Most human infectious diseases, especially recently emerging pathogens, originate from animals, and ongoing disease transmission from animals to people presents a significant global health burden. Recognition of the epidemiologic circumstances involved in zoonotic spillover, amplification, and spread of diseases is essential for prioritizing surveillance and predicting future disease emergence risk. We examine the animal hosts and transmission mechanisms involved in spillover of zoonotic viruses to date, and discover that viruses with high host plasticity  were more likely to amplify viral spillover by secondary human-to-human transmission and have broader geographic spread. Viruses transmitted to humans during practices that facilitate mixing of diverse animal species had significantly higher host plasticity. Our findings suggest that animal-to-human spillover of new viruses that are capable of infecting diverse host species signal emerging disease events with higher pandemic potential in that these viruses are more likely to amplify by human-to-human transmission with spread on a global scale. Emerging, re-emerging, and endemic zoonotic diseases continue to place a substantial burden on global health, particularly where dense human populations and pressures on environmental and economic resources are greatest. Over one billion cases of human zoonotic disease are estimated to occur annually, and novel emerging zoonoses have resulted in hundreds of billions of dollars in economic lossesAfter centuries of documented outbreaks, we have now begun to unravel the mechanisms underlying disease transmission from animals to people. Here, we focus on zoonotic viruses, which are the most frequently emerging human pathogen, constituting less than 15% of all known species of human pathogens, but over 65% of pathogens discovered since 1980Through systematic evaluation of data reported in the scientific literature on zoonotic viruses, we identify several key virus characteristics and transmission mechanisms that are synergistic to zoonotic virus spillover, amplification by human-to-human transmission, and global spread. The majority (94%) of zoonotic viruses described to date n = 162) are RNA viruses, which is 28 times higher  than the proportion of RNA viruses among all vertebrate viruses recognized, indicating that RNA viruses are far more likely to be zoonotic than DNA viruses, as has been reported among human pathogens2 are RNAEmerging pathogens have been noted for their ability to infect a range of animal hosts5789Based on data published to date, transmission of zoonotic viruses to humans occurs by direct or indirect contact with wildlife in a diverse array of interconnected animal-to-human interfaces, with little overlap with viruses transmitted primarily by vectors . ZoonotiOnce animal viruses have spilled over into humans, human-to-human transmission of zoonoses facilitates sustained spread of disease with a rapidity and reach infeasible for zoonotic viruses requiring contact with animal hosts for each transmission opportunity. Human-to-human transmissibility was described for 20% of zoonotic viruses investigated here . We find111415We further characterized zoonotic virus capacity for spread by categorizing viruses according to geographic range in a single country (16%), >1 country in 1–3 World Health Organization-defined (WHO) regions (55%), or ≥4 WHO regions (29%), and used ordinal logistic regression to evaluate characteristics of viruses in broader range categories. We find viruses were more likely to be in broader geographic range categories with increasing host plasticity . Among aWild animals were implicated as a source of disease spillover to humans for the vast majority of zoonotic viruses, further substantiating the concept that the diversity of wildlife on our planet has provided a rich pool of viruses, a fraction of which have successfully adapted to infect humans. Our findings indicate that high viral host plasticity is an important trait that is predictive of pandemic potential of viruses in the zoonotic pool, not only because wide host range was common among viruses that have spilled over from animals to humans, but also because this trait was associated with increased human-to-human transmission and spread on a global scale. Reporting bias must be considered in the interpretation of any association based on data reported in the literature, and the relationship between human-to-human transmissibility and host plasticity could be biased by increased research effort for viruses that have been shown to be transmissible among humans. However our analyses identified a strong linear relationship between host plasticity and likelihood of human-to-human transmissibility, and we estimate zoonotic viruses found in 10 host orders are 12 times more likely to be human-to-human transmissible than zoonotic viruses found in only one animal host order. Human-to-human transmission of viruses with high host plasticity is consistent with the hypothesis that evolutionary selection for viruses with greater ability to adapt rapidly to new hosts co-selects for viruses capable of effective intraspecies transmission in the new host. Evolutionary selection of viruses capable of infecting a wide range of hosts has been a key hypothesis underpinning disease emergence theory7Human practices facilitating heightened contact between taxonomically diverse animal hosts has likely facilitated selection of viruses with high host plasticity and sharing of zoonotic diseases. Zoonotic viruses reported in domestic animals had a significantly wider host range than viruses not shared by domesticate species. Increased research effort targeting diseases in domesticated species could bias data towards this finding, but we also detected increased host range among viruses transmitted by wildlife kept in similarly confined circumstances. Increased host plasticity among viruses shared by domestic animals supports the concept that the breeding and keeping of taxonomically diverse domesticated species in regular close contact with people for centuries has enabled evolutionary adaptive selection for mutation-prone RNA viruses capable of cross-species transmissionOur finding of significantly higher host plasticity among viruses transmitted by direct contact with wildlife kept as pets or in zoos and sanctuaries provides additional evidence to support the premise that confining taxonomically diverse species in close proximity selects for transmission of adaptable viruses with high host plasticity, even among wildlife. Diverse species of wild animals that are confined in zoos, sanctuaries, kept as pets, and sold at markets are also subject to circumstances that facilitate cross-species virus transmission via intimate contact, particularly for zoonotic viruses already adapted to transmission among domesticated animals. Vectorborne transmission similarly enables opportunities for effective contact across diverse animal hosts, which is consistent with our finding of higher host plasticity among vectorborne viruses. Through this mechanism, vector-borne transmission has facilitated emergence of animal diseases in humans, particularly those from wildlife, and, for viruses with generalist vectors, this transmission route is an effective method for interspecific dispersalHere we provide an epidemiologic picture of the animal-human transmission networks likely to perpetuate future disease emergence, and our findings add to previous efforts to guide global health research geographicallyPeer-reviewed scientific literature was searched for reports on zoonotic viruses transmitted from animals to humans using the Web of Science electronic library database for published reports through 2010. An initial list of zoonotic pathogens was established with database searches for topic keywords  and cross checked with World Health Organization (WHO), Food and Agricultural Organization (FAO), Centers for Disease Control and Prevention (CDC), and the World Organization for Animal Health (OIE) web-based reports, and previously published compilations of human infectious diseases and human emerging infectious disease events3Zoonotic viruses were included in analyses of interfaces and hosts if data were available on the circumstances surrounding virus transmission from animals to humans from 1990–2010 in scientific reports searched as described above  transmission of zoonotic viruses to people. Transmission interfaces involving wildlife were stratified by direct and indirect contact transmission and summarized in categories describing human contact as follows i) wild animals in and around human dwellings, ii) wild animals hunted, iii) wild animals consumed, iv) wild animals kept as pets, v) wild animals housed in laboratories, vi) wild animals sold in markets, vii) wild animals kept in zoos and sanctuaries, viii) wild animal exposure during agricultural activities, ix) wild animal exposure during ecotourism activities, x) wild animal exposure during wildlife management activities in protected areas, xi) virus exposure in laboratory settings (lab pathogen), and xii) virus exposure via contaminated water.For all viruses included in analyses, an extended search was conducted to identify confirmed or suspected hosts serving as a source of spillover as reported in the peer-reviewed literature based on virus detection by molecular assay, serological assay, or virus isolation. Animal species included were implicated in the scientific reports as hosts suspected in animal-to-human transmission of a given virus, either through direct contact, indirect contact or vector-borne transmission. Host species were then classified a priori according to ecological circumstances for human contact , which we expected to modify any potential host-pathogen phylogenetic relationships based on purely taxonomic classification. Stratification of animal host categories according to general circumstances of human contact was also important so that analyses could inform on risk interfaces and intervention strategies. Wild terrestrial host species were then categorized further by taxonomic order, except for orders within the superorder xenarthra, which were collapsed into one category (n = 6 zoonotic viruses). Marine mammal orders were also combined due to sparse data, as marine mammals were implicated in spillover of only 3 zoonotic viruses. Due to the large number of viruses reported in domesticated animals, domestic species were grouped according to taxonomy and stratified by similarity in circumstances for human use of animals and their products; i) cattle, ii) equids, iii) goats, sheep, llamas, alpaca, camels, iv) pigs, v) poultry , and vi) dogs and cats. Virus host range (host plasticity) was calculated as the total count of animal taxonomic orders and ecological groups recognized as hosts involved in animal-to-human spillover for each virus.A literature search was similarly conducted to identify geographic range reported for each virus in humans and animals. Geographic distribution in animals and people encompassed importation to another country by infected persons or animals if secondary amplification by animal-to-human or human-to-human transmission occurred. Zoonotic viruses were further classified according to 3 categories of international spread based on published reports as to whether viruses had been reported within 1) a single country only, 2) more than one country but only 1–3 WHO regions, or 3) more than one country and ≥4 WHO regionsWhile search effort was standardized for all viruses in our approach to the literature review, viruses varied in the number of scientific reports available describing their traits, hosts, and geographic range. All virus, host species, and interface data were summarized as binomial variables for each individual virus, in order to account for a variable number of reports and documented spillover events per virus, and adjust for likely increased research effort for viruses that infect humans and domesticated species. Each virus was designated as the unit of analysis for which we compared viral traits, animal hosts involved in spillover, and human activities noted at the point of spillover.Virus genome category (RNA vs DNA) was compared between zoonotic viruses (n = 162) and all viruses reported to infect humans and other vertebrates minus the zoonotic viruses (n = 956) using Fisher’s exact test. Bipartite affiliation (two-mode) networks were generated for virus-host and virus-interface matrix data to evaluate connectedness between host orders and between high-risk disease transmission interfaces involving wildlife. Betweenness centrality was measured for all viruses to indicate the number of connections with wild and domestic animal hosts in the virus-host network, along with the centrality of each virus within the host network, relative to all other zoonotic viruses. Betweenness centrality for each virus was calculated as the number of geodesic paths that pass through a node, standardized by the total number of virus nodes in the network, multiplied by 100. A unipartite (one-mode) network was generated to illustrate host taxonomic orders and groups connected by shared viruses. Network analyses were conducted in the network analysis platform Gephi, using the force-directed algorithm ForceAtlas2 to generate a virus-interface network displayUnadjusted bivariate relationships between viral family, interface categories, and host taxa were examined using exact statistics. Viral traits and transmission circumstances were further evaluated for multivariable associations with virus host plasticity using Poisson regression to evaluate the influence of putative viral traits and high-risk interfaces on the count of host taxonomic orders and ecological groups reported for each virus. Factors evaluated for their relationship with host range included viral family, general transmission category involving domestic animals, wild animals, or vectors, and specific direct and indirect contact wildlife transmission interfaces. Incidence rate (indicating count of host orders) ratios were estimated for all significant independent factors associated with virus host range in the Poisson model (P < 0.05). Viral traits, general transmission categories, wildlife transmission interfaces, and virus host plasticity measures were similarly evaluated for associations with virus capability for human-to-human transmission. Virus host plasticity, general transmission categories, and wildlife transmission interfaces were also evaluated for associations with international spread using ordered logistic regression adjusting for clustering of random effects within virus family.For all multivariable models, putative risk factors with P < 0.20 in univariable analyses were entered forward stepwise and retained in models if P < 0.05. Correlated variables were not included in the same model but deviance measures were used to evaluate changes in model fit to the data with each parameter independently. In all models, variables with <3 categories were evaluated for difference in magnitude, direction, and significance of effect between categories using the likelihood ratio statistic and similar categories were collapsed. Overall model fit was evaluated using Hosmer-Lemeshow goodness-of-fit test and measures of information criteria. Incidence rate and odds ratios were estimated with 95% confidence intervals. Univariable and multivariable statistical analyses were conducted using STATA 13.1 SE .How to cite this article: Johnson, C.K. et al. Spillover and pandemic properties of zoonotic viruses with high host plasticity. Sci. Rep.5, 14830; doi: 10.1038/srep14830 (2015).


Viral class II MHC epitopes that share a T cell receptor (TCR)-face with host epitopes may activate Tregs capable of suppressing protective responses. We designed an immunoinformatic algorithm, JanusMatrix, to identify such epitopes and discovered that among human-host viruses, chronic viruses appear more human-like than viruses that cause acute infection. Furthermore, an HCV epitope that activates Tregs in chronically infected patients, but not clearers, shares a TCR-face with numerous human sequences. To boost weak CD4+ T cell responses associated with persistent infection, vaccines for HIV and HCV must circumvent potential Treg activation that can handicap efficacy. Epitope-driven approaches to vaccine design that involve careful consideration of the T cell subsets primed during immunization will advance HIV and HCV vaccine development.Despite years of research, vaccines against HIV and HCV are not yet available, due largely to effective viral immunoevasive mechanisms. A novel escape mechanism observed in viruses that cause chronic infection is suppression of viral-specific effector CD4 Despite modern advances in preventing disease by vaccination, persistent viral infections continue to pose a major challenge to vaccine development. The most prominent examples are HIV-1 and HCV infections, which remain two of the largest global public health challenges today. Highly effective medications are now available, but these are still inaccessible to the majority of at-risk individuals mainly due to their cost, and limitations on access to healthcare in the developing world countries where HIV and HCV are most prevalent. Unfortunately, no vaccine candidate against AIDS or hepatitis C is currently nearing market approval. Only a handful of HIV vaccine efficacy trials have been completed, and none have yet been completed for HCV . While amutate their T cell epitopes toward greater homology with the human genome. The existence of several highly homologous T cell epitopes, some of which induce regulatory T cell (Treg) responses, has been confirmed. We postulate that this is an important means by which HIV and HCV evade effective T cell responses, and that failure to account for such epitopes may have contributed to the failure of certain vaccine approaches undertaken to date. Methods for discovering HIV and HCV Treg-activating epitopes and strategies for improving HIV and HCV vaccines are described briefly here.Development of effective HIV and HCV vaccines is lagging because traditional strategies for developing vaccines have failed to overcome the ability of the viruses to evade the human immune response. Many obstacles to vaccine development have been uncovered: (i) extensive viral genetic diversity, enabling HIV and HCV to evade humoral as well as cell-mediated immune responses , (ii) la+ T cell responses are required for control of acute viral infections  face; thus, their usefulness was limited to identifying MHC ligands. While MHC binding is necessary to stimulate a T cell response, it is not sufficient. Hence, immunoinformatic-identified MHC ligands have been screened experimentally for T cell activation to validate predictions. Because Tregs are responsive to HIV and HCV epitopes, T cell assays should be performed to ascertain which T cell subsets are activated. Indeed, Treg-activating epitopes have been discovered using overlapping peptide arrays and tetramers in HCV core, NS3, NS4, and NS5 antigens  and HIV An informatic tool that rapidly screens thousands of candidate epitopes could address the problem of viral immune escape, but also needs to consider the vast variability and degeneracy of TCRs, making prediction extremely challenging. Fortunately, the problem can be significantly reduced by searching directly for virus-encoded human homologs that potentially stimulate natural and inducible Tregs, even if some Treg-activating epitopes are not necessarily human homologs. Specifically, pathogen sequences that bind MHC and share the same TCR-face with human MHC ligands may stimulate pre-existing Tregs that emerged from development of central and peripheral tolerance. Shared sequence patterns on the TCR-face are easily searchable.To better define and rapidly assess viral camouflage epitopes, i.e., those epitopes homologous to human, we developed the JanusMatrix algorithm, which leverages our existing algorithm (EpiMatrix) to define MHC-binding peptide epitopes while searching for cross-conservation at the TCR-face. JanusMatrix can be applied to any viral or bacterial target protein to compare its TCR-faces to others in any genomes of interest. JanusMatrix analyzes the two faces of peptide sequences of pathogen origin for T cell activation potential . MHC-fac+CD25+FoxP3+ Treg number and function in peripheral blood leukocyte cultures derived from an HLA-diverse cohort of HCV-infected patients, but not in cultures derived from patients who spontaneously cleared HCV or from non-infected individuals  molecule sequences that share a TCR-face with a highly conserved epitope located in the HIV envelope protein  unlike HIV, which infects and replicates in Tregs, HCV is primarily hepatotropic and (ii) a better understanding of Treg function in chronic HCV infection could lead to treatments that are capable of balancing the competing needs for sustained effector T cell-mediated immunity and limited tissue damage moderated by Tregs .Finally, we are exploring means to fine-tune the epitope content of HIV and HCV vaccines to induce nuanced T cell responses associated with protection. We believe that careful design is needed to improve efficacy. Cross-reactivity at the individual level owing to HLA and HIV or HCV sequence variation may necessitate the development of personalized vaccines that contain T effector, but not Treg epitopes. While personalized vaccines may seem futuristic, tools are available to design such vaccines. As this technology becomes ever more accessible, there will be an even greater incentive to define the means of personalizing vaccines.Several of the coauthors on this manuscript are employees of EpiVax . Anne S. De Groot and William D. Martin are majority stockholders and Leonard Moise holds stock options. These authors recognize the presence of a potential conflict of interest and affirm that the information represented in this paper is original and unbiased observations.


Over the past decade, cellular therapies have emerged as the new frontier of treatment of several diseases. Furthermore the advent of stem cells as renewable source of cell-substitutes to replenish the beta cell population, has blurred the hype on islet transplantation. Breakthrough cellular approaches aim to generate stem-cell-derived insulin producing cells, which could make diabetes cellular therapy available to millions. However, to date, stem cell therapy for diabetes is still in its early experimental stages. This review describes the most reliable sources of stem cells that have been developed to produce insulin and their most relevant experimental applications for the cure of diabetes.The current treatment approach for type 1 diabetes is based on daily insulin injections, combined with blood glucose monitoring. However, administration of exogenous insulin fails to mimic the physiological activity of the islet, therefore diabetes often progresses with the development of serious complications such as kidney failure, retinopathy and vascular disease. Whole pancreas transplantation is associated with risks of major invasive surgery along with side effects of immunosuppressive therapy to avoid organ rejection. Replacement of pancreatic beta-cells would represent an ideal treatment that could overcome the above mentioned therapeutic hurdles. In this context, transplantation of islets of Langerhans is considered a less invasive procedure although long-term outcomes showed that only 10 % of the patients remained insulin independent five years after the transplant. Moreover, due to shortage of organs and the inability of islet to be expanded  The therapeutic approach for type 1 diabetes (T1D) is based on daily insulin injections, combined with blood glucose monitoring, healthy lifestyle and diet. Administration of exogenous insulin fails to mimic the physiological activity of the islet and disease progression may lead to the development of serious complications such as kidney failure, retinopathies and vascular diseases.Replacement of pancreatic beta-cells represents an ideal treatment that could overcome the above mentioned therapeutic hurdles. Whole pancreas transplantation was performed for the first time in 1966 by Kelly and co-workers [In this context transplantation of islets of Langerhans is a less invasive procedure: after the enzymatic and mechanic isolation from the deceased donor’s organ, islets cells are purified and injected into the recipient’s liver via the portal vein, where they rapidly engraft and re-vascularize. Progresses in islet isolation protocols and immunosuppression therapy allowed restoration of normoglycemia in patients who received islets from multiple donors. The “Edmonton Protocol” is a milestone in this field and it is internationally considered the best procedure to explore feasibility and reproducibility of islet transplantation. Patients affected by T1D underwent islet transplantation in conjunction with a glucocorticoid-free immunosuppressive regimen consisting of sirolimus, tacrolimus,and daclizumab with a good outcome at 12 months of follow-up . BrennanAlthough the Islet transplantation according to the Edmond Protocol showed good results in terms of safety, due to shortage of organs and inability of islet to be expanded ex vivo, this therapy can be offered to a very limited number of patients only . HoweverMoreover, recent data were presented on the outcomes of Pancreas Transplantation procedure after Islet Transplantation failure (PAI) and vice-versa (IAP). Although only a very limited number of patients received these procedure, metabolic outcomes seems promitting, specially in PAI . The potStem cells are characterized by two key features: (i) as unspecialized cells they can divide and renew themselves for long periods without differentiating in other cell types, and (ii) they present the potential to develop into many specialized cell types, under certain physiologic or experimental conditions. Such process can be induced by different signals, either internal, as genes expression, or external, as physical contact, chemical secretions or micro-environmental molecules. The induction leads to cell specialization through the restriction and expression of key genes.Stem cells can be classified in different levels by their plasticity, which is the ability to differentiate into the totality or just a part of cell lineages. The zygote, resulting by gametes fusion, represents a totipotent cell type, since it gives rise to both embryo and placental trophoblast. After four days of mitotic divisions, the zygote grows into a group of cells referred to as blastocyst. Embryonic stem cells (ESCs) deriving from the inner cell mass of the blastocyst are pluripotent, because of their ability to develop into the three germ layers but not into extraembryonic tissues. ESCs were first derived from mouse embryos  while inin vivo, or to expand and differentiate in vitro their progeny [Adult stem cells are undifferentiated cells showing the potential to self-renew and differentiate into one or few specific cell lineages of the tissue in which they are found or “neighbour” tissues, in order to maintain and repair the organ of origin. In situation of serious damages, various adult stem cell types can be recruited at distance to repair injured areas. For these reasons, adult stem cells are considered a suitable source for a wide range of cell therapy applications, due to their ability to differentiate  progeny .in vitro and differentiate into insulin producing cells [Pancreatic ducts, exocrine pancreas and islet of Langerhans are proposed as sources of pancreatic stem/progenitor cells. Although their nature and even their existence were initially subject of controversy in the field of beta-cell replacement for diabetes , pancreang cells , 17. Othng cells –20. Furtng cells –23. Moreng cells , which pin vitro from re-programming of differentiated cells with exocrine functions such as amylase/elastase-expressing pancreatic acinar cells and non-endocrine epithelial cells, this latter resulting from the remaining fraction after islet isolation [Attempts at producing islet cells from acinar cells were performed by Zhou et al. on rodent models. Their studies have provided direct evidence that insulin secreting cells can be generated solation .Human pancreatic islets also contain an unrecognized distinct population of cells that expresses the neural stem cell-specific marker nestin. These promising nestin-positive cells isolated from adult pancreas of rodents and human, retain some proliferative capacity and can differentiate ex vivo into pancreatic exocrine and endocrine phenotypes .Liver and small intestine cells share the same lineage origin of pancreatic cells. Therefore, both have been tested as replacement insulin producing cells for diabetes cellular therapy –33.in vitro trans-differentiation led to hybrid hepatocyte-beta cell phenotype incapable of adjusting insulin levels according to variable glucose concentrations. Therefore, in the years to come further evaluations are required to make liver cells a concrete and viable source for beta cell replacement [To date, most of the approaches are focused on liver as a promising abundant source for the generation of insulin producing cells. Liver and pancreas cells, in vertebrates, derive from the same indistinct pool of progenitor stem cells resident in the extra-hepatic biliary tree  and difflacement .Haemopoietic stem cells are situated in stem cell niches like bone marrow or umbilical cord blood and differ from mesenchymal cells, which are distributed in the entire body and can generate fibroblast, adipocyte, chondrocyte along with other connective cells . Haemopoin vitro and in vivo, a strong immunomodulatory activity which may help avoiding post-transplant rejection. Immunomodulation is mediated by different mechanisms: secretions of soluble factors (e.g. prostaglandin E2 and IL-10), pathways activated by direct contact with T-cell or interaction with dendritic cells [in vitro into islet-like cells and transplanted the cells into streptozotocin-induced diabetic rat. Few years later in a similar attempt, Phadnis SM et al. failed to accomplish a sufficient in vitro differentiation of BM-MSC, while achieved the amelioration of glycemia after injection of immature cells in diabetic mice, showing that pre-induced BM- MSCs (in vitro) completed their maturation into endocrine pancreatic lineage and insulin producing cells in vivo [Ubiquitous mesenchymal stem cells (MSCs) can be isolated from various tissues like umbilical cord, bone marrow, adipose tissue and placental tissues. However, to be considered as MSCs, they must meet specific requirements such as adherence to plastic, differentiation abilities towards bone, cartilage and fat lineage, presence/absence of peculiar surface markers. Some MSCs also express stemness markers of pluripotency like OCT4 and Nanog . Moreoveic cells . Successic cells  who demo in vivo . Other s in vivo , 56; fro in vivo , retain the highest plasticity. ECSs can differentiate to all body cell lineages and are uniquely capable of indefinite self-renewal.in vitro stepwise pancreatic-induction protocols that recapitulated each phase of islet cells development from embryonic stem cell to mature endoderm, pancreatic progenitors and, finally, insulin producing beta-cell like cells [In vivo experiments on immunodeficient diabetic mices revealed that normoglycemia was routinely obtained upon injection of human ESCs-derived beta cells progenitors [in vivo maturation of functional insulin-producing cells that could provide adequate hyperglycemia responsiveness. The factors that allow the efficient beta-cell differentiation in vivo are unknown, but may include key elements that are difficult to reproduce in vitro, such as vascularization and interaction with contiguous tissues [Applications of ES cells to human therapy began with their characterization by Thomson et al. (1998) . In 2001ke cells –69. As agenitors –72. Nota tissues .However, the transplantation of a mixed population of immature human ESC-(hESC)-derived cells is accompanied by serious safety concerns related to the persistence of the tumorigenic risk. Current studies are being addressed to solve the tumorigenic issues through the sorting of the pancreatic progenitors from the rest of the population  and antiAn interesting alternative to ensure safety and functionality of transplanted beta cell precursors and hESCs-derived precursors, is represented by the use of cell-encapsulation procedures. The enclosure of cells within a barrier would allow diffusion of glucose, nutrients, insulin and block larger molecules, cells and antibodies. The physical selective barrier provided by capsules could avoid the continuous subministration of immunosuppressive drugs, thus ameliorating the quality of life of diabetic patients. Additionally, the capsule would protect the patient from any risk of tumorigenicity and allow the removal of the graft in case of malfunctioning, increasing the recipient safety after transplantation. Breakthrough studies have adopted encapsulation methods to transplant human beta cell progenitors obtained from stem cells. Seung-Hee Lee et al. transplanted human beta cell progenitors into a macroencapsulation device (TheraCyte), durable and biologically inert, which can be transplanted into human patient for a year without adverse effects. The study demonstrated the lasting survival, differentiation and function of human beta cell progenitors in the device, with evidence of cell replication and maturation. This approach eventually resulted in the restoration of blood glucose levels. In addition, the device provided protection from allograft rejection for more than 140 days, suggesting that encapsulated β-cells are invisible to the immune system .Placenta is the organ responsible for the correct foetal development throughout all the phases of gestation, and is considered a waste product of birth, but its tissues and cells represent an abundant source of stem cells , 78. Fouin vitro and were capable of normalizing the blood glucose level of streptozotocin-induced diabetic mice for several weeks after cells injection [in vitro development of hAECs 3D spheroids: “epi-spheres” were then successfully induced into glucose-responsive insulin producing cells [in vitro differentiation protocol to induce hAECs into definitive endoderm, pancreatic foregut, pancreatic endoderm and, finally, pancreatic endocrine cells [Moreover the transplantation of placenta-derived stem cells for cellular therapy fully complies with safety requirements as there is no risk of tumorigenesis (teratomas) . In the njection . Recentlng cells . Anotherne cells . The insDespite the numerous and encouraging results presented in the last decade, there is still a long road ahead to set a reliable cellular therapy for diabetes. Scientists from all over the world have to set their current and future studies on the basis of two major challenges of cellular therapy: the safety and the efficiency of the transplanted cells. Unfortunately, a cell source providing a robust response after differentiation, is not always considered safe because of transplant-related collateral effects such as immune rejection or, worse, tumorigenesis. Such challenges could be simplified by the adoption of totipotent ESCs in opposition to adult stem cells. ESCs have the greatest stemness potential, hence differentiation potential towards any lineage, any tissue and cell, but their use raises concerns about safety and ethical issues. Although the differentiation protocols have considerably improved their efficiency the transplant of ESCs-derived cells is still associated with the development of malignant tumours due to incomplete differentiation of the clones. Moreover, cells differentiation may lead to secretome changes and phenotype alterations that can elicit the host immune response compelling the patient to an immunosuppressive regimen. In order to bypass the response from the immune system some researchers are developing and testing various devices to encapsulate the transplanted cells within a physical barrier, which could allow the permeability to nutrients, glucose and secreted hormones, and avoid contact with complex molecules, cells and antibodies . Despitein vitro.For this reasons an increasing number of scientists are now focusing on different sources of stem cells that possess less stemness potential compared to ESCs but are safer and free of ethical concerns, namely adult stem cells. The pancreas itself is a promising source for stem cells: duct cells, acinar cells and stem cell from islets of Langerhans share the same embryological origin of beta cells; therefore they are good candidates for the de-differentiation and re-programming towards an insulin producing phenotype. The obstacles to the extensive use of this source are represented by the invasive collection procedures, the scarcity of the stem cells, the difficulty in isolating and expanding the culture in vitro expansion capacities, and rapid cell dedifferentiation [The derivation of insulin producing cells from hepatocytes , which share with beta cells the same endodermic origin, showed promising outcomes. However, even though these tissues represent an abundant pool of reprogrammable cells, the use of hepatocytes ex vivo is hampered by the shortage of donors, poor ntiation .in vitro expansion, and the promising outcomes of various studies showing the differentiation potential towards non-mesodermic lineages. However, in vivo assays on animal models did not prove to be as solid as in vitro experiments. In most cases transplanted MSC-derived insulin-producing cells failed to afford a stable amelioration in STZ-induced diabetic mice. The controversy concerning in vivo potential for MSC cells to differentiate into insulin producing cells is supported by the postulated inability of mesodermic cells to generate functional endocrine pancreatic cells, which could be properly derived only from endodermic progenitors. Further studies are required to improve the in vitro pancreatic differentiation of MSC and consequently a stable and responsive production of insulin in vivo.Numerous studies described the use of MSC from various tissues, pancreas included, as a widely accessible source of insulin producing cells. The expectations on MSC studies were nurtured by the abundance of cells, the ease in isolation and in vitro expandability and differentiation efficiency. Stem cells isolated from the amnion layer of placenta meet most of the mentioned criteria and the adoption of hAECs in cell therapy and regenerative medicine do not raise any ethical concern, unlike the use of ESCs which is restricted or forbidden in most countries [in vitro evaluation will be necessary to define the therapeutic potential, but there is reason to be optimistic that a sufficient number of efficient insulin producing cells will be available in the near future.Therefore, scientists are continuously seeking for the optimal characteristics of a stem cell source, which may include: post-transplantation safety, wide availability, non-invasive collection procedure, ountries . FurtherAD-MSCs, adipose-derived mesenchymal stem cells; AF-MSCs, amniotic fluid mesenchymal stem cells; BM-MSCs, bone marrow mesenchymal stem cells; hAECs, human amniotic epithelial cells; hAMSCs, human amniotic mesenchymal stromal cells; hCMSCs, human chorionic mesenchymal stromal cells; hCTCs, human chorionic trophoblastic cells; IPF-1/PDX1, insulin promoter facto-1/pancreas and duodenal homebox-1; STZ, streptozotocin; UCB-MSCs, umbilical cord blood mesenchymal stem cells; WJ-MSCs, Wharton’s jelly mesenchymal stem cells


Additionally, therapy with HCQ resulted in significant reduction in vascular endothelial dysfunction with improvement in vascular elasticity and flow patterns and better-preserved vascular wall thickness across multiple vascular beds. More importantly, we found that presence of CKD had no mitigating effect on HCQ’s anti-atherosclerotic and vasculoprotective effects. These beneficial effects were not due to any significant effect of HCQ on inflammation, renal function, or lipid profile at the end of 16 weeks of therapy. This study, which demonstrates structural and functional protection against atherosclerosis by HCQ, provides a rationale to evaluate its use in CKD patients. Further studies are needed to define the exact mechanisms through which HCQ confers these benefits.Cardiovascular disease is the largest cause of morbidity and mortality among patients with chronic kidney disease (CKD) and end-stage kidney disease, with nearly half of all deaths attributed to cardiovascular disease. Hydroxychloroquine (HCQ), an anti-inflammatory drug, has been shown to have multiple pleiotropic actions relevant to atherosclerosis. We conducted a proof-of-efficacy study to evaluate the effects of hydroxychloroquine in an animal model of atherosclerosis in ApoE knockout mice with and without chronic kidney disease. Forty male, 6-week-old mice were divided into four groups in a 2 x 2 design: sham placebo group; sham treatment group; CKD placebo group; and CKD treatment group. CKD was induced by a two-step surgical procedure. All mice received a high-fat diet through the study duration and were sacrificed after 16 weeks of therapy. Mice were monitored with ante-mortem ultrasonic echography (AUE) for atherosclerosis and vascular stiffness and with post-mortem histology studies for atherosclerosis. Therapy with HCQ significantly reduced the severity of atherosclerosis in CKD mice and sham treated mice. HCQ reduced the area of aortic atherosclerosis on  Cardiovascular disease (CVD) is the largest cause of morbidity and mortality, 2 amongTherapeutic interventions conventionally proven to be effective in CVD among patients with normal renal functions, have limited if any efficacy in presence of significant CKD. For example, HMG coenzyme A inhibitors, statins, when studied in CKD and ESKD populations have failed to show a significant reduction in CV mortality–7, even These factors have led to a high CV mortality in CKD that hasin vivo, in vitro, and cohort-based reports over the last two decades show that, in addition to being anti-inflammatory, HCQ has multiple other properties that include beneficial effects on vascular compliance and endothelial function,. Thi. Thien fWe measured the vascular endothelial function through the antemortem measurements of vascular elasticity and blood-flow dynamics for all major vessels on AUE using the M mode and Doppler mode. We observed a significant and progressive reduction in vascular elasticity Figs  and 7 anHCQ is an anti-inflammatory drug and inflammation has been proposed to have a significant role in many complications of CKD including CVD and renal failure progression. We examined the impact of HCQ on inflammation, renal functions, and lipids parameters.We evaluated the impact of HCQ on systemic inflammation by measuring the serum levels of hsCRP. Therapy with HCQ did not affect the levels of hsCRP in mice with CKD . To confOur study demonstrates that HCQ confers a significant functional and histological protection against vascular stiffness and atherosclerosis in presence of uremic milieu. HCQ substantially reduced the severity of atherosclerosis (by ~60%) in these atherogenic mice, improved vascular compliance, and prevented vessel wall thickness. More importantly, the anti-atherosclerotic and vasculoprotective effects of HCQ were not mitigated by and appeared to be equally potent in the presence of uremic milieu.in vivo anti-atherosclerotic efficacy for HCQ in CKD. Previous studies demonstrating that HCQ is safe to use in the presence of advanced CKD thus make clinical trials feasible.The principle outcome of our study is the validation of strong atheroprotective effects for HCQ even in the presence of CKD. HMG Co-A inhibitors (statins), the mainstay for management of atherosclerotic CVD, have strong evidence for their efficacy in animal atherosclerosis models and in human clinical trials in the presence of normal renal function–27. HoweClinically, endothelial dysfunction with resultant vascular stiffness, inflammation, and accelerated atherosclerosis are prominent factors for CVD associated with CKD: Endothein vitro studies have shown that HCQ stimulates vascular endothelial cell NO release[in vivo studies in animal models of lupus, diabetes, and metabolic syndrome have since shown a beneficial impact of HCQ vascular endothelial cell function, as judged by improvement in endothelial dependent vasodilation[Ghigo et al. through their O release. Multiplodilation–34. Evenodilation, 35, 36.Endothelial dysfunction as measured by AUE echography has been strongly associated with the development and severity of atherosclerotic plaque in apoE-/- mice models, 24, andIn our study, HCQ failed to reduce the degree of inflammation as reflected by the serum levels of hsCRP among mice with or without CKD. Retrospective database analyses have suggested a possible beneficial role for HCQ on the lipid profile, with some suggesting an improvement in LDL and HDL values–39. In oin vitro, in vivo, and human cohort data indicate that HCQ has many pleiotropic effects, e.g. reduction in oxidative stress, improvement in insulin sensitivity and metabolic syndrome[Analyses of various  syndrome–19, 22,  syndrome with con syndrome–43 in ti syndrome. More imAlthough our study does not provide mechanistic insight, we believe it is clinically relevant because it proposes a therapy for CVD with a drug that has been in use for several decades, even in patients with advanced CKD and ESKD, 45. Thi


Wound healing requires an orchestrated integration of complex biological and molecular events, which include inflammation, proliferation and remodeling. Wharton’s jelly mesenchymal stem cells seem to promote wound healing and tissue repair. Wharton’s jelly stem cells promote fibroblast proliferation and migration, accelerate re-epithelialization and promote overall wound repair by pcrine signaling. Wharton’s jelly is an advantageous mesenchymal stem cell source because the harvest of this type of stem cells is not painful or invasive and because, beside their effect on wound healing, they seem to have a significant impact on the treatment of keloids. Furthermore, they led to better nerve regeneration, better neuroprotection and less inflammation. Wound healing requires an orchestrated integration of complex biological and molecular events, which include inflammation, proliferation and remodeling . DespiteSkin cell renewal is under the control of mesenchymal stem cells (MSCs). Skin MSCs populate the normal skin niche, remain quiescent and become active after injury, aiding in wound closure . MSC pcrin vitro and in vivo. Preclinical and clinical studies have shown a positive effect of ADSCs on wound healing and have verified them as a suitable cell type for skin substitution [Adipose-derived stem cells (ADSCs) are located between mature adipocytes in the adipose tissue. ADSCs are able to differentiate into a variety of different tissues such as bone, adipose tissue and cartilage both titution ,6.ADSCs can relatively easily be harvested through liposuction or during abdominoplasty, but this still requires a surgical procedure. Researchers thus focused on a more advantageous MSC source, such as umbilical cord-derived Wharton’s jelly . The harMoreover, human umbilical cord Wharton’s jelly mesenchymal stem cells (WJ-MSCs) have been shown to have better clinical utility. WJ-MSCs have unique properties between embryonic and adult stem cells. These cells have low-level expression of embryonic stem cell markers and satisfy the criteria recommended by the International Society of Cytotherapy for MSCs. Stem cell quality, quantity and safety can be optimized when cultured under controlled physiolocal conditions, including appropriate oxygen and nutrition supply as well as waste elimination. These can be realized in bioreactors using dynamic culturing strategies.in vitro conditions and in animal models [WJ-MSCs represent a very efficient stem cell source with reported immunoprivileged, anticancer and antifibrotic characteristics under l models . Due to l models .in vitro and in an in vivo mouse wound healing model, suggesting that human WJ-MSCs may promote wound repair. Indeed, pilot clinical studies have so far indicated that MSCs in general are safe in vivo, and they currently represent the most widely used stem cells in the clinical setting [Indeed, caprine WJ-MSCs have already been shown to promote wound repair with minimal scarring, and the results of a recent study by Arno and colleagues suggest that WJ-MSCs enhance wound healing and tissue repair . WJ-MSCs setting . Accordi setting .In conclusion, Wharton’s jelly is an advantageous MSC source, because the harvest of this type of stem cells is not painful or invasive and because, in addition to their effect on wound healing, they seem to have a significant impact on the treatment of keloids. Furthermore, the cells led to better nerve regeneration , better ADSC: Adipose-derived stem cell; MSC: Mesenchymal stem cell; WJ-MSC: Wharton’s jelly mesenchymal stem cell.The authors declare that they have no competing interests.


Resistance training (RT) is the main method of exercise for improving strength and skeletal muscle mass  compared to no failure HI-RT , providing rationale as to why RT to failure might differently affect muscle adaptations in different populations.Few studies have attempted to directly evaluate the effects of HI-RT to muscle failure or no failure in neuromuscular adaptations . Izquierdo et al.  randomizOn the other hand, Drinkwater et al.  studied The lack of measurements of both fatigue levels and muscle activation in studies that compare HI-RT to failure or no failure hampers comprehension on how these mechanisms contribute to RT-related chronic adaptations. Additionally, no studies have directly compared hypertrophic responses after exercising to failure or no failure. The differences in subjects training status represent another problem, hindering the ability to draw relevant comparisons between studies. As the above studies seem to point, fatigue influence on muscle activation and adaptations can differ greatly depending on the subject training level. Apparently, for individuals untrained in strength, RT to failure is unnecessary for maximizing increases in muscle strength and muscle mass. Conversely, for trained individuals, repetitions to failure might result in increased muscle activation, which could explain the greater increases in muscle strength after protocols performed to muscular failure. However, it remains unclear if this fatigue-induced muscle activation truly reflects chronic increases in strength and muscle hypertrophy, and how differently it affects untrained and trained individuals.Recent studies have pointed muscular failure to be an important factor in order to maximize adaptations when RT is done at low intensities (LI-RT). However, no attempts have been made to directly compare the effects of training to muscle failure versus no failure on neuromuscular adaptations. As consequence, the researchers compare LI-RT with HI-RT, assuming the last as the ideal situation for increasing both muscle mass and strength.Regarding untrained individuals, Mitchell et al.  sought tOn the other hand, Holm et al.  investigWhen it comes to strength trained individuals, results are more consistent. To study the influence of training intensity on muscle activation Schoenfeld et al.  employedConsidering the evidence to date, it seems plausible to assume that fatigue differently affects muscle adaptations when LI-RT is performed to muscular failure by different populations (trained and untrained). For untrained subjects, it is possible that protocols resulting in low fatigue, like the one used by Holm et al. , are unaFinally, some studies suggest that RT to failure for a prolonged period may result in overtraining; higher risk of musculoskeletal injury by repetitive effort; and stronger hemodynamic responses, with pressure peaks near muscular failure  grants #2016/00314-3.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.


Many viruses deliver their genomes into the host cell’s nucleus before they replicate. While onco-retroviruses and papillomaviruses tether their genomes to host chromatin upon mitotic breakdown of the nuclear envelope, lentiviruses, such as human immunodeficiency virus, adenoviruses, herpesviruses, parvoviruses, influenza viruses, hepatitis B virus, polyomaviruses, and baculoviruses deliver their genomes into the nucleus of post-mitotic cells. This poses the significant challenge of slipping a DNA or RNA genome past the nuclear pore complex (NPC) embedded in the nuclear envelope. Quantitative fluorescence imaging is shedding new light on this process, with recent data implicating misdelivery of viral genomes at nuclear pores as a bottleneck to virus replication. Here, we infer NPC functions for nuclear import of viral genomes from cell biology experiments and explore potential causes of misdelivery, including improper virus docking at NPCs, incomplete translocation, virus-induced stress and innate immunity reactions. We conclude by discussing consequences of viral genome misdelivery for viruses and host cells, and lay out future questions to enhance our understanding of this phenomenon. Further studies into viral genome misdelivery may reveal unexpected aspects about NPC structure and function, as well as aid in developing strategies for controlling viral infections to improve human health. Viruses are highly efficient gene delivery devices when compared to alternative approaches such as naked nucleic acid delivery or artificial vectors, such as polyamine or liposome complexes ,2,3. VirViruses that replicate in the intact cell nucleus must transport their genomes through the nuclear pore complex (NPC) embedded in the nuclear envelope ,22,23,24The separation of the viral genome from the capsid is termed uncoating. This critical reaction exposes the genetic information and is key to making progeny virions. It is a stepwise gain of function process, which starts at the cell surface upon virus docking to receptors  and invoMost viruses that replicate in the nucleus are larger than the translocation channel of the NPC, which can accommodate cargo with a maximal size of about 40 nm . Since tThe nuclear pore complex (NPC) serves as both gate and gatekeeper to the nucleus. The NPC is a disk-like structure about 50 × 100 nm in size. NPCs are assembled from multiple copies of around 30 different proteins, called nucleoporins (Nups) ,40,41,42NPCs control the passage of a wide range of cargos through passive diffusion and facilitated translocation ,45,46. PHow the NPC achieves size-selective translocation of cargo across the nuclear envelope has been one of the most intriguing unsolved problems in cellular trafficking. Single-molecule methods are starting to provide details on how translocation works for large substrates on the level of ribosomal subunits and viral cargoes ,51,52. FRecent single-genome tracking experiments have provided a fresh perspective on viral nuclear import by permitting a first glimpse of misdelivery at the NPC . It was The issue is of general interest as misdelivery may represent an ‘Achilles’ heel’ for viruses infecting non-dividing cells. For example, over half of human immunodeficiency virus-1 (HIV-1) reverse-transcribed genomes accumulate as capsid-free dead-end products in the cytosol of primary human macrophages ,57. CoulFor viruses, residence inside cells is generally short-lived. The reasons may be several fold. Viruses are unusual structures that do not normally occur in cells, and are subject to antiviral immunity ,13. As dThe NPC is a dynamic, selective, crowded environment that is constantly loaded with fast moving cargoes. In such an environment, bulky viral genomes push the limits of transport capacity and increase the probability of something going wrong. Probable causes of misdelivery include improper virus docking to NPCs, inefficient translocation of vDNA through NPCs, NPC stress, and innate immunity factors interfering with uncoating or translocation .The first critical step in viral nuclear import occurs when a virus or an uncoated subviral structure reaches a NPC and attaches itself to the surface. This process is governed by a series of intricate protein-protein interactions that are virus-specific, and can be divided into separate phases. Initially, viruses or subviral structures, such as DNA or RNA nucleoparticles target NPCs by association of viral NLS motifs with cellular transport receptors of the importin/karyopherin family ,71,72,73Upon making contact, viruses position themselves correctly with respect to the cytoplasmic opening of the central channel of the NPC so that a released genome can pass through  nuclear import . IAV hasAfter a virus latches onto an NPC, the immediate challenge becomes delivering its genome and accessory proteins into the nucleus. Small viruses, such as parvovirus or hepatitis B virus capsids may enter the NPC largely intact, although little is known about the kinetics, efficiencies, and mechanisms ,88,89. LLarge viruses comprise the vast majority of viruses replicating in the nucleus. These include adenovirus, herpesvirus, HIV, and influenza virus. For large viruses to access the limited space and enter the pore, partial or complete uncoating is required ,96,97,98When nuclear import of adenoviral DNA was quantitatively assessed by click-chemistry labeling and fluorescence microscopy, it was found that only a fraction of the incoming viral genomes were imported into the nucleus . At leasin situ photo-conversion experiments [In recent years, it has been discovered that the stress caused by virus uncoating can lead to a partial breakage of the NPC. Such was shown for HAdV, which is among the largest (~920 Å diameter) and most complex (~150 MDa) of the non-enveloped double-stranded DNA viruses ,105,106.eriments . In addieriments . Intereseriments . The vDNeriments ,59,110. Innate immunity is a collection of generic and immediate cellular defense reactions against pathogens without long lasting effects. It is a multi-branched system that acts as a physical and chemical barrier against intruding pathogens, and involves chemicals that recruit immune cells to the site of infection . It compTo date, several classes of membrane bound and cytosolic receptors have been identified and partially characterized, mapping antiviral blocks at multiple stages in virus entry . Only reAs outlined above, problems encountered during nuclear entry may result in misdelivery, leading to accumulation of capsid-free unwound nucleic acids in the cytosol. In the following section, we consider the possible ways that both viruses and cells respond to such a situation and look at how misdelivered genomes may impact the course of viral infection. The appearance of microbes and viruses has evolved an impressive arsenal of sensors and restriction factors that are strategically pieced together as a unified defense system to provide cell-wide protection against pathogens ,133,134.Viruses can escape or block host innate detection or use it to their advantage ,140. ThiIn terms of masking nucleic acids, viruses may avoid recognition by nucleic acid-sensing receptors through modifying their genomes to remove immune-stimulatory motifs, perhaps utilizing virus-encoded endonucleases/phosphatases , cytosinAnother possible way for viruses to handle misdelivery is to digest mislocalized genomes that would otherwise activate interferon expression. This can be accomplished by hijacking cellular factors, as HIV-1 does with the cytoplasmic exonuclease TREX1 , or possNotably, the ability to utilize host proteins and pathways instead of relying on a limited copy number of viral factors to address misdelivery at the nuclear import stage is likely a critical determinant of infection. At this stage, viruses are only beginning to enter the nucleus and so very few, if any, viral proteins have been synthesized that can serve to antagonize innate or intrinsic factors in this early time of marked vulnerability. The window of vulnerability closes, when imported genomes trigger production of viral components that robustly target nucleic acid sensing pathways . For insUnveiling the precise mechanisms by which viruses import nucleic acids through NPCs is a long-standing goal for cell biologists, virologists and gene therapists alike. The cell biology bears conceptual interest due to the large size, hydrophilic nature and the tractability of the cargo. From a virology standpoint, gaining clues about how viruses manipulate cell-machinery to replicate and spread is critical in moving toward the goal of defining potential antiviral strategies. For gene therapy, this research informs new approaches for targeted delivery of exogenous genes into the nucleus of recipient cells. Recent novel labeling procedures combined with state-of-the-art super-resolution imaging have allowed a new direct view of viral nuclear import. Surprisingly, it was discovered that a significant fraction of incoming viruses fail to import their genomes into the nucleus upon attaching to NPCs . This isFuture work will aim to fill in structural data showing how viruses attach at NPCs and to define what types of structural rearrangements may ensue. The major challenge here is sample preparation, which necessitates capturing properly uncoated virus particles attached to the surface of NPCs. This will require extensive efforts in protocol development for infecting cells, isolating samples, and preserving them for experimentation. One promising avenue is cryo-electron tomography, which requires the additional sample preparation problem of maintaining the integrity of the sample while freezing in thin layers of ice (<1 µm thick) for imaging. Alternatively viral capsid-NPC sub-complexes can be purified and investigated in a divide-and-conquer strategy using traditional structural approaches such as X-ray crystallography, NMR spectroscopy, and/or single-particle cryo-electron microscopy. These experiments have the further limitation of size and flexibility, which may hinder determination of a high-resolution structure. In addition to structural studies, we will need to watch viral nucleic acids as they undergo transport through or misdelivery at NPCs in live-cells to quantify the kinetics and players involved in these processes. Live cell experiments are becoming increasingly possible with the availability of brighter fluorescent probes and new quantitative imaging methods that have high spatial and temporal resolution. Along with other tools such as ectopic expression and gene silencing, structural and live cell imaging experiments will serve to increase our understanding of virus misdelivery at nuclear pores and may reveal how cells guard against foreign DNA at the level of nuclear import.


While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa.Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through  Medicinal treatments have a storied history tied to natural products discovery and development. Natural products derived from plants and animals have been the source of traditional medicine for millennia, and more recently have become major sources of chemical diversity as drug leads, driving research efforts in pharmaceutical drug discovery and development ,2. The a®, based on a venom peptide from the Brazilian viper and used to treat hypertension . Traditionally, the determination of disulfide pairing in proteins/peptides was extremely labor-intensive, applying separation of proteolytic fragments by electrophoresis in one dimension, followed by performic acid oxidation and paper chromatographic separation in the other [Determining disulfide connectivity in venom peptides is a fundamental step in establishing structure–function relationships. The disulfide crosslinks in venom peptides provide the structural scaffolds that are essential for their recognition at specific receptor sites ,132. An he other . A theorhe other ,135. Thehe other ,137.Terebra variegata by MS/MS mapping using a partial reduction and dual alkylation protocol applying TCEP-HCl (Tris(2carboxyethyl)phosphine hydrochloride) as reducing agent and NEM  and IAM (iodoacetamide) as alkylating agents. Dual NEM/IAM alkylation resulted in Tv1 peptide species that were labeled with two, four, or six NEM and IAM groups. The location of NEM and IAM modifications in each of the six partially reduced species was determined by matching the MS/MS b- and y-series ions to theoretical patterns [We have recently determined the disulfide connectivity of teretoxin Tv1 from patterns .13C-HSQC spectrum. Disulfide connectivities were then determined based on the proximity of cysteine residues in the 10 lowest-energy structures and were in agreement with the disulfide bond pattern derived by MS/MS analysis [The solution structure of Tv1 was independently derived using standard homonuclear proton NMR techniques on unlabeled folded synthetic peptide to confirm the disulfide bond connectivity derived from MS/MS C. Protonanalysis . Teretoxin silico approach, the number of peptides that are synthesized and characterized could be significantly reduced to a manageable amount.With the large numbers of putative venom peptides identified recently through NGS approaches, it is prohibitively time-consuming and expensive to structurally characterize each of these peptides using NMR, especially considering that some of the identified peptides might be false positives that represent artifacts of the NGS assembly methods. Bioinformatics algorithms that predict the three-dimensional structures of peptides can be used to narrow down which of many candidate peptide toxins are worthy of experimental characterization  Figure . Venom pab initio from their sequence, including the prediction of a completely new protein fold [The Rosetta algorithm for protein folding has enjoyed considerable success in accurately predicting the three-dimensional structure of proteins ein fold ,139. Rosein fold . CompareOur integrative venomics strategy follows a funneling approach from organismal to molecular biology, starting with the description of terebrid venomous lineages and the characterization of teretoxin peptides, and ending with the identification of specific molecular targets and functions . In the Nereis virens polychaetes [To determine the biological activity of selected synthesized teretoxins, peptides are initially tested on a bioassay using their native prey, polychaete worms (Annelida) . Animal ychaetes ,111. As ychaetes ,146,147.ychaetes ,149. OneVenom peptides typically interact with ion channels and modulate their activities, enabling the investigation of specific ion channels and their function ,151. ForVirtual screening is a well-established computational method for identifying ligands that interact with target proteins. It has been applied successfully even to challenging problems such as finding small molecules that are active at a target protein whose structure is not known, such as a G-Protein Coupled Receptors (GPCRs) . In theoin silico venom peptide models. In this regard, virtual screening can be used in conjunction with high-throughput bioassay screening methods to prioritize which molecular targets to screen against. For example, the Bioluminate software package  largely automates all the steps in the homology model process, including special features that allow more sensitive searches for distant structural homologues of ion channels to use as templates. The entire homology modeling process takes only a few minutes. The venom peptide can then be docked against the model using the integrated PIPER protein–protein docking algorithm. The entire process, including simulation time, takes roughly one hour for a given ion channel or receptor target. While the results of such an in silico screen may not always be entirely accurate, they can be improved by including mutagenesis constraints from the literature if available. Such an effort can thus provide a prioritized list of molecular targets for screening , potentially reducing the time and material necessary to identify the molecular channels targeted by venom peptides.Molecular modeling environments that integrate bioinformatics, homology modeling, and docking algorithms can drastically reduce the time needed to create Our approach to teretoxin molecular target discovery is to apply computational algorithms to model the docking of the peptide of interest to a wide range of potential receptors. The docking poses can be refined with long timescale Molecular Dynamics (MD) simulations of the peptide toxin/receptor pose. If the peptide remains in a well-defined pose over the timescale of hundreds of nanoseconds or several microseconds, it suggests that the teretoxin effectively binds the target receptor protein. These receptors are then selected as the more likely candidates and have the highest priority for further experimental verification. Alternatively, receptors where the peptide never establishes a well-defined pose are considered less likely to be the true target of the peptide and are discarded for experimental testing.®, Byetta®, and Captopril® are all breakthrough drugs derived from animal venom peptides via different routes, decades after their initial discoveries [The estimate of available venom peptides from the reservoir of conoidean snails alone is upwards of one million compounds . Giving coveries ,7,8. Howcoveries ,154. Thein vivo circulation [Native venom peptides have remarkable affinity and specificity for drug targets such as ion channels and GPCRs. Many venom peptides can readily serve as scaffolds for peptidomimetics or pharmacological research tools; however, with a few exceptions, most venom peptides often require derivative versions to be useful as therapeutic leads. A common modification applied to venom peptides is devising derivatives to increase affinity for a specific molecular receptor ,156. Anoculation ,159,160.in silico and their effects on affinity and selectivity of binding to specific receptors can be predicted computationally [in silico method is both inexpensive and rapid compared to alanine scans, ensuring that the number of venom peptides examined can be significantly increased.Traditional methods for identifying specific functional mutations in venom peptides include trial and error alanine walks through each residue of the peptide. Following this approach, 20 potential functional mutations in conotoxin α-GID and 70 mutants of spider peptide GpTx-1 were identified ,162. Howtionally ,165. Thein silico molecular design [Rosetta is one of the most widely used and successful algorithms for r design . In addir design ,167,168.r design ,169. We r design .in silico with Rosetta. By combining Rosetta modeling with evolutionary algorithms, we can optimize the process of identifying random mutation possibilities, focusing only on those that have passed the test of millions of years of evolutionary change while maintaining venom peptide bioactivity.Rosetta on its own will identify potential residues that can be altered to enhance venom peptide specificity. However, if we provide Rosetta with the information accumulated through millions of year of evolution inherent to venom peptide genetic sequences, we can significantly boost its efficiency. Computational algorithms that estimate sequence evolution such as PAML  and HyPhy (Hypothesis testing using Phylogenies), can compute the rate of non-synonymous to synonymous mutations in a given group of sequences, identifying specific sites of the venom peptides under positive selection ,172,173.In venom research, evolutionary algorithms have been primarily used to answer questions about venom peptide evolution ,177,178.The potency and specificity of bioactive peptides have propelled these agents to the forefront of pharmacological research, but delivery of peptides to their molecular target is a major obstacle to their widespread application. We have recently devised a Trojan Horse strategy consisting of packaging a bioactive peptide within a modified protein cage to protect it during transport, and releasing it at the target site, which has proven to be a very promising delivery method  Figure .As mentioned earlier, a major obstacle to the medical application of molluscan venom peptides, and indeed peptides in general, is their poor pharmacokinetic profile. In addition, peptides generally exhibit poor membrane solubility and can be rapidly cleared through the liver and kidneys . FinallyThere are numerous strategies for improving the pharmacokinetic profile of therapeutic peptides. One approach is to stabilize the structure of the peptide itself through such methods as peptide stapling, macrocyclization, or grafting of peptide segments onto a small protein scaffold ,160,188.As an alternative to modifying the peptide, our Trojan Horse strategy involves packaging the peptide of interest within a macromolecular nanoparticle that can deliver it to its molecular target and protect it from degradation during transport. Several types of macromolecules have been investigated as potential drug-delivery nanocontainers including liposomes , naturalSalmonella typhimurium bacteriophage P22 as a nanocontainer for the delivery of ziconotide  across the blood-brain barrier (BBB) [Our recently developed peptide drug delivery method repurposes the procapsid from the er (BBB) . Similarer (BBB) . By modier (BBB) ,198. Amoer (BBB) ,197,199.VLPs have a number of significant advantages compared with other macromolecules: First, they are generally uniform in size and composition and possess defined architectures—traits that can allow for precise control of pharmacological properties. Second, as proteins, they are biodegradable by endogenous cellular pathways, reducing the ability to accumulate in an organ. Also, as gene products, VLPs can be produced relatively easily and in high yields using standard molecular biology protocols. Finally, a plethora of tried-and-tested protein modification techniques are available for manipulating the interior and exterior of proteinaceous VLPs such as molecular cloning, standard and unnatural amino acid mutagenesis, protein bioconjugation, and directed evolution .in vivo; and (3) induced disassembly of the capsid and release of the cargo under physiological conditions  (®), across in vitro and in vivo BBB models. Briefly, the cell-penetrating HIV-Tat peptide (YGRKKRRQRRR) was synthesized, fluorescently labeled, and activated with maleimidopropionic acid (MPA), then conjugated to a P22 nanocontainer preloaded with ziconotide and engineered to feature a surface exposed cysteine residue. P22-Tat nanocontainers translocated the BBB, demonstrating the feasibility of this Trojan Horse strategy [In general, the adaptation of protein cages for drug delivery involves three distinct steps: (1) encapsulation of the pharmacological agent within the viral capsid; (2) targeting of the capsid to the desired site ronment) . Applyinstrategy . At a sistrategy ,199.The next step in the adaptation of VLPs for drug delivery, namely controllable disassembly, remains a challenge, mainly due to the fact that the disassembly mechanism must proceed under physiological aqueous environment, with moderate temperature and neutral pH. Overcoming this obstacle requires an integrated approach at the intersection of chemistry and biology, with a particular emphasis on materials science, structural virology, and protein engineering. Recent advances in bioorthogonal chemistry have led to the identification of numerous reactions that proceed under physiological conditions. We are currently investigating the Ring Opening Metathesis Polymerization (ROMP) triggered by a ruthenium catalyst  for the controlled disassembly of the P22 VLP .cis-cyclooctene, or norbornene [ROMP is a polymerization reaction initiated by a transition-metal catalyst and driven by the release of ring strain in a cyclic olefin such as cyclobutene, cyclopentene, rbornene . The ROMrbornene ,203,204.rbornene . As we hrbornene ,199. We learn-from-nature integrative venomics strategy for the discovery and characterization of terebrid venom peptides (This review describes our peptides . This mu


While the genomic revolution has dramatically accelerated the discovery of disease-associated genes, the functional characterization of the corresponding proteins lags behind. Most proteins fulfill their tasks in complexes with other proteins, and analysis of protein–protein interactions (PPIs) can therefore provide insights into protein function. Several methods can be used to generate large-scale protein interaction networks. However, most of these approaches are not quantitative and therefore cannot reveal how perturbations affect the network. Here, we illustrate how a clever combination of quantitative mass spectrometry with different biochemical methods provides a rich toolkit to study different aspects of PPIs including topology, subunit stoichiometry, and dynamic behavior. Technologies like protein microarrays, phage display and the yeast two-hybrid system permit high-throughput screens for PPIs. However, these approaches rely on in vitro assays or heterologous biological systems. Therefore, it is not clear if PPIs detected by these methods occur in the relevant in vivo context.Proteins do not act in isolation but typically mediate their biological functions by interacting with other proteins . Owing tAffinity purification combined with mass spectrometry (AP-MS) has emerged as a particularly attractive method for PPI mapping . A majorWe argue that q-AP-MS is one of the most powerful technologies to map PPIs in health and disease. The aim of this Mini Review is to briefly explain the general principle of q-AP-MS and to emphasize the versatility of AP-MS to investigate various aspects of PPIs including quantities, topology, subunit stoichiometry, and dynamic behavior. We will begin with a brief introduction to quantitative shotgun proteomics.1). Second, in order to determine the amino acid sequence, the instruments selects individual peptides for fragmentation and records the resulting fragment spectra (MS2). Data generated in this manner is then compared to protein databases for peptide and protein identification  of proteins interacting with small molecules and peptides, it should be generally applicable to a range of ligands, including entire proteins, used as baits.Binding affinity is a particularly relevant quantity with regard to characterizing the interaction between two proteins. Typically, binding affinities are measured using methods such as ITC or SPR assays which require considerable quantities of purified proteins. q-AP-MS experiments can also be designed in a way to provide information about binding affinities : First, The examples described above show that a combination of quantitative shotgun proteomics with various biochemical methods can provide a rich toolkit to explore various aspects of PPIs. This can be employed to (i) identify binding partners with high specificity, (ii) assess the stoichiometry of complexes, (iii) provide information about interaction interfaces, (iv) analyze binding affinities, and (v) study dynamic changes of PPIs upon perturbation. Bearing in mind possible pitfalls , mass spThe 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.


Functional neuroimaging research has identified multiple brain regions supporting reading-related activity in typical and atypical readers across different alphabetic languages. Previous meta-analyses performed on these functional magnetic resonance imaging findings typically report significant between-group contrasts comparing typical readers and readers with reading difficulty or a clinical diagnosis of developmental dyslexia. In order to advance our understanding of cross-linguistic convergence of reading-related brain activations for these reader groups, analyses using activation likelihood estimation were carried out separately for typical and atypical readers who ranged from children to adults. Contrasts were analyzed for tasks involving rhyming or reading of letter or word stimuli presented visually in English, Dutch, Italian, German, French, or Norwegian. Typical readers showed reliable activation in only left lateralized regions, including the inferior frontal area, precentral area and middle temporal gyrus. Atypical readers also showed activation in the left inferior frontal area and precentral region, in addition to significant activations in the right hemisphere, including the superior, medial and inferior frontal regions, lingual gyrus and the inferior occipital area. These results distinguish between typical and atypical reader group activations, showing common and distinct regions of activation when engaged in reading-related activities, extending previous meta-analyses on identifying brain regions relevant to reading to include cross-linguistic analyses for alphabetic scripts. Results support the universality of a signature pattern of brain activation in developmental dyslexia across alphabetic languages. Reading involves translating written symbols to sounds in order to extract meaning. This process is complex, and to be fluent, readers coordinate multiple skills such as word decoding and reading rate effectively and simultaneously so that they can attend to comprehension demands from the text . Given tstruggling readers refers to children and adults fitting one or more of the following criteria: (1) reported familial risk of reading difficulty; (2) received a clinical diagnosis of dyslexia; (3) showed significantly lower performance in reading-related tasks  and the low performance cannot be attributed to impoverished learning opportunity, dysfunctional visual or auditory processing and inferior intelligence.The present study aims to extend these findings by examining cross-linguistic convergence of brain activation patterns for typical readers and struggling readers separately, rather than comparatively. To date, empirical evidence largely contributes to our understanding of brain regions that are engaged in typical readers versus struggling readers, which offers a comparative perspective on brain activations for reading. The focus of these analyses is to identify the common and distinct brain regions that contribute to reading in typical readers and in struggling readers. To expand our understanding of reading brain systems, the current study examines brain systems engaged for typical readers and struggling readers as distinct groups. With the aim to examine cross-linguistic convergence, studies conducted in different alphabetic languages and countries are included in this meta-analysis. These studies have variable inclusionary criteria for struggling readers. In the present paper, the term Converging evidence across functional magnetic resonance imaging (fMRI) studies indicate a distinct brain activation pattern for reading. Early in reading development, children show activations in bilateral regions in temporo-parietal, temporo-occipital, and inferior frontal regions . During In an effort to summarize neuroimaging findings comparing sets of brain regions supporting the processing of reading-related tasks, meta-analysis has been carried out on between-group contrasts comparing typical and struggling readers , 2011. Tdivergent brain activation patterns between-groups. However, these prior meta-analyses do not speak to the convergence of brain activation between-groups. To address this gap in the literature, the present meta-analysis examines brain regions recruited to engage in reading-related tasks separately for typical and struggling readers. Unlike previous meta-analyses, we used meta-analysis with the goal of identifying both common and distinct brain regions in children and adults with varying reading ability across different alphabetic script-based languages. This approach can help identify clusters that can be used in future connectivity analyses to illuminate the interaction between brain regions as an interconnected network that supports reading. Specifically, selecting brain regions that are common to both typical and atypical readers could inform understanding of the common and differential functional networks that are employed in readers with diverse reading capacity. To this end, we included published studies that reported activation coordinates separately for typical and struggling readers. Two meta-analyses were conducted separately for these two groups of readers. Given that children are in the process of developing fluent reading and that engaging in reading-related tasks is effortful, it is expected that typical and struggling readers would recruit left frontal regions when engaging in reading-related tasks. However, we expected typical and struggling readers to demonstrate different activation patterns in posterior brain regions because of struggling readers’ atypical mapping of sound-symbol correspondence.The above meta-analyses focus on between-group contrasts and thus are helpful in understanding Table 1).An initial PubMed database search with search criteria: “dyslexia” or “reading difficulty” and “fMRI” or “PET” yielded 508 potential papers. These papers were then reviewed against a set of inclusion criteria. Papers included in the analysis were published in English, used fMRI or PET methods, used whole brain analyses, reported foci separately for typical and atypical readers who had matched demographics, included visual tasks that involved letter or word stimuli, and involved alphabetic languages. Case studies or studies involving clinical populations  were excluded. Studies reporting region-of-interest (ROI) analyses were also excluded. A final set of 13 papers contributed 131 foci for typical readers and 101 foci for atypical readers (see Table 1). In one study, Using this set of inclusion–exclusion criteria, we were able to obtain two samples of typical and struggling readers with comparable sample sizes and matched demographic characteristics. In total, there were 172 typical readers and 174 struggling readers across the 13 studies . Ten studies examined child and/or adolescent participants, two studies examined adult participants, and one study examined participants from adolescence into adulthood.Table 2, studies 1, 2, 6, 8–10, 12, 13) separately reported more than one contrast for both typical and struggling readers, such as an activation condition  versus baseline  and an activation condition versus a control condition . Almost all studies required a button press ; one study required movement of a joystick (1) and one did not require a motor response (9). In accordance with prior meta-analyses involving participants with dyslexia  and left precentral gyrus (BA 6). Since half of the studies included in the analysis were conducted in English, we further investigated whether the reported regions were biased toward English, which has a higher entropy between phonemes and graphemes relative to other alphabetic languages in the analysis . Because contributing studies are only those that report foci within the boundary of the cluster, additional studies that are not listed may have contributed foci that are located near and just outside of the cluster boundary  and the second largest cluster was in the right insula (BA 18). These two largest clusters were much smaller in volume compared to those reported for typical readers. No study was reported as contributing to the right medial frontal gyrus . The lack of contributing studies indicated no reported focus was within the boundary of this cluster, although as described above, there may be foci reported surrounding the significant cluster. Therefore, the lack of contributing studies does not invalidate the results. In regards to the potential bias of contributing studies based on language of administration, studies conducted in English seem to contribute to larger clusters.Figure 1, with the overlapping regions coded as yellow. Two converging regions were observed between typical and atypical readers: the LIFG (BA 44) and the left superior frontal gyrus (BA 6). Aside from these frontal regions, no converging regions were observed in the posterior brain regions. In terms of regions showing divergent activation patterns, struggling readers recruited bilateral frontal regions, parietal and occipital regions when completing reading-related tasks while typical readers showed robust activation in the inferior frontal and fusiform gyrus (BA 37).Significant activated regions for typical and struggling readers are shown in red and blue, respectively, in The present study examined the distinct and overlapping brain regions in typical and struggling readers when engaging in reading-related tasks. Using ALE, 13 studies involving matching typical and struggling readers were included in the present meta-analyses. Previous work has focused on between-group contrasts of activation, highlighting particular brain regions with hyper- or hypoactivation in struggling readers compared to typical readers. The present report included separate analyses for typical developing and struggling readers. Unlike between-group contrasts of activation, these separate analyses surfaced individual activation patterns for typical and struggling readers across brain regions, respectively. In reporting these patterns separately for each group, we were additionally able to qualitatively compare brain regions across groups of typical and struggling readers. Importantly, in addition to identifying distinct regions of activation, we were able to examine common regions of shared activation between typical and atypical readers. Three major findings were observed: (1) Typical readers showed left lateralized activation in frontal and temporal areas when engaging in reading-related tasks; (2) Struggling readers showed distributed bilateral activation patterns in frontal, temporal, and occipital regions; and (3) Both typical and struggling readers showed activation in the left inferior frontal area and in the precentral area. We consider the implications of each of these results in turn.Table 1 involved phonological processing, it is reasonable to believe that the results observed in the typical readers were indicative of a relatively secure phonological processing system, which is an important component to reading success in alphabetic languages.As a complex behavior that takes years to develop and acquire, reading relies on a set of brain regions to orchestrate coherently. Consistent with previous research and meta-analysis, left lateralized regions were observed to have reliable activation across studies in the typical readers, including inferior and superior frontal regions and associative temporal areas e.g., . This leThe second analysis involving struggling readers showed a bilaterally distributed activation pattern, which is consistent with previous research using empirical functional connectivity analysis e.g., . In addiTable 3), indicating functional convergence of lexical retrieval demands in the tasks across studies.Across all the studies, the participants were matched on demographics and background aside from their reading performance. Therefore, in the present study, we examined activation patterns that were common across the two groups to investigate their convergence and divergence in activation patterns. Two regions that both typical and struggling readers recruited to support reading-related activity were the left inferior frontal regions and the left precentral area. One implication of the significant activation observed in the left inferior frontal region may suggest top-down cognitive control relevant to reading. Recent findings offer empirical support for the importance of executive functions and other neurocognitive functions for reading . There iAnother overlapping region between the two groups was in the precentral area. Aside from the possibility that this region is recruited to prepare behavioral responses during tasks, it is possible that activation in the precentral area is relevant to responsiveness of auditory information . In Monza priori theoretical approach. Such efforts can indicate not only which distinct regions are relevant for reading, but also how regions operate as a network of coactivated regions in time. Understanding the network properties of the typical and atypical reading brain has the potential to inform practice in several ways: mechanisms underlying reading difficulty can be further elucidated; developmental responses in establishing integrity of reading networks can be determined; and different pathways for remediation and compensation can be examined. Targeted questions for further study should investigate the efficacy of different reading programs according to factors related to the program  and to the students .The observation of common regions of activation across the two groups suggested that typical and struggling readers showed convergent activation patterns in anterior regions when engaging in reading-related tasks. However, it was possible that typical and struggling readers have differential magnitudes of activation even though the same brain regions were recruited in reading-related activity , 2011. IThe present analyses were limited in a few ways that can inform future research directions. First, while we included only studies involving reading in alphabetic languages, there may be language-specific considerations that limit our findings. Across different alphabetic languages, heterogeneous entropy measures have been observed between grapheme–phoneme mappings , 2005. WSecond, the present findings are limited by the variance in age groups and diverse experimental and baseline tasks across the included studies. The ages of typical and struggling readers were matched within individual studies, but participant age spanned a wide developmental range within each group of readers. As a result, the meta-analytic results for each group may not correspond to developmental or experiential patterns. As discussed above, qualitative differences in the activation of posterior brain regions were observed across groups. However, we acknowledge that the specific posterior brain regions that are recruited or the degree of activation of these regions is dependent on age, reading experience and tasks .The current meta-analyses investigated regions of activation for typical and struggling readers when they were engaged in reading-related tasks, across alphabetic languages. Building on prior work, we conducted separate meta-analyses for typical and struggling readers and through qualitative comparison identified both divergent and convergent patterns of activation. In line with prior research, typical readers showed left lateralized activation in frontal and temporal areas, while struggling readers showed diffuse activation in bilateral frontal, temporal, and occipital regions. In addition, we found convergent regions of activation in the left inferior frontal and precentral areas. With a highly complex behavior such as reading, the learning experience refines the orchestration of a network of brain regions. Based on the results in the present study, reading difficulty is associated with a disruption in the functional activation patterns of key components in the reading brain network. However, the mechanism underlying this functional disruption and how it relates to behavior requires further investigation, considering a developmental framework. Future research taking a network approach investigating the relationship between brain function, brain structure and behavior will shed light on how typical and atypical reading develops. More importantly, designing innovative interventions or support systems for struggling readers may benefit from understanding the similarity and differences in brain and behavior.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.