Predicting gene functions from genome sequence alone has been difficult, and the functions of a large fraction of plant genes remain unknown. However, leveraging the vast amount of currently available gene expression data has the potential to facilitate our understanding of plant gene functions, especially in determining complex traits. Gene coexpression networks—created by integrating multiple expression datasets—connect genes with similar patterns of expression across multiple conditions. Dense gene communities in such networks, commonly referred to as modules, often indicate that the member genes are functionally related. As such, these modules serve as tools for generating new testable hypotheses, including the prediction of gene function and importance. Recently, we have seen a paradigm shift from the traditional “global” to more defined, context-specific coexpression networks. Such coexpression networks imply genetic correlations in specific biological contexts such as during development or in response to a stress. In this short review, we highlight a few recent studies that attempt to fill the large gaps in our knowledge about cellular functions of plant genes using context-specific coexpression networks. The most alarming fact is that less than 1% of all known genes in important crops like maize and rice have experimentally identified functions3. The less precise orthology-based function assignments are still the default method for newly sequenced plant genomes4. However, despite having a great degree of similarity in sequence or protein domains, genes can evolve for divergent functions5, especially those involved in specialized metabolism (SM)6, leaving younger plant genes less annotated7. Nevertheless, homology-based function annotations are coupled with other experimentally derived manual annotations8 and made available as Gene Ontologies (GOs) for several plant species9. The plant GO and other function annotation catalogs11 provide excellent leads, ora priori evidence, to enhance the development of GCNs that offer a scalable and dynamic framework for prediction of gene functionsin silico.The most important utility of gene coexpression networks (GCNs) is in expanding the current state of functional annotations of plant genes. The time-honored technique of genetic screens and loss-of-function mutant analyses for gene function characterization has obvious limitations12, extraction of densely connected gene communities becomes the next step in gene network analysis. The general idea is that because the genes in each community have a higher degree of coexpression, they are more likely to be under the same regulatory program that dictates their expression and therefore might also be functionally related13. These communities are broadly referred to as “clusters” or “modules” of coexpressed genes.GCNs are constructed by connecting pairs of genes if they have high  correlation in their expression profiles across a large set of samples  legend. 17. These predefined functional gene sets for plants can be obtained from annotation catalogs like the GO18, the MapMan bins19, and other pathway databases20. GSA therefore enables propagation of known function information from already annotated genes within each module to genes yet unannotated. However, this technique has several caveats21, and the strength of gene function prediction in this manner depends largely on the quality and expanse of available annotations for the species under consideration. Additionally, a well-designed statistical framework for detecting familiar DNA motifs, or ade novo motif search, in the promoters of module genes potentially links transcription factors (TFs) as putative regulators of module expression23.Biological insights from the resulting clustering data can be gained by using well-established computational approaches in gene set analysis (GSA). A wide variety of GSA methods are available that can be used to evaluate whether a predicted module is significantly over-represented with genes already annotated for certain functional categories representing biological processes or pathways30. The popular methods, challenges, and caveats associated with construction and use of GCNs are reviewed by others33.The typical steps involved in construction of GCNs are shown in Figure 1. Some studies report pre-computed global plant gene networks in the form of web applications that provide excellent resources and tools for users to query and visualize subnetworks of interest34. Of these methods, weighted GCN analysis (WGCNA)35 has been embraced by plant biologists as the most popular method to identify and analyze gene modules in specific developmental phases of a variety of plant species.Whereas the construction of a GCN is fairly simple and straightforward, partitioning of a coexpression network into seemingly diverse functional modules is not a trivial task. Several approaches of module detection from gene expression are proposed in the literature and were comprehensively evaluated recentlyet al.36 profiled gene expression in different compartments of maize kernels at the filling stage and identified a module specific to the basal endosperm transfer layer (a tissue layer important for nutrient transport during grain filling). This study also found a link to MRP-1 as a likely regulator of this module by analyzingcis-regulatory elements enriched in the promoter of genes in this module36. Similarly, an analysis of modules in developing seeds of two contrasting cultivars of soybean led to the identification of the role of a cytochrome P450 family gene which increases seed size and weight upon overexpression37. In wheat, Wanget al.38 identified a module that showed strong association with spike-related traits. The authors of this study validated this module by characterizing three new member genes that showed altered spike complexity when overexpressed in an elite variety of wheat38. An attempt to identify modules of seed development has also been made by using two cultivars of chickpea with contrasting seed size and weight39 and seeds of two different species of cotton40.In the context of seed development, ZhanFragaria vesca) fruits, Shahanet al.41 created three separate coexpression networks: one represented early-stage floral tissues, another spanned the development of pre-fertilized flower to fruits, and the third represented fruits at the ripening stages41. These networks were helpful in establishing the role of the ghost tissue in iron transport post-fertilization41. To mine these networks, the authors of this study built an interactive web interface, which is of value to other users in the field (www.fv.rosaceaefruits.org).To study the development of woodland strawberry  in a non-exhaustive manner under default settings. It is still unclear whether this is due to the nature of underlying datasets or whether other relevant modules (or subnetworks within the identified WGCNA modules) still exist in these development networks. We believe that a vast majority of plant transcriptomes are still under-utilized in the retrieval of biologically relevant information. Secondary analyses of these datasets can reflect on new knowledge pertaining to the originally assayed biological processes, especially the role of regulatory genes48, which is often overlooked in terms of the potential in typical GCN analysis.These recent studies have highlighted the efficacy of the WGCNA as a quick and easy method to process new, high-resolution developmental transcriptomes and recover plant gene modules. However, assessing the stability of predicted modules and performance in terms of accuracy is highly desirable for generating new hypotheses50. A large number of dense modules would likely separate large metabolic pathways and biological processes into their constituent parts. Intuitively, an important cautionary aspect of such an analysis is controlling the granularity of resulting modules51. Very large modules will fail to answer the fundamental question of why the genes are clustered that way. It becomes difficult to explain the regulatory cohesiveness of large modules, as these will incorporate genes that lie on distant bifurcated branches of large metabolic pathways that show significant levels of correlation in expression52. Conversely, a finer granularity will break the network into too many very small modules (fewer than 10 genes), essentially hampering the statistical process of establishing any functional or regulatory context for these modules, rendering them unusable.In another type of module identification setting, the goal is to cluster a GCN by using a graph clustering algorithm optimized to detect a large number of dense modules. This type of study aims to partition the GCN into many functionally cohesive gene modules, typically in the range of a few hundred modules per network. This is a desirable property of biological network analysis, and several algorithms exist that allow the user to define module size as desired53. For example, modules in the Rice Environment Coexpression Network (RECoN)26 were identified by optimizing the parameters of a graph-based clustering algorithm that works under the principle that functionally related genes have denser connections within a network50. With controlled minimum cluster size and density threshold to partition the network, 1744 dense abiotic stress–associated rice modules were extracted from RECoN. Most of these modules contain genes sharing related GO terms and thus can be used as gene sets (in addition to gene sets from GO and other function databases) in enrichment analysis of new differential expression profiles. The RECoN webtool (available athttps://bit.ly/2BOky7x) also provides a window to mine undiscovered novel modules associated with tolerance to abiotic stresses in rice26.A balance between the number of modules, average module size, and other network topological features such as separation  and density (how well genes within each module form a clique) should be carefully monitored by tuning parameters of the clustering algorithm54. Of these, 29 modules that contain differentially expressed genes in nine previously known male sterile mutant lines are regarded as important to anther development54. This study also compared the rice anther modules to modules in FlowerNet, a GCN for anther and pollen development in Arabidopsis55, and found a significant amount of conservation of gene coexpression between rice and Arabidopsis during anther development.An example of a graph-based clustering algorithm applied to a developmental context in rice is the RiceAntherNet, which delineated 545 modules related to anther and pollen developmentMedicago truncatula and Arabidopsis suggests conserved genetic pathways related to defense mechanisms56. Phylogenetic conservation of coexpression modules was examined in greater detail recently for several model plants7. One of the observations of this study was that genes from the same phylostratum tend to be more frequently connected in GCNs7, suggesting new uses of existing GCNs in extrapolating gene functions of lesser-studied plant species. Moreover, it has been shown that coexpression, rather than physical proximity on chromosomes (biosynthetic gene clusters), is a more reliable signal for predicting genes involved in SM57. Understanding the mechanisms of SM is of great interest in the study of medicinal plants58.GCNs have also been instrumental in the discovery of novel biological phenomena. For example, the conservation of a longevity module betweenAnother desirable characteristic of coexpression network modeling is incorporating information about regulatory interactions in the process of module identification. This type of study leads to the discovery of modular gene regulatory networks (GRNs). GRNs are different from GCNs in the sense that GCNs treat TF and non-TF nodes (genes) similarly whereas GRN involves sophisticated reverse-engineering algorithms that operate on TFs differently. The objective of a GRN is to capture direct, causal edges between TF and their putative targets and filter spurious indirect correlations that naturally arise in coexpression data.et al.59 shows that expression data from TF knockouts or overexpression experiments can be very informative in predicting targets but this type of data is useful mostly for capturing downstream regulatory effects61. On the other hand, time-series expression data can capture temporal dynamics of regulatory networks more systematically63. For example, a time-series transcriptome dataset was used to predict GRNs active for lateral root initiation in Arabidopsis, which revealed genetic cascades involved in positive and negative feedback loops as well as target genes of the AUXIN RESPONSE FACTOR 764. Steady-state expression data have also been very useful in regulatory network analysis and predicting TF function. For example, using a collection of gene expression datasets from Arabidopsis seeds, subnetworks associated with desiccation tolerance (DT) were predicted, leading to the identification of three novel TFs now confirmed for their roles in mediating seed DT65.Biological interpretations of predicted GRNs depend largely on the type of transcriptional dataset used. A meta-analysis by Marbachet al.66 developed a dynamic Bayesian network (DBN)-based framework which showed us a broader picture of the spatiotemporal dynamics of stem cell differentiation in the roots of Arabidopsis. Although DBNs are able to unfold cyclic processes and dependencies in time-course expression data, they are inherently limited by the computational complexity which increases with the number of genes61. The algorithm proposed by de Luis Balagueret al.66 seems to circumvent this limitation by applying DBNs to smaller sets of genes within each spatially distinct coexpressed cluster within the roots. This combination of spatial and temporal expression data in GRN inference established a new role of PERIANTHIA (PAN) as a stem cell regulator66.Recently, de Luis Balaguer68. An earlier meta-analysis of some of the popular approaches suggests integrating predictions from different algorithms to boost the accuracy of the consensus GRN59. This technique was later implemented in Arabidopsis stress datasets to predict an oxidative-stress GRN69 and was further explored in the development of a secondary cell wall biosynthesis network70. The authors of the former study used “coregulated” gene pairs instead of “coexpressed” gene pairs into the k-means clustering framework and recovered 572 stress-related modules69.There are more than a dozen other published algorithms for inference of GRNs from large-scale expression data. Several concepts in GRN inference, available algorithms, and their limitations and applications in plant studies are well summarized by others as a primer to interested researchers71, encouraging the application of standard GRN inference techniques to relatively lesser-studied crop genomes. For example, Xionget al.47 re-analyzed the maize embryo and endosperm development dataset72 and predicted modules and regulators involved in the transport of nutrients to the developing seed47.Because a large sample size can be obtained for Arabidopsis, it has been a favorite model in many currently reported GRNs. However, rapidly expanding data repositories allow selection of interest-appropriate transcriptional datasets73. Furthermore, an integrative analysis of ChIP-seq, mRNA-seq, and miRNA-seq data revealed SEP3 as an upstream regulator ofMIR319a andTCP4, which together form a feed-forward loop to regulate petal development74. In the context of GRNs that manifest during abiotic stresses, Wilkinset al.75 employed the concept of ERGINs  in rice. They integrated chromatin accessibility data (ATAC-seq) with the current state of knowledge about putative regulatory interactions in rice and used these data points aspriors to learn a GRN from expression dataset of five tropical Asian rice cultivars grown under abiotic-stress conditions in the field as well as greenhouses. The study identified regulatory interactions between 113 TFs and 4052 target genes of rice75.Inference of GRNs can be further enhanced in a direct network framework (non-modular GRNs), where the primary goal is to explore regulatory targets of a few TFs of interest in more detail as focused small-scale subnetworks. For example, a combination of transcriptional and chromatin immunoprecipitation data (ChIP-seq) revealed GRN components involved in coordinate regulation of root hair growth in Arabidopsis21, one has to consider that there are several caveats associated with using prior knowledge from GO  for function prediction from GCN modules. Predictions can be very biased toward genes and categories that are extremely well annotated and can be driven solely by other computationally predicted annotations rather than empirical evidence.The current state of accumulated plant gene expression data has immense potential for the discovery of components involved in complex traits. The property of modularity in gene networks can be exploited and gene modules treated as fundamental biological units with dynamic expression and regulatory properties. The techniques of module extraction have proven to be very effective in experimental validations while also suggesting a vast scope for improvement in terms of not only statistical methods used but also how gene networks are perceived and evaluated for research. As noticed by Gillis and Pavlidis76. On the other hand, specifying an overarching biological theme in selection of datasets offers intuitive concepts that can be objectively tested. For example, just like individual transcriptomes, GCNs created to study one particular biological process  can be considered static. Comparison of GCNs constructed from conditionally distinct samples, or differential coexpression analysis, will provide valuable information on how plant systems alter their mechanisms in response to different developmental cues and environmental perturbations78. Moreover, a comparison of sets of modules derived under different contexts should potentially map and distinguish modules that are conserved throughout growth and development from those that are under constant rewiring.Transcriptome datasets integrated in a global manner capture broad, constitutive functional relationships that might not vary much with different tissues or organs, developmental phases, or environmental cues like biotic or abiotic stress79 would be extremely useful for integration with gene lists from genome-wide association study (GWAS) datasets in a systems genetics approach to probe complex agricultural traits81.One major question that remains is how to systematically produce a ranked list of genes most relevant to a given trait/process of interest from these complex interconnected gene relationships in networks. Information buried within hundreds of thousands to sometimes millions of predicted functional relationships is not intuitively tractable for researchers interested in selecting a few actionable candidate genes relevant to a biological process of interest. Research toward development of computational tools capable of using gene networks to systematically enrich gene prioritization pipelines82 and study crop genetics as outlined in recent reviews84.It is important to recognize that gene expression data by itself could have limited potential in deciphering cellular organization, regulated at various levels. However, we are optimistic about the future, as integrating signals from heterogeneous molecular datasets will enable training of smart algorithms to identify genomic patterns of components already known to be associated with a phenotype of interest. The trained models then can be used as predictive tools to discover new genes associated with the phenotype


The development of the nervous system relies on the coordinated regulation of stem cell self-renewal and differentiation. The discovery that brain tumours contain a subpopulation of cells with stem/progenitor characteristics that are capable of sustaining tumour growth has emphasized the importance of understanding the cellular dynamics and the molecular pathways regulating neural stem cell behaviour. By focusing on recent work on glioma and medulloblastoma, we review how lineage tracing contributed to dissecting the embryonic origin of brain tumours and how lineage-specific mechanisms that regulate stem cell behaviour in the embryo may be subverted in cancer to achieve uncontrolled proliferation and suppression of differentiation. Summary: Lineage-tracing work in glioma and medulloblastoma reveals similarities between neuronal development and brain tumours, identifying potential new therapeutic avenues that exploit vulnerabilities in tumour growth patterns. Neurological cancers are among the most feared malignancies. They include medulloblastoma, the most common malignant brain tumour in children, and high-grade glioblastoma, one of the most lethal adult cancers  Louis e. TreatmeFor many years, research has focussed on what different types of neurological tumours have in common with other malignancies and with each other, e.g. the disruption of classic oncogenic and tumour suppressor pathways, but this approach has had little effect on improving survival rates. More promising perhaps is the emerging consensus that brain tumours are maintained by a specific neural or glial cancer ‘stem cell-like’ population that self-renews and gives rise to differentiated progeny . WhetherThe possibility that neurological cancers are ‘locked in’ to a developmental programme and may retain many of the controls that impinge on these cell populations during development opens up new and exciting opportunities for understanding and targeting these cancers. Some of these opportunities are already being exploited in the treatment of paediatric neurological malignancies, where the relationship of cancer cells to spatially and temporally distinct embryonic precursors is better understood . For exaIn this Review, we will consider both paediatric and adult central nervous system tumours through the lens of the developmental biology paradigms that they exploit to maintain a stem/progenitor identity, while at the same time producing both proliferating and differentiating progeny. We will also discuss the extent to which viewing these cancers as diseases of development might reveal new therapeutic approaches, exploiting tissue-specific oncogenes and the aberrant developmental phenotype while sparing normal tissue.in vitro, although how this behaviour relates to their in vivo role remains somewhat unclear  and progenitor cells are more readily transformed than differentiated cell types, and embryonic radial glia cells (RGCs) are more prone to transformation than postnatal stem cells Fig. 1). Overall, it is clear that brain tumours arise from multiple cell types that are distinguishable both by location and by degree of differentiation, although the precise cell of origin is rarely clear. Morphologically similar cancers may yet be shown to arise from different cell populations. Moreover, mature brain cells are not completely resistant to oncogene-mediated transformation, as astrocytes and even mature neurons can dedifferentiate to form gliomas in mice . HoweverWhile tumours can arise from progenitor populations, the extent to which they must revert to a stem cell-like state to overcome restrictions in progenitor cell proliferation as well as lineage restriction is an area of debate. It is clear that progenitors can initiate tumour growth in medulloblastoma and glioma [granule neuron precursors in medulloblastoma and oligodendrocyte precursor cells in gliomas ], while In addition to dedifferentiation, an active impediment to differentiation could also ‘trap’ cells in a proliferative pro-tumourigenic state . MechaniAlthough much evidence points to recapitulation of developmental programmes in the behaviour of brain cancer cells, these could also be undergoing an aberrant regenerative process that, in itself, could exploit mechanisms originally active in adult or embryonic NSCs. This is an exciting and yet poorly investigated topic and future research should focus on understanding the similarities between embryonic neural stem/progenitor cells, adult neural stem/progenitor cells and injury-reactivated cells, and investigate the potential contribution of regenerative responses to brain tumour formation .Although the highly proliferative capacity of fate-restricted progenitors plays a significant role in tumour growth and progression, therapies that target proliferation have drastically failed, mainly because the resident slow-cycling stem-like cells can become reactivated and cause tumour relapse . To deveGenomic analysis of individual human medulloblastomas immediately post-diagnosis and after therapy showed that fewer than 12% of diagnostic genetic events were present in the relapsed tumour sample. Indeed, the genetic clone seen to dominate the tumour was different before and after therapy; close analysis revealed that the dominant clone in relapsed tumours arose from a previous minor clone that was, nevertheless, present at initial diagnosis . SimilarIn a similar vein, new findings have been reported on tumour cell dynamics in glioblastoma using a novel clonal fate mapping approach based on genetic barcoding previously applied to mammary tumour models . By combThe studies discussed in this section not only support the existence of seemingly conserved lineage hierarchies in brain tumours that are reminiscent of a normal developmental programme, but also shed light on the relative contribution of genetic variation and developmental mechanisms to inter- and intra-tumoural heterogeneity. This is an important area of study, as resolution of the clonal dynamics and lineage progression of neurological tumours could provide novel approaches to therapy. As shown in the context of other epithelial tumours , brain tAlthough some oncogenic events are shared across multiple tumour types, distinct genetic lesions associated with specific types of tumours point to intrinsic differences in the way cells of different lineages respond to oncogenic assault. Moreover, lineage-specific transcriptional regulators have also been identified as context-dependent oncogenes and/or tumour suppressor genes, reinforcing the idea that key genes that regulate normal developmental lineages may become deregulated in cancer, subverting their normal function and resulting in uncontrolled proliferation and suppression of terminal differentiation . Such liIn eukaryotes, DNA is wound around a core of nucleosomal histone proteins to form chromatin. Chromatin organization is of fundamental importance in the establishment and maintenance of cell-type specific transcriptional programmes during development and differentiation, and imposes the environment in which tissue-specific transcriptional regulators must act. Not surprisingly, alterations to the chromatin landscape can profoundly impact cell fate decisions in development and cancer. At its simplest level, chromatin remodelling is achieved through the concerted activity of proteins and enzymes that regulate histone methylation, histone acetylation, DNA methylation, and nucleosome tri-dimensional structure and repositioning . The impATRX and DAXX. Interestingly, in addition to mutations at key regulatory residues in histone H3.3, mutations have been reported in ATRX and DAXX , is responsible for H3K27 trimethylation, a repressive mark that is tightly associated with inactive gene promoters. EZH2 is upregulated in various cancers, including medulloblastoma and glioblastoma, and it can act as a critical regulator of neoplastic proliferation, maintenance of stem cell-like features and inhibition of differentiation . For exain vivo , whereasin vitro . Howeverwith Myc . This reKDM6A  and the histone methyltransferases mixed lineage leukaemia, MLL2 (KMT2D) and MLL3 (KMT2C), display inactivating and truncating mutations, suggesting tumour suppressive functions. Interestingly, KDM6A and MLL2 mutations have been found to be mutually exclusive, further reinforcing the likelihood that they regulate similar processes , and less frequently in IDH2, occur in 80% of grade II and grade III astrocytomas and oligodendrogliomas, and are also found in high-grade glioblastomas that have arisen over time from these lower-grade gliomas . IDH mutRecent excitement has accompanied the identification of enhancer regions where multiple transcriptional regulators are bound, and which direct a very high level of gene expression, so-called super-enhancers. Super-enhancers are thought to be essential for maintenance of cell identity , whereasAs described above, the majority of individuals with DIPG carry H3.3 mutations that are often accompanied by a reduction in the levels of PRC2-mediated H3K27 trimethylation. However, novel epigenetic analyses demonstrate that several genes not only retain H3K27 methylation but also showed increased H3K27 acetylation , an epigMany transcription factors with well-characterised roles in neurogenesis and development of the nervous system have subsequently been identified as lineage-specific oncogenes and/or tumour suppressor genes in cancers of the central nervous system (CNS). This may illustrate the close relationship between normal developmental processes and tumourigenesis, and may reflect the influence of stem/progenitor cell positional identity on the response to oncogenic pathways. Indeed, stem and progenitors cells located in discrete brain regions and embedded in different supportive niches possess unique transcription factor codes from patterning processes , as wellSOX2 is a prominent member of the sex-determining region (SRY) box 2 family of proteins that have wide-ranging roles in the developing embryo and in adult stem cells. Although eclipsed in recent years by its identification as a key pluripotency factor, SOX2 has also been extensively studied in the context of its important roles in nervous system development and adult NSC activity. Likely reflecting these activities, SOX2 has emerged as a central player in neurological cancers.SOX2 is often highly expressed in glioblastoma and its knockdown reduces proliferation and tumourigenicity in glioblastoma tumour-initiating cells . MirroriThe main functions of proneural basic helix-loop-helix (bHLH) transcription factors are to specify cell fate, to regulate NSC proliferation, and to drive neuronal differentiation during embryonic and postnatal development . AlthougDuring embryonic and postnatal development, ASCL1 plays an important role in the regulation of NSCs and oligodendrocyte precursors . ASCL1 iASCL1 expression is maintained in NSCs and glioma stem cells in culture, where it is essential for their proliferation and self-renewal in part through activation of Wnt signalling . HoweverTreatment of glioma cells with bone morphogenetic protein (BMP) causes cells to enter a state of reversible quiescence characterised by astrocytic marker expression . In contThe many roles of ASCL1 in stem/progenitor regulation indicate that its activity must be tightly controlled and, indeed, post-translational regulation by phosphorylation has been described. For example, multi-site phosphorylation by extracellular signal-regulated kinase ERK biases Ascl1-positive progenitors towards a proliferative glial program responsible for astrocytoma initiation . Moreovein vitro  with OLIG2 (but not with ASCL1) in differentiated glioblastoma generates cells capable of initiating tumours with high efficiency . MoreoveATOH1 is expressed in granule neuron precursors of the postnatal cerebellum and is highly expressed in SHH-type medulloblastomas  Salsano. AlthougRegulation of Atoh1 protein expression and stability is crucial for lineage progression and granule neuron precursor differentiation; Atoh1 destabilization and degradation, which coincides with NeuroD1 upregulation, is a key requirement for progression down the granule neuron lineage . Thus, pMuch attention has been paid to the unpredictable heterogeneity of brain tumours and their aggressive growth characteristics, which have been used to explain their general resistance to treatment . HoweverASCL1 and ATOH1 are predominantly expressed in embryonic and postnatal neurological development, and make appealing targets for therapy, although attention should be paid to the residual function of these genes in the small population of adult NSCs (Indicative of an underlying hijacking of neurodevelopment programmes, a number of transcriptional regulators of developmental neurogenesis act as lineage-specific oncogenes in CNS cancers. Genes such as ult NSCs  and to pult NSCs , and poiAlthough killing cancer cells is almost always the goal of current therapies, if CNS cancers arise from a dysregulation or stalling of developmental processes, an exciting possibility emerges that reactivation of a programme of differentiation will ultimately generate post-mitotic cells, and thus halt tumour growth . This idin vivo remains to be tested fully. Another area in its infancy and yet to be explored fully in the CNS is the concept that changing the tumour microenvironment may lead to changes in behaviour of the tumour cells themselves; by manipulating the niche, we may shut down the tumour stem cell-like programme. Such a possibility has been suggested by work in other tissues but remains open for investigation in the nervous system (Beyond the promise and obvious challenges of targeting individual transcriptional networks, our understanding of how the wider epigenetic landscape influences fate choice, proliferation and differentiation is constantly improving. A clear goal is to use drugs that can influence the epigenome to change the fate and behaviour of cells in response to the endogenous transcriptional programmes, although the specificity of this approach s system .Overall, it is clear that our understanding of the behaviour of brain tumour cells is growing rapidly and will be further enhanced by understanding how aberrant tumour cell behaviour often represents a reversion to a dysregulated developmental phenotype. If we are to further understand this phenomenon and to exploit emerging vulnerabilities that result in either the death or differentiation of tumour cells, we need to have more engagement between developmental biologists and cancer biologists. After all, in many ways cancer is ‘development gone wrong’, so developmental biologists are as well placed as any scientists to help understand and treat these devastating diseases.


Secondary metabolites (SM) produced by fungi and bacteria have long been of exceptional interest owing to their unique biomedical ramifications. The traditional discovery of new natural products that was mainly driven by bioactivity screening has now experienced a fresh new approach in the form of genome mining. Several bioinformatics tools have been continuously developed to detect potential biosynthetic gene clusters (BGCs) that are responsible for the production of SM. Although the principles underlying the computation of these tools have been discussed, the biological background is left underrated and ambiguous. In this review, we emphasize the biological hypotheses in BGC formation driven from the observations across genomes in bacteria and fungi, and provide a comprehensive list of updated algorithms/tools exclusively for BGC detection. Our review points to a direction that the biological hypotheses should be systematically incorporated into the BGC prediction and assist the prioritization of candidate BGC. Amycolatopsis orientalis, is considered a last-resort drug for Gram-positive bacterial infections and life-threatening diseases such as severe colitis caused by Clostridium difficile. Salinosporamide A was first isolated and characterized from Salinispora tropica in 2003 and acts as a potent anticancer reagent that has entered several clinical trials for various types of cancers, including melanoma, pancreatic, and lung cancer , many of which play vital roles in medicine, such as antibiotics and anticancer reagents. For instance, erythromycin, azithromycin, and penicillin are beneficial antibiotics that treat several bacterial infections in lungs, middle ears, and sexually transmitted diseases  and linezolid, the first commercially available oxazolidinone antimicrobial (2000); no new structural classes of antibiotic were introduced to the market . We described clearly in this review that the biological background of BGC prioritization can be more complex than just the resistance genes. We also discuss to which extent these hypotheses might be useful for the computation of BGC prioritization in different genera. Not only do we provide (1) the most complete collection of the biological hypotheses associating with BGC formation and (2) the most updated list of bioinformatics tools exclusively for BGC prediction, our review points to a direction that future BGC prediction tools should be incorporated with the biological hypotheses, leading to the prioritization of candidate BGC for the generation of bioactive compounds.Here, we summarize three hypotheses—based on the observation that some BGCs contain duplicated or resistance genes and the phenomena that some microbes can acquire resistance related genes by horizontal gene transfer; therefore, these hypotheses provide clues for prioritizing BGCs through bioinformatics analysis tools.The resistance hypothesis states that within the BGC there is at least one gene conferring resistance against the potentially harmful secondary metabolites that the organism produces. The resistance mechanism can be categorized into three notable strategies, i.e., target-based strategies, drug efflux, and enzyme deactivation  inhibitor in multiple fungal genomes by screening for homologues of DHAD near a BGC. The research group further expressed the BGC and confirmed the secreted natural product to be aspterric acid. It was shown that the resistance element, the astD gene, encodes a modified DHAD with narrower entrance to the active site, thus exerting inhibitory effects on aspterric acid.The duplication hypothesis arises from the notion that many antibiotics’ common target sites, such as the ribosome, are also found in the producers. Hence, to protect itself, the producer harbors a copy of the target sequence with a slight modification to induce resistance against the antibiotic it produces by providing excess targets or proteins with greater binding affinity to the SM Fig. b. Take Sn et al.  employedHorizontal gene transfer (HGT) is a widely recognized event that happens frequently among bacteria as a driving force to gain genetic advantage Davies . It is pActinobacteria, many of which are notable secondary metabolite producers. Among 320,263 genes laterally acquired by Streptomyces lineages, a large proportion is genes functioning in SM and xenobiotic metabolism  and nonribosomal peptide synthase pathways (NRPS). A study by Ziemert et al. . However, this approach was soon demonstrated to miss out several BGCs of unknown classes. The algorithm has since been improved by many different strategies, such as looking for BGC-like patterns via data training (ClusterFinder) or a phylogenomics approach (EvoMining). Despite differences in computational approaches, all these tools result in a large number of potential BGC predictions, many of which are uncharacterized, necessitating the laborious wet laboratory work to verify the “omics” forecast. The biggest challenge is now no longer to detect BGCs but to prioritize the experimental procedures for BGCs with the most valuable biomedical potentials.Salinispora strains by Tang et al.  enzyme, lies within kanamycin-BGC. AAC(6′) can inactivate kanamycin to protect the organism from the lethal effect of kanamycin  , resistance elements) or a built-in database  BGC also harbors the gliT gene, which encodes for gliotoxin oxidoreductase, an enzyme that converts gliotoxin into a less toxic compound  gene cluster in Aspergillus clavatus, where at least five genes were laterally acquired from an ancestor of Magnaporthe grisea  the presence of resistance genes, (ii) duplicate genes, and (iii) evidence of horizontal gene transfer. It has provided selective criteria for certain species to target antibiotic-producing BGCs where target modification resistance is employed but has not been quite applicable to other species. In general, there seems to be no specific set of rules to highlight BGCs in all species: the more criteria added, the more confident the prediction is.In the future, multiple screening criteria might be included to increase the accuracy of predictions. Another plausible approach is to base the search on function-guided rules. For example, antibiotic seekers will look for resistance elements in BGCs.


Holocene climate variability in the Mediterranean Basin is often cited as a potential driver of societal change, but the mechanisms of this putative influence are generally little explored. In this paper we integrate two tools–agro-ecosystem modeling of potential agricultural yields and spatial analysis of archaeological settlement pattern data–in order to examine the human consequences of past climatic changes. Focusing on a case study in Provence (France), we adapt an agro-ecosystem model to the modeling of potential agricultural productivity during the Holocene. Calibrating this model for past crops and agricultural practices and using a downscaling approach to produce high spatiotemporal resolution paleoclimate data from a Mediterranean Holocene climate reconstruction, we estimate realistic potential agricultural yields under past climatic conditions. These serve as the basis for spatial analysis of archaeological settlement patterns, in which we examine the changing relationship over time between agricultural productivity and settlement location. Using potential agricultural productivity (PAgP) as a measure of the human consequences of climate changes, we focus on the relative magnitudes of 1) climate-driven shifts in PAgP and 2) the potential increases in productivity realizable through agricultural intensification. Together these offer a means of assessing the scale and mechanisms of the vulnerability and resilience of Holocene inhabitants of Provence to climate change. Our results suggest that settlement patterns were closely tied to PAgP throughout most of the Holocene, with the notable exception of the period from the Middle Bronze Age through the Early Iron Age. This pattern does not appear to be linked to any climatically-driven changes in PAgP, and conversely the most salient changes in PAgP during the Holocene cannot be clearly linked to any changes in settlement pattern. We argue that this constitutes evidence that vulnerability and resilience to climate change are strongly dependent on societal variables. Patriarche ,which we use to examine the changing relationship over time between potential agricultural productivity and settlement location.Past climatic changes are often cited as drivers of societal change in the Mediterranean Basin e.g., –7). Whil. Whil7])Potential agricultural productivity (PAgP) is strongly related to climatic factors  while also more directly relevant to human activity than raw climatic variables. Although PAgP is largely a function of these climatic variables, using a derived variable allows moving beyond simplistic threshold approaches to more realistic assessments of the spatiotemporally variable and not necessarily linear consequences of climate change. By exploring if and how settlement location was related to PAgP in different periods of the Holocene, we approach the problem of relating diachronic cultural patterns to climatic changes empirically. Using a site location analysis approach in which we statistically summarize geographic variables and PAgP within buffers around archaeologically-documented settlements, we explore a) how settlement locations relate to spatially- and temporally-variable PAgP, b) whether changes over time in PAgP had any apparent effect on settlement locations, and c) the implications of the reconstructed variability in PAgP and past agricultural practices for understanding the vulnerability and resilience of Holocene Mediterranean populations to climate changes.Site location analysis and related approaches have a long history in archaeology  and whiructions . By focu2, from the Mediterranean to the headwaters of the Durance River in the Alps and from the Rhone into the southern Alps. While these patterns are not necessarily reflected precisely within the study area on which we focus here, the relationship of these broad patterns to the data for our study area suggests that our study area is, in general terms, representative of regional trends.Before turning to empirical analyses, we contextualize our data with a brief overview of the broad archaeological patterns that can be described in Provence throughout the Holocene–i.e., a synthesis that aggregates across a topographically diverse landscape of >30,000 kmPatriarche chronological assignments (in brackets) for sites within our study area, however, reflect the France-wide chronology employed in the Patriarche database, which does not precisely match an up-to-date chronology for Provence. Discussion of chronological issues, as well as additional detail on the archaeology of Holocene Provence, can be found in The date ranges in the discussion below reflect synthesis of research in Provence ,22. Althth millennium BCE the inhabitants of Provence were exclusively Mesolithic–i.e., mobile foragers, with correspondingly modest population densities ), a reduction of human activity and population was accompanied by an overall increase in forest coverage [Panicum miliaceum], and the beginnings of olive [Olea europea] cultivation in the region [Patriarche]) the density of human occupation in Provence was at its lowest, following a notable decrease relative to previous phases. In contrast, the Late Bronze Age  is characterized by notable demographic growth and the emergence of new sites )Arguments linking climate and culture change are much more common in the eastern than the western Mediterranean e.g., ,5,7,56).,7,56.5,7This debate has been less salient in the western Mediterranean, where arguments for climatic influences on cultural phenomena are less common. Nevertheless, cultural trajectories have been linked to Holocene climate in the western Mediterranean in various ways, including both climate impacts on inhabitants and anthropogenic contributions to regional environment and climate; here we focus on the former. Berger  and Berg2  by integrating empirical and modeled data. These include modern geographic data, climate-sensitive agro-ecosystem modeling of PAgP with annual resolution, and archaeological site data from the Early Neolithic through the Gallo-Roman period. For the study area of approximately 1400 km2 , we incomodern geographic data, primarily a 30m digital elevation model (SRTM30 ) and terhigh-resolution  Holocene paleoclimate data produced by downscaling a 0.5° centennial-step modeled dataset derived from inverse modeling of pollen data for the Mediterranean throughout the Holocene , producPatriarche database, which provides coordinates, site classification, and chronological information for approximately 2250 sites (excluding those of uncertain date) in the study area.Archaeological site data spanning the period between 7250–1450 BP  from the Centennial scale episodes of aridity are reflected in the subset of data from Guiot and Kaniewski’s Mediterranean-wide Holocene model  that we Since a significant way in which climatic variables impact human inhabitants is through their effects on subsistence production (cf. ), we focAfter excluding sites of uncertain date and filtering the database of sites for occupational and agricultural sites–i.e., those most likely to be responsive to PAgP–we characterize the immediate catchment of each site by summarizing a series of variables for all pixels within a 200m circular buffer  for inhabitants of the region in different periods, we employ both synchronic and diachronic analyses, comparing locations of distinct types of sites within periods and settlement locations across periods. PAgP of aggregated site catchments for each period is also compared to PAgP for the landscape as a whole during that period, contextualizing estimated land use within the universe of possibilities available to inhabitants. Further methodological detail and consideration of uncertainties is given in not to identify the predictors of site location, but rather to explore to what degree PAgP–itself variable over time and space–was an important factor (or not) in site establishment, persistence, and abandonment during different periods of pre- and proto-history. As discussed below (Section 5), this provides a means of empirically assessing the human consequences of climate dynamism.Relating PAgP to settlement patterns echos previous archaeological attempts to relate settlement patterns to agricultural potential e.g., , 70), bu, bu70]),Synchronic analyses–i.e., investigation of sites from single archaeological periods–serve to check that we are not simply describing general changes in site location preferences that may covary with, but not be directly related to, PAgP. These analyses take two forms. First, where distinct categories of sites are located differently with respect to PAgP, and where the two groups vary independently from one another, we infer that the locations of settlement/agricultural sites are likely responding to particular rather than general imperatives. Second, we examine the distribution of sites against not only PAgP, but also against the other geographic variables that we have summarized within each buffer. Covariance amongst these  makes teasing apart their influences difficult, but where settlement distributions are more restricted with respect to one than to the other we argue that some selection is evident. Active selection may be more strongly argued still in the cases of newly established or abandoned settlements, though the smaller sample sizes of these categories limit their utility in some periods.Diachronic analyses focus on whether changes in settlement location between periods are correlated with changes in PAgP (absolute yields and variability in yields). Chronological resolution–period length–is again a limiting factor, as the averaging of PAgP across long periods minimizes variability , and as data about site size is inconsistently reported we exclude that also. Nevertheless some patterning is strong enough that it likely transcends problems of site preservation or chronological limitations, and the presence of sites from all periods in most areas suggests that taphonomic problems are at least not effacing particular periods from the landscape. Within the study area, the most salient diachronic pattern is a marked MBA abandonment; resettlement of the area began in the LBA and a florescence of sites followed in the LIA and continued into the Gallo-Roman Period.terminus ante quem of the end of the EBA, this apparent offset from the regional pattern may also in part be an artifact of data resolution). The regional population decline in the EIA is reflected locally in the slower pace of new settlement relative to the LBA. Local chronology (i.e. the periodization used in the Patriarche dataset) is not sufficient to resolve regionally-apparent fluctuations during the LIA and Gallo-Roman periods )Even with the limited modeling that we have employed here, and with the limitations in chronological resolution described in Section 4.3, we have been able to demonstrate that inhabitants of Holocene Provence were primarily exploiting the parts of the landscape with higher potential agricultural productivity. The notable exception to this pattern is the marked shift spanning the MBA-LBA-EIA, when other imperatives apparently drove inhabitants to less productive areas.mechanisms linking climate and cultural changes.This case study demonstrates that downscaled paleoclimate data in conjunction with agroecosystem modeling has the potential to shed light on the human consequences of climatic changes. The spatially-explicit and diachronic calculation of a climate-sensitive variable that directly measures an impact on human communities of climate dynamism constitutes a mechanism that can link climate variability with cultural change–or, as in the MBA-LBA-EIA anomaly that we have examined here, suggest that other factors are likely more significant. With this method established, expanding its application to larger, more complete, and more precise datasets becomes manageable. Such efforts have the potential to address fundamental questions about if, how, and when climatic changes impacted past inhabitants by enabling the proposal and evaluation of specific S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.S5 Text(DOCX)Click here for additional data file.S1 Fig(JPG)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 Fig(JPG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


To the Editors:1 Infants are, therefore, initiated on lifelong treatment after an initial positive result before the confirmatory test result is provided.1 Confirmatory testing is critical to verify patient identity  and exclude false-positive results .2 A primary challenge is that in some countries in sub-Saharan Africa, less than 10% of infants with initial positive test results receive a confirmatory test.Since 2010, WHO has recommended that HIV virological testing be used to diagnose HIV infection in infants and children below the age of 18 months and that ART be started without delay upon the first positive result while a second specimen is collected to confirm the initial positive virological test result.4 For example, in a setting with mother-to-child transmission rates of less than 5%, the positive predictive value of a highly sensitive nucleic acid-based technology decreased to nearly 70%.5 Fortunately, newer assays currently in use in the field have higher specificities with less dramatic decreases in positive predictive values.The potential for a false-positive result is of increasing concern as prevalence or transmission rates decrease.8 reaching as low as 2% in a high burden, treatment-experienced setting.13 This reduction in transmission can have a considerable impact on the positive predictive value of infant diagnostic assays, even those with nearly 100% sensitivity and specificity. It is important to note that this issue is primarily predicated on the prevalence/transmission rate in the tested population and not a technological shortcoming. Current assays on the market for infant diagnosis typically have specificities of greater than 98%;17 thus a more perfect assay is unlikely to significantly improve the positive predictive value in settings with low prevalence/transmission rates in the tested population.Mother-to-child transmission rates have decreased considerably over recent years as option B+ and Treat All policies have been implemented,18 whereas South Africa has introduced an indeterminate range that requires further testing before a definitive test result is provided and treatment is initiated.19Currently, most programs and laboratories interpret undetectable test results by the nucleic acid-based technology as negative and detectable test results as positive, relying on thresholds of detection provided by the manufacturers; however, there is increasing concern on how to interpret test results with low levels of viremia, particularly in the context of increasing exposure to antiretroviral treatment and prophylaxis. To date, there has been limited guidance on how to interpret low levels of viremia in test results. However, historically, one would hesitate to initiate lifelong treatment based on detection of the equivalent of very few viral copies per milliliter a true positive result. In this context, different approaches have been considered: guidelines in the United States suggest that infants should not be considered HIV-positive unless they have the equivalent of 5000 viral copies/mL or higher,20 Qualitative diagnostic assays do not always provide viral copies, but instead the polymerase chain reaction cycle threshold when amplification is observed. The cycle threshold is inversely correlated to the amount of virus in a sample.An indeterminate range is a range of viral copy equivalents that would be too low to be accurately diagnosed as HIV infection.22 The systematic review of 32 studies using an indeterminate range21 found 14,753 non-negative test results, of which 2436  were indeterminate. Furthermore, one study that reported the final diagnoses of indeterminate cases found that 76% of infants with an initial indeterminate test result were negative upon retesting, suggesting that these infants were not HIV-positive despite the initial non-negative test result. These data indicate that in countries not implementing an indeterminate range to support result interpretation of infant diagnosis test results, 12.5% of non-negative results could be false positive on initial testing and those infants are potentially put on lifelong treatment unnecessarily.To provide guidance on how to interpret diagnostic test results with low levels of viremia, a systematic review, meta-analysis, and cost-effectiveness model were developed.21 Data provided included only patients with an initial non-negative test and a second test result from a new sample. To better understand the most accurate indeterminate range, true positivity and false positivity were calculated across various proposed indeterminate thresholds based on the initial test result  using the Roche TaqMan v2 technology.22 Furthermore, as the prevalence/positivity/transmission rate at each testing time point decreases, the cost-effectiveness of an indeterminate range increases and is more cost-saving than no indeterminate range.The cost-effectiveness model compared the standard interpretation (no indeterminate range) with a variety of indeterminate range options and concluded that implementing an indeterminate range is far more effective than the standard of care across a variety of cycle threshold ranges.23 Sixty percent or more respondents in each group preferred providing or receiving a full explanation of the meaning of an indeterminate result; however, several thought it would be critical to minimize confusion and concerns for caregivers. Implementing an indeterminate range would be equitable, as doing so would improve the quality of test results provided to caregivers and clinicians and, most importantly, prevent infants from being put on lifelong treatment unnecessarily.A survey provided to program managers (n = 85), health care workers (n = 146), and people living with HIV (n = 587), established that over 85% of respondents in each group found implementation of an indeterminate range to be acceptable to prevent unnecessary lifelong treatment.24 Therefore, before the health care facility is contacted to request that the mother and baby return to the facility for collection of a new sample, any indeterminate test should be repeat-tested on the same sample using additional available dried blood spots or remaining whole blood.25Most countries already apply a national standard operating procedure when testing errors are encountered . Furthermore, a new study suggests that repeating an infant test on the same sample, if and when available, will resolve most (>95%) indeterminate test results.Implementing an indeterminate range will support more accurate nucleic acid-based early infant diagnosis: It is likely that fewer infants would be put on lifelong treatment unnecessarily as most false positives would fall within the indeterminate range and receive additional testing before diagnosis rather than being identified as positive. However, some challenges remain to be resolved. First, the cycle threshold values provided by infant diagnostic assays on the market vary and cannot be directly applied between technologies or assays. Ideally, each assay manufacturer should implement the indeterminate range directly into their proprietary software. Test result readouts would then be positive, negative, or indeterminate. This would be particularly beneficial for point-of-care assays operated by lower cadres of staff to simplify result interpretation. For laboratory-based technologies, rapid determination of the equivalent cycle threshold or viremic values will allow for laboratories to implement the indeterminate range and subsequent repeat testing before software integration.26 However, no evidence informs and justifies differences in indeterminate range values based on the time of sample collection, and further research is needed. In addition, more research would be valuable on the feasibility of implementation and best messaging for health care workers and caregivers/mothers as well as optimal standard operating procedure for indeterminate test results.Furthermore, additional considerations may be necessary for countries using plasma as the sample type for infant testing rather than whole blood or dried blood spots, because the latter sample types typically capture and amplify intracellular nucleic acids that may increase the detected levels of virus. The age at which the child is tested and the timing of transmission may also affect the viral loads, because infants diagnosed at birth can have lower viral loads.8 early infant diagnosis is growing increasingly complex. Continued testing throughout and after the exposure period is critical: Infant diagnosis should no longer be considered a one-time process.Countries, particularly those with low transmission or infant diagnosis positivity rates, should strongly consider implementing an indeterminate range for better management of HIV-exposed infants. Furthermore, many of the issues presented here highlight the critical need for all infants with detectable virus to receive repeat testing on the same sample and for those who are clearly positive, confirmatory testing with a new sample. Finally, as mother-baby retention during the postnatal period is challenging and the proportion of transmission during the postnatal period is increasing,


Cellular therapy of the corneal stroma, with either ocular or extraocular stem cells, has been gaining a lot of interest over the last decade. Multiple publications from different research groups are showing its potential benefits in relation to its capacity to improve or alleviate corneal scars, improve corneal transparency in metabolic diseases by enhancing the catabolism of the accumulated molecules, generate new organized collagen within the host stroma, and its immunosuppressive and immunomodulatory properties. Autologous extraocular stem cells do not require a healthy contralateral eye and they do not involve any ophthalmic procedures for their isolation. Mesenchymal stem cells have been the most widely assayed and have the best potential to differentiate into functional adult keratocytes in vivo and in vitro. While embryonic stem cells have been partially abandoned due to ethical implications, the discovery of the induced pluripotent stem cells (iPSC) has opened a new and very promising field for future research as they are pluripotent cells with the capacity to theoretically differentiate into any cell type, with the special advantage that they are obtained from adult differentiated cells. Cellular delivery into the corneal stroma has been experimentally assayed in vivo in multiple ways: systemic versus local injections with or without a carrier. Encouraging preliminary human clinical data is already available although still very limited, and further research is necessary in order to consolidate the clinical applications of this novel therapeutic line. The stroma constitutes more than 90% of the corneal thickness. Many features of the cornea, including its strength, morphology and transparency are attributable to the anatomy and properties of the corneal stroma . Many diIn the last few years, interest for cellular therapy of the corneal stroma using mesenchymal stem cells (MSCs) from either ocular or extraocular sources has gained a lot of interest; studies show that MSCs are capable of differentiating into adult keratocytes in vitro and in vivo . SeveralConsidering existing scientific evidence, it seems that all types of MSCs have similar behavior in vivo Table , and thuCorneal stroma stem cells (CSSCs) are a promising source for cellular therapy as the isolation technique and culture methods have been optimized and refined ; presumaHuman adult adipose tissue is a good source of autologous extraocular stem cells as it satisfies many requirements: easy accessibility to the tissue, high cell retrieval efficiency and the ability of its stem cells (h-ADASCs) to differentiate into multiple cell types  . This ceBone marrow MSCs (BM-MSCs) are the most widely studied MSCs, presenting a similar profile to ADASCs, but their extraction requires a bone marrow puncture, which is a complicated and painful procedure requiring general anesthesia.Umbilical MSCs (UMSCs) present an attractive alternative, but their autologous use is currently limited as the umbilical cord is not generally stored after birth.Embryonic stem cells have great potential, but also present important ethical issues. However, the use of iPSC technology  could soFinally, it is important to remark that the therapeutic effect of MSCs in a damaged tissue is not always related to the potential differentiation of the MSCs in the host tissue as multiple mechanisms might contribute simultaneously to this therapeutic action for example, secretion of paracrine trophic and growth factors capable of stimulating resident stem cells, reduction of tissue injury and activation of immunomodulatory effects, in which case the direct cellular differentiation of the MSCs might not be relevant and could even be non-existent , 24, 25.We will review the different types of stem cells  that have been proposed for the regeneration of the corneal stroma as well as the current in vitro or in vivo evidence. Finally, we will review the different surgical approaches that have been suggested (in vivo) for the application of stem cell therapy to regenerate the corneal stroma.Park et al. reported that human BM-MSCs differentiate in vitro into keratocyte-like cells when they are grown in specific keratocyte differentiation conditions . They deHuman ADASCs (h-ADASCs) cultured in vitro Fig.  under keThe differentiation of h-ADASCs in functional human keratocytes has also been demonstrated in vivo, for the first time, in a previous study by our group using the rabbit as a model . These cHuman MSCs isolated from neonatal umbilical cords have exhibited similar differentiation behavior to other types of MSCs when transplanted inside the corneal stroma in vivo, expressing keratocyte-specific markers such as keratocan without inducing immune or rejection responses . Liu et Current experience with these human pluripotent stem cells for the corneal stroma regeneration is much more limited. Chan et al. reported that differentiation of these cells into a keratocyte lineage can be induced in vitro, demonstrating an upregulation of keratocyte markers including keratocan .To the best of our knowledge, no in vivo studies with these cells have been performed in the field of regenerative medicine for the corneal stroma. The use of these cells also raises many ethical issues, and together with the lack of in vivo data, discourages their current use in a clinical setting.As already discussed, the use of embryonic stem cells has been partially abandoned because of ethical concerns and especially since the discovery of iPSCs , which aRegarding their application in the regeneration of the corneal stroma, human iPSCs have shown the capability to differentiate into neural crest cells (the embryonic precursor to keratocytes). By culturing them on cadaveric corneal tissue, it promotes their keratocyte differentiation by acquiring a keratocyte-like morphology to express markers similar to corneal keratocytes . It has The limbal palisades of Vogt form a niche that contains both limbal epithelial stem cells (LESCs) and corneal stromal stem cells (CSSCs) . CSSCs eAll these experimental data have raised interest in this novel cell-based therapy for corneal stromal diseases, however, before its application in clinical practice, its efficacy and safety need to be well proven in human clinical trials, while other limitations such as the high laboratory costs and potential therapeutic efficacy differences among different donors have to be given serious consideration.All these types of stem cells have been used in various ways in several research projects in order to find the optimal procedure to regenerate the human corneal stroma. Corneal MSC implantation has been assayed and studied by direct intrastromal transplantation or after implantation from the ocular surface, intravenously and the anterior chamber where cellular migration within the stroma is to be expected. Different cellular carriers have been analyzed in order to enhance the potential benefits of this therapy.subconjunctival injections of BM-MSCs in diabetic mice and reported an increased corneal epithelial cell proliferation as well as an attenuated inflammatory response mediated by tumor necrosis factor-α-stimulated gene 6 (TSG6) [Surface implantation of MSCs would be the optimal approach for ocular surface reconstruction and corneal epithelium/limbal stem cell niche regeneration (not the aim of this review). However, surface implantation of MSCs would still play a role in the prevention or modulation of anterior stromal scars after an ocular surface injury . As discussed previously, MSCs secrete paracrine factors that enhance corneal re-epithelialization and stromal wound healing . Thus, t6 (TSG6) .nanofiber scaffolds. They reported that BM-MSCs grown on these scaffolds can enhance re-epithelialization, suppress neovascularization and local inflammatory reaction when applied on an alkali-injured eye in a rabbit model, and these results were comparable to those obtained with limbal epithelial stem cells, and both were better than those obtained with ADASCs [Holan et al. suggested MSC application on the ocular surface using h ADASCs . The samh ADASCs .Topical application of a suspension of autologous ADASCs has been reported in an isolated clinical case report where authors describe the healing of a neurotrophic ulcer that is not responsive to conventional treatment [reatment . The lacfibrin glue [Finally, Basu et al. suggested the delivery of MSCs using rin glue . They rerin glue .Direct in vivo injection of stem cells inside the corneal stroma has been assayed in some studies, demonstrating the differentiation of stem cells into adult keratocytes without signs of immune rejection. In our study, we also demonstrated the production of human ECM by immunohistochemistry when h-ADASCs were transplanted inside the rabbit cornea . As expen = 5). No signs of inflammation or rejection were observed, confirming all previous evidence reported in the animal model. We also demonstrated, by anterior segment optical coherence tomography (AS-OCT), the production of new collagen in the area of the MSC implantation  strong inflammatory responses induced on their biodegradation and ii) nearly all polymer materials cause a nonspecific inflammatory response .Recently, several corneal decellularization techniques have been described, which provide an acellular corneal ECM . These sIn our opinion, the implantation of MSCs together with decellularized corneal ECM would be the best technique to effectively restore the thickness of a diseased and severely weakened human cornea because the implantation of MSCs alone only achieves a limited new ECM formation and thickness restoration , 15. MorDecellularized tissues have the drawback of requiring specific laboratory equipment, although eye banks could potentially do it and deliver such grafts to different clinical centers. Keratophakia  was described by Barraquer in 1964, but was abandoned due to the unpredictability of the refractive outcome and the relatively high frequency of interface haze development . The lacDemirayak et al. reported that BM-MSCs and ADASCs, suspended in phosphate-buffered solution (PBS) and injected into the anterior chamber after a penetrating corneal injury in a mouse model, are able to colonize the corneal stroma and increase the expression of keratocyte specific markers such as keratocan, with a demonstrated increase in keratocyte density by confocal microscopy . ConversSystemic use, by intravenous injection, of MSCs has also been tested. Intravenous injection of BM-MSCs in mice after an allograft corneal transplant was be able to colonize the transplanted cornea and conjunctiva (inflamed ocular tissues) but not the contralateral ungrafted cornea, simultaneously decreasing immunity and significantly improving allograft survival rate . Yun et A critical question for future clinical trials to further assess the feasibility of cellular therapy of the corneal stroma, is whether the use of autologous MSCs is really necessary and whether allogenic MSCs could achieve the same benefit without any risk of inflammation or rejection. If we consider all published evidence in the animal model where human MSCs were implanted in the corneal stroma, despite being a xenogeneic transplant, no signs of rejection or inflammation have ever been reported –13. ThisExosomes are nano-sized extracellular vesicles that originate from the fusion of intracellular multivesicular bodies with cell membranes and are released into extracellular spaces . They haIn conclusion, cellular therapy of the corneal stroma is a novel treatment modality for stromal diseases, which even though further studies are still required in the form of clinical trials with larger sample sizes in order to definitely establish its safety and efficacy for different stromal diseases, the initial results obtained from the first few pilot clinical trials are encouraging. In our opinion, the creation of cellular banks storing and expanding stem cells or their exosomes, and their shipping and delivery to the different clinical centers for their use may be the future for this promising treatment modality, although prior to this, there is still a lot of research work to be undertaken.


Despite general and wide-ranging negative effects of coral reef degradation on reef communities, hope might exist for reef-associated predators that use nursery habitats. When reef structural complexity is lost, refuge density declines and prey vulnerability increases. Here, we explore whether the presence of nursery habitats can promote high predator productivity on degraded reefs by mitigating the costs of increased vulnerability in early life, whilst allowing for the benefits of increased food availability in adulthood. We apply size-based ecosystem models of coral reefs with high and low structural complexity to predict fish biomass and productivity in the presence and absence of mangrove nurseries. Our scenarios allow us to elucidate the interacting effects of refuge availability and ontogenetic habitat shifts for fisheries productivity. We find that low complexity, degraded reefs with nurseries can support fisheries productivity that is equal to or greater than that in complex reefs that lack nurseries. We compare and validate model predictions with field data from Belize. Our results should inform reef fisheries management strategies and protected areas now and into the future. Despite wide-ranging negative effects of coral reef degradation on reef communities, hope might exist for reef-associated predators that use nursery habitats. This study uses size-based ecosystem models of coral reefs to assess the effects of the presence and absence of mangrove nurseries. As the human population continues to grow, the food security and livelihoods of millions of people in tropical coastal communities depends on the continued productivity of coastal fisheries. For many, coral reef fish provide a major source of dietary protein, as well as livelihoods, a way of life, and cultural identity ,2. Yet, While loss of reef habitat quality is a significant concern, it is not the only driver of reef fish productivity. The presence of nursery habitats, such as mangroves and seagrass beds, can enhance the biomass of multiple fish species –15. TheyThe survival of recruiting fish to coral habitats is contingent on their ability to find refuges from predation, typically in crevices and branching corals . Some prIn early life, complexity is beneficial and promotes survival when predation risk is high . But as For non-predators, low habitat complexity does not offer the same benefits—food is likely to be equally or indeed less available and abundant when structure is lost. Nursery habitat use will still reduce post-settlement mortality, but if the cost of increased predator abundance on the reef outweigh this benefit, then nurseries might not promote productivity and could potentially have a negative impact on survival for the guild as a whole. Additionally, any negative effect is likely to be higher when habitat quality and therefore refuge availability is low.Here, we explore the interaction between nursery use and reef habitat quality for predator, herbivore, and total fisheries productivity in the Caribbean. Specifically, we ask whether mangrove nurseries can buffer losses of fisheries productivity as reef habitats decline. To do this, we combine empirical data on nursery habitat use with a field-tested ecosystem model that explicitly captures habitat complexity ,30,31.−2, versus 0.10 herbivores m−2). Predictions are consistent with published empirical studies that utilised 131 reef sites across the Caribbean, and showed that nurseries enhanced the abundance of nursery-using herbivores but reduced that of non–nursery-using species and the assemblage overall [Models predict that mangroves have positive effects on the biomass of predators  and negligible or negative effects on the biomass of herbivores  . Negativ overall .Our models predict that the biomass of both predators and herbivores responds more strongly to habitat complexity than to nursery availability .Fisheries productivity is defined here as the product of growth and abundance for fish of a size range targeted by fisheries. Our models predict that for predators, fisheries productivity increased in the presence of mangrove nurseries. However, a striking result is that mangrove access more than offsets the deleterious impacts of habitat degradation on fisheries productivity for this guild. In other words, the increased productivity resulting from nursery availability is such that degraded reefs with mangroves have equal or greater productivity than healthy reefs without . For herThe predicted impact of nurseries on productivity is greater than their impact on biomass, for which patterns are mostly driven by habitat complexity. Not surprisingly, the apparent disparity occurs because of growth and turnover. Low complexity habitats with mangrove nurseries have a lower standing stock biomass of predators, but on average, those that are present grow faster in the model  due to a greater availability of prey, resulting in higher turnover rates that contribute to productivity. Given that predatory species are primary targets for most coral reef fisheries and generally have a higher market value than herbivorous species, our results suggest that mangrove availability decreases the vulnerability of reef fisheries to habitat degradation and specifically to declines in structural complexity.To explore whether fishing modified our observed patterns, we simulated a modest but uniform level of fishing on model systems and examined the consequences for predator fish biomass . The addp = 0.95). Although the overall biomass of carnivores was similar in the sites lacking mangroves , the relationship with rugosity was positive, explaining 30% of variance (p = 0.012). Importantly, fishing pressure was lower in these regions because fishers tend to use mangrove islands for temporary camps and shelter and therefore create greater pressure in the mangrove-rich reefs [The model also predicts that the positive impact of rugosity on fish biomass will tend to weaken, albeit remain, when the system is fished . We sougch reefs . Thus, tp = 0.59, n = 10). Thus, it is unlikely that the absence of a functional relationship between rugosity and biomass can be attributed to relatively intense fishing at reefs with higher habitat complexity.The absence of a relationship between rugosity and predator biomass in mangrove-rich areas is consistent with theory. This is because (1) predators fare relatively well at low rugosity when mangroves allow fishes to circumvent the early predation bottleneck and (2) some of this high productivity at low complexity is manifest as elevated biomass because fishing prevents saturation of predators. That means that a portion of the elevated prey production remains as unconsumed biomass and tends to ‘level out’ the relationship with rugosity. This process of biomass accumulation may be exacerbated by supplemental individuals migrating from mangroves in seasonal bursts and therefore contributing higher biomass that has not yet been consumed and converted into productivity higher up the food chain. We do, however, consider whether variable levels of fishing among mangrove-rich sites could be confounding our interpretation of the absence of a rugosity–biomass relationship. While absolute fishing levels have not been quantified, the variability in fishing intensity is far greater among the six regions (particularly between mangrove-rich and mangrove-poor regions) than among sites sampled within each region, which are easily visited by the same fishers. In contrast, the variability in rugosity occurs among sites within a region; a linear model of rugosity among reefs in the three mangrove-rich regions found no significant differences . Note that there was no significant confounding difference in the rugosity between the mangrove-rich and mangrove-absent treatments . The enhanced variance among mangrove-rich systems was not associated with rugosity; rather, it reflects the combined effects of variable strength of mangrove-reef nursery habitat connectivity among sites [Another point of concordance between our model predictions and empirical data can be obtained by comparing the variability of predator biomass between reefs with access to mangrove nurseries versus those without. The model predicts that variance in predator biomass among complex reefs with mangroves is greater than that among complex reefs that lack nurseries  in Belize (data sourced from ) provideOrbicella reefs within each of six regions in Belize. Half of the sites surveyed had strong mangrove connectivity, whilst half lacked mangroves. At each site, fish biomass was quantified using belt transects (see Model predictions relating to nursery and complexity effects on fish biomass were compared to field surveys from at least three S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Yet important questions remain. This review summarizes recent findings, primarily mechanistic insights on the essential cellular and molecular players involved gained within the murine experimental cerebral malaria model. It also highlights recent developments in (a) cell-cell communication events mediated through extracellular vesicles (EVs), (b) mounting evidence for innate immune memory, leading to “trained“ increased or tolerised responses, and (c) modulation of immune cell function through metabolism, that could shed light on why some patients develop this life-threatening condition whilst many do not.Complications from malaria parasite infections still cost the lives of close to half a million people every year. The most severe is cerebral malaria (CM). Employing murine models of CM, autopsy results,  Plasmodium, seven species are able to infect humans to cause malaria. The two major species are Plasmodium vivax and Plasmodium falciparum, with the former accounting for the most cases and the latter being responsible for the majority of deaths , have provided important clues. ECM and HCM have more than twenty documented shared pathological features . In both patients and mice, neurological signs progress from seizures, ataxia and paralysis to coma and eventual death, and both are associated with brain oedema and hemorrhages.A comprehensive appraisal of the histopathology associated with CM or experimental animal models of CM, as well as its changes following characterized interventions, constitute a valid experimental approach to identify targetable disease development stages, cellular or molecular players. This could eventually lead to the identification of adjunctive therapies. Investigating the histopathology and pathophysiology of HCM  in patients has ethical and technical constraints since pathological analysis of brain and most other tissues besides blood are limited to post-mortem observations. Thus, models of experimental cerebral malaria (ECM), caused by infection of susceptible mice with features , 6. In bfeatures , albeit features , and fouIn this review, we summarize the consensus and controversies surrounding the sequence of cellular events leading to the development of vascular neuropathology in experimental CM (ECM) and their correlates in human CM (HCM). Numerous aspects of the similarities and differences between HCM and ECM have been reviewed , 6, 9–13+ and for a late accumulation of CD8+ T cells while for other accumulating cell types, there are still controversies as to direct pathogenic roles  activation such as ICAM-1, E-Selectin, CD36, and von Willebrand Factor (vWF), that all can serve as adhesion molecules for IE. Platelets are now considered key contributors to CM by providing an alternate, indirect mechanism for IE cytoadhesion . This isin vitro , 22. TheMalawian  and SoutMalawian  patientsin vitro  and in v and lymphotoxin  . High leed human  or murined human  brain miSubsequent to the accumulation of IE and platelets, the intravascular accumulation of leucocytes is considered a key step in the development of ECM  and in s+and CD4+) cells in the brain of infected mice have major pathogenic roles. Supporting evidence has been obtained through anti-CD4 or anti-CD8 antibody depletion strategies —previously shown to specifically target Ly6Chi monocytes via induction of their sequestration in the spleen  have been suggested to have distinct roles in CM. While expansion of ILC2 prevents ECM via a mechanism dependent on M2 macrophages and T regulatory cells , there i+T cells or of monocytes that has shown therapeutic effectiveness. The highly effective late stage success of the combination treatment with IMP and established anti-malarial drugs  in immunopathology was adduced from experiments in which we attempted to understand the effector mechanisms of TNF in microvascular damage. Among these was the demonstration that TNF dose-dependently enhanced the release of microvesicles by endothelial cells . In paraortality .in vitro generated endothelial microvesicles even trigger CM-like lesions in naïve recipients  to the additional modulatory role of cell intrinsic metabolic pathways on immune functions (cellular immunometabolism) [reviewed in ].For example, it has been increasingly appreciated that T cell activation results in metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to meet the increased energetic and biosynthetic demands for T cell expansion and effector functions , 88. It Significant metabolic changes such as hypoglycaemia and lactic acidosis as well as perturbations in amino acid metabolism have long been observed in severe malaria in both ECM –97 and HHowever, the recent developments in our understanding of immunometabolism mentioned above have called for a reassessment of hypotheses on mechanisms leading to CNS metabolite changes during CM development. It had been observed for some time that dietary restriction could prevent development of ECM, although only in a prophylactic setting , 104. SeBecause metabolic reprogramming can occur in response to changes in nutrient and oxygen availability, and in response to cytokines or other immune receptor stimulation, it presents the opportunity to therapeutically target these mechanisms at different levels, such as through dietary intervention. For example, pre-exposure to a high-fat diet reduced ECM via a mechanism that involved antioxidants . AnotherCandida albicans-derived ß-glucan-induced trained immunity  in classical malaria-resistance genes cannot explain interethnic differences, epigenetic modifications or different chromatin landscapes were observed on specific host gene promoters or host genes that provide resistance to or susceptibility to malaria –121. In in vitro . Additionfection , 124. Apnfection –127. A r malaria .in utero) malarial exposure. Modulation of trained immunity would also influence the clinical development of rationally designed malaria vaccines. Finally, this knowledge may help re-train innate cells during or before the disease to prevent severe complications. One could imagine that EVs may provide a way to achieve this by shuttling a designed cargo of miRNAS, epigenetic modifying enzymes or metabolites.Much work remains to be done in order to integrate the interactions between immunologic signals, metabolic changes and epigenetic modifications with long-term changes in “innate immune memory” in response to malaria. Deciphering these mechanisms could have far reaching implications. It could help predict the risk of developing CM in individuals with previous (including + T cell binding to endothelial cells, and finally, immunometabolism. These successes have renewed hopes that the mouse model of ECM will continue to bring novel ideas/concepts which then will need to be confirmed or infirmed in HCM. For example, the characterization of retinal pathology in ECM (The ECM model has produced a wealth of information on CM pathogenesis in mice with the aim to find an adjunctive therapy for HCM but its validity has been questioned. However, several investigators have provided a critical and evidence-based defense of this model , 128–131y in ECM  and (133y in ECM –137 (andy in ECM , and foly in ECM , 139.In addition, it should be emphasized that the massive reduction in malaria burden achieved in the last two decades was mostly a result of prevention strategies rather than treatment. In this context, knowledge gained from ECM studies should not be seen as limited to the design of therapies but could also guide the expansion of our arsenal for HCM prevention. In particular, understanding changes in innate immune memory preconditioning and metabolic status in populations with high or low incidence of HCM could present opportunities for prevention through environmental or dietary interventions.This review was co-written by FS and GG.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.