counterparts in the ability to learn a serial reaction time task . However, Ragert and colleagues noted that older adults demonstrated reduced sequence specific learning as compared with younger individuals. Importantly, older adults showed altered interlimb coordination, measured as the time cost related to hand switch , when compared with young adults during performance of the bimanual task . While Bhakuni and Mutha reported no change in the hand switch cost with skill learning in both older and young individuals, Hoff et al. found that the hand switch cost in older adults declined to levels that matched younger individuals. In a study of animals, Chen et al. found that increased neonatal iron supplementation resulted in the depletion of striatal dopamine and reduction of motor performance on rotarod and open field tests in aging, but not in young, rats.The interaction between age and interlimb coordination during skill learning was investigated using a bimanual serial reaction time task . Poor unimanual hand function in older adults has also been shown to result from increased variability in motor output, e.g., forces exerted on an object  have lower force variability during a dynamic stability  test as compared with healthy older adults . Importantly, the authors found that the reduced force variability correlated with greater severity of motor impairment assessed using UPDRS motor scores. Overall, these studies suggest that it is crucial to understand the relationship between motor output variability and hand function with aging when associated with other health-related problems. Yu et al. reported that older women as compared with older men have a higher prevalence of severe stroke, hypertension, obesity, and hyperlipidemia. De Bruin et al. developed a novel task to assess the effects of aging on visuospatial abilities, but failed to find any age-related decrement. Lastly, Kimura et al. studied the effects of aging on performing speedy corrective movements when the target jumps during the reach. The authors inferred that older adults potentially have intact reflexive, but impaired volitional, corrective responses to target jumps during the reach.Other studies in this special section quantified sensorimotor integration processes using unimanual tasks in elderly individuals. Specifically, Valero-Cuevas and colleagues used a novel behavioral paradigm, the Strength-Dexterity (SD) test, which provides information about the sensorimotor integration processes through quantification of the dynamic control of fingertip forces. They found that sensorimotor ability assessed using the SD test significantly contributes to hand function in the aging population, in addition to strength and coordinated upper extremity function  power, in young and older adults. The authors found a significant increase in alpha power over sensorimotor cortices during sustained inhibition of a learned complex finger movement sequence in young adults, but not in older adults. This finding suggests that older adults have impaired inhibitory mechanisms necessary for the temporal control of movement, and that the EEG alpha power can be a sensitive tool to detect any age-related deterioration of inhibitory neural mechanisms.In addition to behavioral metrics, metrics that quantify structural and functional changes in the brain will be useful to understand the effects of aging on the sensorimotor system. The studies compiled within this special issue are intended to promote discussion on the sensorimotor deficits that elderly individuals face and will encourage further behavioral and neurophysiological studies to investigate the specific neural mechanisms.PP drafted manuscript, PP and KC edited and revised manuscript, PP and KC approved the final version of 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.


Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as “effectors” is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function. Fungal plant pathogens are economically important due to the threat they pose to the production and yield of agricultural crops. It has been estimated that global agricultural production suffers an average annual loss of 15% due to plant diseases Oerke, . To reduThe lifestyles of plant pathogenic fungi are highly diverse and utilize distinct strategies to interact with the host plant. For example, necrotrophic fungi infect and kill host tissue and extract nutrients from dead host cells. Biotrophic fungi colonize living host tissue and obtain nutrients from living tissue; whereas hemibiotrophic fungi display two phases during the infection process; first is an initial biotrophic phase followed by a necrotrophic stage  and microbe-associated molecular patterns (MAMPs). During infection, PAMPs present within the plant apoplast and are recognized by membrane-localized pattern recognition receptors (PRRs), which initiates the first reaction of defense called PAMP-triggered immunity (PTI)  receptor-like kinase, recognizes the highly conserved N-terminus of bacterial flagellin (flg22) and activates the MAP kinase pathway to induce expression of defense response genes  lines results in an impaired response to chitin elicitors , flagellin, and fungal cell wall components such as polysaccharides and chitin  and/or triggering defense responses (avirulence factors: Avr). These proteins can be grouped into two classes based on their target sites in the host plant Kamoun, , 2007. APhytopthera infestans has been shown to be required for translocation into potato cells  was shown to be directly recognized by the Arabidopsis NLR RPP1 through association with the C-terminal LRR  the presence of a secretion signal, (ii) being encoded by genes that are induced in planta, (iii) exhibiting similarity to haustorial proteins, (iv) small and cysteine rich, (v) the presence of a known effector motif or a nuclear localization signal, (vi) being encoded by genes with long intergenic regions, (vii) the presence of internal repeats, (viii) a lack of PFAM domains, excluding those associated with pathogenicity. The authors then used clustering algorithms to group protein families of rust pathogens and rank them according to the likelihood of being effectors. Using this approach they were able to identify approximately eight families of effector contenders. This pipeline was also utilized in a study conducted on M. lini, where 200 potential effector candidates were identified  was characterized through protein purification of tomato apoplastic fluids during compatible/incompatible fungal-plant host interactions using polyacrylamide gel electrophoresis (PAGE), reverse-phase HPLC and EDMAN N-terminal sequencing , one of the first effectors, termed SIX1 (Avr3), was identified and sequenced using mass spectrometry (MS)  tomato line. Other studies conducted in the FOL isolate followed suite, where identification of 14 additional SIX proteins were determined through the analysis of the xylem sap proteome of infected tomato plants using two-dimensional gel separation and mass spectrometry  or a compatible (susceptible) interaction. The fundamental principle explaining the outcome of this interaction is based on the gene-for-gene model Flor, . This moL. maculans has an unusual genome structure compared to other pathogenic fungi in that it contains alternating GC and AT-rich blocks termed GC- and AT-isochores. The GC-isochore regions are enriched with housekeeping genes, whereas the AT-isochores are relatively gene-sparse, enriched in TEs which are truncated and further reduced by repeat-induced point mutations (RIP). The AT-isochore harbors ~122 genes encoding small secreted proteins  is another important mechanism by which effector diversification occurs in fungal pathogens. One particular study conducted on in planta effector localization, or protein-protein interactions.Unlike bacterial and oomycete effectors, a limited number of fungal effectors have been functionally characterized. The primary obstacles in fungal effector research derive from difficulties associated with manipulating fungi in a laboratory setting, in particular obligate biotrophs such as rust fungi. Adding to these challenges is the fact that many effector mutants display no associated phenotype, likely due to functional redundancy, poor assay systems or the inability to accurately measure small changes in phenotype. However, technologies such as bimolecular fluorescent complementation (BiFC)  is a small (178 amino acids) secreted effector of U. maydis and related smut fungi that has been shown to accumulate in the apoplast. Doehlemann et al.  a direct interaction between the Pep1 protein and the maize defense peroxidase POX12 was established. It appears that Pep1 is able to suppress plant defense mechanisms through scavenging reactive oxygen species (ROS)  is a secreted, apoplastic effector of U. maydis that is required for virulence; studies have shown that a mutation within the pit2 gene results in attenuation of tumor formation  is part of the largest cluster of effectors identified in U. maydis and was shown to be responsible for anthocyanin induction during biotrophic phase and have a role in virulence , a maize protein kinase that regulates the anthocyanin biosynthetic pathway  is recognized as an organ-specific effector that induces tumor expansion specifically on maize leaves , a factor that has been shown to act in cell cycle progression in yeast  effector that has been shown to target the cytosolic rice R-gene -dependent signaling pathways, which was also previously shown to occur upon recognition of AvrLm1 by Rlm1 -Cas9 system was originally identified in bacteria and archaea as a defense mechanism to prevent the invasion of foreign DNA from phage or plasmids (Barrangou et al., Saccharomyces cereviseae (DiCarlo et al., Trichoderma reesei (Liu et al., M. oryzae (Arazoe et al., Neurospora crassa (Matsu-ura et al., P. sojae (Fang and Tyler, Aspergillus niger for cas9 and sgRNA expression (Nodvig et al., Saccharomyces cerevisiae (Cong et al., T. reesei (Liu et al., Arabidopsis (Endo et al., CRISPR/Cas9-based technologies have emerged as an effective way to generate unmarked mutations in several organisms (Sander and Joung, Clearly, the CRISPR/Cas9 “craze” has led to a plethora of useful tools that target not only single genes, but multiple gene targets, which will be extremely useful for effector research. The most valuable attribute of the CRISPR/Cas9 systems is its versatility, where the tools can be used to specifically target genes for mutagenesis in many organisms. Certainly, this system will transform and improve biological research, leading to many new discoveries in gene regulation and function. Nevertheless, this system is still in its infancy and as such several aspects need to be refined, including off-targeting and expression of the components within the desired host.We have gained a wealth of knowledge toward understanding plant-pathogen interactions over the past several decades. Research has shown that effector proteins play a significant role in the ability of fungal pathogens to establish a compatible interaction with the host plant, and in some cases have confirmed their involvement as virulence factors. We have working methods for developing mutations within single genes, and with the introduction of CRISPR/Cas9 technology, we now have the capability to target multiple genes without the use of markers. This system will significantly improve our ability to target redundant or related effector genes at one time, perhaps providing a definitive or visual phenotype that will provide insight into effector function. Despite our improved knowledge in this area, some important factors remain unresolved. Many fungal effectors have been shown to function primarily within the plant cytosol, yet lack translocation signals within the primary amino acid sequence. This brings into question how effectors are targeted to and taken across the plasma membrane of the plant cell? Although some host targeting sequence motifs have been identified in oomycetes, most fungi do not share this common feature. Moreover, the low sequence homologies among these fungal proteins make it difficult to predict similar features. However, the rapid development of next-generation sequencing tools and new bioinformatics algorithms and pipelines will provide new whole-genome information for both biotrophic and hemibiotrophic fungi. Given the large numbers of effector candidates, research will be a challenge. However, with the continuous development of new tools and techniques we will undoubtedly move toward exceptional opportunities for investigating the roles of fungal pathogen effectors in plant-pathogen interactions.Concept of article by WD. Also went through all research papers and finalized the review. CS did the undertaking of researching on the topic and writing the review. TD and MB read the manuscript and gave input on improvements.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.


In recent years, tumor budding in colorectal cancer has gained much attention as an indicator of lymph node metastasis, distant metastatic disease, local recurrence, worse overall and disease-free survival, and as an independent prognostic factor. Tumor buds, defined as the presence of single tumor cells or small clusters of up to five tumor cells at the peritumoral invasive front  or within the main tumor body , are thought to represent the morphological correlate of cancer cells having undergone epithelial–mesenchymal transition (EMT), an important mechanism for the progression of epithelial cancers. In contrast to their undisputed prognostic power and potential to influence clinical management, our current understanding of the biological background of tumor buds is less established. Most studies examining tumor buds have attempted to recapitulate findings of mechanistic EMT studies using immunohistochemical markers. The aim of this review is to provide a comprehensive summary of studies examining protein expression profiles of tumor buds and to illustrate the molecular pathways and crosstalk involved in their formation and maintenance. The hallmark of malignant disease, namely the ability of a tumor to disseminate and colonize distant sites, requires an arsenal of cellular characteristics. In colorectal cancer and a growing number of solid tumors, epithelial–mesenchymal transition (EMT) is proposed as a fundamental mechanism for epithelial cells to acquire such a “malignant” phenotype , 2. TumoFrom a morphological point of view, tumor buds tend to appear more atypical than their counterparts in the main tumor body [hence the previous term “tumor dedifferentiation” ] Tumor bAs tumor buds are visualized in a histological “snapshot,” studies using immunohistochemistry have been pivotal in improving our understanding of tumor buds and their protein expression profiles . A distiIt is well-recognized that “colorectal cancer” encompasses fundamentally different molecular phenotypes following various pathways of carcinogenesis . As a coActivation of WNT signaling leads to stabilization of membranous/cytoplamasmic β-catenin and its translocation to the nucleus. Located at the cell membrane, β-catenin complexes with E-cadherin and is crucial for maintaining cell–cell adhesion and epithelial cell polarity . HoweverThe functions of proteins encoded by WNT target genes confer characteristics of a malignant mesenchymal phenotype. Proteins involved in the degradation of the extracellular matrix, such as MMP-9 and Cathepsin B have been shown to be overexpressed in buds . SeveralOther cell adhesion proteins such as EpCAM have been implicated in the budding process, with loss of membranous expression identified in tumor buds . EpCAM iModulators of Wnt signaling have also been detected in tumor buds, such as the AAA+ protein family member pontin , which hThe stem-cell concept is centered on the notion that tumor progression is driven by a primarily undifferentiated population of tumor-initiating cancer cells. After initially being described in acute myeloid cancers, cancer stem cells (CSCs) have been identified in a myriad of solid tumors including colorectal cancers. CSCs display aggressive features such as increased invasiveness, chemoresistance, and the ability to mediate angiogenesis and resist apoptosis, with the ability to re-differentiate at metastatic sites . It woulThere is accumulating evidence indicating that the driving force of colorectal cancer progression may not be attributable to tumor cell proliferation alone. Generally, it is thought that EMT-derived tumor cells are hypo-proliferative, underlining the significance of aggressive cellular machinery to exert their malignant properties . The celAs the hypo-proliferative nature of tumor buds is gaining recognition, it may be speculated that in order to survive migration through stroma, tumor buds must confer of essential survival mechanisms. In fact, single epithelial cells detached from the extracellular matrix are programed to undergo a certain form of cell death termed anoikis . In addiCancer cells frequently exploit growth factor signaling from the surrounding microenvironment  to drive tumor progression –63. WellThe tumor suppressor gene RKIP has been linked to EMT on several levels, for one as inhibitor of the Ras–Raf–MEK–ERK signaling cascade at the level of Raf . In addiThe neurotrophic tyrosine kinase receptor TrkB has been linked to EMT via RAS/MAPK-dependent Twist–Snail signaling and has been demonstrated to be a potent and specific suppressor of anoikis , which iChemokines, integral for cell migration and trafficking, are widely expressed by cells of the lymphatic and hemopoietic systems. The chemokine CXCL12 binds to its receptor CXCL4, activating subsequent intracellular pathways involved in chemotaxis, cell survival, and gene transcription . As CXCRThe homeobox transcription factor Cdx2 encodes a transcription factor specific to intestinal differentiation, which is essential for development and homeostasis of gut epithelium . Recent Few studies have examined the expression of other markers of epithelial differentiation in tumor buds. For instance, Harbaum et al  demonstrIt has also been postulated that signals derived from surrounding mesenchymal cells in the tumor microenvironment may play a significant role in facilitating a pro-budding phenotype , 79. ForThe interplay between epithelial and stromal components has also been underlined by studies examining bone morphogenetic protein (BMP) antagonists , 81, hypTumor budding is thought to represent the morphological correlate of EMT in colorectal cancers and has been strongly linked to adverse clinicopathological features and poor overall and disease-free patient survival. These consistent associations indicate that tumor budding has a strong value as a prognostic indicator, and it has been proposed that budding should be an integrated category in pathology reports .in situ hybridization. Immunohistochemistry as a semi-quantitative method may be especially prone to subjectivity, and staining intensity greatly depends on laboratory methods (In an attempt to contribute to our understanding of tumor buds, previous studies have mainly immunohistochemistry to discriminate properties unique to tumor-budding cells. The main reason for this is that immunohistochemistry enables the actual identification of tumor buds for evaluation. To our knowledge, there is currently no method of extracting tumor buds from fresh tumor tissue, presenting huge hurdles for molecular studies specifically geared at tumor buds. Therefore, our understanding of the biology of tumor buds is essentially restricted to protein expresssion profiles (as visualized in Figure  methods . Such isTherefore, although the molecular background of colorectal cancers appears to play an important role in budding, much remains to be investigated in terms of genetic profiles of tumor buds and how various molecular pathways are taken advantage of by these cells to maintain their malignant phenotype and drive tumor progression. Novel areas of interest include the interaction of tumor buds with cancer-associated fibroblasts and inflammatory cells in the tumor microenvironment  and the 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.


Microbes in the gastrointestinal tract are under selective pressure to manipulate host eating behavior to increase their fitness, sometimes at the expense of host fitness. Microbes may do this through two potential strategies: (i) generating cravings for foods that they specialize on or foods that suppress their competitors, or (ii) inducing dysphoria until we eat foods that enhance their fitness. We review several potential mechanisms for microbial control over eating behavior including microbial influence on reward and satiety pathways, production of toxins that alter mood, changes to receptors including taste receptors, and hijacking of the vagus nerve, the neural axis between the gut and the brain. We also review the evidence for alternative explanations for cravings and unhealthy eating behavior. Because microbiota are easily manipulatable by prebiotics, probiotics, antibiotics, fecal transplants, and dietary changes, altering our microbiota offers a tractable approach to otherwise intractable problems of obesity and unhealthy eating. The struggle to resist cravings for foods that are high in sugar and fat is part of daily life for many people. Unhealthy eating is a major contributor to health problems including obesity Conflict over resource acquisition and resource allocation can occur as a result of conflict between different genetic interests within an organism. For example, genetic conflict between maternal and paternal genes is hypothesized to play a role in the unusual eating behavior that characterizes the childhood genetic diseases Beckwith–Wiedemann syndrome and Prader–Willi syndrome. These syndromes are characterized by altered appetite and differences in infant suckling that can result from overexpression of genes of paternal or maternal origin, respectively Microbial genes outnumber human genes by 100 to 1 in the intestinal microbiome, leading some to propose that it is a “microbial organ” that performs important functions for the host, such as nutrient harvesting and immune development Prevotella grows best on carbohydrates; dietary fiber provides a competitive advantage to BifidobacteriaBacteroidetes has a substrate preference for certain fats Akkermansia mucinophila, thrive on secreted carbohydrates provided by host cells. Other butyrate producing microbes, e.g. Roseburia spp., fare better when they are delivered polysaccharide growth substrates in the diet. Specialist microbes that digest seaweed have been isolated from humans in Japan Individual members of the microbiota, and consortia of those microbes, have been shown to be highly dependent on the nutrient composition of the diet. Lactobacillus casei in participants whose mood was initially in the lowest tertile There is circumstantial evidence for a connection between cravings and the composition of gut microbiota. Individuals who are “chocolate desiring” have different microbial metabolites in their urine than “chocolate indifferent” individuals, despite eating identical diets Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 alleviated psychological distress Lactobacillus rhamnosus (JB-1) to mice, not only does it reduce their stress-induced corticosterone hormone levels, but it also makes them more dogged: L. rhamnosus (JB-1) fed mice swim longer than the control fed mice when put in a glass cylinder filled with 15 cm of water and no means of escape L. rhamnosus (JB-1) Toxoplasma gondii suppresses rats' normal fear of cat smells, often to the detriment of the rats, but to the benefit of the microbes that are ingested into their new feline host. T. gondii infected rats are reported to become sexually aroused by cat urine T. gondii at the expense of the fitness of the rat.There are many other examples of microbes affecting their hosts' mood and behavior, mostly from animal studies . ButyratLactobacillus appear to reduce anxiety, colonization of the gut with the pathogen Campylobacter jejuni increased anxiety-like behavior in mice Although certain One potential mechanism by which dysphoria can influence eating involves bacterial virulence gene expression and host pain perception. This mode of manipulation is plausible because production of virulence toxins often is triggered by a low concentration of growth-limiting nutrients. Detection of simple sugars and other nutrients regulates virulence and growth for a variety of human-associated microbes L. acidophilus NCFM, administered orally as a probiotic, increased intestinal expression of cannabinoid and opioid receptors in mouse and rat intestines, and had similar effects in human epithelial cell culture One route to manipulation of host eating behavior is to alter the preferences of hosts through changing receptor expression. One study found that germ-free mice had altered taste receptors for fat on their tongues and in their intestines compared to mice with a normal microbiome Gut microbes may manipulate eating behavior by hijacking their host's nervous system. Evidence shows that microbes can have dramatic effects on behavior through the microbiome-gut-brain axis Evidence suggests that the vagus nerve regulates eating behavior and body weight. For example, blockade or transection of the vagus nerve has been reported to cause drastic weight loss Together these results suggest that microbes have opportunities to manipulate vagus nerve traffic in order to control host eating. Intriguingly, many practices that are known to enhance parasympathetic outflow from the vagus nerve, e.g. exercise, yoga, and meditation, are also thought to strengthen willpower Escherichia coli, Bacillus cereus, B. mycoides, B. subtilis, Proteus vulgaris, Serratia marcescens, and Staphylococcus aureusB. subtilis appears to secrete both dopamine and norepinephrine into their environment, where it interacts with mammalian cells. Transplant of the microbiome from a male to an immature female mouse significantly and stably increases testosterone levels in the recipient Microbes produce a variety of neurochemicals that are exact analogs of mammalian hormones involved in mood and behavior B. infantis 35624 raises tryptophan levels in plasma, a precursor to serotonin Certain probiotic strains alter the plasma levels of other neurochemicals. Lactobacillus strains, reduced hunger-inducing hormones AgRP (agouti related protein) and neuropeptide Y in the hypothalamus Appetite-regulating hormones are another potential avenue for manipulation of mammalian eating behavior. In mice, treatment with VSL#3, a dietary supplement consisting of a mixture of Bacteroides thetaiotaomicron is found on host mucus, where it scavenges N-glycated oligosaccharides secreted by goblet cells in the gut. B. thetaiotaomicron induces its mammalian host to increase goblet cell secretion of glycated carbohydrates A. muciniphila, also increases the number of mucus producing goblet cells when inoculated in to mice Faecalibacterium prausnitzii, a non-mucus-degrading bacterium that is co-associated with B. thetaiotaomicron, inhibits mucus production by goblet cells Several commensal bacteria are known to induce their hosts to provide their preferred nutrients through direct manipulation of intestinal cells. For example, Evolutionary conflict between the gut microbiome and host may be an important contributor to the epidemic of obesity. In a landmark paper, Backhed and colleagues showed that mice genetically predisposed to obesity remained lean when they were raised without microbiota Lactobacillus probiotics have been reported to reduce fat mass and improve insulin sensitivity and glucose tolerance, although these effects are not universally reported for all Lactobacillus species Bifidobacterium breve inhibited weight gain in mice given a high fat diet in a dose-dependent manner L. rhamnosus GG and Bifidobacterium lactis along with dietary counseling, reduced abdominal fat at 6 months post-partum The addition of probiotics  to the diet tends to decrease food intake, consistent with the hypothesis that greater gut diversity may limit microbial control over eating behavior. Some Prebiotics , probiotics, antibiotics, fecal transplant, and diet changes are potential strategies to alter the microbiota. In addition to the proposal that microbiota transplantation should result in adoptive transfer of food preferences Raising an experimental animal on a simple diet with few types of foods, should select for microbes that specialize on those foods. Our hypothesis as to the microbial origin of food preferences predicts that these microbes will influence their host to choose the foods upon which they specialize. An alternative hypothesis, that food cravings result from nutrient shortages If microbial control is mediated through the vagus nerve, then microbial signals should interfere to some extent with the physiological regulation coordinated by the vagus nerve. Vagal tone can be easily measured through respiratory sinus arrhythmia Certain features of microbial ecology, such as population size, would be expected to influence a microbe's capacity to manipulate the host. Microbial communities with low alpha (intrasample) diversity might be more prone to overgrowth by one or more species, giving those organisms increased ability to manufacture behavior-altering neurochemicals and hormones. By comparison, in microbial communities with high alpha diversity any single microbial species will tend to occur at lower abundance. Highly diverse gut microbiotas tend to be more resistant to invasion by pathogenic species than less diverse microbiotas Besides affecting cravings for specific nutrients, conflict between host and microbiota is expected to impact satiety and overall calorie consumption because optimal energy intake is likely to differ between the host and members of the gut microbiota. Excess energy delivered to the gut, beyond what is optimal for the host, might provide energy substrates for microbial growth, permitting certain species to bloom, potentially overwhelming inhibition by competitor organisms and the immune system. Energy excess is predicted to reduce diversity as a result, leading to a vicious cycle of reduced diversity, increased manipulation and chronic energy excess. Such a positive feedback mechanism could drive long-term changes in satiety, harming the host by causing obesity. Experimental increases in gut microbiota diversity are expected to change the satiety setpoint, favoring decreased food intake by the host S. aureus uses the accessory gene regulator system (AGR) of quorum sensing to regulate toxin and other virulence genes. When S. aureus reaches high density, AGR switches from expression of genes involved in colonization and attachment to those involved in tissue invasion One explanation for the health benefits of intestinal diversity is the inhibition of quorum sensing microbes from achieving a quorum. Quorum sensing is a cell–cell communication system used by many gut bacteria to regulate density-dependent conditional strategies, including virulence factor expression and changes in growth. For instance, the common human commensal and pathogen There has been little work to study the co-evolution of the microbiome and their host genomes socially contagious (e.g. through changes in norms) as the authors of the social network study suggest One intriguing implication of microbially induced cravings is that preferences for certain foods may be contagious There are a number of existing hypotheses for the prevalence of obesity and our cravings for unhealthy foods, including addiction/lack of willpower, environmental mismatch, and nutrient shortages. A microbial cause is not mutually exclusive of other alternatives such as nutrient deprivation. In this section, we review each of these alternative hypotheses. We find that none of these hypotheses is completely consistent with the data on cravings, food preferences, and obesity.Conventional wisdom often blames unhealthy eating on a lack of willpower. However, binge eating is not just a matter of mental control Food preferences are thought to arise from a complex interaction between genes, environment, and culture. The modern food environment is vastly different from that of our evolutionary ancestors: the human ancestral diet is thought to contain foods far lower in salt, simple carbohydrates, and saturated fat than the typical Western diet Despite the intuitive appeal of this hypothesis, a number of food preferences and cravings are not in accord with its predictions. For example, one of the most common modern cravings involves a food that ancient hominids never knew and which fulfills no nutritional requirement: chocolate A similar hypothesis proposes that cravings result from nutrient shortage Modern biology suggests that our bodies are composed of a diversity of organisms competing for nutritional resources. Evolutionary conflict between the host and microbiota may lead to cravings and cognitive conflict with regard to food choice. Exerting self-control over eating choices may be partly a matter of suppressing microbial signals that originate in the gut. Acquired tastes may be due to the acquisition of microbes that benefit from those foods. Our review suggests that one way to change eating behavior is by intervening in our microbiota.It is encouraging that the microbiota can be changed by many interventions, hence facilitating translation to the clinic and public health efforts. Microbiota community structure changes drastically within 24 hours of changing diet Competition between genomes is likely to produce a variety of conflicts, and we propose that one important area, impacting human health, is in host eating behavior and nutrient acquisition. Genetic conflict between host and microbiota – selecting for microbes that manipulate host eating behavior – adds a new dimension to current viewpoints, e.g. host-microbiota mutualism


For decades sequential sampling models have successfully accounted for human and monkey decision-making, relying on the standard assumption that decision makers maintain a pre-set decision standard throughout the decision process. Based on the theoretical argument of reward rate maximization, some authors have recently suggested that decision makers become increasingly impatient as time passes and therefore lower their decision standard. Indeed, a number of studies show that computational models with an impatience component provide a good fit to human and monkey decision behavior. However, many of these studies lack quantitative model comparisons and systematic manipulations of rewards. Moreover, the often-cited evidence from single-cell recordings is not unequivocal and complimentary data from human subjects is largely missing. We conclude that, despite some enthusiastic calls for the abandonment of the standard model, the idea of an impatience component has yet to be fully established; we suggest a number of recently developed tools that will help bring the debate to a conclusive settlement. Most modern accounts of human and monkey decision-making assume that choices involve the gradual accumulation of noisy sensory evidence from the environment in support of alternative courses of action. When the evidence in favor of one response option accrues to a threshold quantity a decision is reached and the corresponding action is initiated . In particular, decision makers might rely on a dynamic criterion when the signal-to-noise ratio is poor. With a weak signal one must accumulate evidence over an extended period to make an accurate decision. To avoid the prohibitively high costs associated with extended accumulation, decision makers could adopt a dynamically decreasing decision threshold  or an unacceptably high error rate (i.e., the signal-to-noise ratio is too low; Shadlen & Kiani, When quantitative models are proposed they are typically tested against only a few data sets as proof-of-concept evidence for the validity of the model. This approach is a prerequisite for theoretical progress but it necessarily restricts the generality of the model by testing it across only a narrow range of experimental tasks, procedures, and even species. Recently, we quantitatively compared static and dynamic DDMs in a large-scale survey of behavioral data sets that spanned a range of experimental paradigms and species, and across independent research laboratories (Hawkins, Forstmann, Wagenmakers, Ratcliff, & Brown, The suggestion that dynamic decision criteria maximize reward rate in environments with a poor signal-to-noise ratio implicitly raises the question of how decision makers infer the current signal strength. If the signal remains constant throughout the decision process, a simple solution is to incorporate elapsed time as a proxy for signal strength into the decision variable (Hanks et al., How should a time-variant signal-to-noise ratio inform threshold settings? A static decision criterion is highly insensitive to signals that vary throughout a trial, increasing the probability of an erroneous decision. A sensible approach might be to place greater weight on information presented later in the decision process, which can be achieved with a dynamic decision criterion. The distance between a dynamic decision threshold and the decision variable will decrease as more time passes, irrespective of the current state of the evidence accumulation process. This increases the likelihood of momentary sensory evidence leading to a threshold crossing (Cisek et al., In support of this proposal, evidence that varies throughout a trial can induce prominent order effects. For example, when a bias for a response option appears early in a trial it does not influence human and monkey decision times (Cisek et al., Taken together, formal analyses indicate that whether static or dynamic decision criteria are the optimal decision strategy depends critically on whether two components of the decision environment are fixed or variable within- and between-trials: the reward for a correct choice and the signal-to-noise ratio. When both the reward for a correct decision and the signal-to-noise ratio are constant across trials, the static thresholds DDM maximizes reward rate (for an extensive review see Bogacz et al., It remains unclear however, whether human and monkey decision makers actually use the optimal threshold settings under the different environmental conditions. Although there is some evidence that humans can optimize reward rate there does not seem to be a consensus yet as to whether reward rate maximization is the only goal. Most studies that suggest reward rate as the cause of a dynamic decision criterion do not actually manipulate or even control rewards. However, a number of studies that systematically manipulated rewards showed that increasing sampling costs can cause a dynamic criterion Pitz, . AnotherEven though sequential sampling models make elaborate assumptions about the processes underlying decision-making, behavioral studies – the most common source of data for model comparison – cannot take advantage of this wealth of discriminating information. In fact, different models often make indiscernibly similar behavioral predictions and thus only data on the physiological implementation of the decision process Fig.  might alThere is considerable evidence for the neural implementation of DDMs, for instance from single-cell recordings of monkeys performing experimental decision-making tasks (Forstmann, Ratcliff, & Wagenmakers, Another method of contrasting DDM decision processes with physiological data relies on measurements of the aggregated activity of large neuron ensembles in human subjects, such as EEG, MEG, and fMRI. This line of research is motivated on the assumption that the activity of neuron populations control behavior, not single neurons (Deco, Rolls, & Romo, Taken together, physiological evidence from monkeys, and to a lesser extent from humans, supports the suggestion of a dynamic decision criterion. As time passes, less evidence is needed for decision commitment because an urgency signal increasingly drives neural activity toward the decision threshold. However, comparisons of such neural activity patterns and generalizations across species are complicated because measurements differ in a number of ways. Not only is the mapping between primate and human brain activity uncertain (Mantini et al., Sequential sampling models are one of the most prominent and comprehensive frameworks for understanding human and monkey decision-making. For nearly four decades, decision behavior has been successfully explained by a standard model that assumes decision makers set a quality criterion before engaging in the decision process and maintain the same criterion throughout. In recent years this assumption of a static criterion has been challenged and a number of authors have suggested that decision makers become increasingly impatient as decision time increases, gradually lowering their quality criterion.Models with a dynamic decision criterion have been motivated on two grounds. Firstly, decision makers aiming to maximize their reward rate should theoretically adopt a dynamic decision criterion in dynamic environments. Indeed, studies in which the signal-to-noise ratio or the reward for correct decisions varied between or within decisions have shown that models with a dynamic decision criterion can account for the behavior of humans and primates. However, the conclusion that dynamic environments automatically imply a dynamic decision criterion is not uncontested. Many studies purporting such a conclusion did not systematically manipulate the variability of the decision environment. Moreover, quantitative comparisons of how well models with dynamic and static decision criteria can account for data are often missing.The second main motivation for models with a dynamic decision criterion are single-cell recording studies in behaving monkeys and EEG studies in humans showing patterns of neural activity that are most consistent with a dynamic decision criterion. However, the currently available evidence is equivocal. Neural data from human decision makers are sparse, and theoretical and empirical work linking neural activity at different scales and behavioral outcomes is still missing.To conclude, the recent developments have led to some enthusiastic responses that have called for models with an impatience component to replace the standard model (Shadlen & Kiani, Future progress in establishing a solid evidence base for models with dynamic decision criteria will critically hinge on careful experimentation in combination with rigorous theoretical analysis. Behavioral and electrophysiological studies will need to systematically manipulate the degree to which a decision environment is dynamic, closely controlling the costs and rewards for decisions and carefully varying the range of signal-to-noise ratios of stimuli. Such environments should be presented to both humans and monkeys, and their behavioral and physiological responses should be compared to models with static and dynamic decision criteria using Bayesian model comparison techniques, which allow researchers not only to determine the best fitting model but also to quantify the uncertainty associated with their conclusions (Jeffreys, A recently developed experimental approach that mitigates the need for computationally intense model fitting (Hawkins, Forstmann, et al., Physiological data will play a pivotal role in discriminating models. Sequential sampling models often make different assumptions about the processes giving rise to decision-making yet predict very similar or even identical behavior (Ditterich, In sum, the idea of increasing impatience in decision-making has been suggested sporadically throughout the history of sequential sampling models but has seen a tremendous surge in interest over the last years. Although theoretical arguments make a compelling case for impatience, the empirical support from monkey and human data is less clear. Future studies will have to address this problem further and recent developments promise a more conclusive settlement to the debate sooner rather than later. For the time being, we conclude that the idea of impatience has provided novel theoretical impulses, yet reports of the demise of the standard drift diffusion model are greatly exaggerated.