There is growing interest in the complex relationship between the nervous and immune systems and how its alteration can affect homeostasis and result in the development of inflammatory diseases. A key mediator in cross-talk between the two systems is nerve growth factor (NGF), which can influence both neuronal cell function and immune cell activity. The up-regulation of NGF described in inflamed tissues of many diseases can regulate innervation and neuronal activity of peripheral neurons, inducing the release of immune-active neuropeptides and neurotransmitters, but can also directly influence innate and adaptive immune responses. Expression of the NGF receptors tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) is dynamically regulated in immune cells, suggesting a varying requirement for NGF depending on their state of differentiation and functional activity. NGF has a variety of effects that can be either pro-inflammatory or anti-inflammatory. This apparent contradiction can be explained by considering NGF as part of an endogenous mechanism that, while activating immune responses, also activates pathways necessary to dampen the inflammatory response and limit tissue damage. Decreases in TrkA expression, such as that recently demonstrated in immune cells of arthritis patients, might prevent the activation by NGF of regulatory feed-back mechanisms, thus contributing to the development and maintenance of chronic inflammation. The discovery of nerve growth factor (NGF) more than 60 years ago is associated with embryonal development and the differentiation of peripheral neuronal cells. Studies by Levi-Montalcini in chick embryos clearly demonstrated that the production of this diffusible factor in the target organ was responsible for the survival of peripheral neurons during development. The purification of NGF, its biochemical characterization and the production of neutralizing antibodies made it possible to demonstrate in a number of animal models, both in vitro and in vivo, that NGF is essential for the survival of sympathetic and sensory neurons during nervous system differentiation . The relSo why refer to inflammation and immune responses in a review dedicated to nerve growth factor? The production of NGF is tightly controlled in all regions of the organism innervated by sensory and sympathetic neurons  because its alteration profoundly modifies the organism’s physiology. Nevertheless, a significant increase in NGF synthesis in inflamed tissues has been described in patients and animal models of inflammatory diseases ,24,25,26Studies on inflammatory and autoimmune diseases, which are characterized by an abnormal activation of immune cells and increased production of cytokines, have revealed a localized increase in NGF at the sites of inflammation. Enhanced NGF levels were initially found in the cerebrospinal fluid of multiple sclerosis patients, and it was shown that an increase in NGF closely follows the course of the disease . The synThus, the normally low basal production of NGF is enormously up-regulated during inflammatory response. It is reasonable to hypothesize that, during the inflammatory process and because of tissue damage, there is a release of mediators that modify the local concentration of NGF. A number of studies have shown that cytokines involved in inflammation, such as IL-1β, TNF-α and IL-6, are promoters of NGF synthesis in a variety of cell types ,50,51,52The distribution of NGF receptors in different areas of lymphoid organs suggests that local NGF production and its accumulation in afferent lymphoid vessels not only regulate the innervation of peripheral neurons but can also have an immunomodulatory activity. The expression of NGF and of the Tropomyosin Receptor Kinase A (TrkA) seems to be relevant for the differentiation of lymphoid organs, since structural abnormalities have been described in NGF receptor-deficient mouse lymphoid organs ,62. ThesThe biological effects of NGF are mediated by its binding to two classes of receptors: p75 Neurotrophin Receptor (p75NTR), a 75 kDa glycoprotein that is a member of the TNF-receptor superfamily , and TrkThe expression of these receptors is not restricted to the nervous system, but a number of studies have shown NGF receptor distribution in primary lymphoid organs , where the differentiation of immune cells from multipotent stem cells occurs .The thymus is a primary lymphoid organ in which T-cells differentiate, and it is active throughout childhood until puberty . The thyThe expression of TrkA and p75NTR is modulated in the rat embryo with mRNA levels of TrkA decreasing and of p75NTR increasing in the stroma of the thymus  during rIn bone marrow, a heterogeneous population of stromal cells provides the correct microenvironment for hematopoietic stem cell survival and differentiation in myeloid and lymphoid lineages. In human and rat, stromal cells with dendritic features expressing both TrkA and p75NTR appear in the fetal bone marrow before the hematopoietic activity begins ,84. CD34Similarly to what has been described in bone marrow, there is also a gradient of expression of NGF receptors during embryo development in the bursa of Fabricius ,89, the The expression of NGF receptors also characterizes secondary lymphoid organs  where antigen-presenting cells activate lymphocyte responses and effector cell differentiation.The spleen is composed of two different tissues: the white pulp, responsible for the production and growth of immune and blood cells, and the red pulp, which filters microorganisms and cellular debris from the blood and removes older erythrocytes from circulation . The expIn lymph nodes and in mucosa-associated lymphoid tissues, TrkA is present in follicular dendritic cells, in blood vessel walls, in cryptic tonsillar epithelium and in several monocyte-derived cells including epithelioid and multinucleated Langhans’ cells and interdigitated reticular cells ,94,95. TThe picture emerging from all these studies is that the expression of NGF receptors characterizes both stromal and immune progenitors and is finely regulated during development and post-natal life. Thus, the local production of NGF not only directly and actively modulates innervation patterns and fiber density in lymphoid organs , but canThe expression of TrkA characterizes hemopoietic stem cells and is highest in the more undifferentiated cells, declining during lineage differentiation ,86. ThesIn B- and T-lymphocytes, undifferentiated monocytes and macrophages, the basal expression of TrkA is strongly up-regulated after antigenic or inflammatory stimulation when functional activity is necessary ,113,114.The expression of p75NTR has been described for many immune cells but there is still little information concerning changes in p75NTR during immune cell differentiation and activation ,116. A rThe innervation of primary and secondary lymphoid organs constitutes the anatomical link between the nervous and immune systems. The integrity of this connection is essential to maintain a regulatory function of the nervous system on immune response: experiments of chemical sympathectomy, denervation of lymphoid organs or vagotomy ,34,119 hThrough its effects on neuronal cells, NGF can indirectly regulate the immune response in a variety of manners. It can affect neuronal plasticity in the inflamed tissue by increasing innervation density, axonal terminal sprouting and dendritic arborization.In inflamed tissues there is a marked alteration in nerve fiber distribution, with an increased terminal sprouting leading to hyper-innervation of the injured tissues.Inflammation leads to an increased sensitivity to pain and intense stimulation and firing of the nociceptors, which induces the release of neuropeptides and neurotransmitters from nerve endings in close contact with the cell membranes of keratinocytes, mast cells, macrophages, Langerhans cells and endothelial cells ,128,129 NGF not only influences fiber density and dendritic sprouting in the inflamed tissues, but may also affect the local immune response by directly regulating the production of neurotransmitters and neuropeptides, which, as a growing body of findings is showing, have direct effects on immune cells and are involved in the development of many inflammatory diseases ,29,30,97The presence of NGF receptors on cells other than neuronal cells opens new perspectives regarding the range of action of NGF. The first indication of an immunoregulatory effect of NGF was observed in 1977 by Aloe and Levi-Montalcini, who showed that injecting neonatal rats with NGF resulted in an increase in the number and size of mast cells . It has NGF regulates the survival of different immune cells in a similar manner to its effect in neurons. It promotes the survival of hematopoietic stem cells , eosinopNGF also potentiates the proliferative response of T- and B-cells to several mitogens  and moduTaken together, the available in vitro data suggest that NGF potentiates the activity of innate immune cells and influences B- and T-cell mediated responses. However, the mechanism by which NGF modulates the immune response in vivo is still not fully understood.At present, despite the well-documented increase in NGF concentrations measured in the inflamed tissues of many inflammatory diseases, little is known of the effects of increased levels of NGF or of the activation of its receptors during inflammatory response. Currently available data suggest that the action of NGF is more complex in vivo than it is in vitro.In animal models and in humans, the administration of NGF alone without a more specific inflammatory stimulus, while inducing activation of local sensory hypersensitivity, does not activate any inflammatory response ,174,175.However, the intraventricular administration of NGF after the induction of experimental autoimmune encephalomyelitis (EAE) in marmoset  or mouseTaken all together, the results obtained in different models of inflammation indicate that in vivo NGF administration ameliorates tissue inflammation, suggesting a direct influence of NGF on antigen presentation, down-regulation of inflammatory cytokines and up-regulation of the anti-inflammatory cytokine IL-10.Although some discrepancies exist in the effects of NGF in vitro and in vivo, it seems reasonable to hypothesize that NGF is part of an endogenous mechanism that, while activating innate immune responses, also activates pathways necessary to dampen inflammatory response and limit tissue damage.It should be kept in mind that inflammation is an adaptive response whose main aim is to restore homeostasis . One keyA growing body of data suggests that NGF is a key regulator of the cross-talk between the immune and nervous systems. While the effects of NGF on neuronal cell survival, differentiation and phenotype maintenance are fairly well-known, the effects of NGF on immune cells have still not been completely defined. Further investigations are needed to establish why NGF is synthesized in vivo in the inflamed tissue and how the activation of its receptors can influence the cellular and molecular mechanisms triggered during the inflammatory response. What has already been demonstrated in neuronal cells and is now emerging in studies on immune cells and inflammatory diseases is that NGF definitely controls a multitude of effects, some of them even contradictory. The diverse biological activities of NGF on immune cell populations can be explained by considering the differential expression of NGF receptors during different states of cell differentiation and activation. The complexity of NGF action and its signaling can be better understood if we consider them as being correlated with the need for differing types of regulation in a variety of immune cell types in order to maintain correct homeostasis. Alteration in NGF synthesis and modification of the expression of its receptors may influence physiological responses and be involved in the pathogenesis of inflammatory diseases.A better understanding of the physiological role of NGF and its receptors in regulating immune responses could be of groundbreaking importance with translational implications for human diseases.


Physiological structure and function of cells are maintained by ongoing complex dynamic adaptive processes in the intracellular molecular pathways controlling the overall profile of gene expression, and by genes in cellular gene regulatory circuits. Cytogenetic mutations and non-genetic factors such as chronic inflammation or repetitive trauma, intrinsic mechanical stresses within extracellular matrix may induce redirection of gene regulatory circuits with abnormal reactivation of embryonic developmental programmes which can now drive cell transformation and cancer initiation, and later cancer progression and metastasis. Some of the non-genetic factors that may also favour cancerization are dysregulation in epithelial-mesenchymal interactions, in cell-to-cell communication, in extracellular matrix turnover, in extracellular matrix-to-cell interactions and in mechanotransduction pathways. Persistent increase in extracellular matrix stiffness, for whatever reason, has been shown to play an important role in cell transformation, and later in cancer cell invasion. In this article we review certain cell regulatory networks driving carcinogenesis, focussing on the role of mechanical stresses modulating structure and function of cells and their extracellular matrices. Oral squamous cell carcinoma is a malignancy of oral epithelial cells (keratinocytes) characterized by uncontrolled cell proliferation, increased cell survival and disruption of local tissue structure, with malignant keratinocytes invading the underlying connective tissue through the basement membrane. The acquisition of a malignant phenotype is accompanied by several changes in cellular properties including alterations in cell shape, rearrangement of the cytoskeleton, dysregulation in cell-to-cell communication and adherence, and loss of tensional homeostasis. These changes in the function and structure of cells are caused by major reprogramming of gene-expression profiles, by genetic mutations and by non-genetic microevironmental factors including chronic inflammation and trauma, and by altered mechanical properties of extracellular matrix (ECM) –3.The biomechanical characteristics of cancer cells and their immediate microenvironment, and the physical interactions between these cells and their extracellular matrices play important roles in cancer initiation and progression , 4, 5. TReciprocally, tension and contractile forces generated by the intracellular actin filaments of the cytoskeleton in response to external biomechanical stimulation are transferred to the ECM, with abnormal remodelling of its three dimensional organization . In the In this article we review certain cell regulatory networks driving carcinogenesis, focusing on the role of mechanical stresses that modulate normal function and structure of cells and their ECM, and promote carcinogenesis.The dynamic cross-talk between numerous genes, and their organization within the gene regulatory network will determine cell structure and function . In turnThe intracellular molecular pathways form a network, and each pathway in the network may be activated by one or more biological agents in the microenvironment , 9. ThisThe larger system of the gene regulatory network comprises many regulatory circuits, each circuit having a particular profile of gene expression. The configuration of the specific circuit and interactions between the genes within the circuit determine whether a regulatory circuit remains stable, or is driven to adapt to one of multiple accessible stable attractor states in the network, each of which has a distinct profile of gene expression and is associated with an orderly biological behaviour , 9.Within the genetic landscape of multiple stable attractor states, a cell can switch from one stable attractor state to another in response to stimuli from the microenvironment, for example from a proliferative to a differentiative programme. Furthermore, mutations in certain key regulatory genes can induce changes in the profile of the gene network, resulting in cell transformation and cancer development , 9. HoweThus, the multiple discrete stable attractor states represent pre-existing circuits of gene expression each associated with a different preprogrammed biological behaviour, and according to the built-in rules the switch to a particular stable attractor state will depend on the accessibility of potential target attractors within the gene landscape .The regulatory network that controls tensional homeostasis probably comprises several different attractor states, depending upon the structural architecture of the cells and their ECM. While a normal tensional attractor state maintains the physiological structure of cells and tissues, stiffer matrices with altered tensional homeostasis can switch from a physiological tensional circuit to an aberrant tensional circuit promoting a cellular change to a malignant phenotype , 11. SucPreset circuits of gene expression (stable attractor states) include some developmental programs that are normally shut down post-developmentally; but random genetic mutations may induce rewiring of gene-regulatory systems, making developmental circuits re-accessible and re-activated, thus providing ‘cancer stem cells’ with renewed capacity to initiate cancer development. Chronic inflammation or trauma as well as other stimuli from the microenvironment might also promote the transition to a preset circuit of gene expression associated with cancer . The canAcquisition of a malignant phenotype is accompanied by changes in cellular structure and function caused by reorganization of the cytogenetic regulatory network with the re-programming of gene-expression profiles . Thus, sThe cytoskeleton is a complex of elements comprising actin filaments and intermediate filaments under tension, and of microtubular elements that resist compression, together maintaining the shape and mechanical stability of the cell and its nucleus. These interconnecting cytoskeletal elements are anchored to focal adhesion proteins and integrins on the inner side of the cell’s plasma membrane, and to other specialized proteins on the outer side of the nuclear membrane. Nesprins, the outer nuclear membrane proteins connect the nucleus to the cytoplasmic cytoskeleton, and interact with inner nuclear proteins sun as sun 1, sun 2 and laminins , talin, vinculin, Src, paxillin and tensin, linking the ECM to the cytoskeleton Fig. . The extThe focal adhesion domains thus mediate interactions between cells and their matrices, with ECM stresses promoting integrin clustering, expression and activity, and assembly and maturation of focal adhesion proteins with their consequent stabilization , 22, 30.The strains that develop in integrins and in other mechanosensory molecules in the focal adhesion domains are generated by both intracellular and extracellular stresses , 19. ExtContraction of the intracellular Rho-ROCK-induced stress fibers further promotes clustering and strengthening of integrins and other molecules in the focal adhesion domain . ContracAlteration in the biochemical composition and structural architecture of the ECM together with an aberrant increase in its stiffness can cause dysregulation in the intracellular contractile forces which may disrupt cell-to-cell and cell-to-ECM adhesion .Under physiological conditions, a positive biomechanical feedback loop is created, with ECM stresses inducing intracellular stresses, mediating cellular function and biological behaviour; and reciprocally, the intracellular stresses inherent in the cytoskeleton and stress fibers influence remodelling, function and biomechanical properties of the ECM .The functional activities of the cells are thus influenced by the complex adaptive network that regulates the ECM and cellular mechanotransduction pathways. The elements of this complex system include the structural architectures of both the ECM and the cytoskeleton, which impart the inherent mechanical tension and stiffness characteristic to the cells and their matrices. The arrangement of these elements is dynamic, with particular intracellular molecular signalling pathways being activated according to the biomechanical stimuli received from the microenvironment, and as the stimuli change, so do the intracellular signalling pathways, in order to elicit a homeostatic biological response , 22.Development of cancer is associated with increased stiffness of the local ECM that can then transmit a greater portion of any intrinsically generated or extraneous mechanical stress to the cells, transducing the microenvironmental biomechanical stimuli into intracellular biochemical signals. These signals increase intracellular mechanical stresses which then modulates cellular genes expression, promoting cell proliferation, survival and migration , 31, 33.Low-grade repetitive injury is a local factor that can induce a long-term increase in the stiffness of the ECM, and in the dynamics of cell-to-cell and cell-to-matrix adhesion and of cytoskeletal organization with a consequent increase in cell stiffness, proliferation, survival and migration further promoting cancerous transformation of cells , 22, 36.Transformed cells have an altered profile of intermediate filaments, of cytoskeletal structure and of cell shape compared to normal cells  and the The transmitted mechanical stresses, via the changed and stiffer cytoskeletal structure, can be transmitted to the nucleus causing nuclear deformation with configurational changes in chromatin, thus influencing gene expression which independently of the biomechanical-induced biochemical changes have the potential to promote cancerization Fig.  7, 19]., 19.7, 1The mechanical properties of cancer cells change during cancer progression, with metastatic cells having left the site of the primary tumour to migrate through a different type of extracellular microenvironment, becoming less stiff than they were after initial transformation, to allow penetration of the basement membrane and underlying tissue , 37, 38.In the context of cellular circuits of gene expression and cancerization, it has become evident that extracellular mechanical stresses generated by an aberrant increase in ECM stiffness has the capacity to induce rewiring of the epithelial mesenchymal transition (EMT) developmental cellular circuit , 40. EMTIn addition to stiff matrices and other mechanical stresses, TGF-β, Notch, Wnt and Hedgehog signalling pathways can also activate EMT transcription factors , 42. WitIt appears that there are different modes of cancer cell migration that are governed by different genetic programs, based on the specific characteristics of the cell-to-cell junctions, cell-to-ECM adhesions, the tension of the cytoskeleton and the patterns of remodelling of all these elements. The modes of cell migration change over-time in response to microenvironmental biomechanical cues .Cancer initiation and progression are associated with pathological changes in structure and function of cells and of their ECM, and with disruption of tensional homeostasis characterized by abnormal mechanical stresses within cells and their extracellular matrix. The aberrant process of mechanotransduction which converts biomechanical stimuli into abnormal intracellular biochemical signals is thought to play an important role in early cellular transformation.


The genome-scale cellular network has become a necessary tool in the systematic analysis of microbes. In a cell, there are several layers  of the molecular networks, for example, genome-scale metabolic network (GMN), transcriptional regulatory network (TRN), and signal transduction network (STN). It has been realized that the limitation and inaccuracy of the prediction exist just using only a single-layer network. Therefore, the integrated network constructed based on the networks of the three types attracts more interests. The function of a biological process in living cells is usually performed by the interaction of biological components. Therefore, it is necessary to integrate and analyze all the related components at the systems level for the comprehensively and correctly realizing the physiological function in living organisms. In this review, we discussed three representative genome-scale cellular networks: GMN, TRN, and STN, representing different levels  of a cell’s activities. Furthermore, we discussed the integration of the networks of the three types. With more understanding on the complexity of microbial cells, the development of integrated network has become an inevitable trend in analyzing genome-scale cellular networks of microorganisms. Haemophilus influenzae Rd , transcriptional regulatory network (TRN), and signal transduction network (STN). The most well-studied large-scale biological network is GMN, which is a fundamental framework in systems metabolic engineering . With thenzae Rd , the curenzae Rd . The maienzae Rd . The TRNenzae Rd . On the enzae Rd .From a system point of view, different kinds of biological networks are not working alone, but cooperate with each other to undertake their functions. Integrated network studies will build a more realistic model by investigating the interacting relationships and interacting effects among organism’s different information processing components in its system. This kind of models has an important sense to the theoretical research of living systems and the construction of genetic engineering strains . In thisin vivo experiments  accuracy in predicting gene essentiality -2,3-butanediol, cellulase Egl-237, and isobutanol]. The results have provided important guidance for the in vivo experiments  and the related target promoters which function in the activation or repression of gene transcription. In this definition, the intracellular signals that regulate TF activities or any other additional mechanisms that may influence the expression of genes were excluded, as well as the upstream environmental. Although the development of TRN is not as mature as that of GMN, the current TRN construction is more and more standardized and automated. The detailed construction method of the TRN in microorganism can be seen in this paper . The netBacillus as an example, in B. subtilis (DBTBS), which is constructed in 1999 for collecting the information of experimentally characterized TFs, and they nearly doubled the information in DBTBS. B. subtilis and presented some lessons from the construction processes. B. subtilis to calculate the activity of TFs with a new combination of composition analysis based on a large number of known transcriptome data and experimental data of B. subtilis. They predicted 2258 new regulatory interactions and recalled 74% previously known interactions with this model. The accuracy of predicted new regulation edges was 62% (391/635) (B. subtilis reconstructed in 2008 by integrating the regulation information in DBTBS. They demonstrated that atomic regulons (ARs), which are the sets of genes with the same expression profile, are the effective references for improving the regulatory networks by finding the closely correlated genes in the ARs. The expanded model contains the regulatory information for 2500 of the 4200 genes in B. subtilis 168  . Faria eilis 168 . In addiion data .Salmonella typhi, a pathogen causing typhoid fever. Typhoid fever, which is a frequently happened disease in human, was mainly caused by the typhus toxin secreted by S. typhi. Typhoid fever toxin is expressed uniquely by intracellular bacteria with unknown regulatory network. S. typhi and developed an algorithm called FAST-INSeq to identify the genes and mutants which influence the expression of typhoid toxins. This network can help to understand the expressional regulation of typhoid fever toxin in S. typhi, which would contribute to the treatment of typhoid fever  involved in metabolic reactions affect the activities of certain TFs or signal transduction pathways. On the other hand, enzyme-catalytic metabolic reactions are regulated by other genes or proteins, and the expression of enzymes is different in different environmental conditions. In recent decades, the integrative modeling of metabolic-regulatory networks has become an important research area in the modeling of microorganisms .E. coli (iMC1010) based on the information derived from literature and databases. The network contains 906 metabolic genes and 104 regulatory genes, which regulate the expression of about 53% genes (479/906) in the E. coli metabolic network. This model is capable of predicting the previously unknown TFs, which play important roles in regulating metabolic processes, and interactions between metabolites and TFs  has been used to integrate transcriptional regulatory with metabolic networks. rFBA used the Boolean logic to link transcriptional control to the metabolic process, which permits only on/off states of the network components  and signaling (kinase-substrate pairs) parts. This network was used to investigate the negative genetic interactions and the genes in the negative genetic interactions closely related to the toxicity . Therefore, it is theoretically possible to merge these three types of cellular networks into one integrated network. While actually, lots of elements should be considered in the integration process, such as the logics and computability. On a small scale network integration, Figure 2). They used this approach to construct an integrated network model of E. coli which combines a FBA based central carbon metabolic-regulatory network with an ODE based model of carbohydrate-uptaking-controlling network. They compared the prediction of E. coli single gene perturbation disturbance phenotypes and wild-type for diauxic growth on glucose/glucose-6-phosphate and glucose/lactose using rFBA and ODE methods. They found that iFBA is capable of identifying the dynamics of three transporters and three internal metabolites which cannot be predicted by rFBA alone. Furthermore, iFBA obtained different and more accurate phenotype predictions in the wild-type simulations and single gene perturbation simulations than the ODE model, which indicates that iFBA is an improvement over either individual rFBA or ODE method in network integration  in metabolic, regulatory, and signaling networks . idFBA has been applied to a representative small network of S. cerevisiae, in which metabolic, regulatory, and signaling activities have been included. Finally, idFBA got similar results with an equivalent kinetic model in the prediction of the influence of the extracellular environment on the cellular phenotypes. The advantage of idFBA is that it is capable of solving a linear programming problem without the detailed kinetic parameters, which makes it a possible approach for the genome-scale integration of metabolic, regulatory, and STNs , flux Variability Analysis (FVA), TRAnscription-based Metabolic flux Enrichment (TRAME) and FBA, which were sequentially used to calculate the gene expression caused by the genetic or environmental perturbations, the flux balance bounds modified by the predicted gene expression, the metabolism-transcription interactions, and the optimized objective function under the modified flux bounds. With this methodology, the metabolism, transcription, and signal transduction information were integrated into one computable model. The application of this methodology on E. coli showed that the integrated network has a more powerful capability in phenotype prediction than the approaches using metabolic network alone  has output many important biological insights in novel pathways, regulatory, and metabolic mechanisms. Given the fact that these networks are highly interconnected, the analysis of the integrated networks is expected to supply more novel understanding on biological behaviors which cannot be achievable using the biological information processing-specific network models alone. From biological information processing-specific networks to integrated network, it is an irresistible trend of the analysis of cellular networks. The integrated networks may provide better answers to the issues such as how transcription-regulatory interactions redirect flux distribution in a metabolic network; how a environmental or genetic disturbance influences the phenotype of an organism; or giving more accurate suggestions to the experiment designs and driving biotechnology applications. As lots of information is required in the reconstruction of a large-scale integrated networks, high-throughput experiments will play an increasingly significant role in the network integration. With the development of sequencing technology in recent years, many other types of cellular molecules involved in the regulatory process has been identified with high throughput experiment, and their related cellular networks have been studied, such as the network of mRNA, microRNA , lncRNAsThe study was approved by College of Life Sciences, Tianjin Normal Univeristy.DW, LZ, and QW collected the references. EW and JS contributed in the guideline and revision of the manuscript. TH analyzed the reference. TH and DW wrote the paper.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.


Although our knowledge of chromatin organization has advanced significantly in recent years, much about the relationships between different features of genome architecture is still unknown. Folding of mammalian genomes into spatial domains is thought to depend on architectural proteins, other DNA-binding proteins, and different forms of RNA. In addition, emerging evidence points towards the possibility that the three-dimensional organisation of the genome is controlled by DNA topology. In this scenario, cohesin, CCCTC-binding factor (CTCF), transcription, DNA supercoiling, and topoisomerases are integrated to dictate different layers of genome organization, and the contribution of all four to gene control is an important direction of future studies. In this perspective, we review recent studies that give new insight on how DNA supercoiling shape chromatin structure. The three-dimensional structure of the genome is essential to dictate its biological function. Within the nucleus, the linear sequence of DNA is spatially arranged in a defined manner that allows effective control of gene expression. Transcriptional output depends on a complex interplay between proteins acting within the promoter region of genes and through long range chromosomal interactions. Gene regulation by chromosomal interactions has become evident in the most recent years, thanks to advances in imaging and chromosome conformation capture (3C)-based techniques, which have revealed how chromosomes are folded at different scales. Even though these observations have started to provide answers regarding how gene regulation is achieved in the dense chromatin environment of the nucleus, the mechanisms and factors orchestrating chromosome organization remain unclear.Interphase chromosomes are compartmentalized, and the spatial positioning of genomic segments affects ongoing DNA transactions. The first whole-genome chromatin conformation capture Hi-C,  experimeS. pombe are oriented in a way that transcription direction points outwards from the centre of the domain so that the boundaries are flanked by genes in a convergent orientation [How does cohesin travel along the chromatin fibre extruding the DNA through its ring? Although cohesin is an ATPase, and the cohesin-related complex condensin has been shown to create DNA loops and translocate along DNA in an ATP-dependent manner in vitro ,26, therentation .The transcription-dependent sliding of cohesin along chromatin could be caused by sterical/architectural constrains due to the sizes of the polymerase and cohesin complexes. Alternatively, the torque introduced into DNA by the moving RNAP could be another determinant of cohesin re-localization. The transcribing RNAP induces axial rotation in the transcribed DNA, which should promote polymerase rotation relative to the DNA. However, the rotation of RNAP is hindered due to the viscous drag or the tethering to nuclear structures. Due to this, transcription will change the structure of the DNA helix, causing it to become over-twisted (positively supercoiled) in front of the polymerase, and under-twisted (negatively supercoiled) behind the polymerase ,30. TopoIn addition to this, supercoiling could also constitute a barrier to the movement of proteins traveling along the helix, as suggested by the observation of polymerase stalling in response to supercoil accumulation . This haThe ability to read out the topology of the genome is progressing fast, but the contribution of DNA topology or the role of DNA relaxing enzymes to genome organization has received less attention. Below, we will discuss recent experimental studies and dynamic simulations showing how DNA topology might alter the properties of the chromatin fibre, thus shaping nuclear architecture.Supercoiling is a fundamental property of DNA and can assume toroidal or plectonemic configurations A. It is RNA-sequencing experiments have shown that many enhancers are transcribed. Topoisomerases have been found to be associated to enhancers where they promote transcriptional activation through regulatory interactions with enhancer specific factors, and by stimulating production of RNA transcripts (eRNAs) derived from enhancer sites ,55. The One implication of this hypothesis is that negative supercoiling and active transcription would be expected to correlate. In this regard, it has been reported that DNA supercoiling is organized into large domains of about 100 kb in size with specific topological features: those with a negatively supercoiled, transcription permissive environment, and those that are positively supercoiled, more compacted, and more likely to be silent . These sC. elegans and A. thaliana, which have no known CTCF homologs [Even though in mammals architectural proteins such as CTCF and cohesin seem to have a key role in high-order chromatin organization, emerging evidence suggests that in lower eukaryotes, prokaryotes, and plants, all of which lack CTCF, transcriptional activity and DNA topology are likely to play a similar role in establishing TAD-like domains. Topological domains could be transcription- and topoisomerases-dependent and composed of supercoiled regions forming plectonemes. This is the case of the bacterial genome, which is organized in chromosomal interaction domains (CIDs) determined by supercoiling and transcription, whose structures and features resemble eukaryotic TADs . Modellihomologs . The mecDrosophila, but CTCF does not have a role in TAD formation. Applying Hi-C and Hi-ChIP  to the  of TADs . Even th of TADs , their o of TADs ,70, A/B Drosophila protein HP1a nucleates into foci that display liquid properties during the formation of heterochromatin domains [By increasing contact frequency between distal regions of the chromatin fibre, gene activation and transcription-generated supercoils could promote formation of the A compartment, which will in turn exclude heterochromatic repressed regions forming the B compartment. It is also possible that other types of factors would drive the physical interactions between genes in the same transcriptional state. Clustering of proteins and phase separation events may play a role in the establishment of compartmental domains, as recently described for the formation of super-enhancers  and hete domains . Overall domains , thereby domains . It is p domains  and woul domains .Altogether, this suggests that in organisms that lack architectural proteins such as CTCF, the transcriptional state of chromatin is a critical factor in the formation of compartmental domains, while the organization of mammalian chromosomes is established via a combination of such compartmental domains and point-to-point CTCF-cohesin interactions. In other words, compartmental domains represent the primary mechanism underlying 3D chromatin organization, and architectural proteins are responsible for additional interactions that establish the complex architecture of the mammalian nucleus.Understanding how the genome is organized has become a topic of intense interest and an increasingly growing number of studies, and analyses of the interplay between interphase chromatin and architectural proteins have revealed key features of the 3D structure. Nevertheless, in recent years it has become evident that the structure of the DNA helix in itself is also part of the regulation. By modulating the dynamic rearrangement of the chromatin fibre, DNA supercoiling can control a DNA-based process across long distances. This observation invites the consideration of supercoiling and topoisomerases as active players in domain organization. Our understanding of this phenomenon is still elusive, and improvements of techniques to map DNA supercoiling, topoisomerase activity, and chromatin looping, and their integration with the study of phase-separated systems, are needed to eventually clarify the interrelationships between DNA topology, chromatin topology, and genome function.


Pneumonia is a leading cause of death from infection in the United States and across the globe. During pulmonary infection, clear resolution of host inflammatory responses occurs in the absence of appreciable lung damage. Neutrophils are the first wave of leukocytes to arrive in the lung upon infection. After activation, neutrophils traffic from the vasculature via transendothelial migration through the lung interstitium and into the alveolar space. Successful pulmonary immunity requires neutrophil-mediated killing of invading pathogens by phagocytosis and release of a myriad of antimicrobial molecules, followed by resolution of inflammation, neutrophil apoptosis, and clearing of dead or dying neutrophils by macrophages. In addition to their antimicrobial role, it is becoming clear that neutrophils are also important modulators of innate and adaptive immune responses, primarily through the release of cytokines and recruitment of additional waves of neutrophils into the airways. Though typically essential to combating severe pneumonia, neutrophil influx into the airways is a double-edged sword: Overzealous neutrophil activation can cause severe tissue damage as a result of the release of toxic agents including proteases, cationic polypeptides, cytokines, and reactive oxygen species (ROS) aimed at killing invading microbes. In extreme cases, the damage caused by neutrophils and other innate immune mediators become the primary source of morbidity and mortality. Here, we review the complex role of neutrophils during severe pneumonia by highlighting specific molecules and processes that contribute to pulmonary immunity, but can also drive progression of severe disease. Depending on the identity of the infectious agent, enhancing or suppressing neutrophil-mediated responses may be key to effectively treating severe and typically lethal pneumonia. Pulmonary infection with bacterial, viral, or fungal pathogens can lead to the massive infiltration of innate immune populations, primarily polymorphonuclear leukocytes (PMNs), into the airways and alveolar spaces. Defined as pneumonia, this inflammatory response is aimed at controlling and clearing invading pathogens, but can severely compromise pulmonary function, resulting in significant mortality. Pneumonia lags only heart disease as the greatest burden of disease worldwide, and is the leading cause of death from infection in the United States Mizgerd, , 2008. PKey to the progression of pneumonia are neutrophils, the cell type most associated with the development of severe disease. Neutrophils migrate directly to the site of infection, where they accumulate in significant numbers and unleash a torrent of antimicrobial factors aimed at controlling and clearing infection. Though neutrophils are essential mediators of pulmonary immunity, they can also contribute to the development of severe disease. The very molecules and processes that make neutrophils such potent innate immune responders can cause a great deal of host tissue damage, and can contribute significantly to disease pathology and severity. In this review, we discuss the complex role neutrophils play in the pathogenesis of severe pneumonia, examining their function as mediators of both pulmonary immunity and pulmonary damage. We highlight key molecules and processes employed by neutrophils to destroy invading pathogens, and the unintended consequences that can result from their dysregulation. The processes that govern neutrophil-mediated immunity are wide-ranging and complex, each with an extensive literature beyond the scope of this article. We touch briefly on a handful of well-established factors shown to contribute to both clearance of invading pathogens and the onset of pulmonary damage that defines the pathogenesis of disease. What emerges is an intriguing and complex story, where a key innate immune population is essential to combating infection, but can also be responsible for pathogenesis and mortality of the very disease it is tasked with combating.3 , secondary (specific), tertiary (gelatinase) granules, and secretory vesicles  is a key component of the innate immune response to infection. The ROS can be potent antimicrobials, likely through the oxidation of proteins, nucleic acids, lipids, and a number of other molecules , which is thought to be primarily responsible for antimicrobial activity, as well as hypochlorous acid (HOCl), hypobromous acid (HOBr), and hydroxyl radical (.OH)  to kill invading microbes , and inhibition of neutrophil-mediated killing of pathogens. Neutrophils can play an important role in inducing severe tissue damage in the lung. Thus, a balance must be maintained by employing adequate neutrophil-mediated immune responses without initiating the tissue destruction that can come from their hyper-activity. Further, the timing of neutrophil influx is crucial, as timed depletion studies have revealed that neutrophils can be protective early post-infection, but detrimental when present during later disease stages (Bou Ghanem et al., The article was conceived of and written by RP.Publication of this article was funded by NIH/NIGMS award P20-GM103625.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Perception, cognition and consciousness can be modulated as a function of oscillating neural activity, while ongoing neuronal dynamics are influenced by synaptic activity and membrane potential. Consequently, transcranial alternating current stimulation (tACS) may be used for neurological intervention. The advantageous features of tACS include the biphasic and sinusoidal tACS currents, the ability to entrain large neuronal populations, and subtle control over somatic effects. Through neuromodulation of phasic, neural activity, tACS is a powerful tool to investigate the neural correlates of cognition. The rapid development in this area requires clarity about best practices. Here we briefly introduce tACS and review the most compelling findings in the literature to provide a starting point for using tACS. We suggest that tACS protocols be based on functional brain mechanisms and appropriate control experiments, including active sham and condition blinding. Technological and ethical constraints have forced the study of human cognition to rely on non-invasive electrophysiology and neuroimaging techniques to reveal the neural correlates of perception, cognition and behavioral functions. Through electrochemistry and neuronal cytology, we have learned that neurophysiological dynamics determine neural function , averaging over a full cycle, the tACS current omits the directional voltage component Figure . Other mThe advantage of tACS, unlike other types of NIBS, is that it enables manipulation and entrainment of intrinsic oscillations through the injection of sinusoidal currents Paulus, . The phaThe most prominent mode of neuroplasticity, long-term potentiation , but purely non-retinal stimulation of the visual cortex remains somewhat controversial  stimulation of the left hemisphere can increase risk-taking behavior (Sela et al., Our goal was to clarify the benefits of tACS on neural investigations. Despite many methodological advances, unraveling the neurophysiological and oscillatory complexity of cognitive function requires investigation at multiple scales from cells, to animal neurophysiology, and finally, to clinical trials.Investigators studying cognition through psychophysical measures suggest a vital role played by system-level information integration, such as audio-visual integration (Shams et al., Neuromodulation has widespread potential in investigations of the functional role of oscillatory neural information. The role of phase and frequency information has already been documented in neuropathologies such as schizophrenia (Perez et al., Here we have introduced strictly controlled tACS protocols, methodological precautions, and guidelines for future tACS implementation. Recent developments in tACS scholarship, which are only partially presented, signal the intersection of numerous neurocognitive specializations, focused on the central role of oscillatory activity in brain function, cognition and behavior.AVT and KY compiled literature for this review. KY provided mentorship on methods, theory and prose. AVT wrote the manuscript and provided illustrations.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.


Skin rash is a side effect of drugs that inhibit epithelial growth factor receptor (EGFR) as a part of targeted therapy of cancer. Its appearance and severity correlates with survival. Minocycline, an oral tetracycline antibiotic, is recommended as treatment  of the rash, though infection is seen in only one-third of the patients. Minocycline has additional anticancer properties such as poly(ADP-ribose) polymerase inhibition. It is proposed that such properties contribute to the efficacy of EGFR inhibitors and can also explain the positive correlation between grade of rash and survival as patients with higher grades of rash are more likely to receive minocycline. Early concurrent administration of minocycline is recommended in patients planned for EGFR therapy while awaiting trials proving this hypothesis. Inhibition of epithelial growth factor receptor (EGFR) with either monoclonal antibodies  or tyrosine kinase inhibitors  is approved for the treatment of some subsets of cancers of colon, lung, pancreas, and head and neck. One of the major side effects of these drugs is an acne-form skin rash . MultiplMinocycline, in addition to being an antibiotic, has multiple other actions . It is aInterestingly, minocycline was chosen for the treatment of skin rash in the first randomized trial  for its anti-inflammatory rather than anti-infective effects .Minocycline also appears to enhance the antitumor effects of 5-FU in tumor CT-26 xenograft mice . TherefoInterestingly, the recently published Pan Canadian Rash trial  on the uIn the meantime, the use of minocycline should be recommended in patients on EGFR inhibitors even before the appearance of skin rash [in line with current recommendations ], for a Drs. AV and BB: conceived, wrote, and approved.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.


Gratitude is a complex emotional feeling associated with universally desirable positive effects in personal, social, and physiological domains. Why or how gratitude achieves these functional outcomes is not clear. Toward the goal of identifying its' underlying physiological processes, we recently investigated the neural correlates of gratitude. In our study, participants were exposed to gratitude-inducing stimuli, and rated each according to how much gratitude it provoked. As expected, self-reported gratitude intensity correlated with brain activity in distinct regions of the medial pre-frontal cortex associated with social reward and moral cognition. Here we draw from our data and existing literature to offer a theoretical foundation for the physiological correlates of gratitude. We propose that mu-opioid signaling (1) accompanies the mental experience of gratitude, and (2) may account for the positive effects of gratitude on social relationships, subjective wellbeing, and physiological health. The intentional practice of gratitude brings our attention to the positive aspects of our lives, and empirical research indicates that this can lead to many powerful benefits .We created a database of 48 such stories from Holocaust survivors, describing receiving aid that ranged from relatively inconsequential to life-saving. Some aid met an acute need but did not require much effort on behalf of the benefactor  while other aid required tremendous benefactor effort, but was not useful to the beneficiary . Finally, some stories described life-saving aid that also put the benefactor in tremendous danger, such as sheltering a Jewish fugitive in ones' family home.Research subjects were instructed to imagine themselves as the beneficiary in these stories, to feel the emotional context of the story as deeply as possible, and to imagine what it would feel like to receive the aid described. During this time, we collected functional Magnetic Resonance Imaging (fMRI) data, and after each story, participants were asked to rate how much gratitude they felt on a scale of 1–4. Their own ratings of gratitude were correlated in a general linear model with the BOLD signal activity during their reflection time. We hoped the results would identify core brain regions active during the mental experience of gratitude, and we predicted that these brain regions would be among those known to support moral reasoning and social cognition.Indeed, a set of brain regions whose activity correlated with the intensity of reported gratitude was identified. These regions are located in the medial prefrontal cortex (mPFC), and include the peri-genual anterior cingulate cortex, and the more anterior portion of the mPFC. More generally, the event of receiving aid in our study elicited robust activity compared to baseline, implicating multiple overlapping social, stress-related, and somatosensory brain regions  afferent sensory nerves that are selectively activated by body-temperature, stroking touch Grandi, . C-T fibThe relaxing effect of C-T fiber activation has been attributed to the release of oxytocin and endogenous mu-opioid peptides  exhibited much higher rates of allogrooming than controls  provoked by stories in which a stranger saved the protagonists' life at great personal risk. This alone is not sufficient grounds to claim involvement of the stress-reducing MOR system, but it is reasonable to expect that recognizing one's life is in danger produces stress; and it is similarly reasonable to expect that realizing that a stranger has gone out of their way to save your life would replace this stress with an intense feeling of gratitude.consistent with established effects of the MOR system.Of course, not all gratitude-provoking situations involve stress-relief, but previous studies have noted that subjects report decreased levels of stress following the experimental induction of gratitude Krause, , 2010. TKEY CONCEPT 3The mu-opioid system is centrally involved in the restoration or improvement of homeostasis, as evidenced by its roles in analgesia, positive affect and reward, social motivation, long term affective bonds, and relaxation from stress. Meanwhile, the experience of gratitude is characterized by positive affect, reciprocal prosocial motivation, long term affective bonds, and subjective reports of stress reduction. Qualitative and behavioral parallels between the experience of gratitude and the effects of the mu-opioid system suggest mu-opioid involvement in the experience of gratitude, which could offer a physiological account of many of the positive effects of gratitude. Future studies are necessary to test this hypothesis.The purpose of the current article has been to account for the positive effects of gratitude by exploring its underlying physiological correlates. To this end, we propose that large-scale activation of the MOR system during the mental experience of gratitude may account for the social, individual, and health effects of gratitude that have been observed. Specifically, we propose that the social effects of gratitude may be mediated largely by the MOR system in accordance with the Brain Opioid Theory of Social Attachment  facilitates basic research investigations of the physiological correlates of emotion, and (2) suggests that intentional mindfulness of our emotional state may improve behavioral decision-making.By definition, the power of gratitude extends beyond the individual. A primary function of primate allogrooming is settling tensions and improving social group cohesion, and in human societies a similar effect is likely wrought through moral codes, religious systems, and philosophical appeals to humanity that advocate the intentional practice of gratitude. We nod to Seneca in contending that an empirically-driven resurgence of the intentional practice of gratitude could have significant positive implications for the harmony of the human civilization, from interpersonal relationships to international diplomacy.MH outlined and wrote the manuscript. GF wrote and edited the manuscript. JK, HD, and AD each provided writing, feedback and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To assess the predictive role of education and frequency of reading andwriting habits (FRWH) on the cognitive flexibility, inhibition and planningabilities of healthy elderly individuals.Fifty-seven healthy adults aged between 60 and 75 years with 2 to 23 years offormal education were assessed as to the frequency with which they read andwrote different types of text, as well as their number of years of formaleducation. Executive functions were evaluated using the Hayling Test and theModified Wisconsin Card Sorting Test (MWCST).Weak to moderate positive correlations were found between education, FRWH andthe number of categories completed in the MWCST, while negative correlationswere identified between these variables and the number of perseverative andnon-perseverative errors on the task. Only the FRWH was significantlycorrelated with the number of failures to maintain set. Speed and accuracyon the Hayling Test were only correlated with participant education. Botheducation and FRWH significantly predicted performance on the MWCST, and thecombination of these two variables had a greater predictive impact onperformance on this task than either of the two variables alone. Variabilityin scores on the Hayling Test was best accounted for by participanteducation.In this sample of elderly subjects, cognitive flexibility was sufficientlypreserved to allow for adequate performance on verbal tasks, but may havebenefitted from the additional stimulation provided by regular reading andwriting habits and by formal education in the performance of more complexnon-verbal tasks. In fact, someauthors claim that reading and writing habits may contribute more significantly thanthe number of years of formal education to neurocognitive performance.21 In light of these findings, it hasbeen suggested that the frequency of reading and writing habits (FRWH) may enhancethe effects of education on cognition,9 especially in individuals with higher educationallevels.10 However, moststudies on the association between education, FRWH and cognition have focused ongeneral intellectual ability rather than specific cognitive components. Furthermore,the literature on cognitive reserve tends to be biased toward the study of dementia,and therefore, focuses predominantly on memory and IQ rather than other cognitivefunctions.18Recently, reading and writing habits have also been found to exert an impact onneurocognitive performance. The quality of what is read and written by participants,their reading proficiency,22 Theseare multimodal and multifactorial abilities involved in the processing of bothverbal24 and visuospatial stimuli,25 although their role in the latterhas been far more widely explored than in the former.Given their complexity, relevance to activities of daily living and close interactionwith most cognitive components, the executive functions (EF) are among the mostextensively studied cognitive functions in the current cognitive and clinicalneuropsychological literature. The EF consist of goal-oriented cognitive functionssuch as problem solving, planning, resistance to distraction, cognitive flexibility,initiation and inhibition.27 Performance on the Hayling Test relies heavily oninhibitory control, cognitive flexibility, and verbal processing, both for thegeneration of context-appropriate responses (in part A of the task) as well as forthe suppression of semantically primed words (in part B of the task). This task hasbeen successfully used in the assessment of several neurological populations, andhas produced especially interesting results when used in combination with testsinvolving different input and output modalities, such as the Wisconsin Card SortingTest. This and other related tasks, such as the Modified Wisconsin Card Sorting Test(MWCST),28 rely moreheavily on the use of visuospatial planning, behavioral inhibition and rulemaintenance.The involvement of EF in verbal processing has traditionally been studied throughtasks requiring lexical-semantic processing, such as verbal fluency and the HaylingTest.30 Additionally,like other non-verbal tasks, card sorting tests involve the mental simulation ofoutcomes. This process is closely related to EF in that it requires the selection ofa specific stimulus, the inhibition of surrounding distractors and inappropriateresponses, as well as the ability to shift between possible cognitivestrategies.31Card sorting tests have been widely used to assess executive components such ascognitive flexibility, planning and categorization. Successful performance on thesetasks requires the involvement of several cognitive processes such as workingmemory, processing speed and set-shifting.30 is strongly influenced by age.32 Age-related inhibition impairment has been foundto influence several other cognitive abilities, such as attention,learning33 andmemory.34 Inhibitorycontrol also appears to be impacted by education level.26 However, no studies have assessed the influence ofthe FRWH on inhibitory control using tasks such as the Hayling Test.Studies of the association between EF and individual and sociocultural variables havefound that the inhibition of prepotent responses30 This EF is intimately related to cognitive flexibility, and hasbeen described as the ability to alternate between problem-solving strategies orinterrupt ineffective behaviors in favor of more adaptive ones.35 Like inhibitory control, cognitiveflexibility has also been found to be positively correlated with education byseveral studies investigating the association between sociocultural variables andexecutive performance.36In addition to inhibitory control, another key executive component discussed in theliterature consists of set-shifting.37 however, theinfluence of the FRWH and its association with that of education has only beenscarcely studied. One investigation found that older individuals with highereducational attainment and reading levels as well as better vocabulary make moreefficient use of their neural resources than those with lower educational andreading levels, and poorer vocabulary.38 The authors also found that these variables were associatedwith performance on measures of EF. A second study has also found that higherreading levels were associated with improved executive performance.39 Lastly, a third study assessed therelationship between cognitive functioning and the FWRH and found that a higherfrequency of reading and writing combined with higher educational levels wereassociated with improved executive functioning.10 However, it is important to note that each of theaforementioned studies focused on different executive components, such that thefindings precluded identification of which specific components are more influencedby the FRWH.The occurrence of age-related executive decline has been well documented in theliterature;Although a large number of studies have focused on the assessment of EF, fewinvestigations have assessed the impact of sociocultural factors other thaneducation on these cognitive abilities. This is especially true for the FRWH, whichhas only been scarcely studied in the current literature. Therefore, the goal of thepresent study was to assess the predictive role of sociocultural variables, such aseducation and the FRWH, on the cognitive flexibility, initiation, inhibition,processing speed and planning abilities of healthy elderly individuals, as assessedby the MWCST and the Hayling Test.Sample. Fifty-seven healthy adults  aged between 60and 75 years and with 2 to 23 years of formal education were included in the sample.Participants were recruited by convenience from community, workplace and universitysettings. Only Brazilian-born and native speakers of Brazilian Portuguese, with nouncorrected sensory deficits or any signs of dementia according to the Mini-MentalState Examination,41 were included in the study.Participants with a current or prior history of alcohol-related problems, illicitdrug or benzodiazepine use were excluded from the sample. The sociodemographic dataof the sample is given in Material and method. Participants took part in individual assessmentsessions lasting approximately 45 minutes each. The present study was approved bythe Research Ethics Committee of the institution at which it was conducted, and allparticipants provided written consent prior to enrollment in the study. Thefollowing instruments were used for data collection:Sociodemographic and health questionnaire43 - This is a self-report instrument used to collectdata on variables such as gender, age, years of formal education, socioeconomicstatus and FRWH, as well as clinical conditions which may influence performance oncognitive tasks, and would, therefore, imply participant exclusion . The questionnaire includesthe CAGE scale, which screens for symptoms of alcohol dependence 28 - The task involves a deck of 48cards on which geometric figures varying in number, shape and color are printed.Participants must match the cards according to a particular set of rules, which theymust identify based on accuracy feedback following each response. The MWCST assessesplanning, abstract reasoning, learning, rule maintenance, and cognitive flexibility.In the present study, the total number of categories completed, the number ofperseverative and non-perseverative errors, as well as failures to maintain set wereanalyzed.Hayling Test27 -Adapted to Brazilian Portuguese by Fonseca, Oliveira, Gindri, Zimmermann &Reppold.45 This is atwo-part assessment instrument, consisting of two sets of 15 sentences in which thelast word is omitted. The participant is asked to complete each sentence as quicklyas possible with a word that logically fits the sentence (Part A) or with a wordwhich is semantically unrelated to the sentence (Part B). Cognitive functionsassessed by this instrument include processing speed, initiation, inhibitory controland cognitive flexibility. In the present study, the latency of responses to thestimuli in Parts A and B of the test, the number of correct and incorrect responses,as well as the difference between the time taken to complete each part of the testwere analyzed.Data analysis. Data were analyzed using the SPSS software package,version 17.0. Kolmogorov-Smirnov tests were used to identify deviations from normaldistributions, and Pearson and Spearman coefficients were used to assess therelationship between parametric and nonparametric variables, respectively. Theseresults were used to construct a multiple linear regression model withneuropsychological performance as the dependent variable and education and FRWH asindependent factors. Results were considered significant at 5%.Participant scores on the Hayling Test and MWCST are given in The results of correlations between performance on the two tasks and participants'education and FRWH are shown in The results in Education and FRWH accounted for 28.9% of the variability in the number of completedcategories in the MWCST, 19.8% of the number of perseverative errors and 13.7% ofthe non-perseverative errors on the task. Interestingly, the combination of thesetwo variables had a greater predictive impact on performance on this task thaneither of the two variables alone (data not shown). On the other hand, thevariability in scores on the Hayling Test was best accounted for by participanteducation, which accounted for 14.7% of the speed and 11% of the accuracy in part Aof the task, and 11.9% of the accuracy in part B of the instrument, as well as 7.6%of the discrepancy between the time taken to complete both parts of the test.The aim of the present study was to assess the predictive impact of the socioculturalfactors education and FRWH on the EF of healthy elderly. Cognitive flexibility andplanning were assessed by the MWCST, a predominantly visuospatial task, whileinitiation, inhibition, processing speed and cognitive flexibility were evaluatedusing the Hayling Test, a two-part verbal assessment instrument. Although scores onboth tasks appeared to be influenced by the number of years of formal education andFRWH, the combination of these two variables appeared to have a more significantimpact on the EF underlying performance on the MWCST.46 severalcognitive-communicative processes remain stable over time.47 However, we believe that if a discourse processingtask had been used as an assessment tool for verbal EF, its increased complexity andthe associated increase in cognitive demand  may havesuffered a greater influence from the FRWH.48These findings suggested that, while education had a more significant influence onverbal initiation, inhibition and cognitive flexibility, both education and the FRWHwere found to influence planning, working memory, short-term memory and cognitiveflexibility, as assessed by a non-verbal instrument. As such, our hypothesis thatthe two sociocultural variables influence performance on both tasks, but have agreater impact on Hayling Test scores due to the verbal nature of its stimuli, wasonly partially confirmed. Although both variables had some influence on performanceon the Hayling Test, only education was able to accurately predict the variabilityin its scores. The relative linguistic simplicity of the Hayling Test may haveaccounted for these results, since sentence completion may be considered anautomatic language task for elderly individuals. This hypothesis is supported bystudies which show that, although some language abilities may decline withage,49 Other studies have also foundcorrelations between performance on highly complex discourse tasks with significantexecutive demands and scores on the WCST, suggesting that, even though the latter isa non-verbal task, it relies on the same cognitive abilities underlying verbaltests.23On the other hand, on the MWCST, the two variables were found to have a greaterpredictive impact when analyzed in combination rather than individually. Previousstudies involving the same neuropsychological instruments have found that both ageand education influenced performance on the tasks, with elderly individualsperforming worse than younger participants, and subjects with higher educationlevels obtaining superior scores than those with lower educationalattainment.39 The only study which has assessed reading andwriting habits in the same way as the present investigation used differentinstruments to assess EF, namely, verbal fluency and simple problem-solvingtasks.10 These limitationspreclude the comparison of the present findings with those of other investigationsin the literature.However, no studies have assessed the combined impact of education and the FRWH onperformance in these tasks among elderly individuals. Furthermore, the few studieswhich have looked into the beneficial influence of reading habits on EF did not usethe same assessment tasks as the present study, and assessed reading level based onproficiency tests rather than on the reported frequency of reading andwriting.The FRWH is an ecological measure of cognitive stimulation and allows for anassessment of current intellectual activity, whereas education levels only indicatethe duration of previous periods of continuous cognitive stimulation, which may haveoccurred long before participants are assessed as part of a neuropsychologicalstudy. Although the FRWH may not have an especially relevant impact on less complexverbal tasks, it may predict performance on more complex or non-verbal tasks, asshown by the present findings. The Hayling Test, which predominantly involvessentence completion, is simpler for both discourse and verbal fluency tasks, both ofwhich rely more heavily on executive processes, and would therefore be more likelyto be influenced by the FRWH.In conclusion, it appears that, in this sample of elderly subjects, cognitiveflexibility was sufficiently preserved to allow for adequate performance on verbaltasks, mainly due to their decreased reliance on complex verbal executiveprocessing. However, for the successful completion of complex non-verbal tasks,elderly participants may have benefitted from the additional stimulation provided byregular reading and writing habits. Although education still seems to be the mostrelevant sociocultural factor for EF in old age, the present findings suggest thatthe FRWH must be taken into account in combination with the education variable whenmore complex cognitive functions are considered.The present study provided preliminary evidence of the impact of socioculturalfactors on EF in aging, highlighting the role of education in simpler tasks and thecombined influence of this variable and FRWH in more complex ones. The presentfindings must be interpreted in light of some limitations, such as the inherentdifficulties in assessing participant education, and the problems associated withmeasuring the frequency and quality of reading and writing habits. However, in spiteof these limitations, our findings showed that the importance of the quality ofelderly individuals' educational background and of continuous cognitive stimulationthrough reading and writing after exposure to formal education must be furtherexplored. Additionally, the interaction between these factors in both healthyelderly and clinical populations should be investigated in future studies.


The family of constraint-based methods, such as flux balance analysis, has been fruitfully used to translate genome sequences in predictive, genome-scale modeling platforms. Although these techniques have been initially developed for analyzing single, well-known model organisms, their recent improvements allowed engaging in multi-organism in silico analyses characterized by a considerable predictive capability. In the face of these advances, here we focus on providing an overview of the possibilities and challenges related to the modeling of metabolic interactions within a bacterial community, discussing the feasibility and the perspectives of this kind of analysis in the (near) future.Bacteria have evolved to efficiently interact each other, forming complex entities known as microbial communities. These “super-organisms” play a central role in maintaining the health of their eukaryotic hosts and in the cycling of elements like carbon and nitrogen. However, despite their crucial importance, the mechanisms that influence the functioning of microbial communities and their relationship with environmental perturbations are obscure. The study of microbial communities was boosted by tremendous advances in sequencing technologies, and in particular by the possibility to determine genomic sequences of bacteria directly from environmental samples. Indeed, with the advent of metagenomics, it has become possible to investigate, on a previously unparalleled scale, the taxonomical composition and the functional genetic elements present in a specific community. Notwithstanding, the metagenomic approach  The advent of high-throughput sequencing platforms (NGS) represents one of the most significant milestones in the field of microbial ecology. The possibility of determining genomic sequences directly from environmental samples, circumventing the culturability issues related to most of the bacterial species, allows to investigate the composition of the microbial communities from taxonomical (microbiome) and functional (metagenome) point of view. Metagenomics, in particular, can be used to identify the metabolic potential of a microbial community in terms of the presence of genes encoding enzymes involved in specific metabolic pathways.Thalassiosira pseudonana to a bacterium from the Roseobacter clade has been demonstrated  or “simpler” environments resembling a Petri-dish context  that are often difficult to reconcile with detailed microbiological and metagenomic data. Indeed, (i) the very same microbial strain can have many different functional abilities  , which rGiven the relative simplicity of this kind of analysis and the close relationship with the biology of model organisms, this approach (with slight variations) has been widely used in bioengineering, physiology, and genome-scale synthetic biology . For exaClostridium difficile infections , well-characterized systems. On the other hand, the enzyme-soup approach relies on simplistic assumptions and limited a priori knowledge of the system under study, making it suited for analyzing complex microbial communities (such as the gut microbiota). Overall, current approaches for community metabolic modeling can be divided into: (i) quantitative methods, having a high predictive potential but being limited to simple systems due to parameterization and/or a priori knowledge required and (ii) large-scale methods, providing mostly qualitative insights but applicable to complex microbial communities.A defining feature of community modeling is the sharp increase in complexity with respect to single-organism CB analyses. In other words, the simplistic assumptions at the basis of FBA  become challenging when applied to model multi-organism metabolic interactions. This made necessary the development of innovative approaches, which are briefly described in C. difficile, performed integrating longitudinal metagenomics data with the network expansion method  can be exploited to improve the biological significance of the predictions obtained in face of the increasing complexity, the development of novel innovative methods overcoming the current limitations is indeed a priority. This includes also (i) the combination of different approaches to obtain hybrid methods optimizing the trade-off between quantitative predictions and scale of the systems and, (ii) the development of integrative frameworks to better combine meta-omics data with metabolic reconstructions. An example of the latter is the dynamic modeling of gut microbiota composition to identify bacteria inhibiting n method .Another technical challenge limiting the application of CB methods to complex communities is the quality of GEMREs that can be used. In fact, prediction of metabolic fluxes maximizing a defined objective function requires high-quality metabolic reconstructions , or comparative approaches relying on orthologous genes with “reference” organisms for which high-quality GEMREs are available. Either way, the obtained draft-quality GEMREs require additional refinement steps to fill potential reaction gaps. Perhaps the most notable example of such large-scale analysis is the metabolic reconstruction of 773 human gut microbes using a semi-automatic comparative metabolic reconstruction method . AlthougAltogether, this points to the need of (i) established protocols such as  to develFigure 1). Several efforts have been directed at characterizing the interactions between bacterial pathogens and their host, aiming at designing probiotic formulations to recover damaged communities . For this reason, the automatic generation of models from genomic and metagenomics data is a mandatory step to increase the resolution power of the community model, especially in natural environment where, in principle, every single cell takes part in maintaining the homeostasis of a particular niche.Rational design of engineered microbial communities can be translated into specific services . However, this requires the precise annotation of metabolic functions to the species present in the communities, and this can be not feasible, especially when complete and annotated genomes are missing. Despite the recent progress in genome reconstruction from metagenomes , the gapOn the other hand, while genomics and metagenomics have provided many insights into the role of bacteria in determining potential functional features of a given environment, they both provide a static snapshot of a community, thus failing to deliver a dynamic and fully functional representation. Consequently, obtaining an accurate dynamic model of a community would require longitudinal metagenomics or, more in general, methods to infer growth dynamics of single bacterial species from metagenomics . HoweverFigure 1). As ‘omic sciences become more and more affordable and sensible, their integration into community-level metabolic models is mandatory to achieve a systems level understanding of these biological entities. This highlights the necessity of a working scheme designed to handle large-scale, community-level reconstructions and to derive quantitative insights.All these factors force to discuss about which assumptions made in the context of single-organism metabolic modeling can be still tolerated when trying to accurately infer the (metabolic) dynamic of a bacterial community. Indeed, each framework proposed hitherto takes into account different aspects of microbial interactions leaving to researchers the burden of choosing the kind of model that best fits their needs; this decision should be primarily based on the information available as well as on the resolution level that is possible to achieve (All the authors contributed in conceiving, preparing and revising the manuscript. All the authors approved the manuscript and agreed to be accountable for all aspects of the presented work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.