The plant cell wall has a diversity of functions. It provides a structural framework to support plant growth and acts as the first line of defense when the plant encounters pathogens. The cell wall must also retain some flexibility, such that when subjected to developmental, biotic, or abiotic stimuli it can be rapidly remodeled in response. Genes encoding enzymes capable of synthesizing or hydrolyzing components of the plant cell wall show differential expression when subjected to different stresses, suggesting they may facilitate stress tolerance through changes in cell wall composition. In this review we summarize recent genetic and transcriptomic data from the literature supporting a role for specific cell wall-related genes in stress responses, in both dicot and monocot systems. These studies highlight that the molecular signatures of cell wall modification are often complex and dynamic, with multiple genes appearing to respond to a given stimulus. Despite this, comparisons between publically available datasets indicate that in many instances cell wall-related genes respond similarly to different pathogens and abiotic stresses, even across the monocot-dicot boundary. We propose that the emerging picture of cell wall remodeling during stress is one that utilizes a common toolkit of cell wall-related genes, multiple modifications to cell wall structure, and a defined set of stress-responsive transcription factors that regulate them. The plant cell wall is a complex structure that fulfills a diverse array of functions throughout the plant lifecycle. In addition to maintaining structural integrity by resisting internal hydrostatic pressures, the cell wall provides flexibility to support cell division, a biochemical scaffold that enables differentiation, and a pathological and environmental barrier that defends against stress  are polysaccharides, the structure, and biosynthesis of which have been extensively reviewed in recent times  or from the pathogen cell wall  as a result of degradation  are callose, arabinoxylan and cellulose. Effective papillae that are successful in preventing the penetration attempts of Bgh contain significantly higher concentrations of these polysaccharides compared to ineffective papillae. The papillae are layered with an inner core consisting of callose and arabinoxylan, and an outer layer containing arabinoxylan and cellulose. The association of arabinoxylan and cellulose with penetration resistance opens new targets for the improvement of papillae composition and generation of lines with improved disease resistance. Previous studies described candidate gene expression profiles during papillae formation and discussed their likely functions in defense  gene family in the synthesis of papillary callose , EXPANSIN A6 (EXPA6), XTH10, and XTH32 responded to at least one stress, while 12 genes including CELLULOSE SYNTHASE LIKE G2 (CSLG2), ARABINOGALACTAN PROTEIN 2 (AGP2), BETA GLUCOSIDASE 46 (BGLU46), a 1,3-β-glucanase, and EXPANSIN A8 (EXPA8) responded in the same way to all three. This indicates that common transcriptional responses, and possibly downstream effects on cell wall composition, are employed in the response to distinct stresses.As mentioned previously in this review, plants are often exposed to multiple stresses concurrently, including those that are both abiotic and biotic, and the effects of these are not necessarily simply additive , and lew2-2, showed higher tolerance to osmotic stresses, induced by exposure to NaCl and mannitol, and drought stress compared to wild type plants. Other components of the plant cell wall influence plant tolerance to chilling or frost. For example, Taketa et al. -β-glucan  catalytic motif. Taketa et al. -β-glucan is usually a major component of this structure in the grasses. This hypothesis was strengthened by the work of Cu et al. -β-glucan specific antibody. Interestingly, -β-glucan content in cereal grain appears to be particularly sensitive to environmental conditions, although it is unclear whether this variation depends only on modified HvCslF6 function. Swanston et al. , an enzyme which hydrolyses pectin, and change the composition of the plant cell wall. Consequently, rice plants overexpressing OsBURP16 showed less tolerance to drought , with wild type plants showing 42% survival rate compared to <10% for OsBURP16 overexpression lines. Measuring levels of H2O2, an indicator of stress, showed that OsBURP16 overexpression lines were also more susceptible to salt stress than wild type plants.Transgenic plants have also been used to validate the role of candidate genes potentially involved in cell wall production/modification and abiotic stress. XET/XTH enzymes are typically thought to have a role in cell wall loosening and therefore cell expansion  are often involved in the response to different stresses. However, the inherent complexity of the wall and the large number of genes involved in its synthesis and modification means that many details remain unclear in regards to the genetic and biochemical basis for cell wall responses to stress. Despite the obvious difficulties involved in comparing experiments between different species, stresses and tissues, in the final section of this review we have revisited public transcriptome datasets to highlight broad similarities between different stress types, and consider whether more attention might be focussed on putative cell wall-related genes that have been overlooked previously.The previous sections of this review summarized research conducted on various aspects of cell wall reinforcement and modification during pathogen infection and abiotic stress. Cell wall reinforcement in the form of papillae is a relatively common mechanism that determines the outcomes of infection. However, given the diversity of biotic stresses that can be exposed to a plant, any commonalities in papillae formation would likely be accompanied by a range of distinct cell wall-related defense responses. The same might be expected for different abiotic stresses such as extreme temperature, salinity and flooding. In terms of the overlap between biotic and abiotic stresses, a recent study in Arabidopsis showed that ~25% of the cell-wall related transcripts that responded to fungal infection, herbivory, or drought showed a similar response in each treatment  domains associated with the CAZy database annotations were used to identify barley carbohydrate-related genes present on the 22K Barley1 GeneChip. The normalized transcript levels for each carbohydrate-related gene from Arabidopsis and barley were compared following each stress (relative to untreated controls) within each experiment and represented as a log(2)-fold induction. As might be expected from the previous sections of this review, many cell wall genes showed pronounced responses to the different stresses.Gene expression data is available from the Plant Expression Database . Hierarchical clustering was used to arrange gene families according to similarity in pattern of gene expression Figures  Eisen e. Figuresglucan synthase-like genes of the GT48 CAZy family is hard to support, given the large number of gene families that appear to be upregulated across most stresses. Even though the individual genes induced within each experiment are different, the clustering of CAZy families across the experiments suggests that there is a similar defense response mounted irrespective of the exact stress type. We can see examples of biotrophic fungi inducing similar responses to necrotrophic fungi, drought stress inducing similar responses to cold stress and even examples across different tissues with nematodes in roots compared to whitefly infested leaves.Two different clustering methods have been considered. Within each species the gene families can be clustered based on the correlation of their transcript profile across each experiment. Second, the experiment datasets can also be clustered based on the correlation of the gene family transcript profiles. It is interesting to note that the abiotic stresses generally form a cluster together, as do the biotic stresses, even though there is a vast difference in the type of stress within each cluster Figures . The dogClustering the response of CAZy families identifies activities that appear to be generally upregulated across most of the experiments, and therefore cell wall or carbohydrate components may be altered similarly during the interaction. Figure Twelve CAZy gene families are up-regulated (on average) across abiotic and biotic stresses in Arabidopsis and barley. These include polysaccharide degrading and modifying enzymes such as pectin methylesterase (CE8), carbohydrate esterase (CE10), and glycosyl hydrolases  which target a range of polysaccharides and oligosaccharides containing 1,3-β-glucan and chitin. Some of these CAZy families have already been implicated in stress-responses as pathogenesis-related (PR) proteins. GH17 genes have been classed as 1,3-β-glucan degrading PR-2 proteins (Leubner-Metzger and Meins, Arabidopsis and barley, including GT1, GT8, GT61, GT75, and GT92. The GT1 family includes a large number of genes with a wide range of putative functions including UDP-glucuronosyltransferase activity. By transferring sugars to a wide range of secondary metabolites, UGTs increase the stability and solubility of aglycones and therefore modify their bioactivity and effectiveness as regulators of the defense response (Lim and Bowles, f substrate for arabinoxylan, arabinogalactan protein, and pectic polysaccharide biosynthesis (Hsieh et al., Blumeria graminis f.sp. hordei (Chowdhury et al., The role of the CAZy glycosyltransferase families during the defense response is less characterized than the hydrolytic enzymes. Remarkably, there are five CAZy GT families that are upregulated on average across abiotic and biotic stresses in Whether members of these CAZy families have specific or similar effects on cell-wall targets can be addressed by characterizing the function of the underlying genes. For example, up-regulated expression of the GT8 and GT61 families highlights a potential role of pectin and xylan synthesis in the plant stress response across both species. However, these families contain members that are involved in many different processes and it is important to assess the expression and function of each gene in more detail. Figure The basis for this review was to consider cell wall and polysaccharide-related activities that influence biotic and abiotic stress responses, and highlight those that might fulfill a common function in promoting remodeling of the cell wall as a direct response to abiotic stress or pathogen attack. Genetic and transgenic evidence suggests that modification of specific cell wall activities has a pronounced effect on stress tolerance. In several cases, similar gene families appear to modulate the effect of distinct biotic and abiotic stresses within and across different species, implying that common mechanisms may have been recruited to target seemingly disparate stress types. This is supported by broader whole-transcriptome analyses, which indicate similar responses of individual cell-wall related genes and even CAZy families to different abiotic and biotic stresses. Whether these overlaps in gene expression lead to similar changes in cell wall structure has yet to be confirmed in most cases, particularly in the case of pectins and xylans which show distinct differences in abundance between monocot and dicot models. Indeed, the functions of many cell wall-related genes have yet to be reported during normal growth and development, let alone during stress responses. This highlights a need to further extend genome editing technologies toward entire CAZy families, and to develop methodologies for chemical cell wall analysis that are high-throughput and capable of being targeted toward single cell-types.KH, AL, MT, NS, and JC conceived this review, drafted and revised the manuscript, approved the final version prior to publication and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.KH would like to acknowledge funding from the Scottish Government Research Program. AL, NS and JC were supported by grants from the Australian Research Council. MT was supported by an ARC Future Fellowship.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.


Genetic skeletal diseases (GSDs) are an extremely diverse and complex group of rare genetic diseases that primarily affect the development and homeostasis of the osseous skeleton. There are more than 450 unique and well-characterised phenotypes that range in severity from relatively mild to severe and lethal forms. Although individually rare, as a group of related genetic diseases, GSDs have an overall prevalence of at least 1 per 4,000 children. Qualitative defects in cartilage structural proteins result in a broad spectrum of both recessive and dominant GSDs. This review focused on a disease spectrum resulting from mutations in the non-collagenous glycoproteins, cartilage oligomeric matrix protein (COMP) and matrilin-3, which together cause a continuum of phenotypes that are amongst the most common autosomal dominant GSDs. Pseudoachondroplasia (PSACH) and autosomal dominant multiple epiphyseal dysplasia (MED) comprise a disease spectrum characterised by varying degrees of disproportionate short stature, joint pain and stiffness and early-onset osteoarthritis. Over the past decade, the generation and deep phenotyping of a range of genetic mouse models of the PSACH and MED disease spectrum has allowed the disease mechanisms to be characterised in detail. Moreover, the generation of novel phenocopies to model specific disease mechanisms has confirmed the importance of endoplasmic reticulum (ER) stress and reduced chondrocyte proliferation as key modulators of growth plate dysplasia and reduced bone growth. Finally, new insight into related musculoskeletal complications (such as myopathy and tendinopathy) has also been gained through the in-depth analysis of targeted mouse models of the PSACH-MED disease spectrum. Qualitative  defects in cartilage structural proteins result in a diverse range of both recessive and dominant genetic skeletal dysplasias . These sThe pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) disease spectrum: the generation and study of genetic mouse models. Originally believed to be different but clinically related phenotypes, it was gene and mutation identification in the 1990s that demonstrated that PSACH and some forms of MED were allelic diseases resulting from mutations in COMP  region and the carboxyl-terminal domain (CTD) (7 (p.Asp469del), which accounts for 30% of all PSACH, and the recurrent p.Thr585Met missense mutation in the CTD  ,4. Type in (CTD) . The mut the CTD ,4. The p repeat T region aCompD469dntiation . Proteomntiation   selected for mouse model generation was the first EDM5 mutation to be identified and is an archetypal example  domain of matrilin-3 cause moderate to severe forms of MED . The var example .Matn3V194D mice were normal at birth but developed short-limbed dwarfism that was characterised by a final reduction in tibia length of 12–13%  of which only the female demonstrated a clinical phenotype and both founders died prematurely , which is characterised by the upregulation of binding immunoglobulin protein (BiP) (a sentinel marker of UPR) and a broad range of chaperones, foldases and protein disulphide isomerases  is symbolic of classical UPR; however, this prolonged ER stress does not result in a C/EBP-homologous protein (CHOP)-mediated increase in apoptosis , CompD469del (decrease in types IX and XIV) and CompT585M  cartilage, which further implied disruption to ECM organization as a consequence of Comp and Matn3 mutations that correlated with the observations of altered ECM morphology detected by electron microscopy  and extracellular defects (ECM disruption) to the initiation and progression of growth plate dysplasia is experimentally challenging. Therefore, to directly test the role of ER stress in growth plate dysplasia and reduced long bone growth, independent of disruptions to the cartilage ECM, a novel transgenic approach was taken. This approach was previously established and validated as an innovative tool to dissect disease mechanisms in metaphyseal chondrodysplasia type Schmid, which can be caused by missense mutations in the gene encoding type X collagen .Rdw) was targeted primarily to resting and proliferating chondrocytes using the Col2a1 promoter   . Moreoventiation . Finallyntiation ,35, whicMATN3 mutations, such as Arg121Trp, which are associated with marked interfamilial variability in the radiographic phenotype of patients  onto a C57BL6/J inbred background has been shown to increase the severity of the phenotype by further disrupting chondrocyte proliferation, apoptosis and matrix deposition  or in an entire tissue and will provide a powerful technique for determining the role of ER stress in a broad group of inherited connective tissues diseases.The development and deep phenotyping of ‘ER-stress phenocopies’ ,31 is a In conclusion, these different genetic mouse models  of the PSACH-MED phenotypic spectrum have recapitulated key aspects of disease pathology and identified new fundamental mechanisms that have both temporal and spatially context within the cartilage growth plate. The acquisition of this new knowledge would not have been possible through the use of cell culture models, which do not recapitulate the dynamic properties of the cartilage growth plate.


Sets of genes harboring insertions–deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions–deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values . NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins.The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed  Colorectal carcinoma (CRC) is a universally prevalent cancer and a leading cause of death in the world. The overwhelming heterogeneity of cancer has always been the focus of many genetic studies. This complexity is notable at macroscopic, microscopic, and molecular levels. Colorectal cancer can be sporadic, or may show familial predisposition that is due to identifiable single gene mutations with Mendelian or near Mendelian segregation such as Hereditary Non-Polyposis Colorectal Cancer and Familial Adenomatous Polyposis. A remaining percentage of colorectal cancers are not familial but have an inherited tendency . It has hMLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase e (POLE) mutations. In other studies, integrative analysis suggested an important role for MYC-directed transcriptional activation and repression. Epigenetic modifications like altered methylation events and modifications involving small non-coding RNAs have all been described in the pathogenesis of colorectal cancer  or otherwise upon request from the corresponding author.Here, we present the results of comparisons of genetic alterations and mutational signatures between cases and controls, followed by the results of enrichment analysis. The striking feature in this family is the issue of identical by state (IBS) sharing of variants observed in the cancer tissues as shown below, where comparison of cases to controls did not conform to the expected identical by descent (IBD) sharing especially among siblings. In accordance with culture in this part of the country, consanguinity is common but an Figure 1. Tumor tissue samples are labeled P17 and P61, and controls are labeled P39, P26, and P84. The relations between individuals are shown in the pedigree . The family of individuals P39, P26, P61, and P17 is related to a common grandparent. Individual P84 is a distant relative of the family and hence not shown in the pedigree.The study examined whole exome sequences of samples from five subjects chosen from a Sudanese family with a strong family history of hereditary colorectal cancer (as well as other tumors and diseases) by convenience non-probability sample selection. Two patients  were selected from two different branches of the extended family. Cancer patients had a histopathological diagnosis of Microsatellite Instability-High moderately differentiated colorectal adenocarcinoma. A diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) was made based on Bethesda Criteria. Three controls were selected to comprise a sibling to one patient, a second closely related individual, and a third distantly related control. The family pedigree is shown in Table 1 and Figure 2). We utilized unsupervised learning to find hidden structure in unlabeled data (blinding the phenotypes) using gene-based approach . We used non-metric multidimensional scaling in four dimensions to look for a pattern of gene affection by exonic variants between the five samples . When we studied INDELs, the first dimension completely separated cases from controls with tight sub-clustering of tumor and control samples; the second dimension perfectly separated the two cases while the third and fourth dimensions reflected the variability between the controls . The fact that multiple dimensions provided complete separation of cases and controls – without including the phenotypes in the clustering matrix – strongly implies that the observed pattern of gene hits in INDELs is significantly deviated from simple randomness. Hierarchical clustering of the distance matrix provided comparable results. When analyzing SNVs, clustering of controls was less evident . Except for the third dimension, which likely reflected IBD sharing of alleles resulting in a separation pattern of samples comparable to the pedigree, P84 showed a behavior similar yet intermediary to cancer samples in the first and second dimensions, deviating from expected clustering with the two other controls. We can argue from this preliminary analysis that a gene-based signature of exonic INDEL hits is a reasonable possibility in tumorigenesis. However, a distinct pattern in each tumor is clearly seen as well.There were higher numbers of variants in tumor samples (hMSH2 loss of function truncating mutation (rs63749917) was found, consistent with Lynch syndrome. Although generally there were higher numbers of variants  in tumor samples (Table 1), the number of non-synonymous mutations was consistent with figures from other studies in accordance with evolutionary conservation of the functional protein-coding part of the genome and the higher mutation rate of neutral non-coding regions. However, the overall number of damaging variants in the tumor tissues  was significantly higher with a stepwise increase in damaging mutations. Sample P39 showed the lowest amount of damaging SNVs (hence considered here as a true control) and tumor sample P17 exhibited the highest number while control sample P84 was located in an intermediary position . The number of INDELs was also highest in the two tissue samples and displayed a unique feature of ordered mutation pattern shown below in GO enrichment. Although two controls  have shown similar features of possible genome instability – indicated by number of INDELs – comparable to those in tumor samples , there was no orderly pattern of enrichment in the INDELs gene sets. Besides, none of them developed symptoms of cancer over the past decade, indicating again that the pattern of mutations is a necessary feature of cancer development and pathogenesis.In both tumor samples, p-value 5e-6). To test the reliability of this finding, we compared A–G and C–T transitions ratios in inferred IBS somatic variants (described below) which revealed excess in A/G and C/T ratios as well (Table 2).An interesting feature seen in this family is the sharing of excess variants more than expected by IBD for tumor tissues. Unsupervised clustering of alleles in all variation positions showed deviation from expected allele sharing with a clear separation of tumor samples from control that did not reflect the relations perceived from the pedigree. The two tumors shared more variants than what is expected from familial allele sharing, while the difference between closely related individuals, for instance between the two siblings, patient P61 and control P39, was large. The explanation of this sharing is cumbersome and one of the few possible mechanisms for such ordered allele change is DNA editing and site directed DNA transposition. DNA and RNA editing are ancient mechanisms that originated possibly as early as the emergence of Euokarya. We investigated the possibility of editing which was recently reported to occur in cancer tissues through members of the APOBEC family of enzymes  by compaPOLE was in this set of genes along with POLE4, POLD1, POLI, POLQ, and REV1. This set of genes showed a dense interaction network with multiple protein–RNA interactions centered on ELAVL1/HuR, and remarkable patterns of enrichment. Particularly, some miRNA , transcription factors , GO terms  and pathways  showed significant enrichment. We compared A–G and C–T transitions ratios in inferred IBS variants. This subset of variants showed very little number of novel variations but reserved the excess in A/G and C/T ratios (Table 2).It is difficult to identify IBS with precision in a small number of samples in a consanguineous family as we argued. However, we adopted a unique inference approach based on simple comparison between tumor samples, control samples, and TCGA Colonic Adenocarcinoma somatic mutations (see Methods) to identify variations with a high likelihood of being IBS between tumor samples. The concept was based on identifying variations present in both tumor samples and at least one TCGA sample (considered a positive control) provided that the variations are not seen in any of our controls (negative controls). We identified slightly more than 1100 variations, very likely to be somatic IBS variants between tumor samples, corresponding roughly to more than 900 genes (Supplementary Data: Data Sheet 1). Many of these genes were related to cancer. For instance Although cancer is a multistep complex process, Mendelian-like cancer syndromes are thought to be caused by a limited set of tumor suppressor genes (TSG) germ line mutations. In both tumor (and sometimes control) samples, there was a number of mutations in known cancer-related genes. We verified cancer mutational signatures through gene ontology enrichment and network analysis. We used GO term enrichment, pathway enrichment, and interaction network analysis to identify the critical genetic alterations involved in the tumorigenesis process as well as to highlight possible genetic circuits of predisposition.Table 3). Individually, patients and controls showed variable enrichment patterns. Yet in the cancer samples, ECM receptor interaction, ABC transporter, and olfactory transduction were maintained, while controls P84, and P26 showed enrichment in ECM-receptor interaction and glycosaminoglycan degradation respectively. Control P39 had no significant enrichment in any pathway. The Wnt/B-catenin is an important pathway in cancer and particularly associated with colorectal tumors. Mutations may dictate not only the prognosis, and type of cancers in an individuals but population profiles as well. Binding of LRP and FZ in the canonical Wnt pathway disrupts the destruction complex for catenin degradation and thus mutations in these genes (as in tumor sample P17) would cause it to accumulate along with the mutations in destruction complex pathway. Sample P61 showed a CTNNB1 mutation that has been reported to cause the same accumulation leading to cancer. RNF43 mutations were encountered in both cases while DVK1 and FZD8 are mutated in P61. Individual mutations in AXIN2 and CSNKIE in sample P17 may similarly contribute to such effects. Enrichment in the olfactory receptor family, which was seen, is probably not due to the size of the gene family as claimed by some authors  particularly in the non-synonymous mutations.Tested against KEGG database, enriched pathways unique to the tumors included ABC transporters, Lysine degradation, MODY (maturity-onset diabetes of the young), Pantothenate and CoA biosynthesis, cell cycle, and cell adhesion as well as olfactory receptors . This may further supplement the evidence that INDEL pattern is not at all random in colorectal cancer tissues.ELAVL1/HuR, a hub protein of remarkable centrality interacting with over 2000 proteins in the human proteome, together with another central hubs: UBC, TP73, and NFKB1 (Supplementary Data: Image 2). The direct interactions between ELAVL1 and mutated genes in tumor samples are shown in . Although ELAVL1/HuR and UBC are not mutated in our samples, they were extensively interacting with a large number of input genes. To address whether such interaction particularly the centrality of the ELAVL1/HuR is an artifact of the large interacting partners or true interactions we compared the percentage of interactions in genes having non-synonymous SNVs and genes with synonymous SNVs to test the randomness of interactions. The synonymous set had no significant interactions and ELAVL1/HuR was interacting with only less than five nodes (Supplementary Data: Image 3). We queried The Cancer Genome Atlas database for case records with comparable clinical and pathological phenotypes to our studied samples . We looked at exonic somatic mutation data for four patients we could identify . In two cases, ELAVL1/HuR had relatively few interactions. Instead, another hub protein, TP53, showed marked centrality (Supplementary Data: Images 4–7).We used two different interaction databases to study our gene sets (Reactome and VanBuren Lab Cognoscente). To study the genetic and protein interactions of mutated genes (genes harboring high impact or damaging mutations) with other molecules , we used VanBuren Lab online tool Cognoscente that draws genetic interaction networks between input genes and also highlights potential genetic interactions with other intermediates. The advantage of such network approach is the insights it gives into the potential biological mechanisms involved that are not based directly on mutations within the gene sets. We used all genes affected by exonic or splice site variants (INDELs and damaging SNPs defined by ConDel scores) in both tumor samples as input. The most spectacular feature was the interaction of SNVs and INDELs with the RNA-binding protein NFKB1, HDAC2, PIK3R1, TCF7L2, ITGAV, TRAF2, and CDC27 were top central nodes in sample P17, while in sample P61, NFKB1, ACTN2, PPP2R1B, RELA, PIK3CB, SIRT1, and CAMK2B were central nodes. Overlap of interaction networks of tumor samples showed an interesting feature of increased sharing around central nodes (Supplementary Data: Image 8). As well, clusters of related genes  were also prominent. NFKB1, TCF7L2, TGFBR2, NCOR2, CDC27, ACTN2, PIK3R1/2, PPP2R1B/2A, PABPC1, TBP along with different MAPK and STAT proteins were centrally shared. NFkB1 was the most central protein in direct PPI network, and both cases in our study had NFkB1 mutations . Bearing in mind the intricacies of NFkB interactions, it is almost impossible to predict the change in the signaling pathways based on mutational testing alone. However, the centrality of NFkB1 in the interaction networks of mutated genes is not something to overlook.We utilized a different approach to draw only direct PPI networks for genes affected by all (not only damaging) exonic/splice site variants, for each sample, based on Reactome database. Network centrality was investigated using three different methods of un-weighted centrality . In tumor samples, we found that WHSC1, ERBB2, PRNP, PABPC1, TNFRSF14, MAP2K3, and UBC; and secondly the evolving complexity of interaction networks from control to tumor samples (Supplementary Data: Image 8) which is consistent with the preliminary assumptions we made on a stepwise pattern of predisposition and ordered mutations. The number of PPI network nodes and edges was higher in colorectal cancer tumor samples. The percentage of genes forming a single major functional interaction network (using Reactome database) in samples P39, P26, P84, P17, and P61 out of the total number of gene sets was 4, 17, 19, 26, and 29%, respectively. This was visually detectable from the topography of interaction networks (Supplementary Data: Image 8).The potential existence of predisposition circuitries is highlighted by two findings: firstly by the network of genetic interactions of genes shared between cancer samples  and controls  where the gene ELAVL1/HuR was central together with candidate cancer genes like Cancer is a group of diseases characterized by multiple phenotypes including abnormal cell growth and the potential to invade or spread to other parts of the body, reflecting the complexity of the biological processes involved. Given the plethora of non-synonymous mutations leading to damaged or altered protein functions in healthy individuals, cancer seems like a probabilistic combination of events targeting the biological processes, that raises the fitness of tumor cells. The question “do somatic events occur randomly or due to ordered mutagenesis?” remains unsolved. The network approach employed in the current analysis combined with an individualized and familial context fostered insight into the stepwise and multiple stages of the carcinogenesis process, and allowed addressing these broad themes of predisposition circuitries and mutation randomness. Particularly, inclusion of familial proband controls allowed inferences on the potential role of germline genetic alteration in pathway and network analysis, compelling a revisit to the concept of pathognomonic cancer mutations in disease predisposition. Hundreds of driver mutations were identified following the introduction of exome sequencing studies. For example, 558 interactions and 717 regulatory connections existed among 163 known cancer genes and 164 predicted driver genes extracted from the database DAVID . March eThe current set of data was used to explore how such molecular events may contribute to the carcinogenesis process in an attempt to shed light on the actual complex etiology of the disease from a network rather than a single gene perspective. One challenging question is the magnitude and threshold of accumulating somatic mutations that will tip off the homeostatic balance toward cancer, bearing in mind the obvious existence of predisposition circuitries among cancer-free controls. Variants were not just in numerical excess in the tumors but targeted fundamental pathways and genes in the cancer process including cellular metabolism, cell cycle, angiogenesis, cell adhesion, and DNA repair: in short all processes required for cell survival and fitness.As both germline and somatic point mutations including splicing are rather conserved given their reported mutation rate, they are unlikely to explain all the biological alterations leading to function in cancer. This renders RNA and DNA editing – both rather uncommon events in the human genome – along with insertions–deletions, likely and plausible culprits of such alterations. These events, however, whether affecting genes or protein networks, seem to be least random in any sense, specially with the notable sharing of variants between the two tumors. Such compelling display of ordered mutagenesis suggests an orchestrating molecule or a mechanism that governs or at least sets the action of the network on course and may explain the shared mutation pattern encountered in tumors. This molecule is envisioned to be a central part of the potential cancer cell. The milieu of this cell functions as a system with an input signal  and converging on complex output signals that dictate the phenotype. The input signal is explanatory to the nature of the perturbation and how the circuits and cascade exhibited by the cancer cell are switched on. The most plausible candidate for the input signals, as we will argue, are oncogenic viruses and primarily in our case Epstein–Barr virus (EBV). A viral infection is envisioned to interact with predisposition networks which include counterpart proteins in various downstream stages .The protein ELAVL1/HuR seems the most plausible candidate for coupling the initial viral input signal, yielding the expected effector and orchestrating role of the system given the repertoire of its interacting partners. ELAVL1/HuR is a hub protein like TP3 and TGF-Beta and is thought to be central and key in the carcinogenesis process. Interestingly TP53 was not implicated in the analysis either directly or indirectly, in contrast to examples from TCGA (Supplementary Data: Images 4–7), and consistent with previous literature on its minor role in breast cancer in this part of the world . The lacThe significance of ELAVL1, which in itself is quite conserved and unaffected by direct genetic alterations, lies in its potential role in combination with other molecules. The role it plays along with viral oncogenesis . The DNA binding domain did not show mutations. It is difficult to perceive why some malignant cells would break a central key to cellular functions like NFKB. Experimental evidence for NFkB role in cancer was demonstrated. Exome sequencing of members of an extended family with colorectal cancer revealed interesting features of stepwise accumulation of mutations leading to tumorigenesis, a shared mutation pattern and predisposition circuitry. We highlighted the importance of central hub proteins in the study of cancer genomics and pathogenesis. RNA binding protein ELAVL1/HuR and NFKB, both of which appeared central in the network analysis, are suspected to work in concert with viral etiology on a background of predisposition circuitry to initiate and guide the pathogenesis of colorectal cancer in a subset of predisposed individuals.® Genomic Purification Kit . Tissue lysates and extraction solutions were prepared according to the Wizard® SV Genomic DNA Purification System protocol. Extracted DNA quantity and quality were assessed using the NanoDrop® ND-1000  spectrophotometry at wavelength spectrum of 220–750 nm and standard 1% agarose gel electrophoresis.DNA was extracted and purified from frozen colorectal tumor tissues (tumor samples) and EDTA-preserved blood (controls) using Wizard® . Library preparation and exome capture were done using NimbleGen Oligonucleotide Libraries . Exome sequencing was performed on Illumina HiSeq2000 platform . The clean reads  were aligned to the reference human genome build hg19 using Burrows–Wheeler Aligner  have an influence on genomic milieu, they were not included in these shortlists for network construction due to the lack of consensus scoring criteria to classify their effect. The goal was to reduce false positives as much as possible. VanBuren Lab online tool Cognoscente  was usedFigure 4 . Cytoscape RINalyzer app  was usedFigure 4 . CytoscaFigure 4  was usedFigure 4  workflowOnline repositories for TCGA data available on TCGA data portal  and cbioThis study was ethically approved by the Ethical and Scientific Committee of the Institute of Endemic Diseases, University of Khartoum. Written informed consent was obtained from all participants, in accordance with the Declaration of Helsinki.SS, MI, OE, HM, MR, LB, FB, AS, MN: conceived the study and the experimental design. MK, WN, MA, UE, AH, OE, SF, FI, KA, MI: designed and carried out the analysis. All authors participated in interpretation of data. MI, MK, WN, wrote the paper. SS and MK contributed equally to this work. All authors revised the manuscript critically and were involved in editing the paper and approval of the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ageing disorders can be defined as the progressive and cumulative outcome of several defective cellular mechanisms as well as metabolic pathways, consequently resulting in degeneration. Environment plays an important role in its pathogenesis. In contrast, developmental disorders arise from inherited mutations and usually the role of environmental factors in development of disease is minimal. Age related macular degeneration (AMD) is one such retinal degenerative disorder which starts with the progression of age. Metabolism plays an important role in initiation of such diseases of ageing. Cholesterol metabolism and their oxidized products like 7-ketocholesterol have been shown to adversely impact retinal pigment epithelium (RPE) cells. These molecules can initiate mitochondrial apoptotic processes and also influence the complements factors and expression of angiogenic proteins like VEGF etc. In this review we highlight why and how AMD is an ageing disorder and not a developmental disease substantiated by disrupted cholesterol metabolism common to several age related diseases. Alu RNA deposits begin to emerge later in life and not during development. These aggregates deposit in the extra-cellular space between Bruch’s membrane and retinal pigment epithelium cells (RPE). Gradual and consistent effects of these aggregates gradually cause degeneration of these cells followed by global atrophy of RPE cells, commonly known as geographic atrophy (GA). Besides, active inflammatory components of these deposits between Bruch’s membrane and RPE, stimulate angiogenic factors  which act on choriocapillary network beneath the Bruch’s membrane and stimulate proliferation to new blood vessels . These newly formed blood vessels can outgrow into the RPE cells and result in disruption of RPE cell integrity and function which is well preserved in early life. Understanding the complexity of mechanisms through genetics, epigenetics, metabolic pathways and risk factors have provided insight about the participation of cellular pathways that resemble aging, but not early or late development. Cells that lose their capacity to divide by a phenomenon called cell senescence undergo ageing. Several impaired cellular processes could lead to cellular and morphological changes in the cell over time in association with environmental factors in complex manner ultimately resulting in ageing. These cellular processes include: metabolic pathways  is described by irreversible vision loss in older age. The disease pathology emerges with the degeneration of macula which forms the central part of retina. The macula consists of photoreceptor (rods and cones) important for central vision. As AMD symptoms appear, characteristic features such as formation of drusen, consisting of active and inactive complement associated inflammatory products, aggregate of lipoprotein, cell debris, oxysterols, oxidized phospholipids and It is evident that the disease pathology of AMD is equally influenced by both environmental as well as genetic factors in a complex manner such that the effects are not manifest early in life. Several environmental factors such as age, sex, diet, smoking and demographic distribution have been reported to be strongly associated with AMD pathology Figure , unlike etc. which are reportedly involved in eukaryotes. Several environmental factors like smoking, stress, dietary habits etc. have been well described in introducing chemical changes on the genome  of the genome which impact the expression pattern of regulatory genes. The fundamental chemical changes include methylation, phosphorylation, acetylation, Similarly, there are several co-morbid conditions which have been found to be associated with manifestation of AMD. Persistent hypertension, heart disease and diabetes, accelerate AMD pathogenesis. Lately, it has also been revealed that Alzheimer’s disease appears to have concurrence with AMD pathology suggesting the role of several common gene loci indicating that their associated pathological mechanisms, underlying cascade of downstream events, converge to cause the two diseases of ageing. This review highlights the role of various candidate genes that play an independent role in progression of AMD and digresses on spectrum of gene association studies which modify AMD pathology in a manner not characteristic of a development disorder.Complement system components: e.g., complement factor H (CFH), component C2, component C3 and complement factor I (CFI). Moreover, TLRs receptor components of innate immunity have also been found to be strongly associated with AMD pathogenesis. Therefore, in general the components of innate immunity have predominant role in progression of AMD.Enzymes involved in metabolic processes: Hepatic Lipase (LIPC), cholesteryl ester transfer protein (CEPT) and Apolipoprotein E (APOE) are involved in different stages of lipid metabolism.Angiogenic factors: VEGF has been reported as the strongest angiogenic factor. Several angiogenic factors which have been reported till now are either associated with AMD or are reported to be associated with degenerative and inflammatory diseases. e.g., VEGF, COL genes, TGFBR1, immediate early response (IER3) etc.etc. play a non redundant role in pathologies of AMD.Proteases: Several proteases have been implicated in AMD pathophysiology including degradation of extracellular matrix. For example, Tissue inhibitor metalloprotenases 3 (TIPM3), IER3 and numerous multifunctional proteins which have several cellular functions like ADMS2, HTRA1 TNFRSF10A and regulatory cellular enzymes likeDICER.Proteins in apoptosis and cellular regulation: e.g., The genes associated with AMD can be conveniently categorized into following groups based on their direct or indirect association reported in various studies:AMD literature is replete with both confirmatory and conflicting reports of several genes and their polymorphisms which have been found to be associated with AMD.CEPT) and ABC binding cassettes A1 (ABCA1) gene have been found to be associated with AMD pathology. Lipoprotein lipase acts when systemic cholesterol levels fall along with the function of ABCA1 leading to formation of APOE, APOA1 and HDL. When cholesterol levels rise, the action of CEPT comes into play which removes the cholesterol ester (CE) from HDL and convert into LDL by enzymatic process. LIPC and LPL both have dual functions i.e., as triglyceride hydrolase as well as in receptor mediated endocytosis of cholesterol at different stages of metabolism.Metabolic processes constitute the normal biochemical pathways in the cell which are involved in transportation and biosynthesis of biomolecules including several regulatory processes of the cells. AMD can be regarded as a disease caused by disruption or abnormal metabolic processes especially lipid metabolism. The metabolic processes which are associated with lipid metabolism are one of the major factors in AMD pathogenesis. Several enzymes which participate in lipid and lipid associated metabolism have been found to be associated with initiation and progression of AMD pathology. Several population based genetic and animal based studies have revealed the role of proteins involved in lipid metabolism and their associated receptors in AMD. Important enzymes which participate in lipid metabolism, including human hepatic lipase gene (LIPC), lipoprotein lipase gene (LPL), cholesterol ester transferase gene (LIPC) is expressed in hepatic cells and adrenal gland whose principal function is to convert intermediate-density lipoprotein (IDL) to low-density lipoprotein (LDL) which have been widely investigated in AMD. The lipids and lipoprotein contents of drusen between Bruch’s membrane and RPE are basically distinct from the plasma contents and are comprised of free and esterified cholesterol, phosphotidylcholine (PC), apolipoproteins B-100, A-I and ApoE  (rs493258). They found LIPC association was strongest for a functional promoter variant, rs10468017, that influences LIPC expression and serum HDL levels with a protective effect of the minor T allele (HDL increasing) for advanced wet and dry AMD.Recent studies have demonstrated composition and sub-structures of drusen comprising of 3.2% dry weight of long fatty acid esterified and non-esterified cholesterol and about 30 different types of proteins, including apolipoproteins (apo E and apo B)  mice in 20 month aged mice. The burden of Aβ in the hippocampal region was higher in Apoe+/+ transgenic mice as compared to Apoe+/− and Apoe−/− mice. This suggests that even though both pathologies implicated similar elements but the final effect are quite opposite  is a plasma protein that plays an important role in cholesterol metabolism by facilitating the transportation of triglyceride and cholesteryl ester from VLDL to HDL or vice versa. Several genetic studies have demonstrated conflicting results of CEPT association with AMD progression. Recently, Yu et al.  reported+2/Fe+2 , one of the oxidized component of cholesterol metabolism, has been found to stimulate angiogenic factors and inflammatory processes  and 27-hydroxycolesterol (27-Hch), respectively  stress  can contribute in the progression of pathophysiology of choroidal neovascularization (CNV) in AMD. VEGF expression, expression of proangiogenic factors, and activation of metalloproteases are believed to play an important role in the formation of CNV in AMD. It is well understood that the biologic activities of VEGF-A, proangiogenic molecules like  and proinflammatory cytokines can be influenced by the interaction of cells with the extracellular matrix (ECM). Cell-ECM interaction, useful for integrity and functionality of cell, are influenced by the expression of these factors . ECM protein channel-ECM communications influence tissue remodeling events, including angiogenesis. Several angiogenic factors have been widely studied as one of the cardial feature of AMD pathophysiology: “neovascularization”. The neovascularization being the consequence of dysregulated action of genes involved in proangiogenesis, angiogenesis, extracellular proteins, and metalloproteases is the central thesis in wet AMD. These effects are additive, rarely affecting development milestones. TGFBR1 also possesses the property to exert proangiogenic effects with its encoded protein forming a heteromeric complex with type II TGF-beta receptors and transducing the TGF-beta signal from the cell surface to the cytoplasm when bound to TGF-beta. This protein is a serine/threonine protein kinase. This gene is expressed in all tissues but more abundantly expressed in brain and heart. This protein is involved in several biological processes like induction of apoptosis, response to hypoxia, epithelial to mesenchymal transition, artery morphogenesis, signal transduction, regulation of transcription, negative regulation of endothelial cell differentiation, positive regulation of cell proliferation, collagen fibril organization etc.Similarly, IER3 has been postulated to be responsible for regulating death receptor mediated apoptosis, interaction with NF-κB pathways, and upregulated at irradiation, ionizing radiation, viral infection and at the time of other cellular responses. Numerous functional data relying on cell culture based studies and knock-out mouse models has revealed the involvement of IER3 expression in immune functions and in the physiology of the cardiovascular system. Arlt et al. showed that deficiency of IER3 expression in mice results in an aberrant immune regulation and enhanced inflammation, hypertension or impaired genomic stability  dephosphorylation of “BH3 only protein Bad” which leads to mitochndrial apoptotic pathway; and (ii) activation of “pro-apoptotic BH3 only protein Bim” ultimately inducing apoptosis by inhibiting the activity of Bcl-2 family members proteins along with microtubultes destabilization , atherosclerosis and AMD can be distinguished by several pathological hallmarks that appear at the time of ageing. Collectively, these studies suggest the imperative role of metabolic processes and various oxidized products which exert their additive effect on both complement and proangiogenic/angiogenic molecules with time. Several such reports indicate that altered cholesterol metabolism leads to pathological changes which results in accumulation of sterols, APOE, cholesterol oxidase and oxidized cholesterols in these diseases of ageing (Kannel et al., In this context, the generality of need to compare the critical mechanisms distinguishing an ageing disorder such as AMD from mechanisms akin to developmental disorders can provide insight for future interventions. Understanding the molecular mechanisms that prevent AMD from being a developmental disorder may therefore lead to effective therapies in future. For this, longitudinal comparative studies between developmental and degenerative disorders are warranted.Writing of manuscript: Kaushal Sharma, Akshay Anand, Neel Kamal Sharma. Editing of manuscript: Akshay Anand. Concept of the manuscript: Akshay Anand.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.


Aedes aegypti is an important vector of viral diseases that impact global health. Insecticides are typically used to manage mosquito populations, but the evolution of insecticide resistance is limiting their effectiveness. Thus, identifying new molecular and physiological targets in mosquitoes is needed to facilitate insecticide discovery and development. Here we test the hypothesis that gap junctions are valid molecular and physiological targets for new insecticides. Gap junctions are intercellular channels that mediate direct communication between neighboring cells and consist of evolutionarily distinct proteins in vertebrate (connexins) and invertebrate (innexins) animals. We show that the injection of pharmacological inhibitors of gap junctions  into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency. In contrast, when applied topically to the cuticle, carbenoxolone was the only inhibitor to exhibit full efficacy. In vivo urine excretion assays demonstrate that both carbenoxolone and mefloquine inhibit the diuretic output of adult female mosquitoes, suggesting inhibition of excretory functions as part of their mechanism of action. When added to the rearing water of 1st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency. Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes. Taken together these data indicate that gap junctions may provide novel molecular and physiological targets for the development of insecticides.The yellow fever mosquito  Aedes aegypti, is the most important vector of the viruses that cause yellow, dengue, and chikungunya fevers in humans. These diseases have spread around the tropical and subtropical world, facilitated by globalization of human societies and climate change  vs. response variable-slope) to determine effective dose or concentration 50% values  of 15.47 ng per mg of mosquito body weight (ng/mg) followed by meclofenamic acid (ED50 = 96.39 ng/mg) and carbenoxolone  followed by carbenoxolone (EC50 = 1587 ppm). We were unable to test the efficacy of mefloquine in this assay, because it is not soluble in water and the amount of DMSO required to keep it in solution (>2%) was toxic to larvae.We assessed the efficacy of the gap junction inhibitors as larvicides by adding them to the rearing water of 1n larvae . MeclofeTo determine if knockdown of innexin mRNA levels affected the survival of adult female mosquitoes, we utilized RNAi. First, we used qPCR to determine the relative expression of each innexin in mosquitoes injected with eGFP dsRNA (3 days post injection). As shown in Injection of an innexin dsRNA cocktail containing dsRNAs for each innexin  resulted in significant knockdown of Inx1 (32 ± 8%), Inx2 (69 ± 3%), Inx3 (51 ± 5%), Inx4 (71 ± 10%) and Inx7 (86 ± 2%) by 3 days after injection compared to expression levels in the eGFP-injected controls . The expst instar larvae  were efficacious , and oner larvae . Thus, oIn support of our pharmacological data, the injection of dsRNA against all 6 innexin mRNAs reduced the survival of adult female mosquitoes over the next 11 days . The redThe toxic effects of the gap junction inhibitors and the innexin dsRNAs on mosquitoes are not surprising given that innexin mRNAs are expressed throughout the mosquito life cycle . FurtherIn the present study, we show that at least two of the pharmacological inhibitors (carbenoxolone and mefloquine) perturb the functions of the excretory system, as indicated by their inhibition of the diuretic capacity of adult female mosquitoes . These tSurprisingly, we did not observe effects of a sub-lethal dose of meclofenamic acid (1.53 mM) on the diuretic capacity of mosquitoes , and higWhen applied topically to the cuticle of mosquitoes, only carbenoxolone showed insecticidal activity, whereas mefloquine and meclofenamic acid had limited and nominal activity, respectively. Thus, carbenoxolone appears to have the capacity to penetrate the cuticle. The structures  and chemDespite nominal topical efficacy against adult female mosquitoes, meclofenamic acid was the most potent larvicide of the three compounds . Carbenost instar larvae of A. aegypti than DNOC (Dinitro-ortho-cresol), which is considered a weak insecticide, and 874,643 times less effective than permethrin, a highly potent insecticide [A. aegypti [Although meclofenamic acid and carbenoxolone were respectively the most effective compounds against larval and adult female  mosquitoes, it is important to emphasize that these compounds are not nearly as potent as conventional insecticides, such as permethrin. For example, meclofenamic acid is 46 times less effective against 1ecticide . Additio aegypti .Additional concerns for both carbenoxolone and meclofenamic acid are their efficacy on mammalian gap junctions ,36. MoreThe present study is the first to demonstrate that three different commercially available gap junction inhibitors exhibit insecticidal activity. Moreover, we show that RNAi-based knockdown of mRNAs encoding gap junctional proteins  leads to increased mortality of adult female mosquitoes. Taken together, these results suggest that gap junctions offer new potential insecticidal targets for mosquito control. However, much progress still needs to be made in terms of discovering compounds with acceptable potency and specificity for mosquito gap junctions. The long evolutionary distance between innexins and connexins leaves us hopeful that an innexin-specific compound can be discovered and developed.


The vomeronasal organ (VNO) is a sensory organ that is found in most terrestrial vertebrates and that is principally implicated in the detection of pheromones. The VNO contains specialized sensory neurons organized in a pseudostratified neuroepithelium that recognize chemical signals involved in initiating innate behavioral responses. In rodents, the VNO neuroepithelium is segregated into two distinct zones, apical and basal. The molecular mechanisms involved in ligand detection by apical and basal VNO sensory neurons differ extensively. These two VNO subsystems express different subfamilies of vomeronasal receptors and signaling molecules, detect distinct chemosignals, and project to separate regions of the accessory olfactory bulb (AOB). The roles that these olfactory subdivisions play in the control of specific olfactory-mediated behaviors are largely unclear. However, analysis of mutant mouse lines for signal transduction components together with identification of defined chemosensory ligands has revealed a fundamental role of the basal part of the mouse VNO in mediating a wide range of instinctive behaviors, such as aggression, predator avoidance, and sexual attraction. Here we will compare the divergent functions and synergies between the olfactory subsystems and consider new insights in how higher neural circuits are defined for the initiation of instinctive behaviors. The mammalian olfactory system is composed of multiple chemosensory subsystems that differ in anatomical location, receptor types, and innervation within the central nervous system  as well  and Fprs , which are localized to the upper sublayer of the basal VNO. This spatial segregation is also maintained at the level of the AOB, defining a tripartite organization of the mouse vomeronasal system  class 1b, also known as m Figure .A third population of Gαo-expressing VSNs expresses Fpr-rs1 Figure . Fpr-rs1H2Mv molecules. Instead, vomeronasal receptors seem to be essential: genetic deletion experiments showed two V2Rs—Vmn2r26 (V2R1b) and Vmn2r81 (V2Rf2)—which are needed by their VSNs to respond to specific MHC peptides  family—has been identified to be detected by V2R-expressing VSNs. The mouse genome contains 24 members of this family of 5–15 kDa peptides expressed in extraorbital, lacrimal, Harderian, and submaxillary glands (exocrine glands) in a sex- and strain-specific manner  and other related lipocalins. MUPs are abundantly expressed in urine, but are also found in other secretions, including saliva, milk, and even the olfactory epithelium  activate basal VSNs. Aggressive behavior toward intruder males was identified to require sensory transduction from basal VSNs. Ablation of either Gαo, Gγ8, and MHC peptides have been shown to alter female reproductive function as detected in the Bruce effect test (Leinders-Zufall et al., These recent advances in the identification of specialized receptors, neural pathways and sensory ligands from the basal VNO layer provide the tools to stimulate, study, and determine the molecular mechanisms that trigger specific behavioral responses.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.


Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective  Rett syndrome (RTT) is a devastating disorder that, because of its incidence, is considered one of the main causes of severe intellectual disability in girls  gene was discovered as the genetic cause of Rett syndrome  of MeCP2, their regulation and their relevance in the pathobiology of RTT, we will analyze in depth the current knowledge of MeCP2 structure and molecular functions and provide bioinformatics and experimental data testifying already established and putative PTMs of MeCP2. Functional studies and animal models used to characterize some of these PTMs will be surveyed, highlighting the major weaknesses in the field and which, in our mind, should be the future challenges for a better comprehension of MeCP2 activities.At a molecular level, there exist more contradictory data. In fact, MeCP2 appears as a multifunctional protein, mainly but not exclusively involved in regulating gene expression. It appears that the structure of MeCP2 together with a series of differential post-translational modifications (PTMs) might justify this functional versatility, possibly occurring through the capacity of the methyl-binding protein to interact with several diverse protein partners  have no apparent phenotype until 3–8 weeks of age, when they start showing gross abnormalities, such as locomotor defects, hindlimb clasping, hypotonia, reduced spontaneous movements, tremors, breathing irregularities and often seizures. Symptoms worsen over time, and the animals die within 6–10 weeks of age. Heterozygous female mice (Mecp2−/+) are viable, fertile and appear normal up to 4–6 months of life, when they start manifesting RTT-like symptoms. Other models with less severe genetic lesions, often mimicking human mutations, have subsequently been generated, and are nicely reviewed in Ricceri et al. . Even though the overall phenotype is milder, the 308/yMecp2 mouse model shares most of the features characterizing the knock-out animals, including learning and memory deficits  results in a robust rescue of the general conditions of the animals, including survival and breathing, while mobility, clasping, and tremors were less reversed. Altogether, these studies demonstrated that Mecp2-related disorders are reversible and, at least in mice, they can be treated even at late stages of disease progression  always causes the appearance of RTT-like phenotypes and premature death  or symptomatic (adult) animals. However, recent experimental results have demonstrated that the inactivation of Once the relevance of MeCP2 had been demonstrated for the central nervous system, it became imperative to define the expression pattern of the protein during brain development and the associated neuro-pathological abnormalities.Concerning MeCP2 protein levels, it is generally accepted that its expression in brain mirrors neuronal maturation. That is, MeCP2 increases when neurons develop dendritic arbors, project axons and establish connectivity , and a smaller head and a longer neck characterized the spines. Overlapping results were obtained observing hippocampal neurons of female RTT patients  derived from RTT patients' fibroblasts showed lower dendritic spine density than control neurons , and long-term depression (LTD). Furthermore, 2-photon time lapse imaging has shown that, at the onset of the disease, Mecp2-null somatosensory cortices display remarkable alterations in the dynamics of dendritic spines; on the contrary, when maturation of the connectivity is complete, no differences in spine dynamics are evident in Mecp2-mutant mice with respect to their wild-type controls . In vitro studies have confirmed the relevance of MeCP2 binding to methylated DNA, suggesting that most, if not all, missense mutations within the methyl-binding domain impair the selectivity of MeCP2 for methylated DNA , the MBD, the intervening domain (ID), the TRD and the C-terminal domain  the highly disorganized structure of MeCP2 endows the protein with multiple functions; (ii) the structured MBD gives the protein its unique capability of interacting with both methylated and hydroxymethylated DNA; (iii) PTMs of MeCP2 are likely to affect directly its binding to DNA and protein partners, therefore contributing to the versatility of MeCP2.in vivo transcription at a distance.Adrian Bird and collaborators originally identified MeCP2 as a nuclear factor capable of binding DNA with at least one symmetrically methylated CpG-dinucleotide  with histone deacetylase activities, and that transcriptional repression of methylated DNA Mecp2-null neurons, but not in glia, where the protein is much less abundant  and to regulate splicing of reporter minigenes. Supporting these data i et al. . This deThus, considering the structural flexibility of MeCP2 and the possibility of PTMs affecting its structure and/or interactions with protein partners, we believe that a better comprehension of MeCP2 functions might be obtained through the study of its diversely modified isoforms and their networking capabilities. Therefore, we will progress critically surveying the actual knowledge on MeCP2 phosphorylation that so far certainly represents the best characterized PTM of the methyl-binding protein.Bdnf promoter III, thereby facilitating transcription  began in 2003 when two independent studies demonstrated that neuronal activation triggers a calcium-dependent phosphorylation of Mecp2 and the consequent release of the methyl-binding protein from To begin with, these studies proposed for the first time that Mecp2 is involved in the control of neuronal activity-dependent gene regulation through its PTMs, and that the deregulation of this process might participate in the RTT pathology. As we will see, this concept still remains valid and has been further strengthened by the development of Mecp2 phospho-defective transgenic mice Table . FurtherS421A/yMecp2, Cohen et al., Mecp2 knock-out mice. However, and possibly in accordance with the mild phenotype of the KI mouse that appears to be defective only in the capability of processing novel experience, so far no pathogenic mutation has been associated with this residue. Of relevance, we still lack insights regarding the molecular consequences of this PTM. As already mentioned, initial data suggested that S421 phosphorylation induces the detachment from specific genes. However, a ChIP-seq approach used to describe the genomic distribution of this specific Mecp2 phospho-isoform in brain of knock-in mice did not reveal a selective detachment of pMecp2 either in resting or stimulated conditions. These results might indicate that Mecp2 affects activity-dependent transcription by changing the molecular partners recruited on DNA  analysis identified that neuronal activity induces also S424 phosphorylation in mouse brain  depending on its differential phosphorylation. Accordingly, although S421 phosphorylation remains for the time being the most widely characterized PTM and pT308 results as the most functionally relevant, several other sites of phosphorylation have been mapped, and their modification is often spatially regulated by specific stimuli. Almost no information is available so far on these PTMs and their regulation during brain development; furthermore, since a role of MeCP2 outside of the nervous system, particularly in glia and microglia, has recently been demonstrated as important for MECP2-related disorders, future studies should also investigate MeCP2 phosphorylation in these cells. MeCP2 phosphorylation has been hypothesized to affect its binding to target DNA sequences: however, no data seem to support this hypothesis in neurons, and none of the phosphorylation events characterized so far globally affect MeCP2 binding to chromatin. It is highly plausible then that the identification and/or characterization of novel phospho-residues of MeCP2 will lead to the discovery of PTMs that generally or selectively influence its binding to chromatin. Furthermore, they should help providing a better comprehension of MeCP2 in physiological and pathological conditions. To this purpose, we suggest that studies should focus first on sites that are conserved by evolution and/or have been found mutated in patients. Thus, we employed two different tools to predict phosphorylation sites  to several lysines. In particular, the sumoylation of lysine 223 is required for the recruitment of HDAC1/2 complexes and mutation of K223 abolishes its gene silencing properties in primary cortical neurons  and their possible involvement in MECP2 related disorders represent urgent challenges.in silico analyses; Isabella Barbiero, Anna Bergo, Chetan Chandola, Mohammad S. Nawaz, Laura Rusconi, Gilda Stefanelli, Marta Strollo, and Maria M. Valente contributed equally in the conception and drafting of the manuscript.Nicoletta Landsberger wrote the manuscript with the assistance of Charlotte Kilstrup-Nielsen; Elisa Bellini generated the figures and the tables; Giulio Pavesi performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The recent exponential increase in our knowledge of cellular and molecular mechanisms involved in carcinogenesis has largely failed to translate into new therapies and clinical practices. This lack of success may result in part from the fact that most studies focus on tumor cells as potential therapeutic targets and neglect the complex microenvironment that undergoes profound changes during tumor development. Furthermore, an unfortunate association of factors such as tumor genetic complexity, overestimation of biomarker and drug potentials, as well as a poor understanding of tumor microenvironment in diagnosis and prognosis leads to the current levels of treatment failure regarding a vast majority of cancer types. A growing body of evidence points to the importance of the functional diversity of immune and structural cells during tumor development. In this sense, the lack of technologies that would allow for molecular screening of individual stromal cell types poses a major challenge for the development of therapies targeting the tumor microenvironment. Progress in microenvironment genetic studies represents a formidable opportunity for the development of new selective drugs because stromal cells have lower mutation rates than malignant cells, and should prove to be good targets for therapy. The incidence of cancer has increased worldwide over the last century. In 2012, 32 million people had cancer within 5 years of diagnosis, 14 million new cases were diagnosed, and 8 million cancer deaths occurred worldwide [During the past decades, an increasing body of work has shed light on the cellular and molecular mechanisms involved in the transformation of normal cells into cancerous cells. However, this research largely fails to improve current clinical practices . PhysiciCellular interactions lie at the core of metazoan physiology and encompass an effective and elegant signaling repertoire that integrates up to trillions of cells in a single organism. These interactions begin during the early stages of embryonic development and coordinate events such as cell proliferation and differentiation in complex tissues, crucial processes for the functional maintenance of whole organs . During Cell-cell interactions primarily occur between the mesoderm, endoderm and ectoderm during embryonic development and between the parenchyma and stromal compartments throughout adult life , 13. In Carcinomas, which by definition arise from epithelial cells, account for nearly 80 % of all human cancers . EpithelReactive stroma has been shown to emerge at the beginning of the disruption of the interactions with the epithelial compartment along prostate malignant transformation  and seemThe TGF-ß superfamily is composed by different growth factors which play important roles in both physiological and pathological processes and, lately, has been associated with cancer development . TGF- ß The EMT seems to represent a breakpoint in enabling malignant cells to invade other tissues and organs. In fact, the EMT is primarily a cellular biological program typically involved in various stages of embryonic morphogenesis and wound healing, however, the wide repertory of molecular tools employed during this process is also used as a subterfuge by transformed cells to acquire skills related with invasion, metastasis and resistance to apoptosis . This prin vitro and in vivo approaches, these authors showed that transformation of human mammary epithelial cells mediated by transfection with HRAS and hTERT increased cell proliferation and survival in vitro. Nude mice inoculated with these cells and mammary fibroblasts grew tumors twice as fast as mice inoculated with transformed cells alone [per se does not confer all of the malignant characteristics observed in tumor cells, in particular those related with cellular invasion [In this complex scenario represented by cellular interactions between stromal and epithelial cells, Weinberg and colleagues elegantly clarified these interplay during disease progression in two reports. In a study using ls alone . This seinvasion , epithelinvasion  and metainvasion , 56. In invasion . Howeverinvasion . Furtherinvasion .PTEN (phosphatase and tensin homolog deleted on chromosome 10) depleted prostate cancer cells up regulated the expression of chemokine receptors chemokine (C-X-C motif) receptors (CXCR) 1, 2 and 4 in the stromal compartment, besides augmenting the release of CXCL-12 and chemokine (C-C motif) ligand (CCL)2 that, in turn, by a paracrine signaling, sustained the aggressive behavior exhibited by malignant prostate cells [PTEN controls cell survival and proliferation through the inhibition of PI3K/Akt intracellular signaling pathway [PTEN is constitutively expressed in cells and figures as one of the genes most frequently mutated in several distinct tumors, demonstrating its importance in physiological processes [PTEN, represent an important axis in the interaction between stromal and malignant cells [Since then, many studies have demonstrated the importance of the microenvironment in tumor progression. Recently, Maxwell and colleagues demonstrated that the release of the chemokine (C-X-C motif) ligand (CXCL)-8 in te cells . The tum pathway . PTEN isrocesses . The stunt cells , 63.In fact, the link between inflammation, cancer development and progression, first proposed by Virchow, is now a widely recognized process  currentlThe simple presence of leukocytes in the tumor mass does not allow discriminating the role these cells are playing in the tumor microenvironment. Leucocyte subsets can display different functions . Macrophages, for instance, display a full spectrum of functions that allow them to perform different tasks in the tumor microenvironment. These cells have been classically classified as M1 or M2 . MacrophOther immune subpopulations have been described as playing roles in tumor progression, such as neutrophils  but are also stimulated by growth factors produced by the leukocytes  in human tumors to estimate Th1/cytotoxic based responses and macrophages presumed to be M1 (if CD8/CD68 ratio is high), or M2 (if the ratio is low) .Other groups take into account not only the function of the cells in the tumor microenvironment, but also different cell localizations, considering the relevance of the capacity of the cells to invade the tumor mass or to stay at the tumor margin , 78. ThiThe immune architecture is not only a function of the immune cells recruited to the tumor site, but also of the intrinsic characteristics of tumor cells. As already mentioned, signaling loops have been extensively described between the tumor cells and immune system cellular components , 67, 71.In addition, it has already been reported that blood and lymph vessels network development also represents a notorious hallmark during carcinogenesis and tumor progression . The pecFinally, the microenvironment has been implicated not only in tumor progression but also in carcinogenesis. Some clues initially originated at the end of the 1990s specifically associated MMP-3 expression and release with the development of breast cancer . FurtherSince genes were identified as key elements in the carcinogenic process, they have become the focus of many cancer research projects. These studies have contributed to our knowledge of genetic alterations involved in disease generation and progression, including polymorphisms, mutations, translocations, recombination, and the regulation of gene expression. Moreover, this body of work has allowed for the development of tools currently used in disease diagnosis, prognosis and treatment . The recIn vitro studies provided further evidence of the consequences of altered genetic expression in breast stromal cells: in co-cultures, up-regulation of CCL18 and CCL2 in CAF promoted breast tumor progression by inducing cellular invasion [Unveiling the molecular constitution of tumors also revealed major differences in gene expression patterns of stroma surrounding normal and cancer tissues. For example, breast carcinoma associated fibroblasts (CAF), when compared to normal breast tissue fibroblasts, alter the expression of genes, in particular those associated with the healing process, such as PLAUR, LOXL2, PLOD2 and SDFR1 . Analyseinvasion .Large scale gene expression analyses performed in prostate tumors demonstrated that approximately 500 genes were up-regulated and 600 genes were down-regulated in tumor stroma . Most diMore recent work utilized the same approach to identify molecular alterations during the progression of esophageal adenocarcinoma. The authors reported that stromal samples from Barrett’s esophagus, a clinical condition caused by the long-term gastroesophageal reflux disease (GERD) that normally precedes the esophageal adenocarcinoma development, exhibits a peculiar inflammatory gene expression pattern, mainly represented by the TGF-β pathway. On the other hand, adenocarcinoma stromal samples overexpressed genes associated with poor prognosis including TMEPA, Thrombospondin 1, and BCL6 . In thisin vitro comparing fibroblasts from normal human lungs and from non-small cell lung-cancer, the latter overexpressed genes involved in the TGF-β and MAPK signaling pathways. These findings were corroborated by parallel ex-vivo analyses of fresh tissue samples, and activation of the signaling pathways correlated with a worse prognosis [TP53). Many studies have extensively explored the roles of TP53 in tumor cells [TP53 in the stroma of different tumor types [In functional studies conducted rognosis . Ovarianrognosis  and colorognosis  also disor cells –114. Moror types –119.Rb gene in mice epithelial cells not only allows tumor formation by prostate cells, but also leads to the loss of TP53 in the stromal compartment, abrogating the homeostasis between epithelial and stromal tissues and ending up with loss of TP53 also in the tumor [TP53 in most of human carcinomas could be preceded by TP53 loss in stromal cells which, in turn, would happen as a consequence of an initial driver genetic event in epithelial tissue, such as Rb gene loss, responsible for not only promoting a permissive microenvironment propitious to initiated cells, but also for contributing to an important event in tumor progression which is TP53 loss. A provocative hypothesis conjectured by Hill and colleagues suggested that oncogenic stress mediated by initiated epithelium would create a selective pressure in the microenvironment that culminates with p53-deficient stromal cells selection [TP53 absence was achieved due to the selective pressure imposed by malignant cells through oncogenic stress or due to the presence of p53-deficient fibroblasts since the beginning of the process. Despite the fact that the precise mechanisms of TP53 loss in stromal cells had not been elucidate, the oncogenic stimulus emerges as a pivotal player in this process, since classical molecular alterations in the epithelial compartment, such as k-Ras mutation, were reported as sufficient to inhibit TP53 expression in the fibroblasts [In this way, data produced by Hill and colleagues showed that conditional deletion of he tumor . These oelection . Of noteelection . The stuelection . A cruciroblasts .TP53 loss in stromal cells has been particularly implicated in tumor progression, once its absence is inversely proportional to SDF-1 cytokine levels that, in turn, are involved with cellular proliferation and migration stimulus [TP53 overexpression in fibroblasts leads to a down-regulation of SDF-1 and consequently attenuate migration and invasiveness processes [TP53 loss in the microenvironment cells is also apparently involved with alterations in the cellular redox state. During in vitro assays, stable silencing of TP53 in fibroblasts resulted in overexpression of Endothelial Nitric Oxide Synthase (eNOS) , as well as in its incremented release. Moreover, these alterations culminated in the transformation and invasion of epithelial cells from the ovaries and oral cavity [Alternatively, the consequence of stimulus . In factrocesses . Moreoverocesses . Furtherl cavity .TP53 status in microenvironment was reinforced by the observation that the melanoma cell line B16F1 inoculated in p53-null mice produces tumors greatly faster than p53-wild type mice [TP53. This scenario propitiated tumor growth by promoting an immunotolerant and permissive microenvironment which was evidenced by IFN-γ- and IL-17A suppression. In addition, the exacerbated release of G-CSF, CXCL1 and IL-6 chemokines creates an inflammatory context that promotes malignant proliferation and cell spread by sustaining the angiogenesis process [In fact, oxidative stress damages DNA in tumor cells, but also in stromal cells, contributing to an even greater disruption of microenvironment homeostasis during tumor progression , 127. Moype mice . Increas process .TP53, the loss of function of other tumor suppressor genes in tumor stroma has severe consequences. For example, studies report that loss of function of genes involved in cell survival and proliferation, such as the APC (Adenomatous Polyposis Coli) and PTEN genes  and NRAS [In other way, some patients with non-small cell lung cancer become refractory to Epidermal Growth Factor Receptor (EGFR) inhibitors due to an EGFR mutation or MET amplification that arises after treatment with Getifinib and Erlotinib , 167. Siand NRAS . Moreoveand NRAS  or alterand NRAS . Additioand NRAS . In factand NRAS . Furtherand NRAS , 175. Inand NRAS , 177.For instance, the presence and function of cells such as M2 macrophages can impact not only the natural progression of the tumor, but has also been demonstrated to impact the tumor response to chemo  and radiPresumably, these reports pushed forward the development of drugs that target the microenvironment Fig. , such asImmune therapies represent a more complex landscape due to the fact that the dual roles of the innate and adaptive immunological responses have yet to be completely understood . Neverthin vitro tumor specific lymphocytes to be returned to the patient, a strategy that leads to massive antitumor responses in a fraction of patients bearing metastatic melanomas [Tumors co-evolve with the immune response avoiding immune surveillance and ultimately leading to tumor progression and metastasis . Tumor relanomas . A more elanomas  or chimeelanomas , 193 speelanomas  and willelanomas , 195. ThResearch on the tumor microenvironment as a target for cancer therapy has only just begun when compared with research on tumor cell targets, and, therefore, the benefits of this approach are not yet clear. The future development of new drugs, which will take into account cancer cells and their microenvironment Fig. , is needGenetic determinism has dominated cancer research and produced invaluable insights. However, the simplistic somatic mutation theory (SMT), which states that genetic instability of malignant cells alone drives disease progression, has proved insufficient to explain tumor behavior and, consequently, to point to new disease therapies. A recent, more dynamic view of tumors suggests that disease progression is promoted by the orchestrated interaction between malignant cells and their surrounding environment. Therefore, the development of more effective new therapies requires a global view that integrates the genocentric and microenvironmental knowledge in a plural approach.