Developmental dyslexia is characterized by an impaired acquisition of fluent and skilled reading ability. Numerous studies have explored the neural correlates of this neurodevelopmental disorder, with most classic accounts strongly focussing on left temporoparietal regions. We will review recent findings from structural and functional MRI studies that suggest a more important role of occipitotemporal cortex abnormalities in dyslexia.Recent findings highlight the role of the occipitotemporal cortex which exhibits functional as well as structural abnormalities in dyslexic readers and in children at risk for dyslexia and suggest a more central role for the occipitotemporal cortex in the pathophysiology of dyslexia.We demonstrate the importance of the occipitotemporal cortex in for understanding impaired reading acquisition and point out how future research might enhance our understanding of functional and structural impairments in the reading network via large-scale data analysis approaches. Developmental dyslexia  designates impaired acquisition of reading skills which is not merely accounted for by mental age, sight defects, or insufficient schooling . Affectephonological deficit hypothesis. It postulates a specific deficit to represent, access, and process speech sounds caused by inherent dysfunctions of cortical areas specialized in phonology and reading. Word reading difficulties differ somewhat across languages since their orthographies vary in the transparency of their grapheme-phoneme mappings. It is therefore easier to read Finnish or German words (transparent orthographies) as compared to English words (opaque orthography) [Besides several auditory, visual, and motor dysfunction hypotheses, the prevalent (and most consistent ) cognitiography) , and thiography) , 10. Sevography) ••. Critiography) .In most classic accounts of brain abnormalities in dyslexia, the importance of the left temporoparietal cortex is especially highlighted, since this brain region has been closely linked with the phonological processing deficits in dyslexia , 14, 15•The occipitotemporal , temporoparietal, and left frontal regions regularly emerge in fMRI studies on dyslexia and are identified as important core regions for reading •, 19–22.By contrast, the OCT shows convergent activation irrespective of reading proficiency as demonstrated in robust activation in child as well as adult readers, which highlights the role of the OCT not only during proficient reading but also during reading acquisition . In a Support for the important role of the left OTC in dyslexia is also provided by training studies on children with reading disabilities, showing that systematic reading interventions (50 min per day) increased not only reading fluency but also neural response in the occipitotemporal cortex . This efTaken together, developmental dyslexia is frequently associated with a decreased specialization for letters and words in the OTC. Decreased neural response during reading-related processes in dyslexia seems to be invariant to orthographic depth and is robustly identified in adult as well as infant reading. Altered activation is also found in prereading studies suggesting early developmental brain alterations prior to reading acquisition •. NotablFirst neuroanatomical assessments of structural brain abnormalities in dyslexia arose from post-mortem investigations . AbnormaSimilar to functional MRI investigations, structural assessments show variations in the neuroanatomy of reading-related regions in at-risk prereaders. Structural alterations prior to reading acquisition include atypically small sulcal basins • and redNRSN1 gene was related to gray matter volume in the VWFA. The genetically associated volume profile of the VWFA (gVWFA) was able to classify readers into dyslexic and control individuals. Furthermore, gVWFA accurately distinguished between later dyslexic and normal reading skills in the prereader sample. In another recent genetic imaging study, a link was found between a genetic risk factor for dyslexia and dyscalculia and decreased volume and brain activation of the fusiform gyrus in the left OTC [Genetic imaging studies have also demonstrated an association between OTC structure and genetic markers of dyslexia . Skeide left OTC .Despite the large number of studies pointing to the importance of structural abnormalities in the OTC for dyslexia, it should be noted that the literature on structural brain abnormalities in dyslexia is plagued by inconsistencies and failures to replicate . ThesDyslexia was early conceptualized as a disconnection syndrome , consistTo date, relatively few fMRI studies have reported findings on impaired functional brain connectivity in dyslexia, despite the clear interest on disordered communication between brain regions. With respect to the left OTC, we have recently shown that this region reveals less connectivity with the left inferior frontal regions ••. ImporThe OTC is consistently identified in studies assessing literacy skills in normal and dyslexic reading. This area is assumed to accommodate a functional region—VWFA—which computes representations of visually presented words irrespective of variances in size, font, location, and case . InitialVWFA [and spoken words [Variable response properties suggest that VWFA activation also contributes to other functions besides the mere visual representation of words. In their “The interactive account of the ventral occipitotemporal contributions to reading,” Price and Devlin  propose VWFA . The othen words , 83. It In summary, the OTC is most consistently identified in infant and adult readers irrespective of orthographic depth and is even shown in fMRI assessments of non-alphabetic writing systems. Dyslexic readers not merely show decreased neural response in the OTC but rather indicate unspecific activation towards a reading material. Although less consistent, several structural MRI assessments highlight the role of structural alterations in the OTC in dyslexic readers. Strikingly, structural as well as functional alterations become evident even before reading acquisition in preschool children and are able to accurately predict later reading disabilities. The central importance of the OTC in reading and reading-related skills like spelling and (rapid) object naming might be one potential explanation for the relatively broad literacy impairments of dyslexia. Connectivity impairments between the OTC and frontal areas might be indicative of a disrupted linkage between orthographic and phonological word representations what in turn leads to inefficient and slow reading performance.Considering the evidence of abnormalities in dyslexia in various research domains ranging from behavioral RT measures to functional brain activation and even genetic factors, it seems unlikely that there is a single mechanistic explanation for dyslexia. Additionally, there is an ongoing discussion about whether there are qualitative differences in the neuronal mechanisms that underlie dyslexia, meaning that dyslexic symptoms might be based upon slightly diverging “neuronal subtypes” of dyslexia. The assumption of different neuronal subtypes might account for the sometimes inconsistent findings produced in this field. To illustrate, it remains up to future studies to show how occipitotemporal abnormalities relate to temporoparietal and frontal activation patterns and why certain tasks elicit decreased neural response in dyslexic readers whereas others do not. Furthermore, alterations in functional activation and structure might also be influenced by comorbid disorders like ADHD and dyscalculia and subclinical traits; at the moment, there is not much brain data directly exploring the relationship between dyslexia and other neurodevelopmental disorders and between reading ability and other skills and traits.Advanced approaches might provide suitable methods to classify dyslexic subgroups in a data-driven manner based on brain function, connectivity, and structure: Such methods are already successfully applied in other psychiatric disorders . Besides


Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. In this Perspective, I propose that future advances in microbiome research will emerge from considering “the lives of microbes”: we need to create methods to explicitly interrogate how microbes exist and interact in native-setting-like microenvironments. This approach includes developing approaches that expose the phenotypic heterogeneity of microbes; exploring the effects of coculture cues on cellular differentiation and metabolite production; and designing visualization systems that capture features of native microbial environments while permitting the nondestructive observation of microbial interactions over space and time with single-cell resolution. This is an exciting time for those of us interested in microbial communities. The technologies available to us now (many hardly imaginable even a decade ago) are allowing new scientific questions to be asked and driving the field forward. For instance, sequencing technologies have provided us with fantastic insights into which microbes are present in specific environments as well as into what their transcriptional states are. However, as important as sequencing approaches remain, these methods typically provide only broad snapshots of the bulk behavior of microbial communities. Developing a deeper appreciation of the factors driving the establishment and stability of microbial communities, as well as a predictive framework to enable their rational perturbation, will require a better understanding of the causal relationships between microbial community members. As I illustrate here, a productive approach to do so is to consider “the lives of microbes.” In other words, we need to develop methods that capture the native features of the environments that these tiny organisms experience in the natural world and that allow us to explicitly interrogate how microbes exist and interact with one another at the appropriate spatial scale: the microscale.Bacillus subtilis  and are developing single-cell imaging systems to address the question of how cells physically partition themselves within microbial communities .In their native environments, microbes most frequently live as members of complex multispecies communities. They are therefore surrounded not only by the neighboring cells that are physically present but also by the metabolic signals that these microbes have secreted. These secreted metabolites are primary drivers of microbial community interactions . More exBacillus cereus, has two structurally distinct activities: killing and biofilm induction , which aims to promote a community of similarly minded researchers who are interested in creating controlled model ecosystems to study microbial communities in response to perturbations.In “thinking like a microbe,” we must remember that microbes typically exist at significantly lower densities and overall cell numbers than are usually examined in laboratory culture. Thus, we must establish methods to investigate populations of cells interacting at the spatial scale at which microbes natively exist: the microscale. Ideally, these study systems would capture some of the physical and chemical complexity of natural systems while still allowing the nondestructive visualization of the spatiotemporal dynamics of individual cells over time. Since my laboratory is focused on soil microbial communities, we are creating such native-setting-like microcosms using an optically transparent soil-like substrate ; in someIn addition to the areas outlined above, technological advances in several areas would dramatically advance our understanding of microbial systems. Our ability to understand microbial interactions would be revolutionized by the ability to detect and identify “wild” and genetically intractable microbes, for instance, by using their distinct autofluorescence signatures. Although this would require significant advances in our optical detection capabilities, it would also substantially increase our ability to examine natural, nonmodel microbial systems. Other revolutionary advances will likely result from our improved ability to combine multiple imaging modalities  to simultaneously detect the spatiotemporal distributions of microbes and their metabolites at the microscale level within microcosms or natural environments. Such methods would allow us to dissect not only which cells are producing metabolites within microbial communities but also which microbes are altering their physiology and metabolism in response to these cues.


Acetylcholine (ACh) signaling shapes neuronal circuit development and underlies specific aspects of cognitive functions and behaviors, including attention, learning, memory and motivation. During behavior, activation of muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) by ACh alters the activation state of neurons, and neuronal circuits most likely process information differently with elevated levels of ACh. In several brain regions, ACh has been shown to alter synaptic strength as well. By changing the rules for synaptic plasticity, ACh can have prolonged effects on and rearrange connectivity between neurons that outlasts its presence. From recent discoveries in the mouse, rat, monkey and human brain, a picture emerges in which the basal forebrain (BF) cholinergic system targets the neocortex with much more spatial and temporal detail than previously considered. Fast cholinergic synapses acting on a millisecond time scale are abundant in the mammalian cerebral cortex, and provide BF cholinergic neurons with the possibility to rapidly alter information flow in cortical microcircuits. Finally, recent studies have outlined novel mechanisms of how cholinergic projections from the BF affect synaptic strength in several brain areas of the rodent brain, with behavioral consequences. This review highlights these exciting developments and discusses how these findings translate to human brain circuitries. Neuromodulation of the neocortex by acetylcholine (ACh) not only shapes neuronal circuit development, but is also crucial for sensory and cognitive behavior, such as sensory detection, attention, learning and memory  injections in neocortical target areas or with antibody staining for cholineacetyl transferase (ChAT) or acetylcholine esterase  to cortical targets (Bloem et al., Similar to the BF to PFC cholinergic system, a topographical organization also occurs between the BF and the parahippocampal cortex (Kondo and Zaborszky, New approaches such as iDISCO that allow volume imaging of the whole cholinergic network in cleared brain tissue will perhaps help to get a deeper insight in the topography of cholinergic projections in the cortex in the near future (Renier et al., The classical view on cholinergic signaling in the cerebral cortex is that it is slow and aspecific, most likely volume transmission (Sarter et al., With optogenetic activation of BF cholinergic projections to cerebral cortex, it is now clear that ACh signaling can occur functionally through direct, point-to-point fast synapses in the cortex (Letzkus et al., Direct cholinergic synaptic transmission is also found in deep layers of the neocortex. When BF inputs are activated by ChR2, prefrontal cortical L6 pyramidal neurons show an inward current that is mediated by nicotinic AChRs (Hay et al., From these studies, the picture emerges that both point-to-point cholinergic synaptic transmission as well as tonic cholinergic transmission exist in the neocortex, which depends on action potential firing regimes of BF neurons. At low firing rates, only nicotinic AChRs are recruited that are predominantly located in synapses. Repetitive activity of BF cholinergic neurons recruits extrasynaptic α4β2* nAChR receptors as well as muscarinic receptors by spillover (Kimura et al., AChR are abundantly expressed in primate as well as rodent neocortex (Metherate, Muscarinic and nicotinic AChRs also show a strong layer dependency in the cell types by which they are expressed (Gulledge et al., Cholinergic modulation can inhibit action potential firing of pyramidal neurons in superficial cortical layers by augmenting GABAergic inhibition through nAChRs and mAChRs (Kimura and Baughman, Nicotinic AChRs are highly expressed across all neocortical regions (Metherate, Both mouse and human neocortical interneurons express functional nicotinic AChRs Figures . In mousIn sensory cortical areas, such as auditory and visual cortex, VIP-positive neurons in superficial layers are recruited by cholinergic inputs that activate nicotinic AChRs (Porter et al., Cholinergic receptors are thus placed in excellent positions to rapidly modulate various inhibitory circuit motifs (Tremblay et al., Cholinergic modulation of cerebral cortical circuits is not limited to transient changes in cellular and synaptic activity. Both short-term and long-term synaptic plasticity can be altered by cholinergic receptor activation by BF inputs. The cellular and sub-cellular location of cholinergic receptors not only affects neuronal circuitry excitability, but it will also determine how glutamatergic synapse plasticity is affected by cholinergic inputs (Verhoog et al., Modulation of synaptic plasticity by muscarinic receptors has been found in hippocampal circuits. Low concentrations of mAChR agonists can modulate plasticity of glutamatergic synapses onto hippocampal pyramidal neurons (Shinoe et al., Over the past decade, we have found that in the rodent PFC, the rules of inducing plasticity of glutamatergic synapses are altered by cholinergic inputs from the BF depending on the cortical layers, resulting from a layer-and cell-type specific expression of nAChRs (Couey et al., In the rodent PFC, despite the robust nAChR modulation of thalamic excitatory inputs (Lambe et al., In the rodent PFC, fast synaptic cholinergic synaptic inputs mediated by nAChR containing β2 and α5 subunits are received by L6 pyramidal neurons (Hay et al., The evidence thus presents us with a landscape of cholinergic modulation of the cerebral cortex that is much more refined than previously considered, both on a spatial as well as a temporal scale. From various retrograde and anterograde anatomical methods it is now becoming clear that the BF cholinergic system specifically targets sub-regions of brain areas, as found in both prefrontal cortical subareas as well as rhinal cortical subareas (Bloem et al., in vivo optogenetic approaches should test what the role of these different signaling modes is in behavior. Activating or inhibiting cholinergic projections on a fast time scale should bring us in a position to unravel how this correlates with attention and memory performance. Disentangling the contributions of fast and slow signaling modes could also provide a novel view on disorders in which cholinergic signaling is affected, such as Alzheimer disease. The binding of Aβ to α7 nAchRs (Nagele et al., The findings that ACh can act both on a slow time scale and in fast synaptic transmission, in both a volume transmission mode and point to point cholinergic synapse level extends our views on how ACh can modulate microcircuit function in the cortex. New HDM conceived the story line of the review. JO, MVB, AL and HDM contributed to the writing of the manuscript. JO made Figure 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.


Despite the emergence of genomics-based risk prediction tools in oncology, there is not yet an established framework for communication of test results to cancer patients to support shared decision-making. We report findings from a stakeholder engagement program that aimed to develop a framework for using Markov models with individualized model inputs, including genomics-based estimates of cancer recurrence probability, to generate personalized decision aids for prostate cancer patients faced with radiation therapy treatment decisions after prostatectomy.We engaged a total of 22 stakeholders, including: prostate cancer patients, urological surgeons, radiation oncologists, genomic testing industry representatives, and biomedical informatics faculty. Slides were at each meeting to provide background information regarding the analytical framework. Participants were invited to provide feedback during the meeting, including revising the overall project aims. Stakeholder meeting content was reviewed and summarized by stakeholder group and by theme.The majority of stakeholder suggestions focused on aspects of decision aid design and formatting. Stakeholders were enthusiastic about the potential value of using decision analysis modeling with personalized model inputs for cancer recurrence risk, as well as competing risks from age and comorbidities, to generate a patient-centered tool to assist decision-making. Stakeholders did not view privacy considerations as a major barrier to the proposed decision aid program. A common theme was that decision aids should be portable across multiple platforms (electronic and paper), should allow for interaction by the user to adjust model inputs iteratively, and available to patients both before and during consult appointments. Emphasis was placed on the challenge of explaining the model’s composite result of quality-adjusted life years.A range of stakeholders provided valuable insights regarding the design of a personalized decision aid program, based upon Markov modeling with individualized model inputs, to provide a patient-centered framework to support for genomic-based treatment decisions for cancer patients. The guidance provided by our stakeholders may be broadly applicable to the communication of genomic test results to patients in a patient-centered fashion that supports effective shared decision-making that represents a spectrum of personal factors such as age, medical comorbidities, and individual priorities and values.The online version of this article (10.1186/s12911-017-0526-1) contains supplementary material, which is available to authorized users. Despite exciting advances toward the promise of genomics-driven cancer treatment , 2, therMany of the nearly half of patients with localized prostate cancer who undergo radical prostatectomy (RP) will either develop a biochemical recurrence, in the form of a rising prostate specific antigen (PSA) blood level, or will be considered to be at high risk of recurrence based on adverse pathological features –10. ThesDecisions regarding ART and SRT after RP are complex for patients and clinicians, since choices can be influenced by an individual’s recurrence risk, medical comorbidities and personal preferences and values. A recent Markov decision analysis model considering length and quality of life for ART versus SRT showed that the preferred treatment strategy for an individual patient is dependent upon the specific probabilities for cancer recurrence, as defined based on a genomic risk classifier assay, as well as death from causes other than prostate cancer . GenomicIn the current report, we describe a stakeholder engagement program that we performed in order to develop a framework for individualized decision aids to assist in the shared decision making process for patients making ART and/or SRT decisions with individualized GC-based recurrence risk estimates. Stakeholder engagement is a critical process for the development and implementation of genomic tests for clinical decision-making, because various stakeholders may each have different perspectives on genomic information . We evalOur stakeholder meeting interview team was comprised of two faculty investigators (TNS and JML) with prior research experience and training in stakeholder engagement. Stakeholders were identified and recruited through direct contact with the research team. Patient stakeholders were identified in a single radiation oncology clinic by their physician (TNS), while other stakeholder groups were identified through informal networking by email and at medical conferences. The team conducted a series of meetings with stakeholders to inform the process of developing a personalized decision aid program.Each stakeholder meeting with investigators included only one class of stakeholders. For example, either prostate cancer patients or urologists, but not both, would be invited to each meeting. This approach was used in order to encourage participation and avoid imbalance in power or educational level between groups. Although this choice may have improved convenience and facilitated conversation, the lack of larger focus group may have limited the range of information obtained from the process. A prepared set of slides was used to kick-off each meeting and provide background information , urological surgeons (5 participants), radiation oncologists (5 participants), genomic testing industry representatives (5 participants), and biomedical informatics faculty members (2 participants). The stakeholder participants were chosen for their experience within their respective group. The industry representatives were all employees of GenomeDx, the company that markets the Decipher GC test. The IRB approval of the stakeholder engagement protocol permitted participation of up to 15 prostate cancer patients, but this was limited to 5 patients after investigators noted that response content had been exhausted with no new themes identified after the initial 3 patient stakeholder sessions.Prior to each meeting, a common slide deck was shared with participants , with one female urologist included. Stakeholders from the genomic testing industry were all employees of GenomeDx.Stakeholders offered comments along a range of topics and perspectives. Stakeholder content was categorized according to 4 subjects: empathy, privacy concerns, design and formatting, and location or context of decision aid delivery to patient Table . The majStakeholders were enthusiastic about the potential value of using decision analysis modeling with personalized model inputs for risk of cancer recurrence and other patient-specific factors such as age, other medical problems and lifestyle. Table We engaged a total of 22 stakeholders, including patients, physicians, biomedical informatics faculty members, and genomic testing industry representatives, to gain perspectives and insights regarding our stated goal of developing a framework to deliver personalized decision aids to assist in the shared decision making process for patients making ART and/or SRT decisions with individualized GC-based recurrence risk estimates. Stakeholders were enthusiastic about the general concept of personalized decision aids based upon a heuristic model of Markov decision analysis modeling using several individualized model inputs for event probabilities. Stakeholders provided valuable insights regarding optimal presentation of model results for the decision aid, which will directly influence the design of the decision aid. Stakeholder insights, summarized in Tables Physicians recognize genomic testing as a valuable tool for clinical care , 27, butAlthough many of the insights from stakeholders shown in Table In conclusion, our stakeholder engagement process included perspectives from a range of stakeholders and provided valuable information that will inform the design of a personalized decision aid program for genomic-based treatment decisions regarding ART and SRT after prostatectomy. The guidance provided by our stakeholders may be broadly applicable to the communication of genomic test results to patients in a patient-centered fashion that supports effective shared decision-making.


Metagenomics has transformed microbiology, but its potential has not been fully expressed yet. From computational methods for digging deeper into metagenomes to study designs for addressing specific hypotheses, the Segata Lab is pursuing an integrative metagenomic approach to describe and model human-associated microbial communities as collections of strains. Metagenomics has transformed microbiology, but its potential has not been fully expressed yet. From computational methods for digging deeper into metagenomes to study designs for addressing specific hypotheses, the Segata Lab is pursuing an integrative metagenomic approach to describe and model human-associated microbial communities as collections of strains. Linking strain variants to host phenotypes and performing cultivation-free population genomics require large cohorts and meta-analysis strategies to synthesize available cohorts but can revolutionize our understanding of the personalized host-microbiome interface which is at the base of human health. Metagenomics  is typicThe reductionist approach of profiling the strain as the fundamental unit of the microbiome from metagenomics is, however, hampered by limitations in computational analysis. My laboratory focuses on solving these challenges to allow characterizing strains in human metagenomes with the same level of resolution as is available for single-isolate sequencing. This would bring the field into the next generation of microbiome studies.I discuss the key questions that, in my opinion, need to be answered in the next few years to exploit the potential of shotgun metagenomics in fields of medicine, ecology, and microbiology.Escherichia coli includes strains that are gut commensals as well as highly pathogenic (Helicobacter pylori are associated with widely different risks for gastric cancer (Eggerthella lenta can inactivate a cardiac drug (Prevotella copri are associated with increased risk for rheumatoid arthritis (Staphylococcus epidermidis seem to be associated with psoriatic skin (Pregenomic and isolate sequencing studies of (opportunistic) pathogens provide overwhelming evidence that many microbial phenotypes are strain specific. The species thogenic  and carcthogenic . Differec cancer . These aiac drug , some surthritis  whereas tic skin . Becausetic skin , 9, I arThere are currently no strict definitions of what a bacterial or archaeal strain is, and this inevitably causes difficulties and misunderstandings. If we were to strictly link a strain with its genome, single-nucleotide variants (SNVs) accumulating over microbial generations in pure cultures would lead to calling a given organism a different strain over time. Moreover, the number of different strains would be too large to be tractable, and errors in high-throughput sequencing would make strain identity not testable. On the other hand, any threshold applied to the number of nucleotide variants allowed between “same” strains would be an arbitrary cutoff, would be suboptimal in most cases, and would not account for events such as gene loss/gain or horizontal gene transfer.It would be ideal to define strains as microbial entities that, despite a limited genetic heterogeneity, have the same phenotype under different conditions. However, this would be impossible to establish in practice. Phylogenetic modeling of single microbial genomes can overcome the need for a strict definition in some cases. But because synonymous mutations and other genetic variations that do not give any evolutionary or ecological (dis)advantages can be present in the same microbiome sample, it is not even clear whether we should talk about strain populations or strain clouds instead of single strains. Proposing a general definition of a strain is outside the scope of this Perspective, but it will be crucial for this line of research to at least define practical and operational definitions for strains.We have recently developed three complementary methods to extract strain-level signatures from metagenomes. They are all based on popular methods used to characterize strains from pure cultures adapted to the complexity of metagenomes and the presence of confounding reads. MetaMLST  exploitsNeisseria in different locations of the oral cavity . Because Illumina sequencing is the de facto standard for metagenomics and because presequencing biases affect strain profiling only minimally, our experience is that it is feasible for a laboratory with sufficient computational infrastructure to process public metagenomes by automatically downloading the raw sequences and running them through strain profilers. For a number of practical and technical reasons, the retrieval of consistent metadata is much more challenging. Manual checking and curation of metadata appear to be the only solution to enable cross-study sample analysis, and we organized our manual curation effort into the curatedMetagenomicData resource . This isStaphylococcus epidermidis to psoriasis-affected skin  pathogens have all been linked with strain-specific genomic characteristics using cultivation-based studies. We hypothesize that the same principles also apply to interaction between members of the human microbiome and our bodies. We found preliminary evidence of strain-level adaptation of ted skin , and we Because the overall intersubject strain-level variability of a microbiome is extremely large, similarly large sample sizes are needed to obtain strong statistical support for strain-level associative multiple hypothesis testing. Resources such as the already mentioned curatedMetagenomicData resource  are crucA considerable fraction of the human microbiome remains hidden from reference-based profiling. Although genomes for new species are being published more frequently, metagenomic assembly offers the potential of reference-free microbial identification. While algorithms able to generate high-quality genomic contigs exist , 25, it I have outlined the key questions that I think we need to address on the road to empowering metagenomics with the resolution needed to generate reductionist and mechanistic hypotheses on the link between the human microbiome and human health. In my laboratory and with the multidisciplinary network of collaborations that we maintain, we pursue these goals by developing tools to accurately profile strains from metagenomes and by scaling strain profiling to many thousands of metagenomes with manually curated metadata.


Genetic eye diseases constitute a large and heterogeneous group. Individual diseases may cause multiple structural/functional anomalies and developmental features. Family history may be suggestive; however, it may also be challenging, particularly in late-onset conditions or in cases of variable expression.In the current era of genetic advances, diagnosis of a genetic eye disease is facilitated by well-established collaboration between ophthalmologists and geneticists, as increasingly more patients will be asking for genetic counseling and prenatal diagnosis in addition to ophthalmologic management. Molecular investigation of a genetic eye disease requires customized analysis and advanced technology in addition to the requisite detailed family history and accurate ophthalmological diagnosis. A common indication for genetic testing is the validation of a preliminary diagnosis made in clinical practice. The need to determine the prognostic implications of the genotype, assessment of the recurrence risk and in particular, the possibility of specific gene therapy in the near future encourages clinicians to pursue genetic research.1 A previously established genotype-phenotype correlation may add to the value, either by providing accurate prognostic information or by indicating which particular mutation to look for. One such example may be electroretinographic supranormal rod response, indicating KCNV2 mutation type cone dystrophy, which can be precisely detected by Sanger sequencing or qPCR.2We present here a baseline algorithm covering common genetic mechanisms in order to outline a basic molecular approach for ophthalmologists. The first step of the flow chart, a prudent clinical examination with complete description of the phenotype, is indispensible for making a precise and accurate preliminary diagnosis . If the Conventional karyotyping reveals microscopically visible abnormalities in chromosome number and structure, as well as translocations and large indels, and is appropriate as the first-tier test in multisystemic congenital abnormalities. Although conventional cytogenetic analysis may be considered as a screening test in such patients, microscopic diagnosis sometimes requires preliminary clinical diagnosis, designed in order to unveil specific deletions or duplications. A classic example is the small 11p interstitial deletion in Wilms tumor and aniridia, which could only be shown via fluorescence in situ hybridization or multiplex ligation-dependent probe amplification.3 Furthermore, single nucleotide polymorphism array may enable the detection of disease predisposition or drug resistance (e.g. age-related macular degeneration).Array comparative genomic hybridization methods are preferred for genetic eye diseases involving copy number variations. One such example is congenital cataract, which has a very complicated phenotype-genotype correlation and shows clinical heterogeneity. Responsible mutations in crystallins, transcription factors and membrane proteins have been reported.1 Genetic testing, theoretically, can also reveal the underlying ocular problem in cases with subnormal vision but otherwise normal ophthalmological examination , or it can define the high-risk group for an ocular disease and factors that prevent/delay any poor prognosis (i.e. early-onset glaucoma).4Next generation sequencing is the most current technology allowing parallel sequencing of many genes and may cover either a spectrum of known genes or all exons of all genes, allowing the discovery of new causative genes. The latter is called whole exome sequencing, and is a popular and practical investigation tool for developmental diseases.5 Ophthalmologists would benefit from a practical flow chart based on a priori assumption of genetic basis for each genetic eye disease. This would not only save time and money but may also lead to practical advances in diagnosis and management.The ultimate aim is to treat the condition. This is crucial in genetic disorders, in which modern treatment suggestions involve replacement of the missing molecular element. Many ongoing trials regarding gene therapies appear to have promising results for future treatment options.


In this review, we summarize the different limitations of currently available culture models, and discuss the challenges that have to be overcome for developing more reliable and translational platforms for the in vitro study of spinal MN degeneration.Pathogenic conditions involving degeneration of spinal motor neurons (MNs), such as amyotrophic lateral sclerosis, sarcopenia, and spinal cord injury, mostly occur in individuals whose spinal MNs are fully mature. There is currently no effective treatment to prevent death or promote axonal regeneration of the spinal MNs affected in these patients. To increase our understanding and find a cure for such conditions, easily controllable and monitorable cell culture models allow for a better dissection of certain molecular and cellular events that cannot be teased apart in whole organism models. To date, various types of spinal MN cultures have been described. Yet these models are all based on the use of immature neurons or neurons uncharacterized for their degree of maturity after being isolated and cultured. Additionally, studying only MNs cannot give a comprehensive and complete view of the neurodegenerative processes usually involving other cell types. To date, there is no confirmed  However, these neurons are literally in their immature state  and their peripheral axons occurs in neurodegenerative disorders  , as wellre state , raisingre state . While tre state . This fiin vivo? At embryonic day (E) 9.5, gradient signals of two morphogens (retinoic acid and sonic hedgehog) created in the neural tube organize topographical positioning of neural precursor cells and induce their differentiation into spinal MNs  to efficiently and promptly execute their energy-consuming tasks, such as neurotransmitter release and vesicle recycling (in vitro manifest a hand-like morphology (growth cones) and are devoted to active regeneration, lengthening in response to neurotrophic factors supplemented in the culture media  the original cause or trigger of the disease (degeneration); (ii) the molecular and cellular cascade responsible for the degenerative process; and (iii) the regenerative mechanisms after the disease onset. An exception may be spinal cord injuries, for which (i) is already known. Studying the process of axonal regeneration in a simple MN mono-culture without muscle cells may sound reasonable, as this is a process prior to spinal MN settlement. However, careful attention still needs to be paid when utilizing embryonic neurons for understanding adult diseases, as their regenerative potential may not be the same as that of adult spinal MNs. The best would be to harvest primary spinal MNs from adult mice. In fact, several protocols detailing the methods for this are available ; howeverin vivo receive all these glial benefits in a spatiotemporally regulated manner based on local biological requirements. This situation is quite different from that of dissociated or differentiated MNs harvested “naked” in a plate and forced to regenerate in culture media supplemented with fixed and spatially homogenous concentrations of trophic factors.In the ventral horn, spinal MNs are supported by glial cells at the structural, trophic, and metabolic levels . Chondroin vitro, the naked neuron mono-culture is still useful for the investigation of certain biological aspects, such as the capture of disease-related cell-autonomous events. A high-throughput drug screening study that has recently been conducted by utilizing the survival of ALS patient iMNs as readout has identified bosutinib, the Src/c-Abl inhibitor approved by FDA for treating patients with chronic myelogenous leukemia, as a potential therapeutic candidate for ALS . The most utilized stem cells for studying MN biology are mouse ES cells and human iPS cells. The main advantages of these cell lines compared to primary neurons are that they can unlimitedly be expanded and stored as homogeneous populations. They are thus suited for studies requiring a large quantity of neurons for repetitive tasks, such as drug screening, proteomics, and biochemistry. On the other hand, primary MNs, which have fully differentiated in vivo before being dissociated from the spinal cord, should possess a more genuine identity of spinal MNs compared with MNs differentiated in vitro from stem cells. In terms of purity and quantity, however, primary cultures often face variable contamination with other cell types, and the number of viable primary MNs obtainable per embryo is much more limited. Of prime importance, the iPS cells are the only source enabling us to study patient-derived MNs in vitro . With this method, freshly dissected thin cross sections of the spinal cord are directly explanted in a dish. Thus, spinal MNs “embedded” in the explant do not undergo harsh procedures of chemical digestion and mechanical dissociation, but regenerate in vitro within a micro-environment resembling the in vivo condition. This culture system would also allow us to minimize the supplemental control of trophic factors as glial cells integrated in the system may supply them upon local requirements. Noteworthy, the freshly prepared spinal cord culture can serve as a ready-to-use platform for investigating neural networks by electrophysiological recording  for establishing healthy mature and fully functional NMJs in compartmented systems are still under development.Knowing the significant applications that an bjective . In addibjective . What ha growing . By allo growing . Another growing . Despite growing , no studin vitro? Difficulty in culturing these “grown-up” neurons is thought to be associated with their phenotypic changes. As animals grow, MN dendrites in the ventral horn become longer and form a more complex structure, which increases the chance of MNs to receive more injuries during the isolation, leading to their excitotoxicity-associated cell death  we will be able to compare and study, for the first time in vitro, spinal MNs from different stages of maturity  or diseases ; and (3) it will serve as a novel cell source for drug screening. Yet, all these exciting applications are based on an assumption that adult spinal MNs plated in vitro continue to retain their “mature” properties previously acquired in vivo. Therefore, following successful isolation, it is important to verify whether these adult neurons remain as expected or retrieve “immature” properties during regeneration in an artificial in vitro environment.What if we could achieve robust regeneration of adult mouse-derived mature spinal MNs ll death . Anotherll death . As an ain vitro models of many other cell types. Each culture model has different advantages and disadvantages; it is not a matter of which one is superior to the other. What is important is to select the one most fitted for individual objectives. The good news is that the range of possible models is widening, thanks to recent advance in technologies. Nonetheless, there is certainly still quite some room for progress, and further challenges have to be addressed toward establishing more reliable and translational in vitro platforms.Spinal MNs cannot truly mature and age alone. They need good supports and influences from multiple neighbors, or say “coworkers,” like CNS glia and peripheral residents, such as Schwann cells and muscle cells. All these cells constituting the neuromuscular system benefit the MNs either directly or indirectly. Incidents or diseases sabotaging their good relationships may disrupt the healthy natural maturation and/or aging of spinal MNs, and often result in motor disorders of various severities. The different limitations that we have raised and discussed here apply not only to spinal MN cultures but also to MB, SJM, DBR, and SK contributed to devising, editing, and revising 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.


Otolithic afferents with regular resting discharge respond to gravity or low-frequency linear accelerations, and we term these the static or sustained otolithic system. However, in the otolithic sense organs, there is anatomical differentiation across the maculae and corresponding physiological differentiation. A specialized band of receptors called the striola consists of mainly type I receptors whose hair bundles are weakly tethered to the overlying otolithic membrane. The afferent neurons, which form calyx synapses on type I striolar receptors, have irregular resting discharge and have low thresholds to high frequency  bone-conducted vibration and air-conducted sound. High-frequency sound and vibration likely causes fluid displacement which deflects the weakly tethered hair bundles of the very fast type I receptors. Irregular vestibular afferents show phase locking, similar to cochlear afferents, up to stimulus frequencies of kilohertz. We term these irregular afferents the transient system signaling dynamic otolithic stimulation. A 500-Hz vibration preferentially activates the otolith irregular afferents, since regular afferents are not activated at intensities used in clinical testing, whereas irregular afferents have low thresholds. We show how this sustained and transient distinction applies at the vestibular nuclei. The two systems have differential responses to vibration and sound, to ototoxic antibiotics, to galvanic stimulation, and to natural linear acceleration, and such differential sensitivity allows probing of the two systems. A 500-Hz vibration that selectively activates irregular otolithic afferents results in stimulus-locked eye movements in animals and humans. The preparatory myogenic potentials for these eye movements are measured in the new clinical test of otolith function—ocular vestibular-evoked myogenic potentials. We suggest 500-Hz vibration may identify the contribution of the transient system to vestibular controlled responses, such as vestibulo-ocular, vestibulo-spinal, and vestibulo-sympathetic responses. The prospect of particular treatments targeting one or the other of the transient or sustained systems is now being realized in the clinic by the use of intratympanic gentamicin which preferentially attacks type I receptors. We suggest that it is valuable to view vestibular responses by this sustained-transient distinction. Vestibular afferents with regular resting discharge constitute a system for signaling sustained vestibular stimuli, such as maintained head tilts.Vestibular afferents with irregular resting activity constitute a system for signaling transient vestibular stimuli.Otolith irregular afferents originate from a specialized region of the otolithic maculae called the striola and form calyx synapses on type I receptors which have fast membrane dynamics.Otolith irregular afferents are selectively activated by high-frequency (~500 Hz) low-intensity BCV and ACS. Regular otolithic afferents do not respond to these stimuli at comparable levels.Ototoxic antibiotics selectively affect type I receptors and thus the transient system.The 500-Hz vibration causes eye movements in humans, and the preparatory myogenic potentials for these movements are the ocular VEMPs which index the activity of the transient system.Vestibular nucleus neurons exhibit transient or sustained responses, as in the periphery.The early recordings of the response of cat single vestibular nucleus neurons to angular acceleration established that different neurons had very different response patterns to identical angular acceleration stimuli –3. Some This review shows how recent evidence about otolithic responses to sound and vibration demonstrates the value of applying this distinction of sustained and transient from the receptors to the behavioral responses. We suggest it is a valuable principle for interpreting the results of vestibular functional tests—focusing attention on different aspects of the response rather than just treating the whole response as uniform. This is especially clear with the otoliths, but even with the canals it is useful to distinguish between responses to the onset of an acceleration, as opposed to responses during maintained accelerations. The paper will not cover particular areas in great detail since it has already been done in many other reviews to which the reader is referred. Instead, we aim to bring together evidence from physiology to highlight the relevance of this evidence for understanding vestibular function testing as used clinically. The first part of this review covers physiological evidence and the second part covers the application of the sustained-transient principle to vestibular responses.The regularity of resting discharge of vestibular primary afferents is associated with a range of characteristics, such as conduction velocity, axon thickness, and a range of response dimensions, such as gain to acceleration, sensitivity to electrical vestibular stimulation [including so-called galvanic vestibular stimulation (GVS)—DC or low-frequency electrical stimulation of the sense organs]. While afferents from all vestibular sense organs are activated by GVS at approximately equal thresholds  irregulaAngular and linear acceleration of the whole animal have been the usual stimuli in studies of vestibular physiology, and it has been shown in a number of species that regular and irregular neurons have different frequency responses—with irregular afferents  typically having an increased gain and increasing phase lead with increasing frequency—interpreted as showing that irregular afferents are responsive to both acceleration and change in acceleration (jerk). The characteristics of regular and irregular neurons have been summarized by Goldberg’s definitive review of vestibular afferent diversity .In summary, first-order vestibular neurons with regular resting discharge comprised bouton and dimorphic neurons which synapse on the barrel-shaped type II receptors mainly in the peripheral zone of the cristae and in the extrastriolar zone of the otolithic maculae. They are characterized by thin or medium-sized, slow conducting axons and with a low sensitivity to head rotation and relatively low sensitivity to GVS. Irregular first-order vestibular neurons comprised calyx and dimorphic neurons, which innervate the central cristae and the striolar zones of the otolithic maculae, synapsing predominantly on the amphora-shaped type I receptors Figure . They arIn addition, some vestibular afferents show a sensitive response to sound and vibration –15. ReceDetailed analysis of the timing of the action potentials of irregular afferents evoked by sound or vibration show they are phase locked to individual cycles of the ACS up to 3,000 Hz) or BCV stimulus   are not activated by ACS or BCV stimuli—at least in response to stimulus levels used in clinical studies of human otolithic function  and regular (sustained) otolithic afferents to various physiological and even behavioral responses, and thus the likely functional roles of these classes of afferents. One other question is of the mechanisms responsible for the regularity of resting discharge, which is now clearly established as being due to membrane characteristics of the afferent neurons –31.In these studies, the usual result is that in animals with intact bony labyrinths, afferent neurons (either regular or irregular) from semicircular canals are not activated or only weakly activated by very intense stimulus levels of 500 Hz BCV and ACS , 27, 28.More recently, it has been found that even in animals with intact bony labyrinths, irregular canal afferents can be activated by very low-frequency BCV and indeed phase lock to 100 Hz BCV . HoweverHow can vestibular receptors and irregular otolithic afferents respond to such very high frequencies? Textbook schematic diagrams of the cristae and otolithic maculae give the impression that each vestibular sense organ is a uniform structure with receptor hair cells of similar height. It is now clear that is the very opposite of what is the case. Each macula and each crista shows complex anatomical differentiation across the surface. The receptor types are differentially distributed with predominantly type I receptors at the striola . The extMost models of otolithic stimulation model displacement of the gelatinous otoconial membrane with otoconia adherent to its upper surface in response to linear acceleration stimulation, e.g., Ref. , 46. In In light of this phase locking up to such high frequencies, we have suggested that the hair bundles of the striolar otolithic receptors are deflected by each cycle of fluid displacement caused by the stapes pumping into the fluid-filled inner ear . This fl−3 to 10−2) that bundles move instantaneously with any fluid movement. In other words, this coupling of fluid motion to hair bundle is so strong that the hair bundle displacement follows the fluid displacement almost exactly  the regularity of resting discharge and (2) the response to sound and vibration determined by receptor mechanisms. These are two different aspects of the afferent response, and the evidence is that they are determined by different factors.(1)Recent evidence shows that regularity of vestibular afferent resting discharge is due to membrane characteristics –31. That(2)We  have putThe sustained–transient distinction is clear at the level of the primary afferent, whereas these complementary afferent systems would appear to be lost centrally , 56–58. in vitro physiological studies, recording from vestibular nucleus neurons in slice preparations, have shown that at the vestibular nucleus there is clear evidence of different neurons with very different temporal responses to identical stimuli , and that these neurons can be characterized by the same sustained–transient distinction as for primary afferents. Type A neurons show tonic characteristics—maintained firing to a step of injected current—whereas type B neurons show a brief high-frequency burst of spikes at stimulus onset  and disorders will differentially affect the sustained and transient systems.The frequency of the acceleration stimulus is one such tool. Sustained and transient systems have different frequency responses to acceleration stimulation —the irrevia surface electrodes on the mastoids has been used in a very large number of studies of human vestibular responses. GVS is a complex stimulus which acts on all vestibular sense organs  recorded in a variety of species with a variety of recording montages –76. ContAnimal studies have shown that the type I receptors are more vulnerable to the effects of ototoxic antibiotics than are the type II receptors –81. In hThere are profound differences in otolith-mediated responses—even eye-movement responses—due to the characteristics of the otolithic stimuli. For example: on the one hand are maintained eye-movement responses to maintained otolithic stimuli, such as maintained roll-tilt. On the other hand, there are brief eye-movement responses to transient otolithic stimuli such as clicks or brief tone bursts Figure . The otoA maintained rolled head position alters the linear acceleration stimulus to the otoliths and causes both eyes to adopt a rolled position in the orbit in the opposite direction to head tilt –84. ThisIn contrast, brief bursts of 500-Hz BCV, which physiology has shown to selectively activate otolithic irregular afferents originating from striolar receptors, cause short-latency stimulus-locked eye movements in guinea pigs  and alsoIt has been argued that irregular neurons comprise the transient system. But how is it that their activation, for example, by sustained vibration, can result in maintained nystagmus? For example, maintained 100-Hz vibration stimulation of one mastoid in a patient with unilateral loss induces a nystagmus during the stimulation (vibration-induced nystagmus). It would appear that such a maintained nystagmus during a maintained vibration stimulus should be due to the sustained system (regular neurons), which the physiology shows, are not activated by vibration.The answer lies in the fact that for irregular neurons every single cycle of the stimulus is the direct stimulus to the receptor/afferent neuron. So a maintained 100-Hz vibration will cause 100 repetitive activations, once per cycle, of irregular afferents. So the average firing rate of irregular neurons will increase, just as would occur during a sustained deflection of the cupula during an angular acceleration, and a sustained nystagmus will result.stimulus onset and offset. Vibration-induced activation of irregular neurons starts on the first cycle of the stimulus and so one would expect any nystagmus to start at the very onset of the vibration. That is the observed result [summarized in Dumas et al. ].Similarly, the vibration-induced neural activation ceases abruptly at vibration offset and so one expects the nystagmus to cease abruptly without any decay or overshoot. That is the observed result .As vibration frequency is increased on successive trials from 30 to 100 Hz, the number of phase-locked spikes/s will increase and so one expects nystagmus velocity to increase in a corresponding fashion. That is the observed result . For theThere are three pieces of evidence in support of such an account:These results are complemented by other evidence from the preparatory potentials of the muscles activated by BCV, called VEMPs. These are recorded in response to brief clicks or bursts of 500-Hz ACS or BCV, which the physiological evidence shows to selectively activate otolith irregular neurons. In response to stimuli which are specific for otolith irregular afferents, these VEMPs are small EMGs recorded by electrodes beneath the eyes [ocular vestibular-evoked myogenic potential (oVEMPs)] or over the tensed sternocleidomastoid muscle [cervical vestibular-evoked myogenic potential (cVEMP)] –93 Figu. The shoSo, in our terms, ocular counter roll is a test of static (maintained) otolith function—how the otoliths respond to maintained roll-tilt stimulus—in contrast to VEMP responses, which are tests of otolith dynamic responses to transient otolithic stimulation mediated by otolith striolar type I receptors and otolith irregular afferents. Regular afferents are not activated by these transient sounds and vibrations, which are such effective stimuli in eliciting otolith dynamic responses.What is the clinical value of making this distinction? Immediately after surgical unilateral vestibular loss, there is asymmetrical OCR , but oveDuring normal head movements, the neural drives from the sustained and transient systems are combined to generate smooth compensatory responses. For example, in response to brief, passive unpredictable horizontal head angular accelerations (a head impulse), they combine to generate a smooth compensatory eye movement—the vestibulo-ocular reflex (VOR). One index of the adequacy of vestibular responses is VOR gain, typically defined as the ratio of the area under the eye velocity record to the corresponding area under the head velocity record during the head impulse. This clinical test is called the video head impulse test (vHIT) , 106. VOAs noted above, histological studies in animals have shown that gentamicin differentially attacks type I receptors, and so ITG presumably selectively attenuates the contribution of type I receptors to generate responses to transient stimuli –81. We hRecently, this ITG procedure for the treatment of patients with Meniere’s disease has been combined with sequential vHIT testing of semicircular canal function  as a monThere is now strong evidence that otolithic inputs contribute to early transient adjustments to human blood pressure control and muscle sympathetic nerve activity (MSNA) –122. TheRecent evidence suggests that type I receptors may even have a role in triggering escape behavior in response to hypoxia, and by implication in sudden infant death syndrome. Under hypoxic conditions, healthy mice show marked escape movements, but following ITG (which most probably disables the transient system selectively)—mice in the same hypoxic conditions show little escape movement –128. TheThe existence of these complementary transient and sustained systems seems to reflect a fundamental principle in the design of robotic control of limb movement. Designers of robotics have adopted a principle for initiation of movement of robots called a PID controller , 130. ThThere is a big advantage in such a combination especially at the onset of a movement, since the acceleration component initiates the responses rapidly. If there had only been a velocity signal at the onset, the early response would have been inadequate. The appropriate combination of acceleration and velocity provides a functionally effective way of dealing with response initiation. In an analogous fashion, we suggest these transient and sustained inputs are combined to yield dynamically stable oculomotor responses  analogous to PID control. Zhou et al.  have shovia changes in firing rate, whereas irregular afferents give information about self motion via precise spike timing . There is corresponding differentiation of neural types and neural responses at the vestibular nuclei. These different systems—sustained and transient—are differentially susceptible to ototoxic antibiotics, have different frequency responses, different sensitivity to galvanic stimulation, and most interestingly differential sensitivity to high-frequency BCV (500 Hz) and ACS. These differential sensitivities are now allowing the differential contribution of sustained and transient systems to vestibular responses, such as eye movements, and possibly other vestibular-evoked responses—such as blood pressure—as well. The prospect of particular treatments targeting one or the other of the systems is now being realized in the clinic by therapeutic treatment of ITG preferentially attacking type I receptors with reported benefit to patients while retaining considerable vestibular function. This paper has highlighted parallels between the response characteristics of vestibular afferents and basic principles of robotic design.The old terms “vestibular stimulation” or “otolith stimulation” are just too vague—we now know that afferent input with very different dynamics conveys a range of different information. The evidence reviewed in this paper has pointed to the value of regarding vestibular responses in terms of the transient and sustained characteristics.This review is dedicated to Bernard Cohen who has made so many pioneering contributions to understanding vestibular function.IC wrote the paper. P-PV contributed the section about central vestibular responses, HM contributed to the section on PID, and CW contributed to the section about clinical testing. All authors reviewed the text and approved the final paper.IC and HM are unpaid consultants to GN Otometrics, Taastrup, Denmark, but have received support from GN Otometrics for travel and attendance at conferences and workshops. For all other authors, the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many microbial communities live in highly competitive surroundings, in which the fight for resources determines their survival and genetic persistence. Humans live in a close relationship with microbial communities, which includes the health- and disease-determining interactions with our microbiome. Accordingly, the understanding of microbial competitive activities are essential at physiological and pathophysiological levels. Here we provide a brief overview on microbial competition and discuss some of its roles and consequences that directly affect humans. The earth is inhabited by a vast quantity of diverse microorganisms. The relationship between them is determined by the fundamental drive of each species and strain to promote its own survival. For instance, some strains may live in tightly associated up to symbiotic relationships, thus heavily relying on their allies. Conversely, others may engage in ferocious competition, resulting in a relentless war to win over finite resources such as nutrients, light or territory.According to Ghoul and Mitri, a strain is competitive if it shows phenotypes that cause a fitness decrease in a competitor strain . via secretion of digestive enzymes 2424891011In the frame of passive competition, a strain may restrict the competitor’s access to nutrients, e.gIn settings of active competition (or "interference competition"), rival cells damage each other actively or through chemical warfare with the final goal to eliminate the competitor 131416The evolutionary emergence and preservation of numerous competition strategies reflect their central role in the microbial ecosystem. But is competition also common? Do microorganisms fight each other exceptionally or routinely? Interestingly, quantification attempts regarding the competitive properties of microbes show, for example, that 25% of gram-negative bacteria possess genes coding for a Type VI secretion system 12021e.g. no consideration of the natural environment or actual expression of identified genes), they do provide relevant insight into competitive dynamics. One of the early studies using this approach pre-estimates excessive competition between many bacterial species but few cooperative interactions In general, it is difficult to address the complexity of a microbial community, which covers many influencing components, ranging from environmental to genetic factors. Accordingly, competitive aspects of such communities are duly intricate. In an attempt to assess the extent of exploitative competition, simulations of competitive activity using theoretical metabolic models of different bacteria have been run based on available sequence data 23An example for the complexity of microbial communities is the human microbiome. Its different microbial societies need to function steadily in order to support the host’s life and health for the final sake of the individual strains’ survival. Hence, ecological stability in the different microbiota in and on our bodies is essential. But how does competition influence the stability of a microbial community? Generally, it increases ecological stability as well as it leads to a local decrease of microbial diversity 27There are three established possible long-term consequences of microbial competiton that are ecologically stable A winner is declared. One competitive strain dominates the community whereas the "weaker" strain goes extinct 29a) A metabolic niche is established. The competitors are able to coexist because they specialize on different resource types and therefore occupy different metabolic niches. For instance, when distinct bacterial isolates of tree-holes (aquatic ecosystems created for example by a deformed trunk) are brought into co-culture, they initially tend to compete with one another. Eventually, however, they diverge in their use of resources (e.g. using each other’s waste products) as they coevolve 31b) Territorial niches are assigned. The competitors separate into different territorial niches or patches. This outcome may happen in solid or semi-solid structures such as the soil, mucus or an agar surfaces. This phenomenon has been extensively investigated for microbial colonies, which start as well mixed competitive populations and finally form clonal patches onto the agar surface 33c) As mentioned above, competition - with some exceptions - reduces diversity but increases local ecological stability. However, the resulting long-term outcomes in each competitive scenario likely depends on the selection pressures of the surrounding environment. Of note, selection may lead to a microbial arms race, competitive exclusion as well as synergistic division of labour, all in the same environment but in different areas Microbial Cell, Cabral et al. address the intestinal microbiome’s ecological stability in a broader sense et al. found Escherichia coli to outcompete and kill the opportunistic fungal pathogen Candida albicansin vitro by secreting a soluble fungicidal factor C. albicans is found in the gastrointestinal flora of most healthy humans and was long thought to exist in our gut merely as a commensal resident or as an opportunistic pathogen. Indeed, C. albicans is responsible for more than 400.000 life-threatening infections per year, worldwide C. albicans within the gut are becoming evident that emerge from its interaction with bacterial communities. On the one hand, it is implicated in the promotion of bacterial host colonization and virulence upon bacterial co-infection C. albicans might have beneficial effects for the host upon intestinal infections with bacterial pathogens through an increase of a protective immune response in the host Interestingly, in this issue of et al. exemplifies the potential of such investigations to uncover possible new treatment strategies or drugs against dangerous infections. In fact, the study of microbial competition (within our bodies and in the environment) engages many areas of biology and medicine. For instance, competition between co-evolving bacterial strains may lead to antibiotic resistance The identification of a novel fungicidal factor by Cabral Charles Darwin noted that "If it could be proved that any part of the structure of any one species had been formed for the exclusive good of another species, it would annihilate my theory, for such could not have been produced through natural selection."


A growing number of studies on the acquisition of lexical tone by adult learners have revealed that factors such as language background, musical experience, cognitive abilities, and neuroanatomy all play a role in determining tone learning success. On the basis of these findings, it has been argued that the effectiveness of tone learning in adulthood depends on individual differences in these factors. However, it is not clear whether similar individual differences play an analogous role in tone learning in childhood. Indeed, relatively few studies have made comparisons between how adults and children learn lexical tones. Here, we review recent developments for tone learning in both adults and children. The review covers tone training in a range of contexts, including in naive listeners, in native speakers of other tone languages, in listeners with varying levels of musical experience, and in individuals with speech and hearing disorders. Finally, we discuss the parallels between adult and child tone learning, and provide recommendations concerning how findings in adult tone training can provide insights into tone learning for children by accommodating the needs of individual learners. In recent years, researchers have developed a sophisticated understanding of lexical tone acquisition in adults. Long-term experiences such as language and music exert a persistent influence that shapes the perception of lexical tone, and has implications for subsequent training and acquisition of non-native tone contrasts. To date, the vast majority of this work has been conducted on adults. In this review, we summarize the adult tone research literature and highlight several of the emerging themes that may guide future research and subsequently elucidate our understanding of tone processing in children.Many of the world’s languages use pitch patterns called lexical tones as a contrastive feature. These tone languages, such as Mandarin, Thai, and Cantonese, use lexical tones to differentiate the meanings of words. For example, the Mandarin syllable /ma/ can mean ‘mother,’ ‘hemp,’ ‘horse,’ or ‘scold’ depending on which of the four Mandarin tones are used. Similar pitch variations are not lexically meaningful in non-tone languages such as English. Such language differences have been shown to have a profound effect on the processing of lexical tones. For instance, native Mandarin Chinese listeners show an advantage when identifying tones , and alsOne possible explanation for how language background shapes tone processing is that tone and non-tone language speakers rely on different acoustic cues when discerning lexical tones. Specifically, language experience has been shown to shape perception of pitch such that listeners attend to pitch information that is meaningful in their native language , therebyEvidence is mixed concerning whether tone language speakers have an advantage when perceiving tones in a non-native language. On the one hand, numerous studies have shown that prior tone language experience improves subsequent perception of non-native tones. For example, Cantonese listeners outperformed Mandarin and English listeners on Cantonese tones, and Mandarin listeners outperformed Cantonese listeners who in turn outperformed English listeners on Mandarin tones . FurtherHowever, there is also evidence that prior tone language experience may interfere with non-native tone perception. For instance, Cantonese listeners were constrained by their native phonology  on a task using Mandarin tones, but similar constraints were not observed in Japanese or English listeners, suggesting that the native phonological system may interfere when perceiving non-native tones . SimilarIt is well-established that the perception of non-native lexical tone contrasts is difficult for adult L2 learners , particuand unimodal exposure , suggesting that auditory attention is necessary for tracking statistical regularities . In a suexposure .Tone word learning studies require participants to accurately perceive lexical tones in order to differentiate newly learned words, often containing the same segments. Although it has been suggested that listeners may be more aware of phonological segments than tones , a word A growing number of studies are taking into account individual differences among learners by examining pretraining abilities , and memory availability, and examining how these interact with training paradigm designs such as those mentioned above. In a study involving native English speakers, pitch identification ability was a better predictor of performance on a Mandarin word learning task than musicality, language aptitude, or general cognitive ability and predicted generalization to new talkers . High stIn a study examining older adults, learning performance was best predicted by declarative memory capacity rather than baseline sensitivity for pitch patterns or working memory capacity . This fiThis summary of the field on tone training in naive learners suggests that those with better pretraining abilities will benefit most from training. Who is a good versus poor learner appears to be dependent on perceptual and neurophysiological differences. It is also likely that differences can be accounted for by differences in cognitive ability  or variaWe have thus far reviewed tone training in naive listeners with no tone language experience. Other studies have examined how varying degrees of L2 experience with a tone language affects processing and learning of lexical tones. First, let us look at studies of non-tonal L1 speakers who are actively acquiring a tone language as their L2.Non-tonal L1 speakers who learn a tonal L2 provide a fascinating opportunity to examine the flexibility of the human perceptual system to attend to acoustic cues that serve a critical linguistic function in only one of their languages. A tone language learner may rely on either F0 height or direction when perceiving pitch, depending on their language background. L1 English–L2 Mandarin speakers, who rely on F0 height in their L1 and F0 direction in their L2, were exposed to four of the six Cantonese tones, with English monolinguals and L1 Mandarin speakers serving as the control groups. L2 learners, as well as the Mandarin controls, were significantly better at discriminating the contour–level tone pairs than the level–level tone pairs. This suggests that L2 experience increased L2 learners’ sensitivity to F0 direction in the perception of unfamiliar tones . It shouSeveral studies have observed that L1 intonation patterns exert an effect on the perception of non-native tones. For instance, when presented with Mandarin tone continua, Taiwanese listeners were more sensitive to between-category differences than within-category, whereas French listeners were equally sensitive to differences across continuum steps . In anotNeural investigations have shown that reliable brain changes follow a semester of Mandarin learning in college . In thisOther training studies have investigated whether tone language speakers possess an advantage when it comes to learning the tones of an unfamiliar tone language. There is indeed evidence that sensitivity to tones in one language may boost perception of tones in another language , such thFurthermore, native tone language speakers are capable of learning new tone contours in adulthood. Studies examining tone learning in adults have confirmed that language background affects both attentive and non-attentive processing of tone contrasts, but processing of pitch is malleable even in adulthood . AdditioThe above evidence suggests that tone language experience brings subsequent tone learning advantages and that the adult brain is capable of learning novel tone contrasts from a foreign tone language. Whether similar advantages arising from prior tone language experience occur in children remains to be seen.Both music and lexical tone place great importance on pitch, and thus a growing number of studies have investigated whether musical expertise results in tone language processing (and ultimately learning) advantages. Experience-dependent bidirectional transfer effects have been observed between speech and music . On the In terms of training, musical or tone language experience are associated with significantly better non-native word learning proficiency of tone-based words, as compared to individuals with neither musical training nor tone language experience . FurtherTherefore, musical experience improves pitch encoding and leads to some lexical tone training advantages, although this is modulated by other factors. Similar effects would presumably emerge in children with music experience, although this warrants systematic investigation.Tone training has also been investigated in individuals with speech and hearing disorders  and hearing impairments . A small but growing body of research has examined the congenital disorder amusia (or tone-deafness) that impairs the ability to perceive pitch in language and music . The genAmusics who are native speakers of a tone language are also impaired in their ability to discriminate and identify lexical and non-speech tones. The majority of these studies focus on amusic Mandarin speakers. In one study, Mandarin amusics experienced difficulty identifying and discriminating Mandarin tones, with some participants exhibiting signs of lexical tone agnosia, that is, an inability to distinguish lexical tones . InteresCochlear implant recipients are also impaired in their ability to perceive pitch, and this has serious implications for speakers of tone languages. For example, Mandarin-native cochlear implant users scored between 30 and 50% on Mandarin tone recognition tests . To addrThe sections of the review thus far have covered studies that have examined tone learning in adults. The remainder of the article will be devoted to covering the work that has examined these abilities in children, and proposing how the field may be advanced by addressing the research questions that have been raised in the adult tone learning literature.Using discrimination tasks adapted for infants, researchers have begun to understand the timeline of the developmental processes that underpin language development, including the development of sensitivity to lexical tones . During Given that infants’ sensitivity to non-native lexical tones diminishes over the first year of life, this raises questions regarding the specific nature of the resulting perceptual constraint and how it interacts with language experience. In a distributional learning study, 5-, 11-, and 14-month-old Dutch infants were familiarized with a unimodal or bimodal distribution of high-level versus high-falling Mandarin tones; the 5-month-olds discriminated both, the 11-month-olds discriminated the bimodal distribution, but the 14-month-olds were not able to discriminate either . SubsequIndeed, studies involving bilingual infants supplement findings from monolinguals and demonstrate that early experience with multiple languages may improve perceptual flexibility. In one such study, 7.5-month-old Mandarin–English bilingual infants were able to recognize English words that were matched in pitch and Mandarin words matched in tone. 9-month-olds recognized English words that were mismatched in pitch or Mandarin words that contrasted in tone. By 11 months, however, infants had learned to correctly recognize English words that were pitch-matched and -mismatched, but only recognized tonal matches in Mandarin . InteresFurther changes continue to emerge as a result of long-term experiences as the child enters school age and progresses toward adolescence and adulthood. In Cantonese children, tone recognition improves from ages 4 to 6, and from ages 6 to 10, at which point children perform as accurately as adults . MandariIn sum, the available evidence suggests that similar long-term experiential effects may be observed in children as in adults. Native language background, complexity of the native tone inventory, and prior music experience all contribute to tone processing in children. It is not yet clear, however, how such experiences interact with the emergence of tone processing abilities in children, and which factors take precedence at which timescales of development.Tone training studies in young children have revealed that they are initially sensitive to acoustic differences between lexical tones, but those children from non-tone language backgrounds gradually learn to ignore such pitch differences as lexically relevant. Infant studies adapt word learning and familiarization tasks so that they are age-appropriate in their attentional and task demands, including how responses are measured and by employing a reduced number of trials. Studies on bilingual infants suggest that bilingualism leads to certain perceptual advantages that may aid tone learning. At 12–13 months of age, Mandarin–English bilingual infants were able to use tone to differentiate newly learned words in Mandarin (but not English), whereas Mandarin monolingual infants were unable to learn the words until 17–18 months even though they were capable of discriminating the tones . At 18 mStudies on children from tone language backgrounds have shown that while they are capable of learning novel tone categories, their perceptual performance continues to develop and improve throughout childhood as they grow. For instance, both 2- and 3-year-old monolingual Mandarin Chinese children struggled to recognize words in the presence of vowel and tone variation, but sensitivity to these features were age-dependent. Specifically, only 2-year-olds performed poorly in word recognition in response to tone variation, while 3-year-olds showed insensitivity to tone variation and were able to use tones to learn new words, although tone 3 words were most difficult to learn . FurtherOther than studies that have looked at training using speech stimuli, there is also evidence that music training in childhood can boost pitch processing. In one such study, 8-year-old Portuguese children who completed music training for 6 months improved their reading ability and pitch discrimination in speech, but those who completed painting training for the same amount of time did not . These fThese studies provide useful starting points, but many questions remain concerning the tone learning abilities of children. Very little attention has been paid to individual differences in child learners and thus it is not clear what makes some children more successful learners than others. Useful variables to consider include native language experience, pretraining tone sensitivity, prior music experience, working memory availability, and neurophysiological differences. By isolating the combination of factors that matter most for successful tone learning, it may ultimately be possible to tailor tone training proactively to the needs of individual child learners, including those with speech and communication disorders or hearing impairments, which we will now review.A small number of studies have examined children with speech and developmental disorders or hearing impairments. These studies offer some clues concerning how the effects of such conditions may be ameliorated by behavioral interventions. A number of these studies have examined tone language-learning children with cochlear implants. Children with cochlear implants performed significantly worse than normal hearing controls in Cantonese tone perception, and their tone perception developed in a pattern differing from normal hearing children . AdditioThere is also some promising evidence that children with cochlear implants benefit from training interventions. Children with newly-fitted cochlear implants participated in a family-oriented music training program that consisted of pitch, note, and rhythm discrimination exercises on an electric keyboard. Children who underwent musical training had more interest in music and, after 24 months, showed greater development in all areas of music perception. However, only modest improvements were observed for speech perception in the musically-trained children relative to controls . In anotRecent studies have begun to shed light on how tone processing is influenced by other speech and developmental disorders that affect children. Work by Although great strides have been made in developing a detailed understanding of how children attune to the native tone system, work on the disorders that affect lexical tone processing is still in its infancy. Encouragingly, there are already signs that children with speech and developmental disorders or hearing impairments benefit from training interventions designed to improve tone processing. Future work should strive to improve the efficacy of such interventions.In this review, we have summarized recent developments in the tone learning literature for both adults and children. The literature covered addresses tone training across a range of adult learners differing in their prior language experience from naive listeners, to L2 tone language learners, to native speakers of other tone languages, to those with varying levels of musical experience, as well as individuals with speech and hearing disorders. Below we relate each of these factors to the most relevant research conducted in infants and children.Evidence from speech perception, word learning, and neurophysiological studies on adults has demonstrated that prior language experience exerts a profound and persistent influence on the processing of non-native lexical tones. Native tone language experience may aid processing of similar tone contrasts, but may also in some cases interfere with the processing of non-native tones . Additionally, speakers of non-tone languages may benefit from similarities to native intonational patterns . In geneAside from native language experience, musicianship is another long-term experience that profoundly alters processing of pitch both in speech and non-speech stimuli. While research on the experiential effects of music on adults is growing rapidly, research into the effects of musical training in childhood on lexical tone processing is still in its infancy; however, the few studies that have been conducted have reported that musical training is beneficial for pitch processing .Several studies on adults present converging evidence that pretraining perceptual abilities account for word learning performance . FurtherStudies on speech and hearing disorders in adults have revealed that several conditions may lead to difficulties in lexical tone processing. Adults with congenital amusia are impaired in their ability to identify and discriminate lexical tones . SimilarIn sum, research into the development of sensitivity to lexical tone has revealed that perception, word learning, and subsequent language development follow a complex schedule that is influenced by long-term experience such as active language exposure and bilingualism. In comparison, work on adults has revealed long-term experiences such as language background and musicianship greatly affect processing of non-native lexical tones, that short-term training can improve non-native tone processing, and that individual differences among learners may predict tone learning outcomes. Exploring the interaction of these factors in childhood tone acquisition and tone learning will advance the field of infant and child lexical tone processing and lead to improved learning and interventions for those encountering difficulties in processing lexical tones.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it 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.


Additionally, retrospective clinical trials have suggested that regional anesthesia for cancer surgery  might reduce the risk of recurrence. Lidocaine, a LA, which can be administered intravenously, is widely used in clinical practice for multimodal analgesia. It is associated with a morphine-sparing effect, reduced pain scores, and in major surgery probably also with a reduced incidence of postoperative ileus and length of hospital stay. Systemic delivery might therefore be efficient to target residual disease or reach cells able to form micrometastasis. Moreover, an in vitro study has shown that lidocaine could enhance the activity of natural killer (NK) cells. Due to their ability to recognize and kill tumor cells without the requirement of prior antigen exposure, NKs are the main actor of the innate immune system. However, several perioperative factors can reduce NK activity, such as stress, pain, opioids, or general anesthetics. Intravenous lidocaine as part of the perioperative anesthesia regimen would be of major interest for clinicians, as it might bear the potential to reduce the risk of cancer recurrence or progression patients undergoing cancer surgery. As a well-known pharmaceutical agent, lidocaine might therefore be a promising candidate for oncological drug repurposing. We urgently need clinical randomized trials assessing the protective effect of lidocaine on NKs function and against recurrence after cancer surgery to achieve a “proof of concept.”Surgical removal of the primary tumor in solid cancer is an essential component of the treatment. However, the perioperative period can paradoxically lead to an increased risk of cancer recurrence. A bimodal dynamics for early-stage breast cancer recurrence suggests a tumor dormancy-based model with a mastectomy-driven acceleration of the metastatic process and a crucial role of the immunosuppressive state during the perioperative period. Recent evidence suggests that anesthesia could also influence the progress of the disease. Local anesthetics (LAs) have long been used for their properties to block nociceptive input. They also exert anti-inflammatory capacities by modulating the liberation or signal propagation of inflammatory mediators. Interestingly, LAs can reduce viability and proliferation of many cancer cells  Thein vitro has also been linked to the inhibition of Src activation by the LAs (v1.5 in colon cancer cells) on the blockade of the voltage-gated sodium channel (VGSC)  , 51. Canl (VGSC)  as well in vitro , 53–57. in vitro .in vitro effects, a very recent study showed encouraging results regarding a possible inhibition of tumor growth by lidocaine in a xenograft model in vivo  and were not compatible with concentrations found after a systemic use of the drug (−8 to 10−6 M) may instead preserve cytotoxicity of isolated human NK cells  cancer might also benefit from an antimetastatic effect, ideally associated with a subsequent increase in recurrence-free and overall survival. However, due to the fact that there is currently not enough clinical evidence to support this hypothesis, we urgently call for clinical trials evaluating the effects of perioperative lidocaine during cancer surgical period to answer the question once and for all: is there a beneficial effect or not?Both authors contributed equally to this work.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.